ABCMdb

ABCC7 p.Gly542*

ClinVar:	c.1624G>T , p.Gly542* D , Pathogenic
CF databases:	c.1624G>T , p.Gly542* D , CF-causing c.1625G>A , p.Gly542Glu (CFTR1) ? ,

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[hide] Insight in eukaryotic ABC transporter function by ... FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19. Frelet A, Klein M
Insight in eukaryotic ABC transporter function by mutation analysis.
FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19., 2006-02-13 [PMID:16442101]

Abstract [show]
With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.

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314 [142] G542X, W1316X G542X and W1316X nonsense mutants fail to generate functional CFTR Cl-channels. Login to comment

[hide] Molecular analysis of the cystic fibrosis gene rev... Mol Hum Reprod. 1999 Jan;5(1):10-3. Lissens W, Mahmoud KZ, El-Gindi E, Abdel-Sattar A, Seneca S, Van Steirteghem A, Liebaers I
Molecular analysis of the cystic fibrosis gene reveals a high frequency of the intron 8 splice variant 5T in Egyptian males with congenital bilateral absence of the vas deferens.
Mol Hum Reprod. 1999 Jan;5(1):10-3., [PMID:10050655]

Abstract [show]
It has previously been shown that defects in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are largely responsible for the condition of congenital bilateral absence of the vas deferens (CBAVD), without associated renal abnormalities, in Caucasian populations. To assess the involvement of the CFTR in CBAVD in a population with presumed low cystic fibrosis (CF) frequency, we have analysed 20 CBAVD males from Egypt for the presence of 12 common Caucasian CFTR mutations and the intron 8 5T splice variant, IVS-5T, known to be a major cause of CBAVD in Caucasian patients. In 16 of the males without associated renal abnormalities only one deltaF508 carrier was identified, but an exceptionally high frequency of the IVS-5T variant was found (14 of 32 alleles or 43.7%), confirming that this variant is involved in many cases of CBAVD, even in populations where CF is rare. CFTR mutations or the IVS-5T variant were found neither in the remaining four patients with associated renal abnormalities nor in the spouses of the 20 CBAVD patients. However, one patient was homozygous for a leucine to proline substitution at amino acid position 541 (L541P) of the CFTR. It is as yet not clear whether this change is involved in CBAVD in this male.

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32 Initially, eight of the males` samples were analysed by a commercial kit allowing the detection of eight mutations in the CFTR gene: ∆F508, ∆I507, G542X, G551D, R553X, 1717-1G→A, W1282X and N1303K (INNO-LiPA CF, Innogenetics, Zwijnaarde, Belgium). Login to comment
52 None of the other individuals showed a mutation except the male with the ectopic pelvic kidney (patient ii in the previous paragraph) who had neither a normal nor a mutated band at the G542X site when studied by the INNO-LiPA CF kit. Login to comment
55 This base substitution probably inhibits the binding of the exon 11 PCR fragment to the normal (and mutant) sequence at the G542X locations in the INNO-LiPA CF kit. Login to comment

[hide] Analysis of the complete coding region of the CFTR... Hum Hered. 1999 Mar;49(2):81-4. Loumi O, Baghriche M, Delpech M, Kaplan JC, Bienvenu T
Analysis of the complete coding region of the CFTR gene in ten Algerian cystic fibrosis families.
Hum Hered. 1999 Mar;49(2):81-4., [PMID:10077727]

Abstract [show]
The spectrum of cystic fibrosis (CF) mutations in the North African population remains poorly known. In order to offer an effective diagnostic service and to determine accurate risk estimates, we decided to identify the CF mutations in 10 Algerian CF families. We carried out a chemical-clamp denaturing gradient gel electrophoresis analysis of the CFTR gene and automated direct DNA sequencing. We identified 5 mutations and we characterized 60% of the CF chromosomes. Taking advantage of the homogeneity of the sample, we report clinical features of homozygous CF patients.

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37 The V754M (G to A at position 2392) mutation has previously been reported to the Cystic Fibrosis Genetic Analysis Consortium by Roger Mountford and seems to confer moderate disease when it is associated either with 1812 - 1G→A or G542X. Login to comment
43 Surprisingly, none of the defined mutations (G542X, R553X, G551D, 1717 - 1G→A) which occur relatively frequently in exon 11 in Caucasian populations was identified in our Algerian population. Login to comment

[hide] The complex relationships between cystic fibrosis ... Hum Reprod. 1999 Feb;14(2):371-4. Dohle GR, Veeze HJ, Overbeek SE, van den Ouweland AM, Halley DJ, Weber RF, Niermeijer MF
The complex relationships between cystic fibrosis and congenital bilateral absence of the vas deferens: clinical, electrophysiological and genetic data.
Hum Reprod. 1999 Feb;14(2):371-4., [PMID:10099982]

Abstract [show]
Congenital bilateral absence of the vas deferens (CBAVD) is found in 1-2% of infertile males and in most male cystic fibrosis (CF) patients. CF and some of the CBAVD cases were found to share the same genetic background. In this study, 21 males with CBAVD had extensive physical and laboratory testing for symptoms of CF. Possible defective cellular chloride transport was measured by interstitial current measurement of rectal suction biopsies. Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation analysis was performed for 10 common CFTR mutations. CF-related symptoms were found in six men. On laboratory testing slightly abnormal liver and pancreatic function was found in seven patients. The sweat test was found to be abnormal in four patients; interstitial current measurement showed defective chloride excretion in 11 patients. CFTR gene mutations were found in 66% of the patients: eight were compound heterozygotes; in six, only one common mutation could be detected. The 5T allele in one copy of intron 8 was found in four men. CBAVD appears to be a heterogeneous clinical and genetic condition. A CFTR gene mutation was found in both copies of the allele or interstitial current measurement showed defective chloride excretion in 14/21 cases. Genetic counselling is clearly indicated for couples seeking pregnancy through epididymal or testicular sperm aspiration and intracytoplasmic sperm injection.

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58 CFTR mutation analysis was performed for 10 mutations: we analysed for the mutations R117H, A455E, ∆F508, 1717-1G→A, G542X, R553X, R1162X, S1251N, W1282X, and N1303K. Login to comment

[hide] Fluorescent chloride indicators to assess the effi... Hum Gene Ther. 1999 Apr 10;10(6):861-75. Mansoura MK, Biwersi J, Ashlock MA, Verkman AS
Fluorescent chloride indicators to assess the efficacy of CFTR cDNA delivery.
Hum Gene Ther. 1999 Apr 10;10(6):861-75., 1999-04-10 [PMID:10223721]

Abstract [show]
Cl(-)-sensitive fluorescent indicators have been used extensively in cell culture systems to measure the Cl(-)-transporting function of the cystic fibrosis transmembrane conductance regulator protein CFTR. These indicators have been used in establishing a surrogate end point to assess the efficacy of CFTR cDNA delivery in human gene therapy trials. The ability to measure Cl- transport with high sensitivity in small and heterogeneous tissue samples makes the use of Cl- indicators potentially attractive in gene delivery studies. In this review article, the important technical aspects of Cl- transport measurements by fluorescent indicators such as SPQ are described, applications of Cl- indicators to assay CFTR function are critically evaluated, and new methodological developments are discussed. The available Cl- indicators have been effective in quantifying Cl- transport rates in cell culture models and in vitro systems such as isolated membrane vesicles and liposomes. However, the imperfect photophysical properties of existing Cl- indicators limit their utility in performing measurements in airway tissues, where gene transfer vectors are delivered in CF gene therapy trials. The low efficiency of gene transfer and the cellular heterogeneity in airway samples pose substantial obstacles to functional measurements of CFTR expression. Significant new developments in generating long-wavelength and dual-wavelength halide indicators are described, and recommendations are proposed for the use of the indicators in gene therapy trials.

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249 Cl2 INDICA TOR APPLICATION TO STUDY CFTR FUNCTION (CONT`D ) Cell or tissue Protocol Cell SPQ Quench Findings/significance type Descriptiona loading ion Ref. Deletion of 19 residues HEK 293- Embryonic kidney cell line Passive Cl Xie et al. in the loop between EBNA 5 mM (1995) TM4 and TM5 12±18 hr eliminated Cl transport Coexpression of the N HeLa Cervical carcinom a (epithelial) Passive I Ostedgaard and C halves of CFTR 10 mM et al. resulted in functional 12±18 hr (1997) Cl channels C orrection of C F phenotype by pharmacological maneuvers Increased [IBMX] L cells Mouse fibroblasts Hypotonic I Yang et al. improved D F508 12 hr (1993) response; reduced temperature improved D F508 and G551D Transfer of purified CHO-CFTR Chinese hamster ovary Passive I Marshall CFTR protein induced FRT-CFTR Fisher rat thyroid epithelial 10 mM et al. Cl2 transport LLCPK1 Pig kidney epithelial 12±18 hr (1994) Overexpression of CFPAC-1 Pancreatic duct adenocarcinoma Passive or I Cheng et al. D F508 with sodium JME/CF15 (D F/D F) Hypotonic (1995) butyrate resulted in 1° culture Transformed CF airway 10 mM detectable Cl2 transport epithelial CF airway epithelial Chemical chaperones Swiss 3T3 Mouse embryo fibroblast Passive Cl Brown et al. (glycerol, D2O, TMAO) 5 mM (1996) corrected the CF 12±18 hr phenotype Aminoglycosid e antibiotics HeLa Cervical carcinom a (epithelial) Hypotonic I Howard et al. overcam e premature 10 mM (1996a) stop codons and 10 min corrected CF phenotype Deoxyspergualin C127-CFTR Mouse mammary epithelial Hypotonic I Jiang et al. delivered D F508 to JME/CF15 Transformed CF airway 10 mM (1998) plasma membrane and IBE-1 epithelial 4 min partially restored Cl2 Transformed intrahepatic transport (D F/G542X) Abbreviations: wt, wildtype; PKA, protein kinase A; TM, transmembrane ; TMAO, trimethylamine -N-oxide. Login to comment
252 The efficacy of aminoglycoside s in promoting read-through in the CFTR nonsense mutations G542X and R553X was shown using Cl2 indicators (Howard et al., 1996a). Login to comment

[hide] Blood immunoreactive trypsinogen concentrations ar... Acta Paediatr. 1999 Mar;88(3):338-41. Lecoq I, Brouard J, Laroche D, Ferec C, Travert G
Blood immunoreactive trypsinogen concentrations are genetically determined in healthy and cystic fibrosis newborns.
Acta Paediatr. 1999 Mar;88(3):338-41., [PMID:10229049]

Abstract [show]
Newborns with cystic fibrosis (CF) have increased blood immunoreactive trypsinogen concentrations. When screening for CF in the newborn by immunoreactive trypsinogen measurement, an abnormally high proportion of healthy deltaF508 carriers is found among false-positive neonates, suggesting that a relationship could exist between immunoreactive trypsinogen concentration at birth and the genetic status. Therefore, this study analysed the possible relationships between neonatal blood immunoreactive trypsinogen concentrations and genotype in 1842 healthy newborns and 111 CF patients detected by a neonatal screening programme. A close correlation was found between immunoreactive trypsinogen and deltaF508: the probability of a healthy newborn being a carrier of this mutation increased regularly with the neonatal immunoreactive trypsinogen concentration. In CF patients, there was a significant difference between deltaF508 homozygotes and deltaF508/X (X = other mutation) compound heterozygotes with respect to the mean neonatal blood immunoreactive trypsinogen concentration. CF neonates with two mutations affecting the nucleotide binding domains of the cystic fibrosis transmembrane conductance regulator protein had significantly higher mean immunoreactive trypsinogen concentrations than patients with one mutation affecting a membrane-spanning domain. The data strongly suggest that the neonatal immunoreactive trypsinogen concentration is, in part, genetically determined, with a wide range of variations, similar to the features which have been shown for the relations between the genotype and clinical phenotypes of CF patients.

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61 Twins or unrelated patients with identical genotype had very similar neonatal IRT concentrations: DF508/I148T (twins), 1040 and 1055 mg LÀ1 ; N1303K/G149R (twins), 1600 and 1725 mg LÀ1 ; DF508/E585X, 900 and 945 mg LÀ1 ; DF508/ G542X, 1535 and 1660 mg LÀ1 ; DF508/L206W, 980, 1090 and 1100 mg LÀ1 . Login to comment
72 In this study, CF newborns with one mutation in an exon encoding for either NBD1 or NBD2 (DF508, G542X, G551D, E585X, N1303K, etc.) and the other affecting one of the MSD (R117H, 574delA, I148T, G149R, L206W, etc.) had significantly lower IRT concentrations than CF neonates with both mutations located in NBD. Login to comment

[hide] Blood concentrations of pancreatitis associated pr... Arch Dis Child Fetal Neonatal Ed. 1999 Mar;80(2):F118-22. Sarles J, Barthellemy S, Ferec C, Iovanna J, Roussey M, Farriaux JP, Toutain A, Berthelot J, Maurin N, Codet JP, Berthezene P, Dagorn JC
Blood concentrations of pancreatitis associated protein in neonates: relevance to neonatal screening for cystic fibrosis.
Arch Dis Child Fetal Neonatal Ed. 1999 Mar;80(2):F118-22., [PMID:10325788]

Abstract [show]
AIM: To determine whether pancreatitis associated protein (PAP) is a marker for cystic fibrosis which could be used in neonatal screening for the disease. METHODS: PAP was assayed on screening cards from 202,807 neonates. Babies with PAP > or = 15 ng/ml, or > or = 11.5 ng/ml and immunoreactive trypsinogen (IRT) > or = 700 ng/ml were recalled for clinical examination, sweat testing, and cystic fibrosis transmembrane regulator (CFTR) gene analysis. RESULTS: Median PAP value was 2.8 ng/ml. Forty four cases of cystic fibrosis were recorded. Recalled neonates (n = 398) included only 11 carriers. A receiver operating characteristic curve analysis showed that PAP above 8.0 ng/ml would select 0.76% of babies, including all those with cystic fibrosis, except for one with meconium ileus and two with mild CFTR mutations. Screening 27,146 babies with both PAP and IRT showed that only 0.12% had PAP > 8.0 ng/ml and IRT > 700 ng/ml, including all cases of cystic fibrosis. CONCLUSION: PAP is increased in most neonates with cystic fibrosis and could be used for CF screening. Its combination with IRT looks promising.

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77 Other genotypes were F508/G542X (n=4), F508/N1303K (n=2), F508/I148T (n=2), F508/R117H, F508/R553X, F508/1717-1G->A, F508/ 1078delT, F508/2789+5G->A, F508/ E1308X (a novel CFTR mutation), R553X/ 394delTT, and N1303K/R553X. Login to comment

[hide] DeltaF508 CFTR protein expression in tissues from ... J Clin Invest. 1999 May 15;103(10):1379-89. Kalin N, Claass A, Sommer M, Puchelle E, Tummler B
DeltaF508 CFTR protein expression in tissues from patients with cystic fibrosis.
J Clin Invest. 1999 May 15;103(10):1379-89., 1999-05-15 [PMID:10330420]

Abstract [show]
Heterologous expression of the cystic fibrosis transmembrane conductance regulator (CFTR) provided evidence that the major cystic fibrosis (CF) mutation DeltaF508 leads to defective protein folding in the endoplasmic reticulum, which prevents its processing and targeting to the cell surface. In this study, we investigated endogenous CFTR expression in skin biopsies and respiratory and intestinal tissue specimens from DeltaF508 homozygous and non-CF patients, using immunohistochemical and immunoblot analyses with a panel of CFTR antibodies. CFTR expression was detected at the luminal surface of reabsorptive sweat ducts and airway submucosal glands, at the apex of ciliated cells in pseudostratified respiratory epithelia and of isolated cells of the villi of duodenum and jejunum, and within intracellular compartments of intestinal goblet cells. In DeltaF508 homozygous patients, expression of the mutant protein proved to be tissue specific. Whereas DeltaF508 CFTR was undetectable in sweat glands, the expression in the respiratory and intestinal tracts could not be distinguished from the wild-type by signal intensity or localization. The tissue-specific variation of DeltaF508 CFTR expression from null to apparently normal amounts indicates that DeltaF508 CFTR maturation can be modulated and suggests that determinants other than CFTR mislocalization should play a role in DeltaF508 CF respiratory and intestinal disease.

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12 Skin biopsies were taken from the right shoulder of 5 healthy volunteers, 4 ∆F508 homozygous CF patients, and 3 CF patients homozygous for the stop mutations R553X (n = 1) and G542X (n = 2). Login to comment
65 Three skin biopsies from patients homozygous for the CFTR stop mutations R553X and G542X and 1 rectal suction biopsy homozygous for an out-of-frame deletion of exons 2 and 3 (biopsy and mutation analysis provided by F. Mekus, Medizinische Hochschule Hannover) that results in a stop in exon 4 were analyzed by CFTR immunohistochemistry 1380 The Journal of Clinical Investigation | May 1999 | Volume 103 | Number 10 Figure 1 CFTR antibodies PAC13, PAC865, MATG1104, and M3A7 detect the characteristic immunoreactive CFTR bands in immunoblot analysis of T84 cells. Login to comment
133 3a, 3b, 4a, 4b, 5a, 5b: ×6,168. ing was demonstrated by common negative controls and the absence of specific signals in skin biopsies homozygous for the stop mutations G542X and R553X (n = 3). Login to comment

[hide] Screening for cystic fibrosis transmembrane conduc... Mol Hum Reprod. 1999 Jun;5(6):587-93. Boucher D, Creveaux I, Grizard G, Jimenez C, Hermabessiere J, Dastugue B
Screening for cystic fibrosis transmembrane conductance regulator gene mutations in men included in an intracytoplasmic sperm injection programme.
Mol Hum Reprod. 1999 Jun;5(6):587-93., [PMID:10341008]

Abstract [show]
The present study was undertaken to evaluate the frequency and nature of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in infertile patients undergoing intracytoplasmic sperm injection. A total of 90 patients were screened for a panel of 10 mutations in the CFTR gene frequently involved in congenital absence of the vas deferens (CAVD); the patients included 14 with azoospermia and CAVD, 39 patients with azoospermia without CAVD (n = 39) and 37 patients with severe oligozoospermia. The length of the polymorphic polypyrimidine tract (allele 5T, 7T and 9T) in the intron 8/exon 9 splice-acceptor site was also determined. In 10 out of 14 patients with CAVD, CFTR mutations were found; nine patients had one DeltaISOdiaDeltaF508 mutation and one patient had two CFTR mutations (N1303K/R117H). Allele 5T was present in eight of these patients. In six patients, 5T was the non-DeltaISOdiaDeltaF508 allele and in two patients there was no known CFTR mutation. None of the CFTR mutations were observed in patients with azoospermia without CAVD or with severe oligozoospermia and the frequency of allele 5T was 3.6% (three out of 78 alleles) and 1.35% (one out of 74 alleles) respectively. Our observation suggests that the CFTR gene is not involved in either spermatogenesis or in the pathology of the genital tract, except for CAVD.

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51 Each patient was tested for the nine most frequent cystic fibrosis-causing CFTR mutations: ∆F508, ∆I507, 1717-1G→A, G542X, G551D, R553X, W1282X, N1303K, 621ϩ1G→T and the three most frequent CFTR mutations involved in CBAVD (∆F508, R117H and the IVS8 polyT). Login to comment
53 The other mutations were detected using either heteroduplex analysis (∆I507), allele specific oligonucleotide (ASO) hybridization (G542X, 1717-1G→A, IVS8 polyT) (Kerem et al., 1990), restriction endonuclease analysis (G551D, R553X, W1282X) (Zielenski et al., 1991) or polymerase chain reaction (PCR)-mediated site-directed mutagenesis (621ϩ1G→T, R117H, N1303K) (Friedman et al., 1991). Login to comment
67 ASO hybridization PCR-amplified DNA (6 µl) of exon 9 or exon 11 was denatured and vacuumblotted onto nylon membrane(Hybond Nϩ, Amersham Pharmacia Biotech, Orsay, France) and hybridized with oligonucleotides which recognize the presence of 5T, 7T or 9T for detection of the IVS8 polyT-stretch, normal or mutated allele for 1717-1G→A and G542X. Login to comment
94 Methods for detecting mutations Mutation Method R117H PCR-mediated-site-directed mutagenesis, HaeII digestion N: 113 ϩ 24 bp R117H: 137 bp 621ϩ1G→T MseI digestion N: 269 ϩ 33 bp 621ϩ1G→T: 215 ϩ 54 ϩ 33 bp IVS8polyT ASO hybridization: hybridization at 50°C 5T: TGT GTG TGT TTT TAA CAG washing at 55°C 7T: TGT GTG TTT TTT TAA CAG washing at 51°C 9T: GTG TGT TTT TTT TTA ACA G washing at 55°C ∆I507 Heteroduplex DNA formation ∆F508 Heteroduplex DNA formation (see Figure 1) 17171G→A ASO hybridization, hybridization at 42°C, washing at 54°C N: TTT GGT AAT AGG ACA TCT CC 17171G→A: TTT GGT AAT AAG ACA TCT CC G542X ASO hybridization, hybridization at 42°C, washing at 49°C N: ACC TTC TCC AAG AAC T G542X: ACC TTC TCA AAG AAC T G551D DpnII digestion: N: 425 bp G551D: 243 ϩ 182 bp R553X HincII digestion N: 239 ϩ 186 bp R553X: 425 bp W1282X MnlI digestion N: 178 ϩ 172 ϩ 123 bp W1282X: 301 ϩ 172 bp N1303K PCR-mediated-site-directed mutagenesis, BstNI digestion N: 266 ϩ 23 bp N1303K: 289 bp The underlining indicates the location of nucleotide substitution in normal and mutated allele. Login to comment

[hide] Proportion of cystic fibrosis gene mutations not d... JAMA. 1999 Jun 16;281(23):2217-24. Mak V, Zielenski J, Tsui LC, Durie P, Zini A, Martin S, Longley TB, Jarvi KA
Proportion of cystic fibrosis gene mutations not detected by routine testing in men with obstructive azoospermia.
JAMA. 1999 Jun 16;281(23):2217-24., 1999-06-16 [PMID:10376575]

Abstract [show]
CONTEXT: Infertile men with obstructive azoospermia may have mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, many of which are rare in classic cystic fibrosis and not evaluated in most routine mutation screening. OBJECTIVE: To assess how often CFTR mutations or sequence alterations undetected by routine screening are detected with more extensive screening in obstructive azoospermia. DESIGN: Routine screening for the 31 most common CFTR mutations associated with the CF phenotype in white populations, testing for the 5-thymidine variant of the polythymidine tract of intron 8 (IVS8-5T) by allele-specific oligonucleotide hybridization, and screening of all exons through multiplex heteroduplex shift analysis followed by direct DNA sequencing. SETTING: Male infertility clinic of a Canadian university-affiliated hospital. SUBJECTS: Of 198 men with obstructive (n = 149) or nonobstructive (n = 49; control group) azoospermia, 64 had congenital bilateral absence of the vas deferens (CBAVD), 10 had congenital unilateral absence of the vas deferens (CUAVD), and 75 had epididymal obstruction (56/75 were idiopathic). MAIN OUTCOME MEASURE: Frequency of mutations found by routine and nonroutine tests in men with obstructive vs nonobstructive azoospermia. RESULTS: Frequency of mutations and the IVS8-5T variant in the nonobstructive azoospermia group (controls) (2% and 5.1% allele frequency, respectively) did not differ significantly from that in the general population (2% and 5.2%, respectively). In the CBAVD group, 72 mutations were found by DNA sequencing and IVS8-5T testing (47 and 25, respectively; P<.001 and P = .002 vs controls) vs 39 by the routine panel (P<.001 vs controls). In the idiopathic epididymal obstruction group, 24 mutations were found by DNA sequencing and IVS8-5T testing (12 each; P=.01 and P=.14 vs controls) vs 5 by the routine panel (P=.33 vs controls). In the CUAVD group, 2 mutations were found by routine testing (P=.07 vs controls) vs 4 (2 each, respectively; P=.07 and P=.40 vs controls) by DNA sequencing and IVS8-5T testing. The routine panel did not identify 33 (46%) of 72, 2 (50%) of 4, and 19 (79%) of 24 detectable CFTR mutations and IVS8-5T in the CBAVD, CUAVD, and idiopathic epididymal obstruction groups, respectively. CONCLUSIONS: Routine testing for CFTR mutations may miss mild or rare gene alterations. The barrier to conception for men with obstructive infertility has been overcome by assisted reproductive technologies, thus raising the concern of iatrogenically transmitting pathogenic CFTR mutations to the progeny.

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28 Analysis for 31 of the most common CFTR mutations found within the white CF population,60 consisting of ⌬F508, W1282X, G542X, G551D, N1303K, R553X, G85E, R117H, S549N, V520F, R334W, A455E, R347P, R1162X, Y122X, S549R, 621+1G→T, ⌬I507, R560T, R347H, 3659delC, Q493X, 1898+1G→T, 711+1G→T, 3849+10C→T, 1717-1G→A, 3849+4A→G, 3905insT, 1078delT, 2183AA→G, and 2789+5G→A. Briefly, the technique involved amplification by polymerase chain reaction61 of the relevant exons, followed by digestion with appropriate restriction endonucleases and acrylamide gel electrophoresis with ethidium bromide staining. Login to comment
35 Two CFTR mutations(⌬F508,G542X)wereidentifiedby analysisusingthe31CFTRmutationpanel (TABLE2).TheIVS8-5Tvariantwasiden- tifiedin5alleles.Twomutations(theiden- tical ⌬F508 and G542X mutations detected by the 31 mutation panel) were identifiedthroughscreeningofallCFTR exons and splice sites. Login to comment
45 (%) Men With 2 Mutations ⌬F508/IVS8-5T 7 (11) ⌬F508/IVS8-5T 1 (10) ⌬F508/IVS8-5T 1 (1.8) ⌬F508/R117H 6 (9) W1282X/IVS8-5T 1 (1.8) ⌬F508/L206W 1 (1.6) G544S/IVS8-5T 1 (1.8) ⌬F508/M952T 1 (1.6) V754M/-741T→G 1 (1.8) ⌬F508/P67L 1 (1.6) R75Q/R258G 1 (1.8) ⌬F508/S549R 1 (1.6) R334W/R334W 1 (1.6) R117H/R117H 1 (1.6) R117H/IVS8-5T 1 (1.6) R347P/IVS8-5T 1 (1.6) N1303K/IVS8-5T 1 (1.6) 1677delTA/IVS8-5T 1 (1.6) R117L/IVS8-5T 1 (1.6) D979A/IVS8-5T 1 (1.6) IVS8-5T/IVS8-5T 1 (1.6) Men With 1 Mutation IVS8-5T/N 10 (16) ⌬F508/N 1 (10) IVS8-5T/N 9 (16) ⌬F508/N 1 (2) ⌬F508/N 6 (9) IVS8-5T/N 1 (10) ⌬F508/N 1 (1.8) G542X/N 1 (2) W1282X/N 2 (3) R75Q/N 1 (1.8) IVS8-5T/N 5 (10) L206W/N 1 (1.6) W1282X/N 1 (1.8) 4016insT/N 1 (1.6) R117H/N 1 (1.8) 2423delG/N 1 (1.8) Men With No Mutations 18 (28) 7 (70) 37 (66) 42 (86) *N indicates that no CFTR mutations or variants were detected. Login to comment
58 (%) 31 Mutation panel† ⌬F508 23 (18) ⌬F508 2 (10) ⌬F508 2 (1.8) ⌬F508 1 (1) R117H 9 (7) W1282X 2 (1.8) G542X 1 (1) W1282X 2 (1.6) R117H 1 (0.9) R334W 2 (1.6) S549R 1 (0.8) R347P 1 (0.8) N1303K 1 (0.8) Extensive screen† ⌬F508 23 (18) ⌬F508 2 (10) ⌬F508 2 (1.8) ⌬F508 1Mutations included in R117H 9 (7) W1282X 2 (1.8) G542X 131 mutation panel W1282X 2 (1.6) R117H 1 (0.9) R334W 2 (1.6) S549R 1 (0.8) R347P 1 (0.8) N1303K 1 (0.8) L206W 2 (1.6)‡ R75Q 2 (1.8)‡Mutations not included in P67L 1 (0.8)‡ G544S 1 (0.9)‡31 mutation panel 1677delTA 1 (0.8)‡ 2423delG 1 (0.9)‡ R117L 1 (0.8)‡ V754M 1 (0.9)‡ 4016insT 1 (0.8)‡ -741T→G 1 (0.9)‡ D979A 1 (0.8)§ R258G 1 (0.9)§ M952T 1 (0.8)¶ IVS8-5T 25 (20)# 2 (10) 12 (11) 5 (5) Detectable mutations 72 (56)# 4 (20) 24 (21)# 7 (7) Detectable mutations missed by 31 mutation panel 33 (46) 2 (50) 19 (79) Detectable non-IVS8-5T mutations missed by 31 mutation panel 8 (17) 0 (0) 7 (58) *Percentages indicate allele frequency. Login to comment

[hide] Detection of five rare cystic fibrosis mutations p... Clin Chem. 1999 Jul;45(7):957-62. Castaldo G, Fuccio A, Cazeneuve C, Picci L, Salvatore D, Raia V, Scarpa M, Goossens M, Salvatore F
Detection of five rare cystic fibrosis mutations peculiar to Southern Italy: implications in screening for the disease and phenotype characterization for patients with homozygote mutations.
Clin Chem. 1999 Jul;45(7):957-62., [PMID:10388469]

Abstract [show]
BACKGROUND: The search for the eight most frequent mutations (i.e., DeltaF508, G542X, W1282X, N1303K, 1717-1G-->A, R553X, 2183AA-->G, and I148T) by allele-specific oligonucleotide dot-blot analysis revealed 78% of 396 cystic fibrosis alleles in Southern Italy. The observation of frequent haplotypes on the unidentified cystic fibrosis alleles suggested that a few mutations could account for a large number of unidentified alleles. METHODS: We screened most of the coding sequence of the cystic fibrosis transmembrane regulator gene by denaturing gradient gel electrophoresis to determine the spectrum of these mutations in 68 unrelated cystic fibrosis patients bearing one or both unidentified mutations. RESULTS: The screening revealed five mutations, R1158X, 711+1G-->T, 4016insT, L1065P, and G1244E, each of which had a frequency of 1.3-1.8% (7% collectively). The 7% increase in the detection rate (85% vs 78%) reduces by >50% the residual risk of being cystic fibrosis carriers for couples who had tested negative by molecular analysis. We therefore designed a second allele-specific oligonucleotide set to analyze the five mutations. Among the patients analyzed, one patient homozygous for the L1065P mutation expressed a mild pulmonary and intestinal form of the disease with pancreatic insufficiency. Two other patients, homozygous for mutations R1158X and 4016insT, both expressed a severe cystic fibrosis phenotype. CONCLUSIONS: Five cystic fibrosis mutations are peculiar to patients from Southern Italy. The method described for their analysis is efficient, inexpensive, and can be semi-automated by use of a robotic workstation. The results obtained in patients from Southern Italy may have an impact on laboratories in other countries, given the large migrations of populations from Southern Italy to other countries in the last two centuries.

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13 A few mutations (i.e., ⌬F508, N1303K, G542X, and R553X) are frequent worldwide; the other mutations are regional or "private" mutations. Login to comment
36 All patients were first analyzed for eight CF mutations, i.e., ⌬F508, N1303K, G542X, W1282X, 1717-1G3A, R553X, 2183AA3G, and I148T (9). Login to comment
39 methods The eight CF mutations (i.e., ⌬F508, N1303K, G542X, W1282X, 1717-1G3A, R553X, 2183AA3G, and I148T) were identified with a semi-automated procedure based on a single multiplex PCR amplification followed by the allele-specific oligonucleotide (ASO) identification we described previously (9). Login to comment

[hide] Frequency of CFTR gene mutations in males particip... Hum Reprod. 1999 Jul;14(7):1833-4. Jakubiczka S, Bettecken T, Stumm M, Nickel I, Musebeck J, Krebs P, Fischer C, Kleinstein J, Wieacker P
Frequency of CFTR gene mutations in males participating in an ICSI programme.
Hum Reprod. 1999 Jul;14(7):1833-4., [PMID:10402399]

Abstract [show]
A higher prevalence of cystic fibrosis transmembrane regulator (CFTR) gene mutations has been suggested both in men affected by congenital aplasia of the vas deferens, and in individuals presenting with reduced sperm quality. In this case, an increased risk for offspring being affected by cystic fibrosis (CF) can be expected in couples who are planning to undergo intracytoplasmic sperm injection (ICSI), since most of the male partners suffer from infertility. In order to determine the risk for these couples more precisely, we offered them a test for the most frequent CF mutations prevalent in the German population. The frequency of mutations within the CFTR gene in the female group was in the same range as expected for the general population (six out of 150). In 10 out of 207 males tested, infertility could be explained by exogenous factors not related to CFTR. Among the remaining 197 males with idiopathic infertility, we detected 13 heterozygotes for a mutation within the CFTR gene. This slightly, but significantly (P = 0.014), elevated rate could indicate that infertile males have, compared with the general population, an increased risk of being a carrier of a CFTR gene mutation.

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13 Materials and methods CFTR screening included the most frequent CFTR mutations in the German population (R347P, ∆F508, G542X, S549I,N,R(A→C), G551D, R553X, N1303K, and 3849ϩ10kbC→T) (Do¨rk et al., 1994) as well as the mutation R117H and the analysis of the IVS8-T haplotype. Login to comment
40 The most obvious difference between the studies lies in the frequency of the mutation G542X, accounting for eight out of 18 mutations detected among 101 males by van der Ven et al. (1996) but not found in any of our probands. Login to comment

[hide] Two buffer PAGE system-based SSCP/HD analysis: a g... Eur J Hum Genet. 1999 Jul;7(5):590-8. Liechti-Gallati S, Schneider V, Neeser D, Kraemer R
Two buffer PAGE system-based SSCP/HD analysis: a general protocol for rapid and sensitive mutation screening in cystic fibrosis and any other human genetic disease.
Eur J Hum Genet. 1999 Jul;7(5):590-8., [PMID:10439967]

Abstract [show]
The large size of many disease genes and the multiplicity of mutations complicate the design of an adequate assay for the identification of disease-causing variants. One of the most successful methods for mutation detection is the single strand conformation polymorphism (SSCP) technique. By varying temperature, gel composition, ionic strength and additives, we optimised the sensitivity of SSCP for all 27 exons of the CFTR gene. Using simultaneously SSCP and heteroduplex (HD) analysis, a total of 80 known CF mutations (28 missense, 22 frameshift, 17 nonsense, 13 splicesite) and 20 polymorphisms was analysed resulting in a detection rate of 97.5% including the 24 most common mutations worldwide. The ability of this technique to detect mutations independent of their nature, frequency, and population specificity was confirmed by the identification of five novel mutations (420del9, 1199delG, R560S, A613T, T1299I) in Swiss CF patients, as well as by the detection of 41 different mutations in 198 patients experimentally analysed. We present a three-stage screening strategy allowing analysis of seven exons within 5 hours and analysis of the entire coding region within 1 week, including sequence analysis of the variants. Additionally, our protocol represents a general model for point mutation analysis in other genetic disorders and has already been successfully established for OTC deficiency, collagene deficiency, X-linked myotubular myopathy (XLMTM), Duchenne and Becker muscular dystrophy (DMD, BMD), Wilson disease (WD), Neurofibromatosis I and II, Charcot-Marie-Tooth disease, hereditary neuropathy with liability to pressure palsies, and defects in mitochondrial DNA. No other protocol published so far presents standard SSCP/HD conditions for mutation screening in different disease genes.

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20 The distribution of analysed known mutations is similar to that of the total number of mutations in the entire CFTR gene: missense mutations account for 35% (G27E, G85E, R117H, A120T, I148T, H199Y, R334W, T338I, R347P, R347H, A455E, M718K, S5449N, S5449I, G551D, R560T, R560S, S945L, S977P, I1005R, R1066C, R1070Q, M1101K, D1152H, S1235R, R1283M, N1303K, N1303H), followed by 28% of frameshift mutations (175delC, 394delTT, 457TAT- > G, 905delG, 1078delT, I507, F508, 1609delCA, 1677delTA, 2143delT, 2176insC, 218delA, 2184insA, 2869insG, 3659delC, 3732delA, 3821delT, 3905insT, 4016insT, 4172delGC, 4382delA), 21% of nonsense mutations (Q30X, Q39X, Q220X, W401X, Q525X, G542X, Q552X, R553X, V569X, E585X, K710X, R792X, Y1092X, R1162X, S1255X, W1282X, E1371X), and 16% of splice site mutations (621 + 1G- > T, 711 + 1G- > T, 711 + 5G- > A, 1717-1G- > A, 1898 + 1G- > A, 1898 + 5G- > T, 2789 + 5G- > A, 3271 + 1G- > A, 3272-26A- > G, 3601-17T- > C, 3849 + 4A- > G, 3849 + 10kbC- > T, 4374 + 1G- > T). Login to comment
34 Intron 19, all 27 exons and their exon-intron boundaries, including the 24 most common mutations worldwide (G85E, R117H, 621 + 1G- > T, 711 + 1G- > T, 1078delT, R334W, R347P, A455E, I507, F508, 1717-1G- > A, G542X, S549N, G551D, R553X, R560T, 1898 + 1G- > A, 2184delA, 2789 + 5G- > A, R1162X, 3659delC, 3849 + 10kbC- > T, W1282X, N1303K) (Cystic Fibrosis Genetic Analysis Consortium 1994), and the 15 most common mutations in our population (I148T, 1078delT, R334W, R347P, F508, 1717-1G- > A, G542X, R553X, 2347delG, D1152H, R1162X, 3849 + 10kbC- > T, 3905insT, W1282X, N1303K), were considered in this study. Login to comment
61 The slots C present wild type (wt) sequences, 1-8 present amplification products from CF patients with the following genotypes: 1 = R553X/R553X; 2 = 1717-1G- > A/wt; 3 = R553X/wt; 4 = G542X/wt; 5 = G542X/1717-1G- > A; 6 = G551D/wt; 7 = R560T/wt; 8 = S549N/wt. Login to comment
92 The technique developed demonstrates excellent single-strand separation and non-radioactive visualisation on polyacrylamide gels, and is time-saving and directly Table 2 Known mutations identified in 198 CF patients analysed investigatively Exon (E) Number of CFTR mutations intron (I) chromosomes Patient`s nationality Highest prevalence ∆F508 E10 212 miscellaneous 3905insT E20 025 Swiss Swiss, Amish, Arcadian R553X E11 020 Swiss, German German 1717-1G->A I10 017 Swiss, Italian Italian N1303K E21 011 Swiss, French, Italian Italian W1282X E20 014 Swiss, Italian, Israelit Jewish-Askhenazi G542X E11 009 Swiss, Spanish, Italian Spanish 2347delG E13 008 Swiss R1162X E19 006 Swiss, Italian, Russian Italian 3849+10kbC->T I19 005 German, French R347P E07 004 Swiss T5 I08 004 Swiss R334W E07 003 Swiss Q525X E10 003 Swiss 3732delA E19 003 Swiss S1235R E19 003 Italian, Turkish G85E E03 002 Italian, Greek I148T E04 002 Austrian, Turkish French-Canadian 621+1G->T I04 002 French French-Canadian 1078delT E07 002 Swiss E585X E12 002 Italian 2176insC E13 002 Swiss, Italian 2789+5G->A I14b 002 Italian Spanish D1152H E18 002 Swiss, French 4016insT E21 002 Turkish Q39X E02 001 Swiss 394delTT E03 001 Swiss Nordic, Finnish R117H E04 001 Swiss A120T E04 001 Swiss G126D E04 001 Swiss 711+5G->A I05 001 Russian M348K E07 001 Italian L568F E12 001 Italian 2183AA->G E13 001 Italian Italian K710X E13 001 Swiss S945L E15 001 French 3272-26A.->G I17a 001 Swiss M1101K E17b 001 Swiss Huttite 3601-17C->T I18 001 Swiss R1158X E19 001 Swiss 4005+1G-A I20 001 Italian applicable to early diagnostic testing, carrier detection and prenatal diagnosis. Login to comment

[hide] Analysis of 31 CFTR mutations by polymerase chain ... J Med Screen. 1999;6(2):67-9. Gasparini P, Arbustini E, Restagno G, Zelante L, Stanziale P, Gatta L, Sbaiz L, Sedita AM, Banchieri N, Sapone L, Fiorucci GC, Brinson E, Shulse E, Rappaport E, Fortina P
Analysis of 31 CFTR mutations by polymerase chain reaction/oligonucleotide ligation assay in a pilot screening of 4476 newborns for cystic fibrosis.
J Med Screen. 1999;6(2):67-9., [PMID:10444722]

Abstract [show]
OBJECTIVES: Molecular biological testing for genetic diseases has grown rapidly, but speed, accuracy, specificity, sensitivity, throughput, and cost become more important as large scale screening is considered. This is a pilot study of an assay for the simultaneous detection of up to 31 cystic fibrosis mutations in a multicentre population based screening of 4476 Italian newborns. METHODS: The assay is a polymerase chain reaction, followed by an oligonucleotide ligation assay (PCR/OLA) and finally a sequence coded separation. It allows the detection of up to 31 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Guthrie spots were used as a source of template DNA. RESULTS: 144 carriers were detected during the analysis of 4476 samples, which translates into a carrier frequency of 1/31.1. Forty two carriers were detected from 1341 samples in Pavia (1/31.9), 53 from 1574 in Turin (1/29.7), and 49 from 1561 in San Giovanni Rotondo (1/31.8). Fifteen different mutations were detected, the most common being delta F508 (0.625). Other common mutations included G542X (16 of 144), which was particularly common in southern Italy (14 of 49), N1303K (8 of 144), and R117H (8 of 144), detected only in the northern centres. CONCLUSIONS: PCR/OLA is a robust, accurate, user friendly method for cystic fibrosis screening of newborns using blood spots in a semiautomated way at a low cost per mutation (0.8 Euro).

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8 Other common mutations included G542X (16 of 144), which was particularly common in southern Italy (14 of 49), N1303K (8 of 144), and R117H (8 of 144), detected only in the northern centres. Login to comment
45 Other mutations found with greater than normal frequency were G542X, which is particularly common in southern Italy (14 of 49 individuals from San Giovanni Rotondo), N1303K (8 of 144), and R117H (8 of 144), which was detected only in the northern centres. Login to comment
46 Table 1 Mutations analysed in the CFTR gene using polymerase chain reaction/oligonucleotide litigation assay/sequence coded separation Mutation Location Nucleotide Result F508 Exon 10 3 bp deletion Deletion of Phe-508 I507 Exon 10 3 bp deletion Deletion of Ile-507 (or -506) Q493X Exon 10 C-1609 →→ T Gln-493 → Stop V520F Exon 10 G-1690 → T Val-520 → Phe 1717-1G → A Intron 10 G-1717-1 → A 3`-splice site mutation G542X Exon 11 G-1756 → T Gly-542 → Stop G551D Exon 11 G-1784 → A Gly-551 → Asp R553X Exon 11 C-1789 → T Arg-553 → Stop R560T Exon 11 G-1811 → C Arg-560 → Thr S549R Exon 11 T-1779 → G Ser-549 → Arg S549N Exon 11 G-1778 → A Ser-549 → Asn 3849+10 kb C → T Intron 19 C-3849+10 kb → T Splice mutation 3849+4A → G Intron 19 A-3849+4 → G Splice mutation R1162X Exon 19 C-3616 → T Arg-1162 → Stop 3659delC Exon 19 1 bp deletion Frameshift W1282X Exon 20 G-3978 → A Trp-1282 → Stop 3905insT Exon 20 1 bp insertion Frameshift N1303K Exon 21 C-4041 → G Asn-1303 → Lys G85E Exon 3 G-386 → A Gly-85 → Glu 621+1G → T Intron 4 G-621+1 → T 5`-splice site mutation R117H Exon 4 G-482 → A Arg-117 → His Y122X Exon 4 T-498 → A Tyr-122 → Stop 711+1G → T Intron 5 G-711+1 → T 5`-splice site mutation 1078delT Exon 7 1 bp deletion Frameshift R347P Exon 7 G-1172 → C Arg-347 → Pro R347H Exon 7 G-1172 → A Arg-347 → His R334W Exon 7 C-1132 → T Arg-334 → Trp A455E Exon 9 C-1496 → A Ala-455 → Glu 1898+1G → A Intron 12 G-1898+1 → A 5`-splice site mutation 2184delA Exon 13 Deletion A-2184; A-2183 → G Frameshift 2789+5G → A Intron 14B G-2789+5 → A Splice mutation Table 2 Summary of cystic fibrosis screening results No of samples analysed Normal subjects Carriers Carrier frequency Turin 1574 1521 53 1/29.7 Pavia 1341 1299 42 1/31.9 San Giovanni Rotondo 1561 1512 49 1/31.8 Total 4476 4332 144 1/31.1 Table 3 Detailed list of mutations detected in the Italian population Centre F508 G542X R347P 2183-AG N1303K 711+1GT 1717-1A R347H R117H 1898+1G 2789+5G W1282X R1162X I507 Other TO 33 2 1 1 5 1 1 2 3 2 2 - - - PV 27 - - 1 2 - 1 - 5 - 1 2 1 1 SGR 30 14 2 1 1 1 - - - - - - - - TO, Dipartimento di Patologia Clinica, Ospedale Infantile "Regina Margherita, Torino; PV, Istituto di Anatomia Patologica, Sezione di Anatomia Patologica, Università di Pavia, Pavia; SGR, Servizio di Genetica Medica and Divisione di Neonatologia, IRCCS Casa Sollievo della SoVerenza, San Giovanni Rotondo, Foggia. Login to comment

[hide] Pulmonary outcome in cystic fibrosis is influenced... Infect Immun. 1999 Sep;67(9):4744-50. Parad RB, Gerard CJ, Zurakowski D, Nichols DP, Pier GB
Pulmonary outcome in cystic fibrosis is influenced primarily by mucoid Pseudomonas aeruginosa infection and immune status and only modestly by genotype.
Infect Immun. 1999 Sep;67(9):4744-50., [PMID:10456926]

Abstract [show]
Whether allelic variants of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) independently contribute to pulmonary outcome in CF patients has not been resolved. We used both cross-sectional and mixed-model longitudinal analyses of data from CF patients that were at least 12 years old to determine the influence on pulmonary function (percent predicted forced expiratory volume [FEV(1)]) of the CFTR gene genotype, gender, mucoid Pseudomonas aeruginosa (MPA) infection status, presence of total opsonic antibody to MPA, and, separately, the opsonic antibody activity specific to the mucoid exopolysaccharide (MEP) surface antigen. Two different factors were independently associated with the lack of MPA infection: a high level of MEP-specific opsonic activity (MSOA), implicating an immunologically based mechanism of resistance to infection, and a lack of any type of opsonic antibody to MPA, indicative of no significant exposure or infection. This latter phenotype was found in a subset of CF patients who carried at least one uncommon CFTR gene allele suggestive of a genetic basis for resistance to infection in this group of older CF patients. For CF patients in whom both CFTR gene alleles were identified by screening for the 12 most common variants (75% of alleles), cross-sectional analysis showed that MPA infection was best correlated with lower percent predicted FEV(1), while genotype (two versus one DeltaF508 CFTR gene allele) and a low level of MSOA were associated with increased risk of infection. A mixed-model analysis of longitudinal spirometric measurements that considered multiple risk factors to derive regression equations was used to determine which clinical parameters had the greatest effect on the annual rate of decline in percent predicted FEV(1). This analysis showed that the CFTR gene genotype only modestly modified the constant (y intercept) of the derived equations, while gender and MPA infection status had the largest effects on annual rates of decline in percent predicted FEV(1). These results indicate that the CFTR genotype is usually not a primary determinant of pulmonary function in most CF patients, but gender and MPA infection status are. Infection status is potentially influenced by both immunologic (a high level of MSOA) and genetic factors, such as carriage of a CFTR gene allele that leads to a diagnosis of CF but still confers resistance to infection that is comparable to that of the wild-type CFTR gene.

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51 Genomic DNA isolated from each subject was evaluated for the presence of any of twelve CFTR gene mutations (⌬F508, G551D, G542X, 621ϩ1G3T, ⌬I507, 1717-1 G3A, R117H, N1303K, W1282X, R560T, R553X, and 3849ϩ10kb C3T) by one of three standard assays (10, 11, 32). Login to comment
101 Of these 12, 9 had one ⌬F508 CFTR gene allele, but the second allele was identified for only 2 of these 9 subjects (N1303K and G542X); the remaining 7 subjects carried a second CFTR gene allele that was not among the 12 most common ones we screened for. Login to comment
102 Two uninfected patients lacking any opsonic antibody were also compound heterozygotes with one of the two alleles not identified and the second allele being either G542X or G551D. Login to comment
118 with overall opsonic antibody titer of Ն5 26 14 Ͻ0.001 a Distribution of non-⌬F508 CFTR gene alleles in the uninfected group: G542X, 3 alleles; G551D, 1 allele; W1282X, 1 allele; N1303K, 1 allele; and not identified, 14 alleles. Login to comment
119 Distribution in the infected group was as follows: G542X, 2 alleles; G551D, 2 alleles; 621ϩ1G3T, 1 allele; and not identified, 7 alleles. Login to comment

[hide] Complex allele [-102T>A+S549R(T>G)] is associated ... Hum Genet. 1999 Jul-Aug;105(1-2):145-50. Romey MC, Guittard C, Chazalette JP, Frossard P, Dawson KP, Patton MA, Casals T, Bazarbachi T, Girodon E, Rault G, Bozon D, Seguret F, Demaille J, Claustres M
Complex allele [-102T>A+S549R(T>G)] is associated with milder forms of cystic fibrosis than allele S549R(T>G) alone.
Hum Genet. 1999 Jul-Aug;105(1-2):145-50., [PMID:10480369]

Abstract [show]
We recently reported a novel complex allele in the cystic fibrosis transmembrane regulator (CFTR) gene, combining a sequence change in the minimal CFTR promoter (-102T>A) and a missense mutation in exon 11 [S549R(T>G)]. Here we compare the main clinical features of six patients with cystic fibrosis (CF) carrying the complex allele [-102T>A+S549R(T>G)] with those of 16 CF patients homozygous for mutation S549R(T>G) alone. Age at diagnosis was higher, and current age was significantly higher (P=0.0032) in the group with the complex allele, compared with the S549R/S549R group. Although the proportion of patients with lung colonization was similar in both groups, the age at onset was significantly higher in the group with the complex allele (P=0.0022). Patients with the complex allele also had significantly lower sweat test chloride values (P=0.0028) and better overall clinical scores (P=0.004). None of the 22 patients reported in this study had meconium ileus. All 16 patients homozygous for S549R(T>G), however, were pancreatic insufficient, as compared with 50% of patients carrying the complex allele (P=0.013). Moreover, the unique patient homozygous for [-102T>A+S549R(T>G)] presented with a mild disease at 34 years of age. These observations strongly suggest that the sequence change (-102T>A) in the CFTR minimal promoter could attenuate the severe clinical phenotype associated with mutation S549R(T>G).

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48 Five patients were compound heterozygotes for the complex allele and another mutation, two with ∆F508 and three with, R334 W, G542X, or S945L, respectively, and one patient was homozygous for the [-102T>A+S549R(T>G)] complex allele. Login to comment
50 5 months - 5 years 1 year 4 months 8 months 1.5 years - - 3 months 3 years 1 year 3 months 3 months 1 month 1 month (age of onset) Meconium ileus No No No No No No No No No No No No No No No No Pancreatic Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes insufficiency Table 2 Clinical characteristics of six patients carrying the complex allele: [-102T>A+S549R(T>G) (ND no data, FEV1 forced expiratory volume in 1 s, FVC forced vital capacity) Characteristic Patient P1 P2 P3 P4 P5 P6 Genotype -102T>A+S549R(T>G) -102T>A+S549R(T>G) -102T>A+S549R(T>G) -102T>A+S549R(T>G) -102T>A+S549R(T>G) -102T>A+S549R(T>G) -102T>A+S549R(T>G) R334 W ∆F508 ∆F508 G542X S945L Geographic background South of France South of Spain South of France North of France North of France South of France Ethnic background Jews of Algeria ND Jews ND North of Africa Jews Gender F F F F M M Current age (years) 34 38 6 31 23 18 Age at diagnosis 6 years 35 years 3.5 years 2 months 4 months 9 years Age of first clinical symptoms 5 years ND 3 years 2 months 4 months 5 years First clinical symptoms Episodic bronchitis Dyspnea Pulmonary disease Pulmonary disease Cough Pulmonary disease Sweat Cl- (mmol/l) ND < 60 122 66 72 85 Height (percentile) 60 50 ND 60 26 30 Weight (percentile) 60 50 ND 40 12 < 25 Shwachman-Kulczycki score 85 ND 85 ND 60 85 FEV1 (% predicted) 30 19.3 89 60 59 89 FVC (% predicted) 50 38.7 96 100 76 115 Lung colonization Yes No Yes Yes Yes Yes Pseudomonas aerug. Login to comment
64 As opposed to 100% of S549R(T>G)/S549R(T>G) patients, 50% of patients carrying the (-102T>A) sequence alter- 148 Table 3 Comparison of clinical features between CF patients carrying the complex allele [-102T>A+ S549R(T>G) and those carrying S549R(T>G) only Feature Patients with [-102T>A+ S549R(T>G)] Patients with S549R(T>G) P value (n = 6) (n = 16) Mean ± SD Median (5th-95th Mean ± SD Median (5th-95th (no. studied) percentile) (no. studied) percentile) Current age (years) 25 ± 11.8 (6) 27 (6-38) 5 ± 3.35 (13) 5 (2-12) 0.0032 Age at diagnosis (years) 9.0 ± 13.2 (6) 4.75 (0.16-35) 0.88 ± 1.0 (16) 0.41 (0.08-3.5) NS Sweat Cl- (mmol/l) 79.0 ± 27.13 (5) 72 (50-120) 117.2 ± 25.1 (14) 120 (70-155) 0.028 Shwachman-Kulczycki score 78.8 ± 12.5 (4) 85 (60-85) 50.0 ± 7.3 (15) 50 (35-60) 0.004 Age at onset of lung colonization 11.5 ± 6.7 (5) 11 (3.5-22) 1.0 ± 1.43 (13) 0.41 (0.08-5) 0.0022 Lung colonization 5/6 (83) 13/16 (81) NS (no. positive/no. studied) (%) Pancreatic insufficiency 3/6 (50) 16/16 (100) 0.013 (no. positive/no. studied) (%) Table 4 CFTR haplotypes for seven [-102T>A+ S549R(T>G)] chromosomes (parentheses indicate unknown phase) P1 [-102T>A+S549R(T>G)] 1 2 1 23 7 1 34 13 1 1 [-102T>A+S549R(T>G)] 1 2 1 23 7 1 34 13 1 1 P2 [-102T>A+S549R(T>G)] (1) (2) (1) 23 7 (1) 34 13 1 1 R334W (2) (1) (2) 17 7 (2) 46 13 1 1 P3 [-102T>A+S549R(T>G)] 1 2 1 23 7 1 34 13 1 1 ∆F508 1 2 1 23 9 1 31 13 1 1 P4 [-102T>A+S549R(T>G)] 1 2 1 23 7 1 34 13 1 1 ∆F508 1 2 1 23 9 1 32 13 1 1 P5 [-102T>A+S549R(T>G)] 1 2 1 23 7 1 34 13 1 1 G542X 1 2 1 22 9 1 33 13 1 1 P6 [-102T>A+S549R(T>G)] 1 2 1 23 1 34 13 1 1 S945L 2 1 2 16 7 2 29 13 1 1 Patient Genotype XV-2c KM-9 J44 IVS8CA IVS8(T)n M470V IVS17BTA IVS17BC 3601-65C/A J3.11 ation were pancreatic insufficient (P = 0.013). Login to comment

[hide] [Respiratory disease in cystic fibrosis: from phys... Rev Mal Respir. 1999 Sep;16(4):495-509. Reynaud-Gaubert M
[Respiratory disease in cystic fibrosis: from physiopathology to therapy. Kinesitherapy and pulmonary transplantation excluded].
Rev Mal Respir. 1999 Sep;16(4):495-509., [PMID:10549060]

Abstract [show]
Respiratory disease is the major cause of morbidity and mortality in cystic fibrosis. Cystic fibrosis was long treated in the pediatric setting, but improved survival has led to the implication of adult pneumology in therapeutic management. Since the gene causing cystic fibrosis has been clone, our knowledge of the pathophysiology of the disease has literally exploded, particularly concerning the deleterious consequences of defective expression and distribution of CFTR (cystic fibrosis transmembrane conductance regulator) protein in chronic lung inflammation and infection. This knowledge has led to an optimization of existing therapeutic strategies and to the formulation of hypotheses for the development of new pharmaceutical reagents, allowing an assessment of disease outcome not only in terms of survival but also in terms of quality of life. Early in vivo clinical trials have been encouraging although the efficacy of gene transfer and expression remain modest and the optimal vector remains to be determined. Different potential pharmacological approaches are being studied in order to correct for defective CFTR function at different levels of gene mutation, and to modulate the disorder in transepithelial ionic transfer. One could expect in the near future to see combinations of complementary genotype-specific drugs used for the treatment of cystic fibrosis after patient genotyping to categorize the type of mutation.

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65 MUTATIONS DE LA CFTR Classification Les progrès récents de compréhension de la pathologie au niveau moléculaire ont permis de classer les anomalies de codage du gène en diverses classes, selon qu`elles affectent la transcription du gène, la translation, le transport ou la structure de la CFTR [14] : - Classe I : défaut de biosynthèse, générant une protéine altérée (tronquée ou aberrante) (ex : mutation G542X présente dans 3,4 % des cas). Login to comment

[hide] A phase I study of adenovirus-mediated transfer of... Hum Gene Ther. 1999 Dec 10;10(18):2973-85. Zuckerman JB, Robinson CB, McCoy KS, Shell R, Sferra TJ, Chirmule N, Magosin SA, Propert KJ, Brown-Parr EC, Hughes JV, Tazelaar J, Baker C, Goldman MJ, Wilson JM
A phase I study of adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator gene to a lung segment of individuals with cystic fibrosis.
Hum Gene Ther. 1999 Dec 10;10(18):2973-85., 1999-12-10 [PMID:10609658]

Abstract [show]
A third-generation adenoviral vector containing recombinant human cystic fibrosis transmembrane conductance regulator (CFTR) gene was delivered by bronchoscope in escalating doses to the conducting airway of 11 volunteers with cystic fibrosis. Assessments of dose-limiting toxicity (DLT), efficiency of gene transfer, and cell-mediated and humoral immune responses to vector administration were performed. DLT, manifest by flulike symptoms and transient radiographic infiltrates, was seen at 2.1 x 10(11) total viral particles. A highly specific assay for gene transfer was developed using in situ hybridization with an oligoprobe against unique vector sequence. Detectable gene transfer was observed in harvested bronchial epithelial cells (<1%) 4 days after vector instillation, which diminished to undetectable levels by day 43. Adenovirus-specific cell-mediated T cells were induced in most subjects, although only mild increases in systemic humoral immune response were observed. These results demonstrate that gene transfer to epithelium of the lower respiratory tract can be achieved in humans with adenoviral vectors but that efficiency is low and of short duration in the native CF airway.

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87 STU DY DESIGN A ND DO SE OF ADEN OVIRA L VECTOR H5.001CBCFTRa Baseline FEV1 Subject Dose (particles) a Region dosed Age/sex Genotype (% Pred) 1 2.1 3 109 Left lower lobe 20/M D F508/unknown 85 2 2.1 3 109 Right lower lobe 21/M D F508/G542X 67 3 7.0 3 109 Left lower lobe 23/F D F508/unknown 71 4 7.0 3 109 Left lower lobe 25/F D F508/R347P 48 5 2.1 3 1010 Left lower lobe 20/F D F508/unknown 89 6 2.1 3 1010 Right lower lobe 26/M D F508/unknown 95 7 7.0 3 1010 Right lower lobe 19/M D F508/unknown 108 8 7.0 3 1010 Left lower lobe 25/F D F508/D I507 70 9 2.1 3 1011 Left lower lobe 47/M D F508/unknown 90 10 2.1 3 1011 Right lower lobe 35/M D F508/R117H 61 11 2.1 3 1011 Right lower lobe 24/F D F508/unknown 107 a The dose is reported in total viral particles in a 7-ml suspension. Login to comment

[hide] Rapid F508del and F508C assay using fluorescent hy... Genet Test. 1999;3(4):365-70. Gundry CN, Bernard PS, Herrmann MG, Reed GH, Wittwer CT
Rapid F508del and F508C assay using fluorescent hybridization probes.
Genet Test. 1999;3(4):365-70., [PMID:10627945]

Abstract [show]
Amplification and fluorescent genotyping of the cystic fibrosis F508del locus was achieved from human genomic DNA in less than 30 min. The hybridization of adjacent fluorescent probes at the mutation site was monitored by resonance energy transfer between fluorescein and Cy5 during heating or cooling. Characteristic curves were obtained for each genotype; the first derivative of these fluorescent curves has a maximum at an apparent hybridization temperature (Tm) that is specific for each probe/allele duplex. The direction and rate of temperature change determines the difference between the apparent Tm and the true equilibrium Tm. One hundred and five sample were genotyped for the F508del cystic fibrosis mutation by heating and cooling curve profiles. These genotypes were validated by allele-specific amplification. Two fluorescein hybridization probes were designed to match the wild-type sequence perfectly from either codons 502 to 513 or from 504 to 511 on the cystic fibrosis transconductance regulator gene of chromosome 7. While genotyping for the F508del, an allele with the F508C base change was detected. For both F508del and F508C variants, the Tm shift from wild type was greater with a 24-mer probe than with a 35-mer probe. Fluorescent monitoring of hybridization probes is a versatile technique that can detect unexpected sequence alterations.

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149 Other clinically significant mutations (e.g., G542X, R553X, R1162X, N1303K, W1282X, G551D, G5151X, etc.) Login to comment

[hide] A pilot study of the effect of gentamicin on nasal... Am J Respir Crit Care Med. 2000 Mar;161(3 Pt 1):860-5. Wilschanski M, Famini C, Blau H, Rivlin J, Augarten A, Avital A, Kerem B, Kerem E
A pilot study of the effect of gentamicin on nasal potential difference measurements in cystic fibrosis patients carrying stop mutations.
Am J Respir Crit Care Med. 2000 Mar;161(3 Pt 1):860-5., [PMID:10712334]

Abstract [show]
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene containing a premature termination signal are expected to produce little or no CFTR chloride channels. It has been shown in vitro, that aminoglycoside antibiotics can increase the frequency of erroneous insertion of nonsense codons hence permitting the translation of CFTR alleles carrying missense mutations to continue reading to the end of the gene. This led to the appearance of functional CFTR channels at the apical plasma membrane. The aim of this research was to determine if topical application of gentamicin to the nasal epithelium of patients with cystic fibrosis (CF) carrying stop mutations can express, in vivo, functional CFTR channels. Nine CF patients carrying stop mutations (mean age 23 +/- 11 yr, range 12 to 46 yr) received gentamicin drops (0.3%, 3 mg/ml) three times daily intranasally for a total of 14 d. Nasal potential difference (PD) was measured before and after the treatment. Before gentamicin application all the patients had abnormal nasal PD typical of CF. After gentamicin treatment, significant repolarization of the nasal epithelium representing chloride transport was increased from -1 +/- 1 mV to -10 +/- 11 mV (p < 0. 001). In conclusion, gentamicin may influence the underlying chloride transport abnormality in patients with CF carrying stop mutations.

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29 Four patients were homozygous for the W1282X mutation, three were compound heterozygous W1282X/ G542X, one patient was compound heterozygous W1282X/3849 ϩ 10kb C→T, and one patient was compound heterozygous W1282X/ ⌬F508. Login to comment
70 Age (yr) FEV1 (% pred) Genotype 1 18 62 W1282X/W1282X 2 46 30 W1282X/W1282X 3 18 97 W1282X/W1282X 4 13 87 W1282X/W1282X 5 17 62 W1282X/G542X 6 23 60 W1282X/G542X 7 28 80 W1282X/G542X 8 38 44 W1282X/3849 ϩ 10kbC→T 9 12 73 W1282X/⌬F508 of gentamicin to increase the frequency of erroneous insertion of nonsense codons, thereby permitting the translation of CFTR alleles carrying missense mutations to continue reading to the end of the gene. Login to comment
84 Quantification studies have shown that after aminoglycoside incubation, the amount of full-length CFTR produced is as much as 25% (in the R553X mutation) to 35% (in the G542X mutation) of that observed in cells transfected with a wild-type CFTR complementary DNA (cDNA) (9, 10). Login to comment

[hide] Partial CFTR genotyping and characterisation of cy... Clin Genet. 2000 Jan;57(1):56-60. Zebrak J, Skuza B, Pogorzelski A, Ligarska R, Kopytko E, Pawlik J, Rutkiewicz E, Witt M
Partial CFTR genotyping and characterisation of cystic fibrosis patients with myocardial fibrosis and necrosis.
Clin Genet. 2000 Jan;57(1):56-60., [PMID:10733236]

Abstract [show]
Myocardial necrosis and fibrosis is a rare complication of cystic fibrosis (CF) causing sudden and unexpected death in infancy due to cardiac arrest. Characteristic morphological lesions are recognisable postmortem. The 18 CF patients with this complication had varied clinical features including mild pulmonary involvement, early onset severe pancreatic insufficiency, and profound electrocardiogram (ECG) changes. In this group of patients, 5 were deltaF508 homozygotes, 1 was deltaF508/ N1303K and 1 was a deltaF508/M compound heterozygote. A pair of affected siblings (deltaF508 homozygotes) were fully concordant for myocardial involvement and for the general course of the disease. The co-existence of a genetic predisposition to myocardial lesions resulting most probably from severe cystic fibrosis transmembrane (CFTR) genotypes (such as deltaF508/deltaF508, deltaF508/N1303K) and deficiency of certain trophic factors necessary for metabolism of the myocardium, are postulated to cause myocardial complications in CF leading to circulatory failure and early death.

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59 The latter was negative for 14 other mutations: DI507, 1717-1GA, G542X, G551D, R553X, R560T, 3849+10kbCT, N1303K, W1282X, S549I, S549N, 621+1GT, 2789+5GA, R117H. Login to comment

[hide] CFTR gene mutations and male infertility. Andrologia. 2000 Mar;32(2):71-83. Stuhrmann M, Dork T
CFTR gene mutations and male infertility.
Andrologia. 2000 Mar;32(2):71-83., [PMID:10755189]

Abstract [show]
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are a relatively frequent cause of male infertility. Depending on their molecular consequences, CFTR mutations may either result in typical cystic fibrosis (CF), one of the most common autosomal recessive disorders, which is characterized by chronic lung disease, pancreatic exocrine insufficiency, an increase in the concentration of sweat electrolytes and male infertility, due to obstructive azoospermia, or in atypical (often monosymptomatic) forms of CF such as congenital absence of the vas deferens (bi- or unilateral), bilateral ejaculatory duct obstruction or bilateral obstructions within the epididymides. All males with idiopathic obstructive azoospermia bear an increased risk for CF offspring. Couples requesting microsurgical epididymal sperm aspiration and in vitro fertilization, e.g. intracytoplasmic sperm injection, should be offered genetic counselling and molecular genetic analysis of the CFTR gene, if male infertility due to obstructive azoospermia is the underlying cause.

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84 CFTR mutations and male fertility Disorder Number of Proportion of Most frequent mutations (%) patients mutated alleles (%) Ethnic origin Reference CBAVD 17 20.6* DF508 (20.6) French Dumur et al. (1990b) CBAVD 25 38.0 DF508 (26.0) Northern European Anguiano et al. (1992) CBAVD 12 41.7 DF508 (20.8) French Culard et al. (1994) CBAVD 49 45.9 DF508 (32.6), R117H (6.1) Caucasians Oates & Amos (1994) CBAVD 47 21.3 DF508 (8.5), D1152H (3.2) Mostly Askenazim Augarten et al. (1994) CBAVD 30 41.7 DF508 (15.0), G542X (6.7), R117H (3.3) Spanish Casals et al. (1994) CBAVD 67 44.8 DF508 (20.9), R117H (4.5), W1282X (3.7) French Mercier et al. (1995) CBAVD 102 65.7+a DF508 (21.6), 5T (21.1), R347H (2.4) Caucasians Chillon et al. (1995) CBAVD 45 75.6+b DF508 (25.6), 5T (25.6), R117H (3.3), W1282X (3.3) French Costes et al. (1995) CBAVD 25 52.0+c 5T (26.0), DF508 (12.0), R117H (6.0) Caucasian Jarvi et al. (1995) CBAVD 70 68.6+d 5T (25.7), DF508 (19.3), W1282X (7.9) Mostly Caucasian Zielenski et al. (1995) CBAVD 101 79.2+e DF508 (26.2), R117H (11.4), 5T (12.9) Mostly German Do¨rk et al. (1997) CUAVD 10 5.0 DF508 (5.0) Spanish Casals et al. (1995) CUAVD 21 19.0 DF508 (9.5), R117H (4.8) Caucasian Mickle et al. (1995) BEDO 7 78.6 DF508 (28.5), 5T (21.4), R117H (14.3) Mostly German Meschede et al. (1997) IASV 16 3.1 I1139V (3.1) Mostly German Meschede et al. (1997) Azoospermia† 17 23.5+c 5T (14.7), R117H (5.9) DF508 (2.9) Caucasian Jarvi et al. (1995) Azoospermia 21 9.5 DF508 (2.4), G551D (2.4), R117H (2.4), G542X (2.4) Caucasian van der Ven et al. (1996) Spermatogenic failure 18 5.5+c G542X (2.8), 5T (2.8) Caucasian Jarvi et al. (1995) Spermatogenic failure 80 8.7 G542X (4.4), DF508 (3.1) Caucasian van der Ven et al. (1996) Spermatogenic failure 75 2.7+f DF508 (1.3), R117H (0.6), 5T (0.6) Dutch Tuerlings et al. (1998) *Testing only for DF508; +testing included the 5T allele; a-f, frequency of the 5T allele in the general population: a5.2%, n=498; b5.3%, n=131; c,dnot determined; e4.8%, n=186; f3.7%, n=212; †azoospermia with normal vas deferens and bilateral epididymal obstruction. Login to comment
95 Most patients are of German origin CFTR genotype (mutation class in brackets) Patients with typical CF (%) Patients with CBAVD (%) DF508 (2)/DF508 (2) 247 (59.4) 0 DF508 (2)/N1303K (2) 17 (4.1) 0 DF508 (2)/R347P (4) 13 (3.1) 0 DF508 (2)/R553X (1) 11 (2.6) 0 DF508 (2)/G542X (1) 11 (2.6) 0 DF508 (2)/G551D (3) 11 (2.6) 0 DF508 (2)/R1162X (1) 10 (2.4) 0 DF508 (2)/3849+10 KbC T (5) 9 (2.2) 0 DF508 (2)/2789+5G A (5) 9 (2.2) 0 DF508 (2)/3272-26 A G (5) 7 (1.7) 2 (2.6) DF508 (2)/1717-1G A (1) 6 (1.4) 0 DF508 (2)/CFTRdel21Kb (1) 5 (1.2) 0 DF508 (2)/R117H (4) 3 (0.7) 21 (26.9)* DF508 (2)/IVS8-5T (5) 2 (0.5) 9 (11.5)* DF508 (2)/other 33 (7.9) 20 (25.6) Other/other 22 (5.3) 26 (33.3) *Including one CUAVD patient each. Login to comment
140 One further aspect, not discussed by van der Ven et al. (1996), was thatbe caused by CFTR mutations was further substantiated by Meschede et al. (1997) who screened the increase in CFTR mutation frequency in males with reduced sperm quality was almost exclusively affected in a subset of CBAVD patients with CFTR mutations (e.g. IVS8-5T), whereas in other casesdue to the identification of mutation G542X on seven of 160 CFTR alleles (4.3%), which is 20 no disturbances of sperm maturation occur. Login to comment
141 times higher than the frequency of G542X in the general population of this area (0.2%). Login to comment
142 G542X is Genotype phenotype correlations particular frequent in Spain and Italy (The Cystic Fibrosis Genetic Analysis Consortium, 1994; The expression of CF disease is highly heterogeneous among different patients. Login to comment
146 The same mutation G542X was present in one of the 18 often monosymptomatic CF. Login to comment

[hide] Branch migration inhibition in PCR-amplified DNA: ... Nucleic Acids Res. 2000 May 1;28(9):E42. Lishanski A, Kurn N, Ullman EF
Branch migration inhibition in PCR-amplified DNA: homogeneous mutation detection.
Nucleic Acids Res. 2000 May 1;28(9):E42., 2000-05-01 [PMID:10756209]

Abstract [show]
A novel method for detection of any mutation located within a PCR-amplified DNA sequence was demonstrated. The method is based on the inhibition of spontaneous DNA branch migration. Partial duplexes produced by PCR amplification of a test and a reference genomic DNA sample anneal to form four-stranded cruciform structures. Spontaneous DNA branch migration results in dissociation of these structures when the test and reference sequences are identical. Any base substitution, deletion or insertion inhibits branch migration and produces stable cruciform structures. When suitable ligands are attached to the PCR primers, the cruciform structures can be detected by standard immunochemical methods. This approach was tested using several commonly occurring mutations within the human cystic fibrosis gene. New methods for increasing the specificity of PCR amplifications are described that were used for successful mutation analysis.

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126 Genotype Without reference DNA With reference DNA Wild-type homozygotes wt/wt 1.4 (1.2) 2.4 wt/wt 1.6 (1.3) 2.1 wt/wt 2.0 (1.5) 2.4 wt/wt 2.1 (1.4) 2.0 wt/wt 1.6 (1.2) 3.2 wt/wt 1.7 (1.4) 1.5 Heterozygotes G542X/wt G→T 41 (42) 38 G542X/wt G→T 73 (90) 66 G551D/wt G→A 83 (84) 93 G551D/wt G→A 99 (69) 76 R553X/wt C→T 92 (84) 57 R553X/wt C→T 105 (95) 61 R560T/wt G→C 130 (123) 70 R560T/wt G→C 109 (135) 111 G551D/R553X G→A/C→T 134 (134) 193 G551D/R553X G→A/C→T 134 (144) 235 Mutant homozygotes G542X/G542X G→T 1.5 (1.4) 174 G542X/G542X G→T 1.4 (1.5) 133 Blank (no target DNA) 1.6 Figure 3. Login to comment
139 Genotype Without εAεAG With εAεAG wt/wt 37 1.4 wt/wt 39 1.4 wt/wt 41 1. wt/wt 41 1.5 G542X/wt 121 126 G551D/wt 100 93 R553X/wt 82 85 R560T/wt 97 96 vi A PCR modification similar to the use of nested primers provided a powerful alternative method for reducing background signals. Login to comment

[hide] Genotype and phenotype in cystic fibrosis. Respiration. 2000;67(2):117-33. Zielenski J
Genotype and phenotype in cystic fibrosis.
Respiration. 2000;67(2):117-33., [PMID:10773783]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene which encodes a protein expressed in the apical membrane of exocrine epithelial cells. CFTR functions principally as a cAMP-induced chloride channel and appears capable of regulating other ion channels. Besides the most common mutation, DeltaF508, accounting for about 70% of CF chromosomes worldwide, more than 850 mutant alleles have been reported to the CF Genetic Analysis Consortium. These mutations affect CFTR through a variety of molecular mechanisms which can produce little or no functional CFTR at the apical membrane. This genotypic variation provides a rationale for phenotypic effects of the specific mutations. The extent to which various CFTR alleles contribute to clinical variation in CF is evaluated by genotype-phenotype studies. These demonstrated that the degree of correlation between CFTR genotype and CF phenotype varies between its clinical components and is highest for the pancreatic status and lowest for pulmonary disease. The poor correlation between CFTR genotype and severity of lung disease strongly suggests an influence of environmental and secondary genetic factors (CF modifiers). Several candidate genes related to innate and adaptive immune response have been implicated as pulmonary CF modifiers. In addition, the presence of a genetic CF modifier for meconium ileus has been demonstrated on human chromosome 19q13.2. The phenotypic spectrum associated with mutations in the CFTR gene extends beyond the classically defined CF. Besides patients with atypical CF, there are large numbers of so-called monosymptomatic diseases such as various forms of obstructive azoospermia, idiopathic pancreatitis or disseminated bronchiectasis associated with CFTR mutations uncharacteristic for CF. The composition, frequency and type of CFTR mutations/variants parallel the spectrum of CFTR-associated phenotypes, from classic CF to mild monosymptomatic presentations. Expansion of the spectrum of disease associated with the CFTR mutant genes creates a need for revision of the diagnostic criteria for CF and a dilemma for setting nosologic boundaries between CF and other diseases with CFTR etiology.

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165 Both studies showed that certain mild alleles (R117H; A455E; 3849+10kbC→T) from class IV or V tend to be associated with significantly lower Cl sweat levels than those for severe alleles ('F508; 621+1G→T; G542X; R553X, etc.). Login to comment

[hide] A novel mutation in the CFTR gene correlates with ... J Med Genet. 2000 Mar;37(3):215-8. Wang J, Bowman MC, Hsu E, Wertz K, Wong LJ
A novel mutation in the CFTR gene correlates with severe clinical phenotype in seven Hispanic patients.
J Med Genet. 2000 Mar;37(3):215-8., [PMID:10777364]

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292 The frequency of this mutation in the Hispanic CF patients attending CHLA is similar to that of G542X, only second to F508. Login to comment

[hide] Correlation between mutations and age in cystic fi... J Med Genet. 2000 Mar;37(3):225-7. Rivard SR, Allard C, Leblanc JP, Milot M, Aubin G, Simard F, Ferec C, de Braekeleer M
Correlation between mutations and age in cystic fibrosis in a French Canadian population.
J Med Genet. 2000 Mar;37(3):225-7., [PMID:10777368]

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292 The frequency of this mutation in the Hispanic CF patients attending CHLA is similar to that of G542X, only second to F508. Login to comment

[hide] Molecular analysis in Brazilian cystic fibrosis pa... Genet Test. 2000;4(1):69-74. Bernardino AL, Ferri A, Passos-Bueno MR, Kim CE, Nakaie CM, Gomes CE, Damaceno N, Zatz M
Molecular analysis in Brazilian cystic fibrosis patients reveals five novel mutations.
Genet Test. 2000;4(1):69-74., [PMID:10794365]

Abstract [show]
We have performed molecular genetic analyses on 160 Brazilian patients diagnosed with cystic fibrosis (CF). Screening of mutations in 320 CF chromosomes was performed through single strand conformation polymorphism (SSCP) and heteroduplex analyses assay followed by DNA sequencing of the 27 exons and exon/intron boundaries of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The frequency of CFTR variants of T-tract length of intron 8 (IVS8 Tn) was also investigated. This analysis enabled the detection of 232/320 CF mutations (72.2%) and complete genotyping of 61% of the patients. The deltaF508 mutation was found in 48.4% of the alleles. Another fifteen mutations (previously reported) were detected: G542X, R1162X, N1303K, R334W, W1282X, G58E, L206W, R553X, 621+1G-->T, V232D, 1717-1G-->A, 2347 delG, R851L, 2789+5G-->A, and W1089X. Five novel mutations were identified, V201M (exon 6a), Y275X (exon 6b), 2686 insT (exon 14a), 3171 delC (exon 17a), and 3617 delGA (exon 19). These results contribute to the molecular characterization of CF in the Brazilian population. In addition, the identification of the novel mutation Y275X allowed prenatal diagnosis in a high-risk fetus.

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6 Another fifteen mutations (previously reported) were detected: G542X, R1162X, N1303K, R334W, W1282X, G58E, L206W, R553X, 6211 1GRT, V232D, 1717-1GRA, 2347 delG, R851L, 27891 5GRA, and W1089X. Login to comment
51 The next most common mutations were: G542X (8.8%), R1162X (2.5%), N1303K (2.5%), R334W (2.5%), W1282X (1.3%), G58E (1.3%), L206W (0.6%), and R553X (0.6%). Login to comment
81 In this study, 16 mutations were identified: D F508, G542X, R1162X, N1303K, R334W, W1282X, G58E, L206W, R553X, 6211 1GRT, V232D, 1717-1GRA, 2347 delG, R851L, 27891 5GRA, and W1089X. Login to comment
84 GEN OTYPES, FREQUENCIES, AN D PRESENCE OF PI FRO M 160 CF PATIE NTS (320 CF CHROM OSOM ES) Number and frequency (%) Genotype Number Frequency (%) of patients with PI D F508/D F508 47 29.40 47 (100%) D F508/G542X 13 8.10 13 (100%) D F508/R1162X 6 3.80 6 (100%) D F508/R334W 5 3.10 3 (60%) D F508/N1303K 3 1.90 3 (100%) D F508/W1282X 2 1.20 2 (100%) D F508/G58E 2 1.20 1 (50%) D F508/L206W 1 0.62 0 D F508/R553X 1 0.62 1 (100%) D F508/R851L 1 0.62 0 D F508/2789 1 5g ® A 1 0.62 0 D F508/3617delGA 1 0.62 1 (100%) D F508/3171delC 1 0.62 1 (100%) D F508/2686insT 1 0.62 1 (100%) D F508/Y275X 1 0.62 1 (100%) D F508/U 22 13.80 14 (64%) G542X/G542X 3 1.90 3 (100%) G542X/N1303K 3 1.90 2 (67%) G542X/R1162X 1 0.62 1 (100%) G542X/U 5 3.10 4 (80%) N1303K/R1162X 1 0.62 1 (100%) N1303K/G58E 1 0.62 0 2347delG/2347delG 1 0.62 1 (100%) R334W/V232D 1 0.62 0 R334W/W1089X 1 0.62 1 (100%) R334W/U 1 0.62 1 (100%) W1282X/U 1 0.62 1 (100%) G58E/U 1 0.62 1 (100%) R553X/U 1 0.62 1 (100%) L206W/U 1 0.62 0 621 1 1G ® T/U 1 0.62 1 (100%) 1717-1G ® A/U 1 0.62 Not known V201M/U 1 0.62 0 U/U 27 16.90 12 (44%) Total 160 100 - U, Unknown CF mutation. Login to comment
88 The second most common mutation, G542X, which occurred in 8.8% of our patients, is also the second most frequently reported in Spanish Mediterranean coastal areas (8.0%) (Casals et al., 1993). Login to comment
104 However, one of our compound heterozygote patients for severe mutations (N1303K/G542X), has PS. Login to comment
110 Unexpectedly, however one compound heterozygote (N1303K/G542X) CF patients (who was referred to above as having PS) was the son of first cousins, which is compatible with the high frequency of CFTR heterozygotes in the population. Login to comment

[hide] Spectrum of CFTR mutations in Mexican cystic fibro... Hum Genet. 2000 Mar;106(3):360-5. Orozco L, Velazquez R, Zielenski J, Tsui LC, Chavez M, Lezana JL, Saldana Y, Hernandez E, Carnevale A
Spectrum of CFTR mutations in Mexican cystic fibrosis patients: identification of five novel mutations (W1098C, 846delT, P750L, 4160insGGGG and 297-1G-->A).
Hum Genet. 2000 Mar;106(3):360-5., [PMID:10798368]

Abstract [show]
We have analyzed 97 CF unrelated Mexican families for mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Our initial screening for 12 selected CFTR mutations led to mutation detection in 56.66% of the tested chromosomes. In patients with at least one unknown mutation after preliminary screening, an extensive analysis of the CFTR gene by single stranded conformation polymorphism (SSCP) or by multiplex heteroduplex (mHET) analysis was performed. A total of 34 different mutations representing 74.58% of the CF chromosomes were identified, including five novel CFTR mutations: W1098C, P750L, 846delT, 4160insGGGG and 297-1G-->A. The level of detection of the CF mutations in Mexico is still lower than that observed in other populations with a relatively low frequency of the deltaF508 mutation, mainly from southern Europe. The CFTR gene analysis described here clearly demonstrated the high heterogeneity of our CF population, which could be explained by the complex ethnic composition of the Mexican population, in particular by the strong impact of the genetic pool from southern European countries.

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17 We previously reported that only 39% of CF chromosomes from Mexican patients with Mestee ethnic background carry the ∆F508 mutation (Orozco et al. 1993) and 7.2% carry the G542X mutation (Villarreal et al. 1996), which is common in the Spanish population (Casals et al. 1997). Login to comment
27 Detection of known mutations ∆F508, G542X, N1303K, ∆I507 and 2869insG were screened by PCR-mediated site directed mutagenesis (PSM) as previously described (Friedman et al. 1991). Login to comment
41 The second most common mutation was G542X, present in 6.1% of CF chromosomes, followed by ∆I507 and S549N (each 2.5%), N1303K (2.06%) and 2055del→A (1.03%). Login to comment
69 First, we tested these patients for 12 mutations selected for the following reasons: five are the most common mutations worldwide (∆F508, G542X, N1303K, G551D and R553X; CFGAC 1994); 362 Table 1 Frequency of the CFTR gene mutations in 97 (194 chromosomes) Mexican patients Mutation Number of Frequency affected alleles (%) ∆F508 79 40.72 G542X 12 6.18 ∆I507 5 2.57 S549N 5 2.57 N1303K 4 2.06 R75X 3 1.54 406-1G→A 3 1.54 I148T 3 1.54 2055del9→A 2 1.03 935delA 2 1.03 I506T 2 1.03 3199del6 2 1.03 2183AA→G 2 1.03 G551D 1 0.51 R553X 1 0.51 1924del7 1 0.51 G551S 1 0.51 1078delT 1 0.51 Y1092X 1 0.51 R117H 1 0.51 G85E 1 0.51 3849+10KbC→T 1 0.51 1716G→A 1 0.51 W1204X 1 0.51 W1098Ca 1 0.51 846delTa 1 0.51 P750La 1 0.51 V754M 1 0.51 R75Q 1 0.51 W1069X 1 0.51 L558S 1 0.51 4160insGGGGa 1 0.51 297-1G→Aa 1 0.51 H199Y 1 0.51 2869insG 0 0 R1162X 0 0 3120+1G→A 0 0 Total 34 145 74.58% aNovel mutations detected in this study Fig.1 Sequencing ladders showing the CFTR novel mutations. Login to comment
74 The G542X mutation has a frequency higher (6.1%) than that reported to the CFGAC, but similar to Spanish patients (Casals et al. 1997). Login to comment

[hide] Genotype-phenotype correlation in three homozygote... J Med Genet. 2000 Apr;37(4):307-9. Kilinc MO, Ninis VN, Tolun A, Estivill X, Casals T, Savov A, Dagli E, Karakoc F, Demirkol M, Huner G, Ozkinay F, Demir E, Seculi JL, Pena J, Bousono C, Ferrer-Calvete J, Calvo C, Glover G, Kremenski I
Genotype-phenotype correlation in three homozygotes for the cystic fibrosis mutation 2183AA-->G shows a severe phenotype.
J Med Genet. 2000 Apr;37(4):307-9., [PMID:10819640]

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446 The clinical data presented for three patients homozygous for the mutation and eight compound heterozygous patients who carry a severe mutation ( F508, G542X, and G1244E) on the other CFTR chromosome indicate that the mutation causes a severe CF phenotype. Login to comment

[hide] Applicability of different antibodies for immunohi... J Histochem Cytochem. 2000 Jun;48(6):831-7. Claass A, Sommer M, de Jonge H, Kalin N, Tummler B
Applicability of different antibodies for immunohistochemical localization of CFTR in sweat glands from healthy controls and from patients with cystic fibrosis.
J Histochem Cytochem. 2000 Jun;48(6):831-7., [PMID:10820156]

Abstract [show]
The hereditary disease cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Understanding of the consequences of CFTR gene mutations is derived chiefly from in vitro studies on heterologous cell cultures and on cells hyperexpressing CFTR. Data from ex vivo studies on human tissue are scarce and contradictory, a fact which is in part explained by secondary tissue destruction in most affected organs. The purpose of this study was to establish conditions under which wild-type and mutated CFTR can be studied in affected human tissue. Sweat glands carry the basic defect underlying CF and are not affected by tissue destruction and inflammation. Therefore, we used this tissue to test a panel of eight different CFTR antibodies under various fixation techniques. The antibodies were tested on skin biopsy sections from healthy controls, from CF patients homozygous for the most common mutation, DeltaF508, and from patients carrying two nonsense mutations. Of the eight CFTR antibodies, only three-M3A7, MATG 1104, and cc24-met the criteria necessary for immunolocalization of CFTR in sweat glands. The labeling pattern in the CF sweat glands was consistent with the postulated processing defect of DeltaF508 CFTR. The antibodies exhibited different sensitivities for detecting DeltaF508 CFTR.

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33 Materials and Methods Tissue Samples Full-thickness skin biopsies were taken from the right shoulder of six healthy volunteers, four ⌬F508 homozygous CF patients, and four patients bearing two nonsense mutations within the CFTR gene (G542X/G542X; nϭ2; R553X/ R553X; and G542X/W1282X). Login to comment
114 No signals are observed with MATG 1104 (1:2400; Ba) in G542X/ G542X tissue; isotype control (Bb). Login to comment
115 G542X/W1282X is shown with cc24 (1:100; Ca; specific peptide competition, Cb); again, no labeling above the negative control is observed. Login to comment
130 Although overreading of stop codons has been reported to occur in mammalian cells (McCaughan et al. 1995), ample evidence exists that the nonsense mutations R553X and G542X of the CFTR gene result in reduced to undetectable levels of mRNA transcripts (Hamosh et al. 1992; Will et al. 1995) and absence of full-length protein (Howard et al. 1996). Login to comment
163 J Clin Invest 95:1601-1611 Gregory RJ, Cheng SH, Rich DP, Marshall J, Paul S, Hehir K, Ostedgaard L, Klinger KW, Welsh MJ, Smith AE (1990) Expression and characterization of the cystic fibrosis transmembrane conductance regulator. Nature 347:382-386 Hamosh A, Rodenstein BJ, Cutting GR (1992) CFTR nonsense mutations G542X and W1282X associated with severe reduction of CFTR mRNA in nasal epithelial cells. Login to comment

[hide] Cytokine dysregulation in activated cystic fibrosi... Clin Exp Immunol. 2000 Jun;120(3):518-25. Moss RB, Hsu YP, Olds L
Cytokine dysregulation in activated cystic fibrosis (CF) peripheral lymphocytes.
Clin Exp Immunol. 2000 Jun;120(3):518-25., [PMID:10844532]

Abstract [show]
Recent studies demonstrate in vivo and in vitro cytokine dysregulation in CF epithelial cells. To see if these abnormalities may be generalized to other cells expressing cystic fibrosis transmembrane conductance regulator (CFTR) but not directly exposed to local inflammation, we studied mRNA transcription, intracellular protein production and extracellular secretion of IL-2, IL-4, IL-5, IL-10 and interferon-gamma (IFN-gamma) from freshly isolated blood mononuclear and CD4+ T cells from CF patients and controls. Cells were activated by phorbol myristate acetate (PMA) and anti-CD3, PMA-ionomycin, or lipopolysaccharide (LPS) and assessed for cytokine mRNA transcription by semiquantitative reverse transcriptase-polymerase chain reaction, intracellular protein production by flow cytometry, and secretion by supernatant ELISA. Cytokine expression was highly stimulus-dependent. CF cells showed higher IL-10 transcription than control cells after maximal activation by LPS (P = 0.01); despite this, cytokine production and secretion were equivalent to controls. CF cells showed lower cellular IL-10 production after PMA-anti-CD3 activation (P = 0.002). CF cells secreted less IFN-gamma than control cells after maximal activation by PMA-anti-CD3 (1836 +/- 273 pg/ml versus 9635 +/- 3437 pg/ml, P = 0.04). IL-2, IL-4 and IL-5 regulation was similar to controls. We conclude that CF mononuclear cells show selective cytokine dysregulation after maximal activation, namely reduced IFN-gamma secretion and increased IL-10 mRNA without increased production or secretion. These findings extend defects described in respiratory epithelial cells to circulating immunoregulatory cells, suggesting a link between CF genotype and cytokine dysregulation.

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30 Except for one patient who was heterozygous for G542x, the remainder (50%) were heterozygous for dF508, with other identified mutations including 3659delC (n 1), G85E (n 2), W1282X (n 1), 1898 1 1 (n 1), 2184delA (n 1), and G542X (n 1); six patients carried an unidentified mutation at the second allele. Login to comment

[hide] Heterogeneity for mutations in the CFTR gene and c... Hum Reprod. 2000 Jul;15(7):1476-83. Casals T, Bassas L, Egozcue S, Ramos MD, Gimenez J, Segura A, Garcia F, Carrera M, Larriba S, Sarquella J, Estivill X
Heterogeneity for mutations in the CFTR gene and clinical correlations in patients with congenital absence of the vas deferens.
Hum Reprod. 2000 Jul;15(7):1476-83., [PMID:10875853]

Abstract [show]
Congenital absence of the vas deferens (CAVD) is a heterogeneous disorder, largely due to mutations in the cystic fibrosis (CFTR) gene. Patients with unilateral absence of the vas deferens (CUAVD) and patients with CAVD in association with renal agenesis appear to have a different aetiology to those with isolated CAVD. We have studied 134 Spanish CAVD patients [110 congenital bilateral absence of the vas deferens (CBAVD) and 24 CUAVD], 16 of whom (six CBAVD, 10 CUAVD) had additional renal anomalies. Forty-two different CFTR mutations were identified, seven of them being novel. Some 45% of the CFTR mutations were specific to CAVD, and were not found in patients with cystic fibrosis or in the general Spanish population. CFTR mutations were detected in 85% of CBAVD patients and in 38% of those with CUAVD. Among those patients with renal anomalies, 31% carried one CFTR mutation. Anomalies in seminal vesicles and ejaculatory ducts were common in patients with CAVD. The prevalence of cryptorchidism and inguinal hernia appeared to be increased in CAVD patients, as well as nasal pathology and frequent respiratory infections. This study confirms the molecular heterogeneity of CFTR mutations in CAVD, and emphasizes the importance of an extensive CFTR analysis in these patients. In contrast with previous studies, this report suggests that CFTR might have a role in urogenital anomalies.

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55 ∆F508 and G542X were the most frequently patients (30 CBAVD, 10 CUAVD) were reported previously (Casals identified CF mutations, but at lower frequencies than in CF et al., 1995). Login to comment
59 Mutations ∆F508 and in CAVD than in CF patients [4.0% versus 0.6% (χ2 ϭ 20.09, G542X (Kerem et al., 1989, 1990) were analysed in all patients, as P Ͻ 0.001) and 3.6% versus 0.3% (χ2 ϭ 28.45, P Ͻ 0.001)they are the most common mutations in Spanish CF patients, 53% respectively].and 8% respectively (Casals et al., 1997). Login to comment
62 Recently, direct analysis of 31 CFTR mutations (PCR/OLA Cystic Fibrosis Assay; Perkin Elmer, Foster City) was 6(5T), ∆F508, G542X, L206W and R117H are the most performed in 30 of these infertile men. Login to comment
70 Thethe vas deferens M470V variant in exon 10 was analysed in 82 patients (98 Mutation CBAVD CUAVD Total alleles M470 and 66 V470), (TTGA)n in intron 6a which (n ϭ 42) 156 alleles (%) 14 alleles (%) 170 alleles (%) presented the higher frequency of seven repeats (129/272 alleles), T854T in exon 14a (72/272 alleles), 4521G→A in 5T 50a (32) 6 (43) 56 (33) exon 24 (62/272) and 3601-65C/A in intron 18 (48/272) were∆F508 40 (26) 3 (21) 43 (25) G542X 10 (6) 1 (7) 11 (6) the most frequent. Login to comment
95 CFTR genotypes in 24 patients with congenital unilateral absenceTable III. CFTR genotypes in 110 patients with congenital bilateral absence of the vas deferens of the vas deferens Mutations IVS8-6(T) n (%)Mutations IVS8-6(T) n (%) Two CFTR mutations 62 (56) Two CFTR mutations 5 (21) ∆F508/- 5T/9T 2 (8)∆F508/- 5T/9T 17 (15) G542X/- 5T/9T 6 (5) G542X/- 5T/9T 1 3732delA/- 5T/7T 1∆F508/L206W 9T/9T 6 (5) ∆F508/D1270NϩR74W 7T/9T 3 (3) L383S/- 5T/7T 1 One CFTR mutation 4 (17)∆F508/R117H 7T/7T 1 ∆F508/P1021S 7T/9T 1 ∆F508/-a 7T/9T 1 3732delA/-a 7T/7T 1∆F508/M952T 7T/9T 1 ∆F508/D110Y 7T/9T 1 Q890R/- 7T/7T 1 -/-a 5T/7T 1∆F508/S50P 5T/9T 1 ∆F508/2751ϩ3A→G 5T/9T 1 Negative CFTR mutations 15 (62) -/- 7T/7T 10 (42)G542X/R117H 7T/9T 1 G542X/2789ϩ5G→A 7T/9T 1 -/- 7T/9T 3 (12) -/- 9T/9T 2 (8)R117H/2789ϩ5G→A 7T/7T 1 R117H/712-1G→T 7T/9T 1 R117H/∆I507 7T/7T 1 aThree carrier patients with renal agenesis. Login to comment
101 Azoospermia was more frequent in CUAVD ∆F508/- 9T/9T 3 (3) patients with CFTR mutations (7/9) than in those without-/- 5T/9T 2 (2) G542X/- 7T/9T 2 (2) mutations (8/13), and sperm concentration was higher in the 2789ϩ5G→A/- 7T/7T 2 (2) latter group. Login to comment

[hide] Repeat administration of DNA/liposomes to the nasa... Gene Ther. 2000 Jul;7(13):1156-65. Hyde SC, Southern KW, Gileadi U, Fitzjohn EM, Mofford KA, Waddell BE, Gooi HC, Goddard CA, Hannavy K, Smyth SE, Egan JJ, Sorgi FL, Huang L, Cuthbert AW, Evans MJ, Colledge WH, Higgins CF, Webb AK, Gill DR
Repeat administration of DNA/liposomes to the nasal epithelium of patients with cystic fibrosis.
Gene Ther. 2000 Jul;7(13):1156-65., [PMID:10918483]

Abstract [show]
The major cause of mortality in patients with cystic fibrosis (CF) is lung disease. Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene product in the airways is a potential treatment. Clinical studies in which the CFTR cDNA was delivered to the respiratory epithelia of CF patients have resulted in modest, transient gene expression. It seems likely that repeated administration of the gene transfer vector will be required for long-term gene expression. We have undertaken a double-blinded study in which multiple doses of a DNA/liposome formulation were delivered to the nasal epithelium of CF patients. Ten subjects received plasmid DNA expressing the CFTR cDNA complexed with DC-Chol/DOPE cationic liposomes, whilst two subjects received placebo. Each subject received three doses, administered 4 weeks apart. There was no evidence of inflammation, toxicity or an immune response towards the DNA/liposomes or the expressed CFTR. Nasal epithelial cells were collected 4 days after each dose for a series of efficacy assays including quantitation of vector-specific DNA and mRNA, immunohistochemistry of CFTR protein, bacterial adherence, and detection of halide efflux ex vivo. Airway ion transport was also assessed in vivo by repeated nasal potential difference (PD) measurements. On average, six of the treated subjects were positive for CFTR gene transfer after each dose. All subjects positive for CFTR function were also positive for plasmid DNA, plasmid-derived mRNA and CFTR protein. The efficacy measures suggest that unlike high doses of recombinant adenoviral vectors, DNA/liposomes can be successfully re-administered without apparent loss of efficacy.

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24 Genotype Dose Sex Age (years) FEV1 (litres) Clinical score A1 ⌬F508/1078delT CFTR M 22.7 3.25 85 A2 ⌬F508/⌬F508 CFTR M 22.9 3.4 90 A3 ⌬F508/G542X CFTR M 28.5 1.75 70 A5 ⌬F508/F945L CFTR M 30.3 3.05 70 A6 ⌬F508/⌬F508 CFTR M 24.4 1.25 70 B1 ⌬F508/⌬F508 CFTR M 17.6 3.8 90 B3 ⌬F508/1898+1(GϾA) CFTR F 16.9 2.15 80 B4 ⌬F508/G551D CFTR M 32.8 1.8 55 B6 ⌬F508/⌬F508 CFTR M 18.8 3.5 95 A4 ⌬F508/G551D Placebo F 16.5 2.65 95 B5 ⌬F508/3659delC Placebo M 16.4 2.8 85 All patients were pancreatic insufficient. Login to comment

[hide] Distribution of CFTR gene mutations in cystic fibr... J Med Genet. 2000 Aug;37(8):E16. Teder M, Klaassen T, Oitmaa E, Kaasik K, Metspalu A
Distribution of CFTR gene mutations in cystic fibrosis patients from Estonia.
J Med Genet. 2000 Aug;37(8):E16., [PMID:10922396]

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7 First, several known mutations were tested directly by the heteroduplex analysis (HA; F508, 394delTT, polyT variants in IVS8), restriction digestion (RD; G551D, R553X, 1811+1.6kbA→G, L206W, 3849+10kbC→T), and amplification refractory mutation system (ARMS, kits from Cellmark Diagnostics, UK; G542X, 621+1G→T, N1303K). Login to comment
66 This is the second most frequent mutation in several Nordic populations, with a relative frequency of 1.9% in Denmark,2 3.2% in Flanders,32 6.5% in Sweden,2 2.2-5.5% in Norway,2 and 30% in Finland.3 It was also found on 1.5% of the CF chromosomes in Russia.32 We did not find any of the mutations more common in European populations, such as G542X (2.6%), N1303K (1.6%), G551D (1.5%), or W1282X (1.0%),4 which is not surprising, as the relative frequency of these mutations is less than 1% in Nordic countries also. Login to comment

[hide] The geographic distribution of cystic fibrosis mut... Eur J Pediatr. 2000 Jul;159(7):496-9. Dawson KP, Frossard PM
The geographic distribution of cystic fibrosis mutations gives clues about population origins.
Eur J Pediatr. 2000 Jul;159(7):496-9., [PMID:10923221]

Abstract [show]
Information regarding three of the more common cystic fibrosis mutations is presented (delta F508, G542X, N13031K) to support the concept of a geography associated with cystic fibrosis mutations. We present the hypothesis that a knowledge of the geography of cystic fibrosis mutations is important for an understanding of genotype-phenotype correlations, gene flow, historical population migration and cystic fibrosis screening. CONCLUSION: A new method of study of mankind's cultural spread is being revealed and the survival of the various mutations supports the concept that they may provide a selective advantage to the carrier.

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12 Eur J Pediatr (2000) 159: 496±499 Ó Springer-Verlag 2000 K. P. Dawson (&) Department of Paediatrics, Faculty of Medicine and Health Sciences, UAE University, PO Box 17666, Al Ain, United Arab Emirates P. M. Frossard Department of Pathology, Faculty of Medicine and Health Sciences, UAE University, PO Box 17666, Al Ain, United Arab Emirates We present here population genetics data that has been gathered about the DF508 mutation and information with regards to two other common mutations, namely G542X and N1303K. Login to comment
45 The G542X mutation G542X, a nonsense mutation, is the second most common mutation after DF508. Login to comment
49 Kerem et al. [10] described the G542X in their major paper on the identi®cation of the CF gene. Login to comment
53 They have produced a fascinating hypothesis that the areas with an elevated frequency of the G542X mutation correspond to ancient sites of occupation by the occidental Phoenicians. Login to comment
56 Thus the evidence to date suggests that the G542X mutation may provide another link in the story of the spread of the CF gene mutations and in the de®nition of their geography [1]. Login to comment
60 Microsatellite markers indicate that the mutation is about 35,000 years old (similar to G542X) and again diusion through Europe from an Asian origin is suggested by these recent ®ndings [8]. Login to comment
104 Loirat F, Hazout S, Lucotte G (1997) G542X as probably Phoenician cystic ®brosis mutation. Login to comment

[hide] Future pharmacological treatment of cystic fibrosi... Respiration. 2000;67(4):351-7. Zeitlin PL
Future pharmacological treatment of cystic fibrosis.
Respiration. 2000;67(4):351-7., [PMID:10940786]

Abstract [show]
Cystic fibrosis (CF) is an autosomal recessive disorder that is caused by over 850 different mutations in the CF gene. It is useful to group these mutations according to the defect that results in the CFTR mRNA or protein. New pharmacological treatments targeted towards specific mutations that are relatively common are being developed. Class I mutations do not produce CFTR protein because of a premature stop signal in the CFTR DNA. These null mutations can be corrected by certain aminoglycosides which cause the aberrant stop signal to be skipped. Mutations leading to a CFTR protein that attains an unstable structure shortly after translation in the endoplasmic reticulum form class II. Class II mutations can be restored to the protein trafficking pathway by manipulation of chaperone protein/CFTR interactions with chemical chaperones or drugs that affect gene regulation such as the butyrates. Production of a CFTR with reduced Cl(-) transport on the basis of abnormal regulation of the chloride channel is the basis of class III. Genistein can overcome this block in regulation. Mutations that partially reduce chloride conductance through CFTR (class IV) can be stimulated with milrinone, which is a phosphodiesterase inhibitor. Finally, mutations that lead to a severe reduction in normal CFTR protein form class V. Increased levels of CFTR could be generated with the butyrates or supplemented with gene therapy. Although most of the reported mutations in CFTR are rare and unclassified, it may be possible to use genotype-phenotype correlations to determine the best approach.

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22 Examples of CFTR mutations organized by classification of the defect in CFTR biosynthesis Type Genotype Phenotype Defect Cell diagram Drugs that may improve phenotype G542X 621+1 G → T 3905insT W1282X R553X 1717-1 G → A PI no CFTR protein no cell surface chloride transport gentamicin G418 Class II [64] 'F508 N1303K (P574H)a (A455E)a PI defective CFTR processing defective CFTR trafficking no cell surface chloride transport chemical chaperones CPX phenylbutyrate deoxyspergualin Class III [64] G551D G551S PI defective chloride channel regulation reduced or absent cell surface chloride transport genistein pyrophosphate Class IV [64, 66] R117H R334W G314E R347P ('F508)a P574H PS reduced chloride conductance reduced levels of cell surface chloride transport genistein milrinone phenylbutyrate Class V [64] 3849+10 kb C → T 2789+5 G → A 3272-26 A → G A455E 3120+1 G → A 1811+1.6 kb A → G 5Tb PS normal CFTR channels reduced numbers of normal CFTR reduced cell surface chloride transport genistein milrinone phenylbutyrate a Some mutants have features of more than one class of defect. Login to comment

[hide] Mutations of the cystic fibrosis gene, but not cat... Am J Gastroenterol. 2000 Aug;95(8):2061-7. Ockenga J, Stuhrmann M, Ballmann M, Teich N, Keim V, Dork T, Manns MP
Mutations of the cystic fibrosis gene, but not cationic trypsinogen gene, are associated with recurrent or chronic idiopathic pancreatitis.
Am J Gastroenterol. 2000 Aug;95(8):2061-7., [PMID:10950058]

Abstract [show]
OBJECTIVE: We investigated whether mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene and cationic trypsinogen gene are associated with recurrent acute, or chronic idiopathic pancreatitis. METHODS: Twenty patients with idiopathic pancreatitis (11 women, nine men; mean age, 30 yr) were studied for the presence of a CFTR mutation by screening the genomic DNA for more than 30 mutations and variants in the CFTR gene. Selected mutations of the cationic trypsinogen gene were screened by Afl III restriction digestion or by a mutation-specific polymerase chain reaction (PCR). In each patient exons 1, 2, and 3 of the cationic trypsinogen gene were sequenced. Patients with a CFTR mutation underwent evaluation of further functional electrophysiological test (intestinal current measurement). RESULTS: No mutation of the cationic trypsinogen gene was detected. A CFTR mutation was detected in 6/20 (30.0%) patients. Three patients (15.0%) had a cystic fibrosis (CF) mutation on one chromosome (deltaF508, I336K, Y1092X), which is known to cause phenotypical severe cystic fibrosis. One patient was heterozygous for the 5T allele. In addition, two possibly predisposing CFTR variants (R75Q, 1716G-->A) were detected on four patients, one of these being a compound heterozygous for the missense mutation I336K and R75Q. No other family member (maternal I336K; paternal R75Q; sister I1336K) developed pancreatitis. An intestinal current measurement in rectum samples of patients with a CFTR mutation revealed no CF-typical constellations. CONCLUSIONS: CFTR mutations are associated with recurrent acute, or chronic idiopathic pancreatitis, whereas mutations of the cationic trypsinogen mutation do not appear to be a frequent pathogenetic factor.

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53 Using the ARMS technology (elucigene CF20, Zeneca Diagnostics, Oxfordshire, UK) all samples were tested additionally for the mutations E60X, R347P, A455E, 1078delT, 2183AA3G, G542X, G551D, N1303K, W1282X, 1717-1G3A, R553X, 621ϩ1G3T, R117H, R1162X, 3849ϩ10kbC3T, R334W, S1251N, and 3659delC. Login to comment

[hide] Effect of genistein on native epithelial tissue fr... Br J Pharmacol. 2000 Aug;130(8):1884-92. Mall M, Wissner A, Seydewitz HH, Hubner M, Kuehr J, Brandis M, Greger R, Kunzelmann K
Effect of genistein on native epithelial tissue from normal individuals and CF patients and on ion channels expressed in Xenopus oocytes.
Br J Pharmacol. 2000 Aug;130(8):1884-92., [PMID:10952679]

Abstract [show]
The flavonoid genistein has been shown to activate a Cl(-) conductance in various cell types expressing CFTR. We examined if similar effects can be observed when genistein is applied to native ex vivo tissues from human respiratory tract and rectum. We further compared the effects when genistein was applied to oocytes of Xenopus laevis expressing CFTR. In oocytes, both wtCFTR and DeltaF508-CFTR were activated by genistein while both cyclic AMP (K(v)LQT1) and Ca(2+) (SK4) activated K(+) channels were inhibited at high concentrations of genistein. Biopsies from nasal polyps and rectal mucosa were obtained from normal individuals (non-CF) and CF patients and in the presence of amiloride (10 micromol l(-1); mucosal side) the effects of genistein were assessed using a perfused Ussing chamber. In non-CF airway epithelia, genistein (50 micromol l(-1); mucosal side) increased lumen negative I(sc) but had no additional effects on tissues pre-stimulated with IBMX and forskolin (100 micromol l(-1) and 1 micromol l(-1); both sides). In non-CF rectal biopsies, in the presence of amiloride (10 micromol l(-1); mucosal side) and indomethacin (10 micromol l(-1); basolateral side), genistein increased lumen negative I(sc) and enabled cholinergic (carbachol; CCH, 100 micromol l(-1); basolateral side) stimulation of Cl(-) secretion indicating activation of luminal CFTR Cl(-) channels. However, after stimulation with IBMX/forskolin, genistein induced opposite effects and significantly inhibited CCH activated I(sc). In CF airway and intestinal tissues genistein failed to induce Cl(-) secretion. Thus, genistein is able to activate luminal CFTR Cl(-) conductance in non-CF tissues and mutant CFTR in oocytes. However, additional inhibitory effects on basolateral K(+) conductance and missing effects in native CF tissues do not support the use for pharmacological intervention in CF.

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26 Eight CF patients presenting with nasal polyps were tested for six common mutations: DF508, R553X, N1303K, G542X, G551D and R347P. Login to comment
30 In all CF patients from whom rectal biopsies were studied DNA analysis was carried out for the following CFTR mutations: DF508; R117H and S108F in exon 4; R347P, R347H, I336K and T338I in exon 7; S549N, G551D, R553X, G542X, Q552X, 1717-1 G?A in exon 11; W1282X and 3905insT in exon 20; N1303K in exon 21 and 3849+10kB C?T in intron 19. Login to comment

[hide] Increased circulating levels of plasma ATP in cyst... Clin Physiol. 2000 Sep;20(5):348-53. Lader AS, Prat AG, Jackson GR Jr, Chervinsky KL, Lapey A, Kinane TB, Cantiello HF
Increased circulating levels of plasma ATP in cystic fibrosis patients.
Clin Physiol. 2000 Sep;20(5):348-53., [PMID:10971545]

Abstract [show]
Recent studies have shown that the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP-binding cassette (ABC) transporter whose mutations are responsible for cystic fibrosis (CF), permeates ATP. However, little information is available concerning extracellular ATP concentrations in CF patients. Thus, the goal of this preliminary study was to determine the circulating levels of plasma ATP in CF patients. Circulating levels of plasma ATP were determined by the luciferin-luciferase assay in both CF patients and healthy volunteer control subjects. The two groups were compared using an analysis of variance. CF genotype and age, which ranged from 7 to 56 years, were also used to compare data by single-blind analysis. With comparable sample numbers, CF patients had statistically higher levels of circulating ATP (34%, P<0.01) when compared by analysis of covariance with the age of the subjects as the cofactor. The CF patients bearing the DeltaF508 genotype had a 54% (n=33, P<0.01) higher plasma ATP concentration compared to controls, while patients bearing other CF genotypes were similar to controls (n=10, P<0.4). We conclude that CF patients have higher circulating levels of ATP when compared to controls. Increased levels of plasma ATP, which is an important autocrine/paracrine hormone in many cell types, may be associated with chronic manifestations of the disease.

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65 other CF mutations including R117H (n 3), G551D (n 1), G542X (n 1) and W1282X (n 1), had an encompassed plasma ATP level of 1á22 0á22 lM (n 10), thus similar to control values (P<0á4). Login to comment
66 Of these genotypes, the G551D mutation had the highest plasma ATP concentration (2á25), while the W1282X, G542X, and R117H mutations had plasma ATP concentrations of 1á6, 0á75 and 0á68, respectively. Login to comment

[hide] Prenatal detection by real-time quantitative PCR a... Clin Chem. 2000 Sep;46(9):1417-20. Costes B, Girodon E, Vidaud D, Flori E, Ardalan A, Conteville P, Fanen P, Niel F, Vidaud M, Goossens M
Prenatal detection by real-time quantitative PCR and characterization of a new CFTR deletion, 3600+15kbdel5.3kb (or CFTRdele19).
Clin Chem. 2000 Sep;46(9):1417-20., [PMID:10973878]

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51 The mutations tested were S549N, S549R, R553X, G551D, V520F, ⌬I507, ⌬F508, Q493X, 1717-1G3A, G542X, R560T, R347P, R347H, 3849ϩ4A3G, W1282X, R334W, 1078delT, 3849ϩ10kbC3T, R1162X, N1303K, 3659delC, 3905insT, A455E, R117H, Y122X, 2183AA3G, 2789ϩ5G3A, 1898ϩ1G3A, 621ϩ1G3T, 711ϩ1G3T, and G85E. Login to comment

[hide] Mode of action and application of Scorpion primers... Nucleic Acids Res. 2000 Oct 1;28(19):3752-61. Thelwell N, Millington S, Solinas A, Booth J, Brown T
Mode of action and application of Scorpion primers to mutation detection.
Nucleic Acids Res. 2000 Oct 1;28(19):3752-61., 2000-10-01 [PMID:11000267]

Abstract [show]
Scorpion primers can be used to detect PCR products in homogeneous solution. Their structure promotes a unimolecular probing mechanism. We compare their performance with that of the same probe sequence forced to act in a bimolecular manner. The data suggest that Scorpions indeed probe by a unimolecular mechanism which is faster and more efficient than the bimolecular mechanism. This mechanism is not dependent on enzymatic cleavage of the probe. A direct comparison between Scorpions, TaqMan and Molecular Beacons on a Roche LightCycler indicates that Scorpions perform better, particularly under fast cycling conditions. Development of a cystic fibrosis mutation detection assay shows that Scorpion primers are selective enough to detect single base mutations and give good sensitivity in all cases. Simultaneous detection of both normal and mutant alleles in a single reaction is possible by combining two Scorpions in a multiplex reaction. Such favourable properties of Scorpion primers should make the technology ideal in numerous applications.

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39 We have used Scorpion primers to detect five common ABCC7 mutations, ∆F508 (11-13), N1303K (15), W1282X (12), G542X (12) and G551D (12,16) (Table 1). Login to comment
60 Mutation site Base change Probe-target mismatch ∆F508 M55115 436-438 CTT del - N1303K M55128 329 C→G C-C W1282X M55127 395 G→A C-A G551D M55116 362 G→A C-A G542X M55116 334 G→T C-T All PCR reactions were carried out on a Roche LightCycler. Login to comment
72 Oligo name Code Oligo sequence MTHFR forward primer BPF 5'-CTGACCTGAAGCACTTGAAGG-3' MTHFR reverse primer BPR 5'-ATGTCGGTGCATGCCTTCAC-3' MTHFR Molecular Beacon MMB 5'-FAM GCGAGTGCGGGAGCCGATTTCTCGC MR-3' MTHFR TaqMan MT 5'-FAM TGCGGGAGCCGATTT TAMRA-3' MTHFR Scorpion MS 5'-FAM CCCGCGGAAATCGGCTCCCGCACCGCGGG MR HEG CTGACCTGAAGCACTTGAAGG-3' ∆F508 normal Scorpion 508S 5'-FAM CCGCGCAAACACCAAAGATGATATTTTCTGCGCGG MR HEG AGTTTTCCTGGATTATGCCT-3' ∆F508 mutant Scorpion 508M 5'-ROX CCGC(F)GCAAACACCAATGATATTTTCTGCAGCGG MR HEG AGTTTTCCTGGATTATGCCT-3' ∆F508 reverse primer 508R 5'-TTGGGTAGTGTGAAGGGTTC-3' ∆F508 forward primer 508F 5'-AGTTTTCCTGGATTATGCCT-3' Hybrid Scorpion HS 5'-FAM CCGCGCAAACACCAAAGATGATATTTTCTGCGCGG MR HEG CTTGGAGAAGGTGGAATCAC-3' N1303K Scorpion N13S 5'-FAM CCCGCGCGGAACATTTAGAAAAAACTTGGATCCCGCGCGGG MR HEG TTTCTTGATCACTCCACTGTTC-3' N1303K reverse primer N13R 5'-CATACTTTCTTCTTCTTTTCTTT-3' W1282X Scorpion W12S 5'-FAM CCCGCGCCTTTCCTCCACTGTTGCGCGCGGG MR HEG ATGGTGTGTCTTGGGATTCA-3' W1282X reverse primer W12R 5'-GGCTAAGTCCTTTTGCTCAC-3' G551D Scorpion 551S 5'-FAM CCCGCGCCTCGTTGACCTCCACTCGCGCGGG MR HEG CTTGGAGAAGGTGGAATCAC-3' G551D reverse primer 551R 5'-AAATGCTTGCTAGACCAATA-3' G551D forward primer 551F 5'-CTTGGAGAAGGTGGAATCAC-3' G551D-DIST Scorpion (90 bases between 3'-end of Scorpion primer and 5'-end of probe target) G90 5'-FAM CCCGCGCCTCGTTGACCTCCACTCGCGCGGG MR HEG CAGATTGAGCATACTAAAAG-3' G542X Scorpion G542S 5'-FAM CCGCGCACCTTCTCCAAGAACTAGCGCGG MR HEG CCAAGTTTGCAGAGAAAGAC-3' G542X reverse primer G542R 5'-AAATGCTTGCTAGACCAATA-3' Table 3. Login to comment
73 Annealing and fluorescence monitoring temperatures used for each locus Loci/test Annealing temperature (°C) Monitoring temperature (°C) ∆F508 48 51 N1303K 44 61 W1282X 49 53 G551D 47 55 G551D-DIST 46 55 G542X 48 53 Unimolecular versus bimolecular test 47 51 Distance constraints G90 Scorpion 46 55 G551DS Scorpion 47 55 MTHFR 58 58 described in Results were 95°C for 30 s, 58°C for 60 s and 72°C for 30 s. Login to comment

[hide] Mutation in the gene responsible for cystic fibros... JAMA. 2000 Oct 11;284(14):1814-9. Wang X, Moylan B, Leopold DA, Kim J, Rubenstein RC, Togias A, Proud D, Zeitlin PL, Cutting GR
Mutation in the gene responsible for cystic fibrosis and predisposition to chronic rhinosinusitis in the general population.
JAMA. 2000 Oct 11;284(14):1814-9., 2000-10-11 [PMID:11025834]

Abstract [show]
CONTEXT: Chronic rhinosinusitis (CRS) is a common condition in the US general population, yet little is known about its underlying molecular cause. Chronic rhinosinusitis is a consistent feature of the autosomal recessive disorder cystic fibrosis (CF). OBJECTIVE: To determine whether mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, which is responsible for CF, predispose to CRS. DESIGN: Case-control study conducted from 1996 to 1999 in which the DNA of CRS patients and controls was typed for 16 mutations that account for 85% of CF alleles in the general population. Chronic rhinosinusitis patients with 1 CF mutation were evaluated for a CF diagnosis by sweat chloride testing, nasal potential difference measurement, and DNA analysis for additional mutations. SETTING: Otolaryngology-head and neck clinic of a US teaching hospital. PARTICIPANTS: One hundred forty-seven consecutive adult white patients who met stringent diagnostic criteria for CRS and 123 CRS-free white control volunteers of similar age range, geographic region, and socioeconomic status. MAIN OUTCOME MEASURES: Presence of CF mutations by DNA analysis among CRS patients vs controls. RESULTS: Eleven CRS patients were found to have a CF mutation (DeltaF508, n = 9; G542X, n = 1; and N1303K, n = 1). Diagnostic testing excluded CF in 10 of these patients and led to CF diagnosis in 1. Excluding this patient from the analyses, the proportion of CRS patients who were found to have a CF mutation (7%) was significantly higher than in the control group (n = 2 [2%]; P =.04, both having DeltaF508 mutations). Furthermore, 9 of the 10 CF carriers had the polymorphism M470V, and M470V homozygotes were overrepresented in the remaining 136 CRS patients (P =.03). CONCLUSION: These data indicate that mutations in the gene responsible for CF may be associated with the development of CRS in the general population. JAMA. 2000;284:1814-1819.

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11 Results Eleven CRS patients were found to have a CF mutation (⌬F508, n=9; G542X, n=1; and N1303K, n=1). Login to comment
30 Analysis of CFTR Genes Genomic DNA samples extracted from the blood of participants were screened for 16 mutations (R117H, 621+1G→T, R334W, R347P, A455E, ⌬I507, ⌬F508, 1717-1 G→A, G542X, S549N, G551D, R553X, R560T, 3849+10 Kb C→T, W1282X, and N1303K) that account for 85% of CF alleles in the white population using the multiplex reverse dot hybridization system (Roche Molecular Systems, Alameda, Calif).16,17 This test also identified the 5T, 7T, and 9T variants of the splice acceptor site in intron 8 and F508C, I507V, and I506V (exon 10) polymorphisms of the CFTR gene. Login to comment
46 Eleven CRS patients were found to have CF mutations (TABLE 1); 9 had the common mutation, ⌬F508, 1 had G542X, and 1 had N1303K. Login to comment
53 Using this technique, we previously demonstrated that at least 96% of mutations in the exons and flanking intron regions of the CFTR gene can be detected.16 A 97% sensitivity was achieved using 115 samples with previously identified mutations16 in this study.Sequenceanalysisofsampleswith anabnormalDGGEpatternidentifiedan R75Q mutation in the N1303K carrier and an L967S mutation in the G542X carrier. Login to comment
59 Patient 1386 had changes in exon 10 (M470V), exon 15 (L967S), and exon 11 (G542X), and pedigree analysis was used to show that the M470V variant and the L967S variant segregated independently of G542X. Login to comment
67 Cystic Fibrosis (CF) Mutations and Cystic Fibrosis Transmembrane Regulator (CFTR) Variants in Chronic Rhinosinusitis (CRS) Patients and Controls* CRS, No. (%) Non CRS, No. (%) P Value (n = 147) (n = 123) CF mutations ⌬F508/+ 1 2 ⌬F508/M470V 7 0 G542X/M470V, L967S 1 0 N1303K/+; M470V/M; R75Q/+† 1 0 (⌬F508/2789+5G→A)‡ 1 0 Frequency of CF carriers 10 (7) 2 (2) .04§ (n = 136) (n = 121) CFTR variants 5T Variant in non-CF carriers࿣ 5T/+ 11 (9) 6 (6) .25§ Codon 470 genotypes among non-CF carriers࿣ M/M 21 (16) 23 (19) M/V 55 (40) 64 (53) .03¶ V/V 60 (44) 34 (28) *Plus (+) indicates wildtype. Login to comment
87 1386 M G542X/M470V, L967S 16 14 + Asthma, Pseudomonas aeruginosa pneumonia§ Normal Ͻ15 Fathered 3 children, genetically confirmed 1379 F ⌬F508/M470V Ͻ10 8 - Normal Normal 31 Problem conceiving 1380 M ⌬F508/M470V 59 2 + Asthma Normal 39 Fathered 2 children 1468 F ⌬F508/M470V 20 2 - Normal Normal 29 Problem conceiving 1509 F ⌬F508/M470V 26 None - Asthma Normal 21 . Login to comment

[hide] The incidence of cystic fibrosis (CF) mutations am... Clin Genet. 2000 Oct;58(4):333-5. Tanackovic G, Barisic I, Gjergja-Matejic R, Hecimovic S, Pavelic J
The incidence of cystic fibrosis (CF) mutations among patients from Croatia.
Clin Genet. 2000 Oct;58(4):333-5., [PMID:11076060]

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5 After DNA isolation (2), we screened the samples for the 16 most common CFTR mutations: DF508, DI507 [heteroduplex analysis (3)] G542X, G551D, W1282X, N1303K, 3849+10kbCT, R553X, 621+1GT, R1162X, 1717-1GA, 2789+ 5GA, 3849+4AG, 1898+1GA, R117H [restriction fragment length polymorphism, (4-7)] and 3905insT [single-strand conformational polymorphism analysis (8)]. Login to comment
7 The presence of six different mutations was observed on 60 CF chromosomes: DF508, G542X, 1717-1GA, R117H, N1303K and R1162X (Table 1). Login to comment
14 The second most common mutation among Croatian CF patients was G542X with a relative frequency of 5.0%. Login to comment
16 The frequency of mutation G542X in two neighbouring countries was, however, lower: Slovenia (3.3%) (17) and Hungary (2.4%) (12, 18). Login to comment

[hide] CF gene and cystic fibrosis transmembrane conducta... J Am Soc Nephrol. 2000 Dec;11(12):2285-96. Persu A, Devuyst O, Lannoy N, Materne R, Brosnahan G, Gabow PA, Pirson Y, Verellen-Dumoulin C
CF gene and cystic fibrosis transmembrane conductance regulator expression in autosomal dominant polycystic kidney disease.
J Am Soc Nephrol. 2000 Dec;11(12):2285-96., [PMID:11095651]

Abstract [show]
Disease-modifying genes might participate in the significant intrafamilial variability of the renal phenotype in autosomal dominant polycystic kidney disease (ADPKD). Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is a chloride channel that promotes intracystic fluid secretion, and thus cyst progression, in ADPKD. The hypothesis that mutations of the CF gene, which encodes CFTR, might be associated with a milder renal phenotype in ADPKD was tested. A series of 117 unrelated ADPKD probands and 136 unaffected control subjects were screened for the 12 most common mutations and the frequency of the alleles of the intron 8 polymorphic TN: locus of CF. The prevalence of CF mutations was not significantly different in the ADPKD (1.7%, n = 2) and control (3.7%, n = 5) groups. The CF mutation was DeltaF508 in all cases, except for one control subject (1717-1G A). The frequencies of the 5T, 7T, and 9T intron 8 alleles were also similar in the ADPKD and control groups. Two additional patients with ADPKD and the DeltaF508 mutation were detected in the families of the two probands with CF mutations. Kidney volumes and renal function levels were similar for these four patients with ADPKD and DeltaF508 CFTR (heterozygous for three and homozygous for one) and for control patients with ADPKD collected in the University of Colorado Health Sciences Center database. The absence of a renal protective effect of the homozygous DeltaF508 mutation might be related to the lack of a renal phenotype in CF and the variable, tissue-specific expression of DeltaF508 CFTR. Immunohistochemical analysis of a kidney from the patient with ADPKD who was homozygous for the DeltaF508 mutation substantiated that hypothesis, because CFTR expression was detected in 75% of cysts (compared with <50% in control ADPKD kidneys) and at least partly in the apical membrane area of cyst-lining cells. These data do not exclude a potential protective role of some CFTR mutations in ADPKD but suggest that it might be related to the nature of the mutation and renal expression of the mutated CFTR.

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52 Genomic DNA samples were screened using the Elucigene CF12 kit (based on Amplification Refractory Mutation System technology; Zeneca Diagnostics, Abingdon, UK), to detect the following 12 CFTR mutations: 1717-1G3A, G542X, W1282X, N1303K, ⌬F508, 3849ϩ10kbC3T, 621ϩ1G3T, R553X, G551D, R117H, R1162X, and R334W. Login to comment
99 Characteristics of the 12 mutations of the CF gene screened for among the patients with ADPKD and the control subjectsa Name Location Nucleotide Change CFTR Domain Consequence R117H Exon 4 G3A at 482 TM2 Arg3His at 117 621ϩ1G3T Intron 4 G3T at 621ϩ1 mRNA splicing mutation R334W Exon 7 C3T at 1132 TM6 Arg3Trp at 334 ⌬F508 Exon 10 3-bp deletion between 1652 and 1655 NBD1 Phe-508 deletion 1717-1G3A Intron 10 G3A at 1717-1 NBD1 mRNA splicing mutation G542X Exon 11 G3T at 1756 NBD1 Gly3Stop at 542 G551D Exon 11 G3A at 1784 NBD1 Gly3Asp at 551 R553X Exon 11 C3T at 1789 NBD1 Arg3Stop at 553 R1162X Exon 19 C3T at 3616 Arg3Stop at 1162 3849ϩ10kbC3T Intron 19 C3T in a 6.2-kb EcoRI fragment 10 kb from 19 NBD2 Creation of a splice acceptor site W1282X Exon 20 G3A at 3978 NBD2 Trp3Stop at 1282 N1303K Exon 21 C3G at 4041 NBD2 Asn3Lys at 1303 a Modified from reference 16. Login to comment

[hide] Type I, II, III, IV, and V cystic fibrosis transme... Curr Opin Pulm Med. 2000 Nov;6(6):521-9. Choo-Kang LR, Zeitlin PL
Type I, II, III, IV, and V cystic fibrosis transmembrane conductance regulator defects and opportunities for therapy.
Curr Opin Pulm Med. 2000 Nov;6(6):521-9., [PMID:11100963]

Abstract [show]
Recent advances in cellular and molecular biology have furthered the understanding of several genetic diseases, including cystic fibrosis. Mutations that cause cystic fibrosis are now understood in terms of the specific molecular consequences to the cystic fibrosis transmembrane conductance regulator (CFTR) protein expression and function. This knowledge has spawned interest in the development of therapies aimed directly at correcting the defective CFTR itself. In this article, we review the molecular defect underlying each recognized class of CFTR mutation and the potential therapies currently under investigation. Opportunities for protein-repair therapy appear to be vast and range from naturally occurring compounds, such as isoflavonoids, to pharmaceuticals already in clinical use, including aminoglycoside antibiotics, butyrate analogues, phosphodiesterase inhibitors, and adenosine nucleotides. Future therapies may resemble designer compounds like benzo[c]quinoliziniums or take the form of small peptide replacements. Given the heterogeneity and progressive nature of cystic fibrosis, however, optimal benefit from protein-repair therapy will most likely require the initiation of combined therapies early in the course of disease to avoid irreparable organ damage.

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22 The nonsense mutations G542X, W1282X, R553X, Q39X, E60X, R75X, L719X, Y1092X, and S1196X significantly reduce the levels of mutant CFTR mRNA to 5 to 30% of wild-type levels [28]. Login to comment
37 Molecular fate of CFTR protein Type of genetic defect and example Class-specific potential therapeutic approach Specific clinical examples I No synthesis Nonsense G542X Frameshift 394delTT Splice junction 1717-1G→A Aminoglycoside readthrough of premature termination site Gentamicin II Trafficking block AA deletion ∆F508 Missense N1303K Manipulation of intracellular folding environment (chemical or molecular chaperones) Phenylbutyrate, CPX III Block in regulation Missense G551D Stimulation of membrane localized mutant channel Genistein, MPB- compounds IV Altered conductance Missense R117H Augmentation of mutant channel conductance Milrinone, adenosine nucleotides V Reduced synthesis of normal protein Missense A455E Alternative splicing 3849+10kbC→T Maximal activation of decreased but functionally normal channels Stimulation of mRNA and protein synthesis ? Login to comment

[hide] Molecular screening of the CFTR gene in men with a... Mol Hum Reprod. 2000 Dec;6(12):1063-7. Jezequel P, Dubourg C, Le Lannou D, Odent S, Le Gall JY, Blayau M, Le Treut A, David V
Molecular screening of the CFTR gene in men with anomalies of the vas deferens: identification of three novel mutations.
Mol Hum Reprod. 2000 Dec;6(12):1063-7., [PMID:11101688]

Abstract [show]
Many studies have shown that congenital absence of the vas deferens (CAVD) is a genital cystic fibrosis transmembrane conductance regulator (CFTR)-mediated phenotype, with a broad spectrum of abnormalities causing male infertility. The genotype of these patients includes mutations in the CFTR gene, e.g. DeltaDeltaF508, R117H and the T5 allele; all of which are commonly found in CAVD. In this study we have screened the entirety of CFTR gene in 47 males with anomalies of the vas deferens: 37 cases of congenital bilateral absence of the vas deferens, three cases of congenital unilateral absence of the vas deferens and seven cases of obstructive azoospermia with hypoplastic vas deferens. Among the 94 chromosomes studied, 65 mutations, of which three are novel (2789+2insA, L1227S, 4428insGA), were identified. The majority of patients (63.8%) had two detectable CFTR gene mutations. Furthermore, high frequencies of the DeltaDeltaF508 mutation (44.7%), the T5 allele (36.2%) and R117H mutation (19.1%) were observed.

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54 The three men with CUAVD wereTotal genomic DNA was isolated from the patients` peripheral blood cells and analysed for mutations in the whole CFTR region and splice compound heterozygotes (G542X/R1070W, ∆F508/R117H, junctions. Login to comment
60 Tworegions (1, 2, 5, 6a, 6b, 7, 10, 11, 12, 14a, 14b, 15, 16, 17a, 17b, 18, 20, 22, 23, 24) were amplified with a GC-clamp primer and six exons mutations were found in 31.9% of patients, 31.9% had one Table I. Summary of the clinical and biological findings of a population of men with congenital bilateral absence of the vas deferens (CBAVD, n ϭ 37), congenital unilateral absence of the vas deferens (CUAVD, n ϭ 3) and obstructive azoospermia (Obs A, n ϭ 7) Patient Phenotype Surgical Age Weight Height Sweat test Other clinical CFTR exploration (years) (kg) (m) (Cl- mEq/l) manifestation genotype 1 CBAVD ϩ 40 63 1.72 72 ∆F508/T5 2 CBAVD ϩ 31 66 1.76 40 L1227S/3272-26A→G 3 CBAVD ϩ 29 ∆F508/T5 4 CBAVD 29 sinusitis -/- 5 CBAVD 32 50 1.60 ∆F508/T5 6 CBAVD 35 64 1.66 ∆F508/T5 7 CBAVD ϩ 28 ∆F508/R117H 8 CBAVD ϩ 34 69 1.80 24 ∆F508/R117H 9 CBAVD ϩ 35 65 1.70 R117H/T5 10 CBAVD ϩ 32 50 1.70 31 asthma ∆F508/T5 11 CBAVD ϩ 26 left hydrocele T5/- 12 CBAVD ϩ 23 left varicocele, G551D/T5 asthma, anosmia 13 CBAVD ϩ 29 ∆F508/T5 14 CBAVD ϩ 36 63 1.64 52 ∆F508/R117H 15 CBAVD ϩ 37 60 1.76 ∆F508/T5 16 CBAVD ϩ 34 70 1.65 24 ∆F508/A1067V 17 CBAVD 35 61 1.73 42 ∆F508/R117H 18 CBAVD 25 72 1.82 86 2183AA→G/T5 19 CBAVD 28 88 1.76 7 -/- 20 CBAVD ϩ 29 ∆F508/T5 21 CBAVD 31 48 epididymite -/- 22 CBAVD 28 ∆F508/T5 23 CBAVD ϩ 32 68 1.76 36 flatulence ∆F508/R1070W 24 CBAVD ϩ 31 64 1.76 39 R1162X/T5 25 CBAVD 30 17 asthma R117H/L375F 26 CBAVD ϩ 36 62 1.70 ∆F508/R1070W 27 CBAVD 30 6 -/- 28 CBAVD 35 85 1.70 R1070W/- 29 CBAVD 39 bronchectasis -/- 30 CBAVD ϩ 29 ∆F508/- 31 CBAVD 31 bronchectasis, -/- deafness 32 CBAVD ϩ 26 asthma, otitis -/- 33 CBAVD ϩ 28 allergy -/- 34 CBAVD 37 36 R117H/- 35 CBAVD 33 -/- 36 CBAVD ϩ 30 64 1.68 R117H/T5 37 CBAVD ϩ 37 71 1.78 31 pancreatitis, 621ϩ1G→T/I980K alcoholism 38 CUAVD 43 62 1.68 40 allergy G542X/R1070W 39 CUAVD ϩ 35 allergy ∆F508/R117H 40 CUAVD ϩ 34 hydrocele L375F/G551D 41 Obs A ϩ 32 26 T5/- 42 Obs A 23 60 sinusitis ∆F508/2789ϩ2insA 43 Obs A ϩ 25 80 sinusitis, chronic ∆F508/4428insGA 44 Obs A ϩ 30 bronchitis -/- anosmia 45 Obs A 29 50 -/- 46 Obs A 29 75 1.77 ∆F508/T5 47 Obs A ϩ 30 82 1.66 -/- mutation and the T5 allele, 10.7% had only one mutation and clinical palpation. Login to comment
73 This mutation creates a stop codon 43 nucleotides 26 ∆F508/R1070W (TG)10T9/(TG)10T7 downstream leading to the deletion of 33 C-terminus amino 16 ∆F508/A1067V (TG)10T9/(TG)10T7 acids of the CFTR protein including the TRL-COOH domain.42 ∆F508/2789ϩ2insA (TG)10T9/(TG)10T7 43 ∆F508/4428insGA (TG)10T9/(TG)11T7 This highly conserved proteic site is a perfect match for the 25 R117H/L375F (TG)10T7/(TG)10T7 binding consensus domain of the Naϩ-Hϩ exchanger regulatory 38 G542X/R1070W (TG)10T9/(TG)11T7 factor (NHE-RF), a cytoplasmic phosphoprotein that may play40 L375F/G551D (TG)10T7/(TG)10T7 37 621ϩ1G→T/I980K (TG)10T9/(TG)10T9 an important regulatory role in CFTR function (Wang et al., 2 L1227S/3272-26A→G (TG)10T9/(TG)12T7 1998). Login to comment

[hide] Pharmacological treatment of the ion transport def... Expert Opin Investig Drugs. 2001 Jan;10(1):1-19. Roomans GM
Pharmacological treatment of the ion transport defect in cystic fibrosis.
Expert Opin Investig Drugs. 2001 Jan;10(1):1-19., [PMID:11116277]

Abstract [show]
Cystic fibrosis (CF) is a lethal monogenetic disease characterised by impaired water and ion transport over epithelia. The lung pathology is fatal and causes death in 95% of CF patients. The genetic basis of the disease is a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel. The most common mutation, DeltaF508, results in a protein that cannot properly be folded in the endoplasmic reticulum, is destroyed and hence does not reach the apical cell membrane. This paper will discuss those pharmacological approaches that are directed at correcting the defect in ion transport. At present, no clinically effective drug is available, although research has defined areas in which progress might be made. These are the following: (1) the drug 4-phenylbutyrate (4PBA) increases the expression of DeltaF508-CFTR in the cell membrane, probably by breaking the association between DeltaF508-CFTR and a chaperone; (2) a number of xanthines, in particular 8-cyclopentyl-1, 3-dipropylxanthine (CPX), are effective in activating CFTR, presumably by direct binding and also possibly by correcting the trafficking defect; (3) the isoflavone genistein can activate both wild-type and mutant CFTR, probably through direct binding to the channel; (4) purinergic agonists (ATP and UTP) can stimulate chloride secretion via a Ca(2+)-dependent chloride channel and in this way compensate for the defect in CFTR, but stable analogues will be required before this type of treatment has clinical significance; (5) treatment with inhaled amiloride may correct the excessive absorption of Na(+) ions and water by airway epithelial cells that appears connected to the defect in CFTR; although clinical tests have not been very successful so far, amiloride analogues with a longer half-life may give better results. The role of CFTR in bicarbonate secretion has not yet been established with certainty, but correction of the defect in bicarbonate secretion may be important in clinical treatment of the disease. Currently, major efforts are directed at developing a pharmacological treatment of the ion transport defect in CF, but much basic research remains to be done, in particular, with regard to the mechanism by which defective CFTR is removed in the endoplasmic reticulum by the ubiquitin-proteasome pathway, which is a central pathway in protein production and of significance for several other diseases apart from CF.

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219 5.6 Gentamicin The aminoglycoside antibiotic gentamicin has been shown to suppress two premature stop mutations (G542X and R553X) [114]. Login to comment

[hide] Update on clinical trials in the treatment of pulm... Expert Opin Investig Drugs. 1999 Nov;8(11):1917-1927. Shah PL
Update on clinical trials in the treatment of pulmonary disease in patients with cystic fibrosis.
Expert Opin Investig Drugs. 1999 Nov;8(11):1917-1927., [PMID:11139834]

Abstract [show]
Cystic fibrosis is a congenital disease resulting from an abnormality of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. A defect in ion transport leads to poor clearance of viscoelastic secretions and a susceptibility to bacterial infection. This initiates a self-perpetuating cycle of infection and inflammation that accounts for the chronic endobronchial sepsis and pulmonary damage observed in patients with cystic fibrosis. Recent studies have attempted to correct the gene defect, enhance the expression and function of the CFTR protein and correct the ion transport defect. Improving the rheological properties of airway secretions, enhancing host defence and controlling inflammation are the other key strategies.

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43 A pilot study has thus far treated eight patients with the G542X mutation with iv. Login to comment
57 (Genetech, Roche, NIH) & University of Cornell Repeated dosing, every 30 days to the Lungs Age > 15 years FEV1 > 40% predicted 26 Degree of transfection, duration and stability of expression Adenovirus vector Phase II University of Iowa Repeated administration to the lung NK NK Degree of transfection, duration and stability of expression Adeno-associated viral vector Phase I Targeted Genetics, Medeva, Stanford University, University of Washington & Boston Children Hospital Aerosol to lung Age > 15 years FVC > 40% predicted CF genotypes: DF508, G551D, W1282X, and G542X 12 Degree of transfection Cationic lipids, Cytofectins Phase I Genzyme, Vical, University of Alabama Lung Age > 18 years FEV1 > 40% predicted NK Degree of transfection Liposomal DNA complexes Phase II NHLI, UK Inhalation to lung Male FEV1 > 70% 16 i) functional correction ii) degree of transfection iii) bacterial adherence NHLI: National Heart & Lung Institute (Imperial College, UK); NIH: National Institute of Health (USA); NK: Not known. Login to comment

[hide] Unilateral renal agenesis associated with congenit... Hum Reprod. 2001 Feb;16(2):282-8. McCallum T, Milunsky J, Munarriz R, Carson R, Sadeghi-Nejad H, Oates R
Unilateral renal agenesis associated with congenital bilateral absence of the vas deferens: phenotypic findings and genetic considerations.
Hum Reprod. 2001 Feb;16(2):282-8., [PMID:11157821]

Abstract [show]
An association between congenital bilateral absence of the vas deferens (CBAVD), normal renal anatomy and cystic fibrosis (CF) gene mutations is well established (CF/CBAVD). We postulate that unilateral renal agenesis (URA) and CBAVD (URA/CBAVD) may have a non-CF mutation-mediated genetic basis that leads to abnormal development of the entire mesonephric duct at a very early stage in embryo development (< or =7 weeks). The physical, laboratory and radiographic findings of men with URA/CBAVD (n = 17) and CF/CBAVD (n = 97) were compared; the fertilization and pregnancy rates in the URA/CBAVD population calculated, and the incidence of renal agenesis in immediate family members and offspring of men with URA/CBAVD analysed. No statistical differences could be identified within any of the above comparisons. The fertilization rate for the URA/CBAVD group was 58.2 +/- 26.3%. Eight infants and two fetuses had normal renal anatomy, while one terminated male fetus had bilateral renal and vasal agenesis. Thirty first-order relatives had normal renal units. Anatomical expression of the reproductive ductal derivatives in men with URA/CBAVD and CF/CBAVD was similar, but the phenotypic outcome of the renal portion of the mesonephric duct was different. The potential for transmission of this fatal anomaly reinforces the need for prenatal ultrasounds with all pregnancies involving URA/CBAVD men.

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60 Therereflects the 26 mutation/5T allele polymorphism assessment at the was no history of maternal diabetes, obvious teratogen exposurepresent time at Boston University Center for Human Genetics: or evidence of known congenital syndromes in themselves, orR117H; 1717-1G→A; G542X; 621ϩ1; S549N; R560T; I507; G551D; their families, that the men in either group could recall. Login to comment

[hide] Comprehensive mutation screening in a cystic fibro... Pediatrics. 2001 Feb;107(2):280-6. Wine JJ, Kuo E, Hurlock G, Moss RB
Comprehensive mutation screening in a cystic fibrosis center.
Pediatrics. 2001 Feb;107(2):280-6., [PMID:11158459]

Abstract [show]
OBJECTIVES AND BACKGROUND: The identities of a cystic fibrosis (CF) patient's CFTR mutations can influence therapeutic strategies, but because >800 CFTR mutations exist, cost-effective, comprehensive screening requires a multistage approach. Single-strand conformation polymorphism and heteroduplex analysis (SSCP/HA) can be an important part of mutation detection, but must be calibrated within each laboratory. The sensitivity of a combined commercial-SSCP/HA approach to genotyping in a large, ethnically diverse US center CF population has not been established. STUDY DESIGN: We screened all 27 CFTR exons in 10 human participants who had an unequivocal CF diagnosis including a positive sweat chloride test and at least 1 unknown allele after commercial testing for the 70 most common mutations by SSCP/HA. These participants were compared with 7 participants who had negative sweat tests but at least 1 other CF-like symptom meriting complete genotyping. RESULTS: For the 10 CF participants, we detected 11 of 16 unknown alleles (69%) and all 4 of the known alleles (100%), for an overall rate of 75% inpatients not fully genotyped by conventional 70 mutation screen. For 7 participants with negative sweat tests, we confirmed 1 identified mutation in 14 alleles and detected 3 additional mutations. Mutations detected in both groups included 7 missense mutations (S13F, P67L, G98R, S492F, G970D, L1093P, N1303K) and 9 deletion, frameshift, nonsense or splicing mutations (R75X, G542X, DeltaF508, 451-458Delta8 bp, 5T, 663DeltaT, exon 13 frameshift, 1261+1G-->A and 3272-26A-->G). Three of these mutations were novel (G970D, L1093P, and 451-458Delta8 bp(1)). Thirteen other changes were detected, including the novel changes 1812-3 ins T, 4096-278 ins T, 4096-265 ins TG, and 4096-180 T-->G. CONCLUSION: When combined with the 70 mutation Genzyme test, SSCP/HA analysis allows for detection of >95% of the mutations in an ethnically heterogeneous CF center population. We discuss 5 possible explanations that could account for the few remaining undetected mutations.

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16 Mutations detected in both groups included 7 missense mutations (S13F, P67L, G98R, S492F, G970D, L1093P, N1303K) and 9 deletion, frameshift, nonsense or splicing mutations (R75X, G542X, ⌬F508, 451-458⌬8 bp, 5T, 663⌬T, exon 13 frameshift, 1261؉1G3A and 3272-26A3G). Login to comment
86 Mutations in the Stanford CF Mutation Database After Screening With the Genzyme70 Assay Mutation n % n % ⌬F508 353 67.11% 353 67.11% Splice mutations 16 3.04% 621ϩ1 G3T 5 0.95% 1717-1 G3A 5 0.95% 2789ϩ5 G3A 1 0.19% 1898ϩ1 G3A 1 0.19% 3849ϩ10 kb C3T 4 0.76% Stop mutations 31 5.89% Q493X 1 0.19% G542X 13 2.47% R553X 4 0.76% R1162X 1 0.19% W1282X 10 1.90% S1455X 2 0.38% Insertions/deletions 9 1.71% 681 del C 1 0.19% 2184 del A 2 0.38% 3859 del C 5 0.95% 3905 ins T 1 0.19% Missense mutations 33 6.27% G85E 4 0.76% R117H 3 0.57% R334W 6 1.14% G551D 14 2.66% R560T 3 0.57% N1303K 3 0.57% Unknown mutations 84 15.97% 84 15.97% Total 526 100.00% 526 100.00% ARTICLES tients with positive sweat tests were selected for SSCP/HA analysis based on clinical status, ethnicity, and previous screening with the Genzyme70 assay. Login to comment
155 PS none - SN3 unk/unk 10-15 PS none GATT6/6, 1001ϩ11 C3T, TG10, T9 homozygous all SN4 unk/unk 25 PI None M470V (10), (GT)11/10 GATT7/7, 1896ϩ152 A/T (I12); 2694T/G (14a), 4521G3A (24) SN5 G542X unk 25 PS none M470V (10), TG11, T7 SN6 unk*/unk* 48 PS P67L (3) 1261ϩIG3A (I13) 1898ϩ174 ins A (I12), 2694T3G (14a) 4521G3A (24) 1812-3 ins T, (I11), homozygous SN7 unk/unk 40 PS None found - Notation as for Table 1. Login to comment

[hide] The molecular basis of cystic fibrosis in South Af... Clin Genet. 2001 Jan;59(1):37-41. Goldman A, Labrum R, Claustres M, Desgeorges M, Guittard C, Wallace A, Ramsay M
The molecular basis of cystic fibrosis in South Africa.
Clin Genet. 2001 Jan;59(1):37-41., [PMID:11168023]

Abstract [show]
The spectrum of CFTR mutations in three South African populations is presented. To date. a total of 192 white patients (384 chromosomes) with confirmed CF have been tested. deltaF508 accounts for 76% of the CF chromosomes in this group, with 3272-26A-->G, 394delTT and G542X occurring at the following frequencies: 4, 3.6 and 1.3%, respectively. A further 11 mutations account for 6% of CF chromosomes. A total of 91% of the CF-causing mutations can now be detected in the South African white population. Haplotype analysis suggests a founder effect in South Africans of European origin for the two common CFTR mutations, 3272-26A-->G and 394delTT. The diagnosis of CF has been confirmed in 14 coloured and 12 black CF patients. In the coloured population, both the deltaF508 and 3120 + 1G-->A mutations occur at appreciable frequencies of 43 and 29%, respectively. In the black population, the most common CF-causing mutation, the 3120 + 1G-->A mutation, occurs at an estimated frequency of 46%. Four other mutations have been detected, resulting in the identification of a total of 62.5% of mutations in this population.

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6 DF508 accounts for 76% of the CF chromosomes in this group, with 3272-26AG, 394delTT and G542X occurring at the following frequencies: 4, 3.6 and 1.3%, respectively. Login to comment
27 Five other mutations were identified in the white population, G542X, R553X, S549N, 621+lGT and N1303K which together account for a further 3% of mutations (5). Login to comment
40 White and coloured patients with unidentified CF mutations were tested for 15 mutations including 394delTT, Q493X, 3272-26A G, 3120+1GA as well as 11 other mutations, R117H, R334W, G542X, G551D, R553X, 621+ 1GT, W1282X, N1303K, 1717-1GA, R1162X, 3849+10kbCT. Login to comment
58 Frequency of CFTR mutations in white CF chromosomes Mutation Number of chromosomes Frequency (%) DF508 291 76 3272-26AG 16 4 394delTT 14 3.6 G542X 5 1.3 R553X 4 1 1W1282X 4 14N1303K G551D 3 0.8 3120+1GA 2 0.5 R117H 1 0.3 Q493X 1 0.3 S549N 1 0.3 621+1GT 1 0.3 1717-1GA 1 0.3 2789+5GA 1 0.3 91Total 349/384 Table 2. Login to comment
59 Genotypes of South African coloured CF patients Genotype Number of patients 2DF508/DF508 DF508/3120+1GA 5 1DF508/G542X DF508/U 2 3120+1GA/R1162X 1 13120+1GA/G551D 3120+1GA/U 1 1U/U 14Total U=unidentified mutation. Login to comment
81 The G542X mutation accounted for 1.3% of mutations. Login to comment
85 The R1162X, G551D and G542X mutations were each found on one chromosome. A total of 82% (23/28 chromosomes) of mutations have been identified. Login to comment

[hide] Frequency of cystic fibrosis transmembrane conduct... Chest. 2001 Mar;119(3):762-7. Marchand E, Verellen-Dumoulin C, Mairesse M, Delaunois L, Brancaleone P, Rahier JF, Vandenplas O
Frequency of cystic fibrosis transmembrane conductance regulator gene mutations and 5T allele in patients with allergic bronchopulmonary aspergillosis.
Chest. 2001 Mar;119(3):762-7., [PMID:11243954]

Abstract [show]
STUDY OBJECTIVE: To assess the frequency of cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in patients with allergic bronchopulmonary aspergillosis (ABPA). DESIGN: Case-control study. All subjects in the study were screened for the presence of 13 mutations in the CFTR gene (R117H, 621 + 1G(-)>T, R334 W, Delta F508, Delta I507, 1717-1G(-)>A, G542X, R553X, G551D, R1162X, 3849 + 10kbC(-)>T, W1282X, and N1303K). Moreover, they were also screened for the presence of the 5T variant in intron 8. SETTING: University hospital and community-based hospital. PATIENTS: Twenty-one white patients with ABPA participated in the study. The presence of CFTR mutations was also investigated in 43 white subjects with allergic asthma who did not show sensitization to Aspergillus fumigatus and in 142 subjects seeking genetic counseling for diseases other than cystic fibrosis (CF). RESULTS: Six patients with ABPA were found to be heterozygous for one CFTR mutation, including Delta F508 (n = 2), G542X (n = 1), R1162X (n = 1), 1717-1G(-)>A (n = 1), and R117H (n = 1). The 5T allele was not detected in ABPA patients. None of the ABPA patients showed sweat chloride concentrations > 60 mEq/L. The frequency of CFTR mutation carriers was significantly higher in ABPA patients (6 of 21 patients; 28.5%) than in control asthmatic subjects (2 of 43 subjects; 4.6%; p = 0.01) and in subjects seeking genetic counseling (6 of 142 subjects; p < 0.001). CONCLUSION: These findings indicate that in patients without a clinical diagnosis of CF, CFTR gene mutations could be involved in the development of ABPA, in association with other genetic or environmental factors.

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6 All subjects in the study were screened for the presence of 13 mutations in the CFTR gene (R117H, 621 ؉ 1G->T, R334 W, ⌬F508, ⌬I507, 1717-1G->A, G542X, R553X, G551D, R1162X, 3849 ؉ 10kbC->T, W1282X, and N1303K). Login to comment
11 Results: Six patients with ABPA were found to be heterozygous for one CFTR mutation, including ⌬F508 (n ‫؍‬ 2), G542X (n ‫؍‬ 1), R1162X (n ‫؍‬ 1), 1717-1G->A (n ‫؍‬ 1), and R117H (n ‫؍‬ 1). Login to comment
42 Genomic DNA samples were screened for the following CFTR mutations: R117H/ exon 4, 621 ϩ 1G-ϾT/intron 4, R334 W/exon 7, ⌬F508/exon 10, ⌬I507/exon 10, 1717-1G-ϾA/intron 10, G542X/exon 11, R553X/ exon 11, G551D/exon 11, R1162X/exon 19, 3849 ϩ 10kbC-ϾT/ intron 19, W1282X/exon 20, and N1303K/exon 21. Login to comment
58 Six patients (patients 1 to 6) were identified to carry one CFTR mutation, including ⌬F508 (n ϭ 2), G542X (n ϭ 1), R1162X (n ϭ 1), 1717-1G-ϾA (n ϭ 1), and R117H (n ϭ 1). Login to comment
99 Sweat Chloride,† mEq/L CFTR Mutation Intron 8 Polythymidine Tract Alleles 1 17 ⌬F508 7T/9T 2 33 ⌬F508 7T/9T 3 6 G542X 7T/9T 4 38 R1162X 7T/7T 5 54 1717-1G3A 7T/7T 6 8 R117H 7T/9T 7 36 - 7T/7T 8 23 - 7T/7T 9 14 - 7T/7T 10 19 - 7T/7T 11 37 - 7T/7T 12 NA - 7T/7T 13 40 - 7T/7T 14 38 - 7T/7T 15 14 - 7T/7T 16 19 - 7T/7T 17 32 - 7T/7T 18 15 - 7T/7T 19 34 - 7T/9T 20 13 - 7T/7T 21 34 - 7T/7T *See Table 1 for expansion of abbreviation. Login to comment

[hide] Visualization of oligonucleotide probes and point ... Proc Natl Acad Sci U S A. 2001 Mar 27;98(7):3940-5. Zhong XB, Lizardi PM, Huang XH, Bray-Ward PL, Ward DC
Visualization of oligonucleotide probes and point mutations in interphase nuclei and DNA fibers using rolling circle DNA amplification.
Proc Natl Acad Sci U S A. 2001 Mar 27;98(7):3940-5., 2001-03-27 [PMID:11274414]

Abstract [show]
Rolling circle amplification (RCA) is a surface-anchored DNA replication reaction that can be exploited to visualize single molecular recognition events. Here we report the use of RCA to visualize target DNA sequences as small as 50 nts in peripheral blood lymphocytes or in stretched DNA fibers. Three unique target sequences within the cystic fibrosis transmembrane conductance regulator gene could be detected simultaneously in interphase nuclei, and could be ordered in a linear map in stretched DNA. Allele-discriminating oligonucleotide probes in conjunction with RCA also were used to discriminate wild-type and mutant alleles in the cystic fibrosis transmembrane conductance regulator, p53, BRCA-1, and Gorlin syndrome genes in the nuclei of cultured cells or in DNA fibers. These observations demonstrate that signal amplification by RCA can be coupled to nucleic acid hybridization and multicolor fluorescence imaging to detect single nucleotide changes in DNA within a cytological context or in single DNA molecules. This provides a means for direct physical haplotyping and the analysis of somatic mutations on a cell-by-cell basis.

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32 Cell lines with mutations at the G542X locus of the cystic fibrosis transmembrane conductance regulator (CFTR) gene (GM11497, heterozygous mutation; GM11496; homozygous mutations), a cell line (CTL2337) with a single C insertion between nucleotides 5382 and 5383 of the BRCA1 gene, and standard HeLa cell lines, were obtained from the American Tissue Type Collection (Corriel Cell Repositories, Camden, NJ). Login to comment
54 A mixture of 500 nM decorator probes (Det3-Cy3 for ⌬F508, Det1c-FITC, Det1d-FITC for G542X, Det4-Cy5 for M1101K) in 2 ϫ SSC, 1% BSA, and 0.1% Tween 20 was applied to the slides. Login to comment
68 Physical mapping of three loci in the CFTR gene region by RCA Locus ODN sequences ⌬508 Probe-primer PRP3 (89): 3Ј-CCCTCTTGACCTCGGAAGTCTCCCATTTTAATTCGTGTCACCTTCTTAAAtttt(CH2)18tttttACGTCATCATGAACATTACACGTTCCAC-3Ј Circle3 (78): GTGGAACGTGTAATGTTCATGATGACGTGCATCCTTGACAGCCGATGAGGCTGGCATCCTTGACAGCCGATGAGGCTG Decorator probe: Det3-Cy3 (24): 5Ј-Cy3-GCATCCTTGACAGCCGATGAGGCT-3Ј G542X Probe-primer PRP1 (89): 3Ј-GAACCTCTTCCACCTTAGTGTGACTCACCTCCAGTTGCTCGTTCTTAAAGtttt(CH2)18tttttATGATCACAGCTGAGGATAGGACATGCGA-3Ј Circle1 (78): CGCATGTCCTATCCTCAGCTGTGATCATCAGAACTCACCTGTTAGACGCCACCAGCTCCAACTGTGAAGATCGCTTAT Decorator probe: Det1c-FITC (18): 5Ј-FITC-TCAGAACTCACCTGTTAG-3Ј; Det1d-FITC (18): 5Ј-FITC-ACTGTGAAGATCGCTTAT-3Ј M1101K Probe-primer PRP4 (89): 3Ј-GACGGTTGACCAAGAACATGGACAGTTGTGACGCGACCAAGGTTTACTCTtttt(CH2)18tttttCTTGTACATGTCTCAGTAGCTCGTCAGT-3Ј Circle4 (78): ACTGACGAGCTACTGAGACATGTACAAGGAGCAGTCCTGT˙CAGCTAGGTCACGGAGCAGTCCTGTCAGCTAGGTCACG Decorator probe: Det4-Cy5 (24): 5Ј-Cy5-GAGCAGTCCTGTCAGCTAGGTCACG-3Ј Bold type: probe sequence; lowercase tttt(CH2)18ttttt: linker; standard type: RCA primer, circle, and decorator ODN sequences. Note that probe-primer and AD-P2-ODNs have polarity reversal and two 3Ј ends. Login to comment
75 Human genomic DNA fibers were stretched from freshly cultured normal peripheral blood lymphocytes and GM11496 cells (homozygous G542X mutation). Login to comment
93 A mixture of 500 nM decorator probes (Det3-Cy3 for ⌬F508, Det1c-FITC, Det1d-FITC for G542X, Det4-Cy5 for M1101K) in 2 ϫ SSC, 1% BSA, and 0.1% Tween 20 was applied to the slides. Login to comment
100 Experiments designed for allele discrimination at the G542X locus did not use the PAC prehybridization step outlined above. Login to comment
101 Instead, the P1 anchor ODN and the pair of P2 AD-ODNs for the G542X locus were cohybridized with the 50-mer non-AD-ODN probes for the ⌬508 and M1101K loci, which acted as reference markers. Login to comment
102 The hybridization, ligation, RCA reactions, washes, and signal detection conditions were carried out as described for the detection of G542X mutations in interphase cells. Login to comment
122 To test the ability of RCA to detect small genomic DNA sequences within interphase nuclei and stretched DNA fibers we chose three 50-bp targets at the ⌬508, G542X, and M101K mutation loci within the CFTR gene. Login to comment
131 In Fig. 2A, the RCA products from the ⌬508, G542X, and M11101K probes were detected by Cy3-, FITC-, and Cy5-labeled decorator ODNs, respectively. Login to comment
136 The overall RCA detection efficiency at the ⌬508, G542X, and M1101K loci averaged 37%, 45%, and 35%, respectively. Login to comment
140 (A) RCA detection of probes targeted to the G542X locus (FITC), the ⌬508 locus (Cy-3), and the M1101K locus (Cy5) in normal human lymphocytes. Login to comment
143 (C) Cohybridization of two PAC clones (extended green and red signals) with ⌬508 (yellow), G542X (green), and M1101K (white) oligomer probes. Login to comment
153 The overall efficiency in detecting RCA signals at the ⌬508, G542X, and M1101K loci were 48%, 44%, and 36% respectively. Login to comment
157 The sequences of the P1 and the two AD-P2 ODNs designed to detect mutations at the G542X locus of the CFTR gene are given in Table 2. Login to comment
172 Detection of the G542X mutation also was done on stretched DNA fibers prepared from normal lymphocytes and the GM11496 cell line homozygous for the G542X mutation (Fig. 3B). Login to comment
176 RCA detection of wt and mu alleles at the G542X locus of the CFTR gene. Login to comment
178 (B) Discrimination of wt and mu alleles of the G542X locus on stretched DNA fibers. Login to comment
183 The wt G542X allele is labeled with Cy5 while the mu allele is labeled with Cy3. Login to comment
184 The merged image (Com) shows a yellow-blue-white hybridization pattern when the G542X allele is wt and a yellow-green-white pattern when G542X is a mu allele. Login to comment
186 It was apparent, however, that wt and mu alleles at the G542X could be readily detected as many fibers showing the hybridization profiles illustrated in Fig. 3B were observed. Login to comment

[hide] Genetic, andrological and clinical characteristics... Int J Androl. 2001 Apr;24(2):73-9. Attardo T, Vicari E, Mollica F, Grazioso C, Burrello N, Garofalo MR, Lizzio MN, Garigali G, Cannizzaro M, Ruvolo G, D'Agata R, Calogero AE
Genetic, andrological and clinical characteristics of patients with congenital bilateral absence of the vas deferens.
Int J Androl. 2001 Apr;24(2):73-9., [PMID:11298840]

Abstract [show]
The possibility of retrieving spermatozoa from the epididymis allows patients with congenital bilateral absence of the vas deferens (CBAVD) to father a child by means of assisted reproduction techniques. This has, however, increased the chance of transmitting a mutated allele of the cystic fibrosis transmembrane conductance regulator (CFTR) gene which increases the risk of generating offspring with cystic fibrosis (CF). Because of the increased heterogeneity of the CFTR locus, the study of a discrete number of mutations, as usually carried out in a diagnostic work-up, is unable to ascertain the presence of a mutation in a relatively high proportion of the patients screened. In an attempt to increase the chance of detecting the presence of CFTR gene abnormalities, 37 patients with CBAVD and one patient with congenital unilateral agenesis of the vas deferens (CUAVD) underwent an enlarged diagnostic protocol, which included screening for the most expected mutations of the CFTR gene in our population, evaluation of the five thymidine (5T) allelic variant, sweat test, respiratory function tests, evaluation of steatocrit, and an accurate evaluation of the history of the patient to search for symptoms commonly found in patients with CF. A single CFTR gene mutation was found in 18 patients (48.6%) with CBAVD and in the patient with CUAVD. The most frequent mutation observed was the Delta F508. Eleven patients (45.8%) had the 5T variant and in five of them it was not associated with any detectable mutation of the CFTR gene. Two female partners were found to be carriers of a mutation, whereas 5 (18.5%) had the 5T variant. As many as 71% of CBVAD patients had the simultaneous presence of at least two signs and/or symptoms suggestive of CF, albeit they were of mild intensity and the patients felt fit and healthy. In conclusion, these results suggested that some patients with CBAVD without CFTR gene mutation or 5T variant, even when their sweat test is negative, may show clinical suspicion of carrying a CFTR gene mutation and therefore are at risk of generating children affected by CF if the partner carries a mutation as well. The screening for mutations and a careful clinical examination may contribute to better identification of patients with CFTR-related CBAVD.

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49 We investigated the following 11 CFTR mutations: DF508, G542X, R553X, N1303K, W1282X, R347P, L1077P, 2183AA ® G, 1717±1G > A, R1162X, and R117H. Login to comment
61 The other mutations found were: W1282X in four patients, G542X in two patients, R347P in one patient and R553X in one patient. Login to comment
63 The patient with CUAVD had the G542X mutation associated with the 5T allele (Table 1). Login to comment
66 Four wives (14.8%) had the 5T allelic variant and of their husbands, two had the alleles G542X or W1282X and two had no mutations. Login to comment
87 38) with CUAVD Patient Age (years) Mutation/ 5T allele Sweat test Steatocrit FEV1 Other clinical features 1 38 R347P/ND Normal ND ND ND 2 29 DF508/ND ND ND ND ND 3 37 ±/ND ND ND ND ND 4 38 ±/ND ND ND ND ND 5 29 DF508/ND Normal ND = ND 6 40 ±/ND Normal ND = ND 7 32 DF508/ND Borderline ND = ND 8 29 DF508/ND ND ND ND ND 9 41 ±/ND Borderline ND ¯ ND 10 32 DF508/± Normal = = RB 11 35 R553X/± Borderline - = RB 12 29 ±/ND Borderline - = Diarrhoea 13 37 ±/ND Abnormal = = Sinusitis 14 36 W1282X/± Normal - = Recurrent bronchitis 15 35 G542X/± Abnormal = ¯ ± 16 34 W1282X/5T Abnormal = = Diarrhoea 17 31 ±/5T Abnormal = = ± 18 22 ±/± Borderline - = Diarrhoea 19 27 G542X/± Abnormal = = Recurrent bronchitis 20 35 ±/± Abnormal - = Recurrent bronchitis 21 33 W1282X/± Abnormal = ND Sinusitis, diarrhoea 22 30 DF508/5T Abnormal - = ± 23 20 ±/± Abnormal = = Sinusitis, diarrhoea 24 39 ±/± Normal = ¯ Asthma, collapse 25 35 ±/5T Normal - ¯ Sinusitis, diarrhoea 26 26 W1282X/5T Abnormal - = ± 27 35 ±/± Normal - = ± 28 30 DF508/5T Normal - = ± 29 29 DF508/ND ND ND ND Collapse 30 35 ±/5T Normal = ¯ ± 31 36 DF508/5T Borderline = ¯ Sinusitis, asthma, collapse, polyps 32 41 ±/5T Normal - ¯ Recurrent respiratory infection 33 39 ±/5T Normal = ¯ Sinusitis 34 27 DF508/5T Borderline - = ± 35 39 ±/± Normal ND ¯ Diarrhoea 36 37 ±/± Normal - = Polyps 37 40 ±/± Abnormal - ¯ Asthma, recurrent respiratory infection 38 29 G542X/5T Borderline - ¯ Diarrhoea ND: Not determined; ±: absence of mutations or clinical features; =: unchanged; -: increased; ¯: decreased. Login to comment
97 As shown elsewhere (Dumur et al., 1990; Anguiano et al., 1992), the presence of DF508 was the commonest mutation observed in patients with CBAVD, followed by W1282X and G542X. Login to comment
98 DF508 is also the most frequent mutation found in patients with CF, followed by G542X and W1282X (F. Mollica et al., unpublished data). Login to comment
114 The patient with CUAVD presented with a G542X/5T genotype and had also an abnormal sweat test, reduced FEV1, steatorrhoea and symptoms of digestive dysfunction. Login to comment

[hide] Inflammation in cystic fibrosis airways: relations... Eur Respir J. 2001 Jan;17(1):27-35. Scheid P, Kempster L, Griesenbach U, Davies JC, Dewar A, Weber PP, Colledge WH, Evans MJ, Geddes DM, Alton EW
Inflammation in cystic fibrosis airways: relationship to increased bacterial adherence.
Eur Respir J. 2001 Jan;17(1):27-35., [PMID:11307750]

Abstract [show]
It is unclear whether inflammation in the cystic fibrosis (CF) lung relates predominantly to bacterial infection, or occurs as a direct consequence of mutant cystic fibrosis transmembrane conductance regulator (CFTR) protein. Interleukin (IL)-8 secretion from CF and non-CF cell lines, and from CF and non-CF human primary nasal epithelial cells incubated with or without Pseudomonas aeruginosa, was measured. Activation of nuclear factor-kappaB (NF-kappaB) in unstimulated CF and non-CF nasal epithelial cells, cell lines and murine tissues was measured by gel-shift assays. No significant difference in basal IL-8 production or NF-kappaB activation was observed between CF and non-CF primary nasal cells. However, CF cells exhibited a significantly (p<0.01) increased IL-8 secretion following P. aeruginosa stimulation. Equalization of the increased P. aeruginosa adherence observed in CF cells, to non-CF levels, resulted in comparable IL-8 secretion. Further, IL-8 production did not differ with mutations which result in either correctly localized CFTR, or in partial/total mislocalization of this protein. Similar levels of NF-kappaB activation were observed in a number of organs of wildtype and CF mice. Finally, IL-8 secretion and NF-kappaB activity were not consistently increased in CF cell lines. Cos-7 cell transfection with plasmids expressing deltaF508 or G551D mutant CFTR protein resulted in increased activation of a p50-containing NF-kappaB complex, but IL-8 secretion was similar to wild-type cells. The authors conclude that the stimulus produced by Pseudomonas aeruginosa is the predominant inflammatory trigger in their models.

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140 Group 1 (n=13) with mistraf- ®cking mutations on both alleles (DF508 homozygotes and a DF508/I507 compound heterozygote), group 2 (n=6) with only one mistraf®cking mutation (DF508 compound heterozygotes, the second mutation being R553X62, 1717-1GAA, 621+1GAT, G551D62), and group 3 (n=3) without mistraf®cking mutations (genotypes being G542X/3849+10kB GAT, G542X/ R533X, 1717-1GAA/G551D). Login to comment

[hide] An alpha1-antitrypsin enhancer polymorphism is a g... Eur J Hum Genet. 2001 Apr;9(4):273-8. Henry MT, Cave S, Rendall J, O'Connor CM, Morgan K, FitzGerald MX, Kalsheker N
An alpha1-antitrypsin enhancer polymorphism is a genetic modifier of pulmonary outcome in cystic fibrosis.
Eur J Hum Genet. 2001 Apr;9(4):273-8., [PMID:11313771]

Abstract [show]
Lung disease is the direct cause of death in over 90% of cystic fibrosis (CF) patients. Excess neutrophil elastase is an important determinant of pulmonary disease in CF. alpha1-antitrypsin (AAT), also known as alpha1-proteinase inhibitor (alpha1PI) is a major modulator of elastase activity. We investigated the hypothesis that an enhancer polymorphism in the AAT gene would contribute to pulmonary prognosis in CF. Respiratory function, chest X-ray scores, bacterial colonisation and infective exacerbation were assessed to evaluate pulmonary disease severity in the CF group. Sixteen patients were found to have the 1237A allele, and 108 the more frequent G allele. Contrary to expectation, the patients with the 1237A allele were found to have better indices of pulmonary disease progression than those without, as indicated by less change in X-ray score (1237A: 0.2+/-0.1; 1237G: 1.2+/-0.1; P = 0.002) and fewer infective exacerbations (1237A: 2.8+/-0.6; 1237G: 4.6+/-0.3; P = 0.03) over the preceding 2 years. Also, a higher proportion of the 1237A (25%) than the 1237G (6.5%) were not colonised by Pseudomonas Aeruginosa (P = 0.04). Prospective monitoring of infections for a further 2 years confirmed a lesser propensity to infection in patients with the 1237A allele. These trends were also observed in a tightly matched sub-set of CF genotypes of similar age and sex, thus confirming that these effects were independent of the CF genotype. These results indicate that this AAT enhancer polymorphism is associated with better pulmonary prognosis in CF. Though the number of CF patients with the polymorphism is small, and these data need to be confirmed in larger studies, they suggest that a cautious approach should perhaps be taken to treatment of CF patients with supplemental AAT.

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66 1237G group: G551D (10); R117H (3); R560T (3); D1507 (2); E60X (2); N1303K (1); 1717-1 (1); 621H (1); G542X (1); POL 400 (1); R352Q (1); RT0F (1); 621+G4T (1); Unknown (15). Login to comment

[hide] Prevalence of cystic fibrosis mutations in Israeli... Genet Test. 2001 Spring;5(1):47-52. Orgad S, Neumann S, Loewenthal R, Netanelov-Shapira I, Gazit E
Prevalence of cystic fibrosis mutations in Israeli Jews.
Genet Test. 2001 Spring;5(1):47-52., [PMID:11336401]

Abstract [show]
The aim of this study was to evaluate the screening policies of cystic fibrosis (CF) in the Jewish population. The prevalence of mutations that account for CF in Israel have been defined in the past by determining the frequency of CF mutations in affected individuals. This study is a population-based study and is, therefore, different from previous patient-based studies. We found that the CF mutations D1152H, W1089X, and 405 + IG-->A were present in some ethnic groups in which no CF patients carrying these mutations were reported. These facts necessitate a reevaluation of the screening policy regarding the ethnic groups in Israel. We studied 9,430 healthy Jewish Israeli individuals of 36 countries of origin. The prevalence of CF mutations was 1:19, 1:19, 1:28, and 1:42 for the Ashkenazi, Sephardi, North African, and Eastern Jews, respectively. CF mutations were identified in 374 (4.0%) individuals. These included 173 (46.3%) carriers of the W1282X mutation; 110 (29.4%) found to carry delF508; 23 (6.1%) who carried G542X; 22 (5.9%) who carried 3849 + 10Kb (C-->T; 20 (5.3%) who carried D1152H; 10 (2.7%) who carried N1303K; 11 (2.9%) who carried 405 + IG-->A; 4 (1.1%) who carried W1089X; and one (0.3%) who carried S549R. No carriers were detected for the 1717-1G-->A, G85E, and T360K mutations, which were tested for in 7,383, 1,558, and 41 individuals, respectively.

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33 These were: delF508 (Kerem et al., 1989), W1282X (Vidaud et al., 1990), G542X, 1717-1G R A, S549R (Kerem et al., 1990), N1303K (Osborn et al., 1991), 3849 1 10Kb C R T (Highsmith et al., 1994), T359K/Q360K (Shoshani et al., 1992), G85E (Zielenski et al., 1991), 405 1 1G R A (Dork et al., 1993), W1089X (Shosani et al., 1994), and D1152H (Highsmith et al., 1993). Login to comment
45 There were 173 (46.3%) carriers of the W1282X mutation; 110 (29.4%) carriers of delF508; 23 (6.1%) carriers of G542X; 10 (2.7%) carriers of N1303K; and 22 (5.9%) carriers of 3849 1 10KbC R T. Twenty (5.3%) were found to carry D1152H; 11 (2.9%) carried 405 1 1G R A; 4 (1.1%) carried W1089X; and 1 (0.3%) carried S549R. No carriers were detected for the mutations 1717-1G R A, G85E, and T360K, which were tested for in 7,383, 1,436, and 41 individuals, respectively. Login to comment
52 Mutations tested for all individuals in the cohort North Total Ashkenazi Sephardi Africa Eastern number of Mutation no. 6850 no. 933 no. 1146 no. 468 carriers W1282X 142 17 8 6 173 delF508 86 12.25 11.5 0.25 110 G542X 20.25 0.5 1.75 0.5 23 N1303K 7.5 1.5 0.25 0.75 10 3849110Kb 17 2 2 1 22 C® T B. Mutations tested for individuals of non-Ashkenazi origin, mixed origin, and of spouses of carriers Type of Total number mutation Number tested Ashkenazi Sephardi North Africa Eastern of carriers D1152H Number tested 1,305 458.25 722.75 280 Carriers 11.5 4.5 3.5 0.5 20 405 Number tested 425.75 372 633.5 119 11G® A Carriers 0.5 1 9.5 0 11 W1089X Number tested 539.25 345 638.5 135 Carriers 2 0.5 1 0.5 4 S549R Number tested 534.5 385.5 686 110 Carriers 0 1 0 0 1 a Ethnic origin was classified according to the country of origin of the four grandparents of each individual. Each grandparent was calculated as contributing a quarter of his/her gene pool and these were summed up for each ethnic origin. Login to comment
74 Therefore, Ashkenazi Jews would have been tested for the five main mutationsonly: delF508,W1282X, G542X, N1303K, and 3849 1 10KbC R T. The mutations D1152H and W1089X would not have been included in the test panel in Ashkenazi Jews. Login to comment

[hide] Gastrointestinal, liver, and pancreatic involvemen... Pancreas. 2001 May;22(4):395-9. Modolell I, Alvarez A, Guarner L, De Gracia J, Malagelada JR
Gastrointestinal, liver, and pancreatic involvement in adult patients with cystic fibrosis.
Pancreas. 2001 May;22(4):395-9., [PMID:11345141]

Abstract [show]
BACKGROUND: The clinical prevalence of cystic fibrosis (CF) in adults continues to rise, with a consequent impact on adult gastroenterology practice. AIM: To characterize the gastrointestinal manifestations of CF in adult patients. PATIENTS AND METHODS: The clinical records of 89 adult CF patients treated at our institution from 1992 to 1999 were reviewed. Patients were distributed into two groups: group A (39 patients), which consisted of patients who were diagnosed with CF at when they were younger than 14 years old and who survived into adulthood; and group B (50 patients), who were diagnosed with CF at the age of 14 years or older. Data on CF genetic mutations, nutritional state, evidence of pulmonary, gastrointestinal, liver, or pancreatic involvement were collected for each patient. RESULTS: The most prevalent genetic mutation in our series was deltaF508, present in 50 patients (56.2%), 29 of whom belonged to group A and 21 who belonged to group B. In group A, the deltaF508 mutation was associated with exocrine pancreatic insufficiency (PI) in 26 of 29 patients (89.6%), whereas in group B it was associated with PI in only four patients (19%). Overall, PI was present in 33 of 39 patients (84.6%) in group A and in eight of 50 patients (16%) in group B. Four patients in group B had experienced previous episodes of acute pancreatitis; two of them had associated PI. Of the 89 patients, 12 (10 in group A) were malnourished. Malnutrition was invariably associated with PI. Hepatic and biliary tree abnormalities were particularly prevalent in patients in group A and was usually associated with PI. Intestinal manifestations were uncommon. CONCLUSIONS: Diagnosis of CF before the age of 14 years is associated with greater gastrointestinal compromise than diagnosis at an older age, particularly with regard to PI. CF carriers of the deltaF508 mutation have an increased risk of developing gastrointestinal manifestations.

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45 The second most prevalent mutation was G542X, present in eight patients. Login to comment
50 In the remaining 14 patients, ⌬F508 was carried with G542X, R1162X, N1303K, L206W, 1717-1G>A, 711+1G>T, or an unidentified mutation. Login to comment
51 In the 10 patients of group A without ⌬F508, the mutations identified were: G542X (present in five), R1162X, Q890X, ⌬I507, 2183A, and 1609-CA. Login to comment
56 5T, G542X, R334W, N1303K, L206W, 3659-C, and G85E were identified in the remaining nine patients. Login to comment
64 Other genotypes present in our series ⌬F508/711+1G>T 2A 5T/5T 1B ⌬F508/5T 2B ⌬1507/- 1A ⌬F508/R117H 2B R1162X/1898+1G>A 1A ⌬F508/R1162X 1A 2183A/- 1A ⌬F508/N1303K 1A 1609-CA/1811+1.6kbA>G 1A ⌬F508/3272-26A>G 1B 1609-CA/R347P 1A ⌬F508/D836Y 1B Q890X/- 1A ⌬F508/1717-1G>A 1A R334W/- 1B G542X/W1282X 1A N1303K/2789+5G>A 1B G542X/2789+5G>A 1B 3659-C/- 1B G542X/P205S 1B G85E/- 1B G542X/D1270N 1B Negative 1A, 20B L206W/- 1B Unknown 2A creatic insufficiency was highly prevalent, affecting 33 patients (84.6%). Login to comment
98 The second most prevalent mutation, G542X, was present in eight patients (8.98%), a prevalence similar to that described in the literature. Login to comment
135 The four patients in our series ⌬F508/G542X all belonged, as expected (28), to group A and three of them had pancreatic insufficiency. Login to comment

[hide] Complete and rapid scanning of the cystic fibrosis... Hum Genet. 2001 Apr;108(4):290-8. Le Marechal C, Audrezet MP, Quere I, Raguenes O, Langonne S, Ferec C
Complete and rapid scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by denaturing high-performance liquid chromatography (D-HPLC): major implications for genetic counselling.
Hum Genet. 2001 Apr;108(4):290-8., [PMID:11379874]

Abstract [show]
More than 900 mutations and more than 200 different polymorphisms have now been reported in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Ten years after the cloning of the CFTR gene, the complete scanning of the 27 exons to identify known and novel mutations remains challenging. Rapid accurate identification of mutated alleles is important for prenatal diagnosis, for cascade screening in families at risk of cystic fibrosis (CF) and for understanding the correlation between genotype and phenotype. In this study, we report the successful use of denaturing ion-pair reverse-phase high performance liquid chromatography (D-HPLC) to analyse rapidly the complete coding sequence of the CFTR gene. With 27 pairs of polymerase chain reaction primers, we optimised the temperature conditions required for the analysis of each amplicon and validated thetest conditions on samples from a panel of 1552 CF patients who came from France and other European countries and who had mutations and polymorphisms located in the various melting domains of the gene. D-HPLC identified 415 mutated alleles previously characterised by denaturing gradient gel electrophoresis and DNA sequencing, plus 74 novel mutations reported here. This new technique for screening DNA for sequence variation was extremely accurate (it identified 100% of the CFTR alleles tested so far) and rapid (the complete CFTR gene could be analysed in less than a week). Our approach should reduce the number of untyped CF alleles in populations and thus decrease the residual risk in couples at risk of CF. This technique may be important not only for CF,but also for many other genes with a high frequency of point mutations at a variety of sites.

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140 For example, if only the ∆F508 and the other most common mutations (G551D, G542X, W1282X, 1717-1 G→A) are sought, the detection rate is 70% and the residual risk is around 1/3. Login to comment

[hide] Improved detection of cystic fibrosis mutations in... Genet Med. 2001 May-Jun;3(3):168-76. Heim RA, Sugarman EA, Allitto BA
Improved detection of cystic fibrosis mutations in the heterogeneous U.S. population using an expanded, pan-ethnic mutation panel.
Genet Med. 2001 May-Jun;3(3):168-76., [PMID:11388756]

Abstract [show]
PURPOSE: To determine the comparative frequency of 93 CFTR mutations in U.S. individuals with a clinical diagnosis of cystic fibrosis (CF). METHODS: A total of 5,840 CF chromosomes from Caucasians, Ashkenazi Jews, Hispanics, African Americans, Native Americans, Asians, and individuals of mixed race were analyzed using a pooled ASO hybridization strategy. RESULTS: Sixty-four mutations provided a sensitivity of 70% to 95% in all ethnic groups except Asians, and at least 81% when the U.S. population was considered as a whole. CONCLUSIONS: For population-based carrier screening for CF in the heterogeneous U.S. population, which is characterized by increasing admixture, a pan-ethnic mutation panel of 50 to 70 CFTR mutations may provide a practical test that maximizes sensitivity.

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109 More strikingly, the five mutations reported to account for 97% of Ashkenazi Jewish chromosomes (⌬F508, G542X, W1282X, N1303K, and 3849 ϩ 10 kbCϾT)16 accounted for only 39/48 chromosomes in this study (81.3%). Login to comment

[hide] Evidence that systemic gentamicin suppresses prema... Am J Respir Crit Care Med. 2001 Jun;163(7):1683-92. Clancy JP, Bebok Z, Ruiz F, King C, Jones J, Walker L, Greer H, Hong J, Wing L, Macaluso M, Lyrene R, Sorscher EJ, Bedwell DM
Evidence that systemic gentamicin suppresses premature stop mutations in patients with cystic fibrosis.
Am J Respir Crit Care Med. 2001 Jun;163(7):1683-92., [PMID:11401894]

Abstract [show]
Here we report the effects of gentamicin treatment on cystic fibrosis transmembrane regulator (CFTR) production and function in CF airway cells and patients with CF with premature stop mutations. Using immunocytochemical and functional [6-methoxy-N- (3-sulfopropyl) quinolinium (SPQ)-based] techniques, ex vivo exposure of airway cells from stop mutation CF patients led to the identification of surface-localized CFTR in a dose-dependent fashion. Next, five patients with CF with stop mutations and five CF control subjects were treated with parenteral gentamicin for 1 wk, and underwent repeated in vivo measures of CFTR function (nasal potential difference [PD] measurements and sweat chloride [Cl(-)] testing). During the treatment period, the number of nasal PD readings in the direction of Cl(-) secretion was increased approximately 3-fold in the stop mutation patient group compared with controls (p < 0.001), and four of five stop mutation patients with CF had at least one reading during gentamicin treatment with a Cl(-) secretory response of more than -5 mV (hyperpolarized). A response of this magnitude was not seen in any of the CF control subjects (p < 0.05). In an independent series of experiments designed to test the ability of repeat nasal PDs to detect wild-type CFTR function, evidence of Cl(-) secretion was seen in 88% of control (non-CF) nasal PDs, and 71% were more than -5 mV hyperpolarized. Together, these results suggest that gentamicin treatment can suppress premature stop mutations in airway cells from patients with CF, and produce small increases in CFTR Cl(-) conductance (as measured by the nasal PD) in vivo.

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61 Nasal cells from a G542X/ ⌬F508 patient (A, B, C) and a ⌬F508/⌬F508 patient (E, F ) in increasing concentrations of gentamicin: (A) G542X/⌬F508 0 ␮gm/mlgentamicin;(B)G542X/ ⌬F508 10 ␮gm/ml gentamicin; (C) G542X/⌬F508 100 ␮gm/ ml gentamicin; (D) G542X/⌬F508 no gentamicin, no primary antibody (control); (E) ⌬F508/⌬F508, no gentamicin; (F) ⌬F508/⌬F508, 100 ␮gm/ml gentamicin. Login to comment
86 STUDY PATIENT DEMOGRAPHICS* Age/Sex Premature Stop CF Subjects Control CF Subjects Genotype P.a FEV1 (% pred) Age/Sex Genotype P.a FEV1 (% pred ) 18/M G542X/⌬F508 (ϩ) 52 23/F ⌬F508/⌬F508 (ϩ) 38 18/M G542X/⌬F508 (ϩ) 76 18/F ⌬F508/⌬F508 (ϩ) 29 15/F W128X/⌬F508 (ϩ) 105 30/M ⌬F508/G55ID (ϩ) 19 28/M G542X/⌬F508 (ϩ) 25 26/F ⌬F508/⌬F508 (ϩ) 53 13/F R553X/621G-T (ϩ) 40 28/F ⌬F508/⌬F508 (ϩ) 34 18.4 yr (5.77)† 59.6 (31.5)† 25 yr (4.69)† 34.6 (11.8)† * Age (yr, mean Ϯ SD), sex (M ϭ male, F ϭ female), bacterial colonization (P.a ϭ Pseudomonas aeruginosa), FEV1 (forced expiratory volume in 1 s, %pred. Login to comment
90 (A) Cells from a G542X/⌬F508 patient (open symbols) and a ⌬F508/⌬F508 patient (closed symbols) were isolated, grown, and loaded with SPQ as described in METHODS. Login to comment
94 A positive response was obtained for 5/44 G542X/⌬F508 cells treated with 10 ␮gm/ml gentamicin and 42 of 44 cells treated with 100 ␮gm/ml gentamicin, and the means of these responding cells are plotted (Ϯ S.E.M.). Login to comment
95 No responding cells were identified in the G542X/⌬F508 cells without gentamicin treatment (n ϭ 40), or any of the ⌬F508/⌬F508 cells (n Ͼ 80 cells tested in each condition). Login to comment
97 (B) The percent of positive cells in each condition are shown for the ⌬F508/⌬F508 (left) and G542X/ ⌬F508 cells (right) in the increasing concentrations of gentamicin. Login to comment
112 To investigate the effects of gentamicin on CFTR biosynthesis in human tissues, nasal cells from a G542X/⌬F508 patient with CF were grown on glass coverslips and exposed to medium with gentamicin at 0, 10, and 100 ␮g/ml for 16 h followed by immunohistochemical staining for surface-localized CFTR. Login to comment
114 Figures 1A-1C identify CFTR antigen at the cell membrane from G542X/⌬F508 nasal cells with increasing gentamicin concentrations. Login to comment
118 In a parallel set of experiments, nasal cells isolated from a G542X/⌬F508 patient with CF were grown on glass coverslips and studied for evidence of halide transport, using the fluorescent dye SPQ. Login to comment
120 Figure 2 shows that G542X nasal cells exposed to gentamicin at 10 and 100 ␮g/ml had increased halide efflux in response to stimulation with a cAMP-elevating cocktail, compared with cells with no gentamicin exposure. Login to comment
122 Similar (immunocytochemical and functional) results have been observed for airway cells derived from other patients with premature stop mutations (G542X/⌬F508 and R553X/⌬F508 ([23] and our unpublished observations). Login to comment
180 Similar observations were made with transient expression systems for other CF-associated premature stop mutations, including G542X, R553X, and R1162X mutations, in addition to W1282X (12, 13). Login to comment

[hide] The use of intraallelic variability for testing ne... Genetics. 2001 Jun;158(2):865-74. Slatkin M, Bertorelle G
The use of intraallelic variability for testing neutrality and estimating population growth rate.
Genetics. 2001 Jun;158(2):865-74., [PMID:11404347]

Abstract [show]
To better understand the forces affecting individual alleles, we introduce a method for finding the joint distribution of the frequency of a neutral allele and the extent of variability at closely linked marker loci (the intraallelic variability). We model three types of intraallelic variability: (a) the number of nonrecombinants at a linked biallelic marker locus, (b) the length of a conserved haplotype, and (c) the number of mutations at a linked marker locus. If the population growth rate is known, the joint distribution provides the basis for a test of neutrality by testing whether the observed level of intraallelic variability is consistent with the observed allele frequency. If the population growth rate is unknown but neutrality can be assumed, the joint distribution provides the likelihood of the growth rate and leads to a maximum-likelihood estimate. We apply the method to data from published data sets for four loci in humans. We conclude that the Delta32 allele at CCR5 and a disease-associated allele at MLH1 arose recently and have been subject to strong selection. Alleles at PAH appear to be neutral and we estimate the recent growth rate of the European population to be approximately 0.027 per generation with a support interval of (0.017-0.037). Four of the relatively common alleles at CFTR also appear to be neutral but DeltaF508 appears to be significantly advantageous to heterozygous carriers.

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253 the recent growth rate of European populations is not TABLE 2 Parameters and results for the CFTR data sets p n N0 ␮ ϭ 0.0001 ␮ ϭ 0.0005 ␮ ϭ 0.001 ␮ ϭ 0.0005 Mutation (ϫ10-4 ) i So (ϫ103 ) (ϫ107 ) (single) (single) (double) (double) ⌬F508 132 2112 59 160.0 20.0 0.0 0.0 6.0 ϫ 10-73 6.3 ϫ 10-21 ⌬F508 132 2112 118 160.0 20.0 0.0 5.5 ϫ 10-91 4.4 ϫ 10-39 1.0 ϫ 10-4 G542X 6.8 116 9 170.6 5.82 8.9 ϫ 10-8 0.012 0.231 0.907 N1303K 5.6 59 7 105.3 5.18 5.9 ϫ 10-4 0.180 0.639 0.977 1717-1G-A 3.2 24 3 75.0 4.38 0.031 0.370 0.695 0.945 R1162X 2.2 68 4 309.1 5.18 3.4 ϫ 10-4 0.082 0.313 0.829 Definitions are as in Table 1, except the mutation rates. Login to comment
271 TABLE 3 Values of ␹2 ϭ -2 log(L0/L*) calculated as described in the text for each allele at CFTR compared to the rest ␮ ϭ 0.00028: ␮ ϭ 0.00028: ␮ ϭ 0.00084: ␮ ϭ 0.00084: Mutation So ϭ 59 So ϭ 118 So ϭ 59 So ϭ 118 ⌬F508 20.3 11.0 19.6 4.6 G542X 5.8 1.4 5.4 0.8 N1303K 8.4 3.8 7.9 3.0 1717-1G-A 3.4 0.5 3.5 1.0 R1162X 1.8 0.6 1.6 0 So was estimated by using a parsimony algorithm to infer because they depend on assumed values of parameters and on a simplified model of past population growth.the minimum number of mutations necessary to gener- The results for CFTR are compatible with those for PAHate the observed haplotypes. Login to comment

[hide] Analysis of the entire coding region of the cystic... Hum Mutat. 2001 Aug;18(2):166. Castellani C, Gomez Lira M, Frulloni L, Delmarco A, Marzari M, Bonizzato A, Cavallini G, Pignatti P, Mastella G
Analysis of the entire coding region of the cystic fibrosis transmembrane regulator gene in idiopathic pancreatitis.
Hum Mutat. 2001 Aug;18(2):166., [PMID:11462247]

Abstract [show]
Many Cystic Fibrosis (CF) carriers have been detected testing some subjects with chronic pancreatitis for a limited number of mutations. The aim of this study was to find out if some subjects with pancreatitis and a CFTR mutation actually carry another, undetected mutation. We screened for 18 CFTR mutations plus the CFTR intron 8 poly(T) tract length a population of 67 patients suffering from idiopathic either acute, or recurrent acute, or chronic pancreatitis. Three of them were diagnosed as affected by CF. Among the others, a subset of 14 (8 CFTR mutation carriers, 4 5T carriers, and 2 sweat chloride borderliners) was selected and analyzed by denaturing gradient gel electrophoresis. Six possibly CF-related mutations were detected: L997F and 3878delG were found in two of the subjects already carrying another mutation, S1235R and L997F in one patient carrying the 5T, and L997F and D614G in the two patients with borderline sweat chloride. Among the 14 selected cases a total of 11 patients carried at least one mutation, and three of them were compound heterozygotes. Though it is debatable whether these three individuals can be considered affected by CF, their pancreatitis is possibly a clinical manifestation of some CFTR-related disease. Hum Mutat 18:166, 2001.

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41 Genetic analysis Phase 1 - Patients were tested for the following mutations: F508del, I507del, R117H, R1162X, 2183AA>G, N1303K, 3849+10KbC>T, G542X, 1717-1G>A, R347P, R352Q, R553X, Q552X, G85E, 711+5G>A, W1282X, 3132delTG and 2789+5G>A, plus the CFTR intron 8 poly(T) tract length. Login to comment

[hide] Adenosine triphosphate-binding cassette superfamil... Biol Reprod. 2001 Aug;65(2):394-400. Larriba S, Bassas L, Egozcue S, Gimenez J, Ramos MD, Briceno O, Estivill X, Casals T
Adenosine triphosphate-binding cassette superfamily transporter gene expression in severe male infertility.
Biol Reprod. 2001 Aug;65(2):394-400., [PMID:11466205]

Abstract [show]
Cystic fibrosis transmembrane regulator (CFTR), multidrug-resistant (MDR)1, and multidrug resistance-associated (MRP) proteins belong to the ATP-binding cassette (ABC) transporter superfamily. A compensatory regulation of MDR1 and CFTR gene expression has been observed in CFTR knockout rodent intestine and in an epithelial cell line of human colon, whereas a high homology and similar anion binding site are shared by MRP and CFTR proteins. To provide better insight into the relationship among the expression behavior in vivo of the three genes in human testis, analysis of MDR1 and MRP gene expression in testicular biopsies was performed and related to the presence of CFTR gene mutations in congenital absence of the vas deferens (CAVD: n = 20) and non-CAVD (n = 30) infertile patients with azoospermia or severe oligozoospermia. A CFTR mutation analysis performed in both groups of patients supported the involvement of CFTR gene mutations in CAVD phenotype (85%) and in defective spermatogenesis (19%). Quantitative reverse transcription-polymerase chain reaction analysis of testicular tissue showed a CFTR-independent MDR1 and MRP gene expression in human testis, suggesting that the mechanisms underlying CFTR gene regulation in testis are different from those in intestine. These findings should contribute to the understanding of patterns of in vivo expression of CFTR, MDR1, and MRP genes in CFTR-related infertility.

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87 Phenotypical and genotypical description of CAVD and non-CAVD infertile patients.a No. patient Phenotype FSH (U/L) Non-CFTR infertility-associated factors Testicular biopsy CFTR mutation M470V polymorphism CAVD infertility 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CUAVD CUAVD CUAVD CUAVD 3.1 7.3 3.1 2.4 1.9 3.5 5.7 4.3 3.6 ND 2.2 4.8 11.3 2.1 ND 7.6 5.3 6.5 3.9 21.4 None None None None None None None None None None None None None None None None None None None Yes 1 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes V232D/V232D F508del/R117H F508del/R117H G542X/2789ϩ5GϾA F508del/D1270N ϩ R74W F508del/D1270N ϩ R74W S945L/R258G F508del/5T F508del/5T L206W/5T R117H/N F508del/N Y1014C/N 5T/N N/N N/N Y1092X/R258G 621ϩ1GϾT/5T Q890R/N N/N M/M M/M M/M M/M M/V M/V M/V M/M M/V M/V M/V M/V M/V M/V M/M V/V V/V M/V V/V M/M Non-CAVD infertility 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 TF (SA) TF (SA) TF (SA) TF (SA) TF (SA) TF (SA) TF (SA) TF (SA) TF (SA) TF (SSO) TF (SSO) TF (SSO) TF (SSO) TF (SSO) TF (SSO) TF (SSO) TF (SSO) TF (SSO) TF (SSO) TF (SA) TF (SA) TF (SSO) OA OA OA OA OA OA OA OA 42.0 15.9 34.8 8.9 26.3 6.4 7.8 15.6 8.7 3.2 3.9 12.6 4.7 1.3 5.6 3.9 6.1 9.3 8.8 19.3 9.6 ND 3.3 5.9 6.6 3.6 1.9 4.2 2.0 4.4 None None None None None None None None None None None None None None None None Yes 2 Yes 2 Yes 2, 3 Yes 4 Yes 5 Yes 6 None None None None None Yes 1 Yes 7 Yes 8 Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes No No No No No No Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes F508del/N R334W/N N/N N/N N/N N/N N/N N/N N/N R75Q/N N/N N/N N/N N/N N/N N/N N/N N/N N/N N/N N/N N/N 5T/5T N/N N/N N/N N/N N/N N/N N/N M/M V/V M/V M/V M/V M/V V/V V/V V/V V/V M/V M/V M/V ND V/V M/M M/V M/M M/V M/M M/V V/V M/V M/V M/V V/V V/V M/V M/V V/V a CFTR mutations and M470V allele are also described for each patient. Login to comment
94 CFTR Analysis We have identified 14 different CFTR mutations (R117H, L206W, V232D, R258G, F508del, G542X, 621ϩ1GϾT, Q890R, S945L, Y1014C, Y1092X, D1270N, 2789ϩ5GϾA, IVS8-6[5T]) in 17 of 20 patients of the CAVD group, giving a CFTR mutation frequency of 85%. Login to comment

[hide] Salt-independent abnormality of antimicrobial acti... Am J Respir Cell Mol Biol. 2001 Jul;25(1):21-5. Bals R, Weiner DJ, Meegalla RL, Accurso F, Wilson JM
Salt-independent abnormality of antimicrobial activity in cystic fibrosis airway surface fluid.
Am J Respir Cell Mol Biol. 2001 Jul;25(1):21-5., [PMID:11472971]

Abstract [show]
The link between the genetic defect in cystic fibrosis (CF) and the recently described breach in pulmonary host defense has focused on the role of salt and water metabolism in the airways. Using a human bronchial xenograft model we demonstrate a salt-independent abnormality in bacterial killing in CF airway surface fluid (ASF). Biochemical characterization implicates the absence or dysfunction of a molecule critical to the constitution of normal bacterial killing. Our study suggests that CF transmembrane conductance regulator (CFTR) deficiency causes a primary abnormality in the composition of ASF that leads to a salt-independent defect in host defense. Importantly, this defect is corrected by adenovirus-mediated gene transfer of CFTR.

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35 R347P, and one ⌬F508/unknown; plus one G542X/unknown, one 621 ϩ 1G Ͼ T/W1282X, and four unknown) and their bacteriology (three Staphylococcus aureus positive, three Pseudomonas aeruginosa positive, and four other positive results). Login to comment

[hide] XV-2c/KM-19 haplotype analysis of cystic fibrosis ... Am J Med Genet. 2001 Aug 15;102(3):277-81. Orozco L, Gonzalez L, Chavez M, Velazquez R, Lezana JL, Saldana Y, Villarreal T, Carnevale A
XV-2c/KM-19 haplotype analysis of cystic fibrosis mutations in Mexican patients.
Am J Med Genet. 2001 Aug 15;102(3):277-81., 2001-08-15 [PMID:11484207]

Abstract [show]
We analyzed 97 unrelated Mexican cystic fibrosis (CF) patients and their first-degree relatives to study the association of XV2C/TaqI/KM19/PstI haplotypes with CF mutations in this population. Haplotype phases could be established in 148 CF and 110 normal chromosomes, and haplotype distributions of normal and CF chromosomes differed significantly (P < 0.001). DeltaF508 and G542X mutations accounted for 56% of CF chromosomes and were found to be associated with haplotype B in 97.2% and 72.7% of chromosomes, respectively. The haplotype distribution of CF chromosomes carrying other rare and unknown mutations was similar to that of normal chromosomes (P > 0.05), haplotypes A and C being the most frequent. This is in accordance with the extensive heterogeneity and the spectrum of mutations reported in Mexican CF patients. We also report the haplotype distribution of all informative chromosomes bearing rare mutations; some were found to be associated with previously reported haplotypes, whereas others were found on different haplotypes. Recombination or recurrence of mutations may explain these different associations, although other intragenic markers must be used to better understand the origin and dispersion of CF mutations in our country. XK haplotype analysis allowed carrier detection among sibs in 24.3% of families, showing that this method may be useful for carrier detection in populations with high allelic heterogeneity.

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43 DF508 and G542X chromosomes (56% of all CF chromosomes) were highly associated with haplotype B (97.2% and 72.7%, respectively). Login to comment
47 Carrier Detection XK haplotype analysis of families with at least one unknown CF allele or with uncommon mutations was TABLE I. Distribution of XK Haplotype on Cystic Fibrosis (CF) and Normal Chromosomes A B C D DF508 N 72 70 (97.2%) 2 (2.8%) G542X N 11 1 (9.09%) 8 (72.72%) 2 (18.18%) Others N 32 13 (40.6%) 5 (15.6%) 9 (28.1%) 5 (15.6%) Unknown N 33 18 (54.5%) 1 (3.0%) 13 (39.4%) 1 (3%) CF chromosomes N 148 32 (21.6%) 84 (56.7%) 22 (14.9%) 10 (6.8%) Normal N 110 54 (49.1%) 8 (7.3%) 35 (31.8%) 13 (11.8%) useful to determine the carrier status of siblings in 18 families (24.3%), as illustrated in Figure 1. Login to comment
51 This difference is mainly due to the high frequency of mutations DF508 and G542X, associated with haplotype B in 97.2% and 72.7% of chromosomes, respectively. Login to comment
72 G542X is the second most common CF mutation worldwide. Login to comment
74 It has been suggested that G542X was introduced into Europe by the occidental Phoenicians 2,000 or 3,000 years ago [Loirat et al., 1997]. Login to comment
75 In the Mexican population, G542X is also the second most common mutation (6.1%), and was found to be most frequently associated with haplotype B (72.2%), but also with haplotypes A (9.09%) and D (18.18%). Login to comment
77 Recombination on the original G542X chromosome. Login to comment
82 The fact that G542X was found in Native American populations of the U.S. Southwest such as the Pueblo [Grebe et al., 1992] supports the latter hypothesis. Login to comment

[hide] Aerosol and lobar administration of a recombinant ... Hum Gene Ther. 2001 Jul 20;12(11):1369-82. Joseph PM, O'Sullivan BP, Lapey A, Dorkin H, Oren J, Balfour R, Perricone MA, Rosenberg M, Wadsworth SC, Smith AE, St George JA, Meeker DP
Aerosol and lobar administration of a recombinant adenovirus to individuals with cystic fibrosis. I. Methods, safety, and clinical implications.
Hum Gene Ther. 2001 Jul 20;12(11):1369-82., 2001-07-20 [PMID:11485629]

Abstract [show]
Cystic fibrosis (CF), an autosomal recessive disorder resulting from mutations in the cystic fibrosis trans-membrane conductance regulator (CFTR) gene, is the most common lethal genetic illness in the Caucasian population. Gene transfer to airway epithelium, using adenoviruses containing normal CFTR cDNA, leads to transient production of CFTR mRNA and, in some studies, to correction of the airway epithelial ion transport defect caused by dysfunctional CFTR. Inflammatory responses to the adenoviral vector have been reported, particularly at high viral titers. We evaluated the effects of adenovirus-mediated CFTR gene transfer to airway epithelium in 36 subjects with CF (34 individuals, 2 of whom received two separate doses of vector), 20 by lobar instillation and 16 by aerosol administration. Doses ranged from 8 x 10(6) to 2.5 x 10(10) infective units (IU), in 0.5-log increments. After lobar administration of low doses there were occasional reports of cough, low-grade temperature, and myalgias. At the highest lobar dose (2.5 x 10(9) IU) two of three patients had transient myalgias, fever, and increased sputum production with obvious infiltrates on CT scan. After aerosol administration there were no significant systemic symptoms until the 2.5 x 10(10) IU dose, when both patients experienced myalgias and fever that resolved within 24 hr. There were no infiltrates seen on chest CT scans in any of the patients in the aerosol administration group. There were no consistent changes in pulmonary function tests or any significant rise in serum IgG or neutralizing antibodies in patients from either group. Serum, sputum, and nasal cytokines, measured before and after vector administration, showed no correlation with adenoviral dose. Gene transfer to lung cells was inefficient and expression was transient. Cells infected with the vector included mononuclear inflammatory cells as well as cuboidal and columnar epithelial cells. In summary, we found no consistent immune response, no evidence of viral shedding, and no consistent change in pulmonary function in response to adenovirus-mediated CFTR gene transfer. At higher doses there was a mild, nonspecific inflammatory response, as evidenced by fevers and myalgias. Overall, vector administration was tolerated but transfer of CFTR cDNA was inefficient and transgene expression was transient for the doses and method of administration used here.

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170 DAY 1 CLINICAL CHARACTERISTICS OF SUBJECTS RECEIVING LOBAR ADMINISTRATION OF VECTOR Age FEV1 Dose Subject Sex (years) (% pred) NIH score Genotype Vector (IU) 1 M 31 2.31/50 69 DF508/DF508 Ad2/CFTR2 8 3 106 2 M 26 3.92/81 87 DF508/R117H Ad2/CFTR2 8 3 106 3 M 23 1.59/38a 67 DF508/DF508 Ad2/CFTR2 8 3 106 4 F 23 1.55/46 65 DF508/R117H Ad2/CFTR2 2.5 3 107 5 M 30 3.19/79 85 DF508/DF508 Ad2/CFTR2 2.5 3 107 6 M 27 4.18/99 87 DF508/W1282X Ad2/CFTR2 2.5 3 107 7 F 33 1.47/50 70 DF508/R3342 Ad2/CFTR2 8 3 107 8 F 28 2.0968 78 G542X/Other Ad2/CFTR2 8 3 107 9 M 15 3.80/94 93 DF508/A455L Ad2/CFTR2 8 3 107 10 F 33 2.47/75 92 DF508/Other Ad2/CFTR2 2.5 3 108 11 M 17 3.82/84 95 DF508/DF508 Ad2/CFTR2 2.5 3 108 12 F 22 1.71/53 77 DF508/Other Ad2/CFTR2 2.5 3 108 13 F 23 1.72/58 85 DF508/DF508 Ad2/CFTR8 2.5 3 108 14 F 19 2.71/61 85 DF508/Other Ad2/CFTR8 2.5 3 108 15 F 35 1.77/63 81 DF508/DF508 Ad2/CFTR8 8 3 108 16 M 38 1.70/41 81 DF508/W1282X Ad2/CFTR8 8 3 108 17 M 27 3.42/69 86 DF508/DF508 Ad2/CFTR8 8 3 108 18 M 15 3.97/85 97 DF508/DF508 Ad2/CFTR8 2.5 3 109 19 F 17 2.66/75 77 DF508/DF508 Ad2/CFTR8 2.5 3 109 20 M 24 3.35/78 93 DF508/DF508 Ad2/CFTR8 2.5 3 109 11 M/9F Average: 25.3 2.64/67.4 81.85 aFEV1 1.77 (42%) at enrollment. as well as vector type and dose for the lobar and aerosol administration groups, respectively. Login to comment
201 DAY 1 CLINICAL CHARACTERISTICS OF SUBJECTS RECEIVING AEROSOL ADMINISTRATION OF VECTOR a Age FEV1 Dose Subject Sex (years) (% pred) NIH score Genotype Vector (IU) 21 F 32 3.63/112 85 DF508/R117H Ad2/CFTR2 8 3 106 22 F 28 3.38/77 91 DF508/other Ad2/CFTR2 8 3 106 23 F 28 1.30/39b 83 DF508/other Ad2/CFTR8 2.5 3 107 24 M 18 3.51/71 96 DF508/other Ad2/CFTR8 2.5 3 107 25 F 37 1.81/61 83 DF508/DF508 Ad2/CFTR8 8 3 107 26 F 18 3.53/92 93 DF508/DF508 Ad2/CFTR8 8 3 107 27 F 27 2.24/77 81 G551D/621-1GT Ad2/CFTR8 2.5 3 108 28a M 25 4.22/93 97 G2111GT/G542X Ad2/CFTR8 2.5 3 108 29 M 15 2.01/85 90 other/other Ad2/CFTR8 8 3 108 30 M 18 4.06/109 96 DF508/3489110kbC-T Ad2/CFTR8 8 3 108 31 M 40 3.81/71 75 DF508/3849110kbC-T Ad2/CFTR8 2.5 3 109 32 F 17 2.29/75 92 DF508/G542X Ad2/CFTR8 2.5 3 109 33 F 21 2.99/89 95 DF508/DF508 Ad2/CFTR8 8 3 109 34 M 15 3.37/95 94 DF508/I507 Ad2/CFTR8 8 3 109 35a M 26 3.45/77 97 G2111GT/G542X Ad2/CFTR8 2.5 3 1010 36a F 35 2.4/74 89 DF508/other Ad2/CFTR8 2.4 3 1010 7 M/9 F 25 3.0/81.1 89.8 aPatient 10 (lobar administration) and patient 36 are one individual; patient 28 and 35 are another single individual. Login to comment

[hide] Intron-8 polythymidine sequence in Australasian in... Eur Respir J. 2001 Jun;17(6):1195-200. Massie RJ, Poplawski N, Wilcken B, Goldblatt J, Byrnes C, Robertson C
Intron-8 polythymidine sequence in Australasian individuals with CF mutations R117H and R117C.
Eur Respir J. 2001 Jun;17(6):1195-200., [PMID:11491164]

Abstract [show]
Compound heterozygotes for a severe cystic fibrosis transmembrane conductance regulator (CFTR) mutation and the R117H or R117C mutation (R117H/C) have clinical presentations that vary from classic cystic fibrosis (CF) to an incidental genetic finding. The aim of this study was to assess the influence of the intron-8 polythvmidine sequence (IVS8) on the relationship between genotype and phenotype of individuals with R117H/C. All individuals with R117H/C known to CF clinics in Australia and New Zealand were retrospectively studied by collecting information on genotype, age, pancreatic status, sweat electrolytes, sputum microbiology and pulmonary function. Forty-one individuals (39 with R117H and two with R117C), 16 on an IVS8-5T background and 25 on an IVS8-7T background were identified. Twelve individuals presented clinically, four were siblings of known R117H/C compound heterozygotes and 25 were detected by newborn screening. Eleven of 14 of the IVS8-5T group (78%) with sweat chloride results available had sweat CI > 60 mmol x L(-1) compared to 5 (20%) of the R117H/7T group (Chi-squared=10.4, p=0.001). Two were pancreatic insufficient, both IVS8-5T. Two IVS8-5T individuals have recently died (aged 43 and 19) and of the 14 surviving IVS8-5T group, 11 (79%) are symptomatic compared to eight (32%) of the IVS8-7T individuals (Chi-squared=6.1, p=0.01). In conclusion, most individuals with R117H/C on a IVS8-5T background have an elevated sweat chloride and clinical cystic fibrosis, which in some cases is severe. Most individuals with R117H/C on an IVS8-7T background do not have clinical cystic fibrosis but should be followed for the development of clinical disease.

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41 Infants with a positive (w60 mmol?L-1 ) or borderline (40 - 60 mmol?L-1 ) sweat chloride and in whom there is an unidentified mutation are referred for an extended mutation analysis which includes: DF508, R117H, G551D, A455E, G542X, N1303K, W1282X, 1717-1, R560T, R347P, R334W, R1162X, S549N, 621z1, 3849z10CwT, and the IVS8 polythymidine sequence. Login to comment
57 Two of the individuals had a second severe mutation (G542X/R117H and G551D/R117H), four had an unknown second mutation and one patient was homozygous for R117H. Login to comment

[hide] Silicon-based biosensors for rapid detection of pr... Clin Chem. 2001 Oct;47(10):1894-900. Jenison R, La H, Haeberli A, Ostroff R, Polisky B
Silicon-based biosensors for rapid detection of protein or nucleic acid targets.
Clin Chem. 2001 Oct;47(10):1894-900., [PMID:11568116]

Abstract [show]
BACKGROUND: We developed a silicon-based biosensor that generates visual, qualitative results or quantitative results for the detection of protein or nucleic acid targets in a multiplex format. METHODS: Capture probes were immobilized either passively or covalently on the optically coated surface of the biosensor. Intermolecular interactions of the immobilized capture probe with specific target molecules were transduced into a molecular thin film. Thin films were generated by enzyme-catalyzed deposition in the vicinity of the surface-bound target. The increased thickness on the surface changed the apparent color of the biosensor by altering the interference pattern of reflected light. RESULTS: Cytokine detection was achieved in a 40-min multiplex assay. Detection limits were 4 ng/L for interleukin (IL)-6, 31 ng/L for IL1-beta, and 437 ng/L for interferon-gamma. In multianalyte experiments, cytokines were specifically detected with signal-to-noise ratios ranging from 15 to 80. With a modified optical surface, specificity was also demonstrated in a nucleic acid array with unambiguous discrimination of single-base changes in a 15-min assay. For homozygous wild-type and homozygous mutant samples, signal-to-noise ratios of approximately 100 were observed. Heterozygous samples yielded approximately equivalent signals for wild-type and mutant capture probes. CONCLUSIONS: The thin-film biosensor allows rapid, sensitive, and specific detection of protein or nucleic acid targets in an array format with results read visually or quantified with a charge-coupled device camera. This biosensor is suited for multianalyte detection in clinical diagnostic assays.

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68 Capture sequences were as follows: For ⌬F508, the wild type was 5Ј-YAC AAC ACC AAA GAT GAT ATT TT-3Ј, and the mutant was 5Ј-YCC GAA ACA CCA ATG ATA TTT TC-3Ј For N1303K, the wild type was 5Ј-YAC GGA TCC AAG TTT TTT CTA A-3Ј, and the mutant was 5Ј-YAC GGA TCC AAC TTT TTT CTA A-3Ј For G551D, the wild type was 5Ј-YAA CTC GTT GAC CTC CAC TC-3Ј, and the mutant was 5Ј-YAA CTC GTT GAT CTC CAC TC-3Ј For G542X, the wild type was 5Ј-YAA CAC CTT CTC CAA GAA CTA TA-3Ј, and the mutant was 5Ј-XAA CAC CTT CTC AAA GAA CTA TA-3Ј antibody immobilization Capture antibodies were diluted to 2 mg/L in 0.1 mol/L sodium bicarbonate buffer, pH 9.3, and 200 nL was spotted using a Hamilton MP2200 pipetting robot equipped with a modified dispense head. Login to comment
79 Input concentrations for the probe sets was as follows: ⌬F508, 150 nmol/L for both the wild type and mutant; G542X, 75 nmol/L for the wild type and 750 nmol/L for the mutant; N1303K, 300 nmol/L for both the wild type and mutant; G551D, 150 nmol/L for both the wild type and mutant. Login to comment
119 One mutation, ⌬F508, is a 3-bp deletion, whereas the other three, G542X, G551D, and N1303K, are SNPs. Login to comment
144 Detection of SNPs in the CFTR gene. A locator for the capture probes specific for the four interrogated mutations is shown. CCD images from experiments using human DNA of defined genotype, subjected to multiplex PCR, as input to the assay as follows: (A), wild type/wild type; (B), ⌬F508/⌬F508; (C), G542X/wild type; (D), G542X/G542X; (E), G551D/ wild type; (F), G551D/G551D. multiplex array format. Login to comment

[hide] Mutations of the cystic fibrosis gene in patients ... Am J Gastroenterol. 2001 Sep;96(9):2657-61. Truninger K, Malik N, Ammann RW, Muellhaupt B, Seifert B, Muller HJ, Blum HE
Mutations of the cystic fibrosis gene in patients with chronic pancreatitis.
Am J Gastroenterol. 2001 Sep;96(9):2657-61., [PMID:11569691]

Abstract [show]
OBJECTIVE: Several studies have reported an increased frequency of cystic fibrosis gene mutations in idiopathic but not in alcoholic chronic pancreatitis. The impact of cystic fibrosis gene mutations on the long-term course of chronic pancreatitis has not been analyzed. The aim of our study was to determine the frequency of cystic fibrosis gene mutations in patients with chronic pancreatitis with long-term follow-up and to see whether patients with mutations have a clinically different natural course compared to those without mutations. METHODS: Eighty two patients with chronic pancreatitis and 11 patients with recurrent acute pancreatitis of our well defined pancreatitis cohort were screened for the 31 most common cystic fibrosis gene mutations. The impact of cystic fibrosis gene mutations on the long-term course of chronic pancreatitis was assessed. RESULTS: A cystic fibrosis gene mutation was detected in five of 49 patients with alcoholic chronic pancreatitis (10.2%; 2.3 times the expected frequency) and in three of 14 patients with idiopathic-juvenile chronic pancreatitis (21.4%; 4.8 times the expected frequency). No mutations were found in the remaining patients with chronic pancreatitis of rare causes, hereditary pancreatitis, and recurrent acute pancreatitis. The frequency of pancreatic calcifications was significantly higher in patients with alcoholic chronic pancreatitis without mutations. This result was not confirmed in patients with idiopathic-juvenile chronic pancreatitis. The duration of pain and the frequency of exocrine and endocrine insufficiency was comparable in both subgroups irrespective of the mutation status. CONCLUSION: Our data indicate a significantly increased frequency of cystic fibrosis gene mutations both in patients with alcoholic and idiopathic-juvenile chronic pancreatitis. The natural course was similar in patients with mutations compared to those without mutations.

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56 Using multiplex PCR, 15 genomic fragments were amplified which contain the following mutations: ⌬F508, ⌬I507, Q493X, V520F, 1717-1G3A, G542X, G551D, R553X, R560T, S549R, S549N, 3849 ϩ 10kbC3T, 3849 ϩ 4A3G, R1162X, 3659delC, W1282X, 3905insT, N1303K, G85E, 621 ϩ 1G3T, R117H, Y122X, 711 ϩ 1G3T; 1078delT, R347P, R347H, R334W, A455E, 1898 ϩ 1G3A, 2183AA3G, 2789 ϩ 5G3A. Login to comment

[hide] Cystic fibrosis phenotype evaluation and paternity... Hum Reprod. 2001 Oct;16(10):2093-7. Josserand RN, Bey-Omar F, Rollet J, Lejeune H, Boggio D, Durand DV, Durieu I
Cystic fibrosis phenotype evaluation and paternity outcome in 50 males with congenital bilateral absence of vas deferens.
Hum Reprod. 2001 Oct;16(10):2093-7., [PMID:11574497]

Abstract [show]
BACKGROUND: Most infertile males with congenital bilateral absence of vas deferens (CBAVD) carry mutations on the cystic fibrosis transmembrane conductance regulator gene and may express mild cystic fibrosis (CF) symptoms. Barriers to paternity for these men can now be overcome by assisted reproduction. Our aims were to investigate the CF-related phenotype and clinical outcome for 50 patients with CBAVD seen at a CF adult centre between 1992 and 1999. METHODS AND RESULTS: The investigation of the patients included screening for 22 CF mutations and identification of the poly-T variant of intron 8, sweat testing, clinical investigation for CF-related extra-genital manifestations, and genetic counselling. CFTR mutations were detected on 56 alleles of the 50 patients. A total of 15 (30%) was compound heterozygote and 26 (52%) heterozygote. In all, 38% of the patients had a positive sweat test. Four patients were diagnosed with typical CF not detected previously. Twenty-one patients became fathers following ICSI (eight cases), artificial insemination by donor or IVF with sperm donor (seven cases) or through adoption (six cases). A mail survey allowed the identification of CF-related clinical symptoms. Information on the occurrence of CF-related symptoms was obtained for 58.5% of patients: in the absence of initial symptoms, no new clinical signs were reported. CONCLUSION: Patients diagnosed with CBAVD need genetic counselling before assisted reproduction. Even when no wish for paternity is expressed, CF gene screening should be associated with at least a sweat test and clinical evaluation because of possible mild forms of CF disease. Medical follow-up did not reveal any new symptoms.

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30 Leukocytes samples were analysed for a series of 22 CF mutations including the five most frequently encountered in our region (The CF Genotype Consortium, 1994): ∆F508, G542X, N1303K, 1717-G-A, 885E; and 17 others: R117H, R334W, R347H, R347P, 556delA, S549N, S549I, S549R, G551D, R553X, R560T, G1244E, S1255X, W1282X, R1283K, 3898ins C, D1270N. Login to comment
40 sCFTR mutation was detected in 56 alleles of the 50 patients: ∆F508 in 30 alleles, R117H in six, D1270N in two, G542X in one, 1717ϩG-A in one, 2789ϩ5G-A in one, R347H in one and the 5T allele in 14. Login to comment
45 Description of the 50 men with CBAVD:CF genotype and phenotype Number age CF SCC CF-related (years) genotype (mmol/l) symptoms 1 29 ∆F508/R117H 59 2 35 ∆F508/R117H 54 3 24 ∆F508/R117H 43 4 33 ∆F508/R117H 39 5 26 ∆F508/5T 90 S/B 6 27 ∆F508/5T 67 S 7 32 ∆F508/5T 55 8 30 ∆F508/5T 51 9 31 ∆F508/5T 44 10 44 ∆F508/5T 38 S 11 36 ∆F508/5T 36 S 12 54 ∆F508/5T 21 S 13 31 R117H/R347H 79 S 14 36 1717G-A/5T 50 S 15 32 5T/5T 77 P/DM 16 27 ∆F508/- 94 S 17 41 ∆F508/- 90 S/B 18 30 ∆F508/- 88 19 30 ∆F508/- 82 S 20 32 ∆F508/- 81 21 25 ∆F508/- 79 22 31 ∆F508/- 79 23 27 ∆F508/- 75 S 24 43 ∆F508/- 70 25 38 ∆F508/- 65 26 34 ∆F508/- 52 S 27 31 ∆F508/- 47 S 28 35 ∆F508/- 40 S 29 26 ∆F508/- 39 S 30 25 ∆F508/- 36 31 33 ∆F508/- 33 32 37 ∆F508/- 28 S 33 36 ∆F508/- 18 S 34 33 G542X/- 45 S 35 37 D1270N/- 116 36 34 D1270N/- 103 S/P 37 46 R117H/- 95 39 37 2789ϩ5G-A/- 100 S 40 27 5T/- 90 S 38 30 5T/- 51 44 38 5T/- 45 41 30 -/- 57 S 42 35 -/- 52 S 43 36 -/- 46 B (tobacco) 45 33 -/- 40 S 46 31 -/- 36 S/asthma 47 32 -/- 28 B (tobacco) 48 28 -/- 28 49 30 -/- 26 50 35 -/- 20 S SCC ϭ sweat chloride concentration. Login to comment
49 Results of assisted reproduction procedures Number CF genotype CF mutation in Assisted reproduction procedure women ICSI IVFSD AID adoption 1 ∆F508/R117H 0 failure success 2 ∆F508/R117H failure 3 ∆F508/R117H failure 4 ∆F508/R117H 0 5 ∆F508/5T 0 6 ∆F508/5T success 7 ∆F508/5T ∆F508/- failure 1 8 ∆F508/5T success 9 ∆F508/5T failure 1 10 ∆F508/5T 0 1 11 ∆F508/5T 0 12 ∆F508/5T failure failure 13 R117H/R347H failure failure 14 1717G-A/5T failure 15 5T/5T 0 16 ∆F508/- ∆F508/- 0 success 17 ∆F508/- 0 1 18 ∆F508/- 0 19 ∆F508/- failure 20 ∆F508/- ∆F508/- 0 success 21 ∆F508/- success 22 ∆F508/- failure 23 ∆F508/- failure 24 ∆F508/- in process 25 ∆F508/- 0 1 26 ∆F508/- failure 27 ∆F508/- failure 28 ∆F508/- success 29 ∆F508/- 0 success 30 ∆F508/- failure success 31 ∆F508/- 0 1 32 ∆F508/- 0 33 ∆F508/- success 34 G542X/- failure failure 35 D1270N/- success 36 D1270N/- 0 37 R117H/- failure 39 2789ϩ5G-A/- in process 40 5T/- ∆F508/- 0 38 5T/- failure 44 5T/- 0 success 41 -/- success 42 -/- failure 43 -/- 0 45 -/- in process 46 -/- 0 47 -/- 0 success 48 -/- failure 49 -/- failure 50 -/- success IVFSD ϭ IVF with sperm donor; AID ϭ artificial insemination by donor. Login to comment

[hide] Analysis of exocrine pancreatic function in cystic... Eur J Clin Invest. 2001 Sep;31(9):796-801. Walkowiak J, Herzig KH, Witt M, Pogorzelski A, Piotrowski R, Barra E, Sobczynska-Tomaszewska A, Trawinska-Bartnicka M, Strzykala K, Cichy W, Sands D, Rutkiewicz E, Krawczynski M
Analysis of exocrine pancreatic function in cystic fibrosis: one mild CFTR mutation does not exclude pancreatic insufficiency.
Eur J Clin Invest. 2001 Sep;31(9):796-801., [PMID:11589722]

Abstract [show]
BACKGROUND: Cystic fibrosis (CF) is the most common cause of exocrine pancreatic insufficiency in childhood. The aim of the present study is to evaluate the correlation between genotype and exocrine pancreatic insufficiency in CF patients. The special emphasis was put on the analysis of mild CFTR mutations. DESIGN: The study comprised 394 CF patients and 105 healthy subjects (HS). Elastase-1 concentrations were measured in all subjects. RESULTS: Severe pancreatic insufficiency was associated with the presence of two CFTR gene mutations (DeltaF508, N1303K, CFTR dele 2,3 (21kb), G542X, 1717-1G-A, R533X, W1282X, 621GT, 2183AAG, R560T, 2184insA and DeltaI507, G551D, 895T) and mild insufficiency with the presence of at least one mutation (R117H, 3171insC, A155P2, 138insL, 296 + 1G-A, E92GK, E217G, 2789 + 5G-A. 3849 + 1kbC-T/3849 + 1kbC-T) genotype resulted in high elastase-1-values. However, in case of patients with genotype DeltaF508/3849 + 10kbC-T, 1717-1GA/3849 + 10kbC-T as well as with DeltaF508/R334W, both high and low elastase-1 concentrations were found. Low E1 values were found in a patient with DeltaF508/R347P genotype. CONCLUSION: Patients who carry two 'severe' mutations develop pancreatic insufficiency, whereas those who carry at least one 'mild' usually remain pancreatic sufficient. However, the presence of one mild mutation does not exclude pancreatic insufficiency.

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5 Results Severe pancreatic insufficiency was associated with the presence of two CFTR gene mutations (DF508, N1303K, CFTR dele 2,3 (21kb), G542X, 1717±1G-A, R533X, W1282X, 621GT, 2183AAG, R560T, 2184insA and DI507, G551D, 895T) and mild insufficiency with the presence of at least one mutation (R117H, 3171insC, A155P2, 138insL, 296 1 1G-A, E92GK, E217G, 2789 1 5G-A. Login to comment
51 Results Among 394 genotyped CF patients, the following mutations on alleles were found (n): DF508 (464), 3849 1 10kbC-T (30), CFTR dele2,3(21 kB) (21), N1303K (15), G542X (12), 1717±1G-A (9), R533X (6), W1282X (6), 621 1 G-T (3), R117H (2), 3171insC (2), A155P2 (2), 2183AAG (2), R334W (2), 895T (2), 296 1 1G-A (2), E92GK (2), 138insL (1), E217G (1), 2789 1 5G-A (1), R347P (1), R560T (1), 2184insA (1), I507 (1), G551D (1). Login to comment
57 CFTR gene mutations were classified as `severe' (E1 , 96 mg g21 ) ± severely affecting pancreatic function (DF508, N1303K, CFTR dele 2,3 (21kb), G542X, 1717±1G-A, R533X, W1282X, 621 1 1G-T, 2183AAG, 895T, R560T, 2184insA, DI507, G551D) and `mild' (E1 . Login to comment
81 500 DF508/3849 1 10kbC-T (17) 1 4 1 6 5 DF508/CFTR dele2,3(21kb) (15) 9 4 2 DF508/N1303K (10) 7 3 DF508/1717±1G-A (7) 5 2 DF508/G542X (7) 4 2 1 DF508/W1282X (5) 4 1 DF508/R553X (3) 3 DF508/R334W (2) 1 1 DF508/2183AAG (2) 2 DF508/R117H (1) 1 DF508/621GT (1) 1 DF508/R347P (1) 1 DF508/2184insA (1) 1 DF508/DI507 (1) 1 3849 1 10kbC-T/3849 1 10kbC-T (3) 3 N1303K/CFTR dele2,3(21kb) (2) 1 1 1717±1G-A/3849 1 10kbC-T (2) 1 1 3171insC/A155P2 (2) 1 1 296 1 1G-A/E92GK (2) 2 R117H/138insL (1) 1 W1282X/3849 1 10kbC-T (1) 1 N1303K/3849 1 10kbC-T (1) 1 CFTR dele2,3(21kb)/3849 1 10kbC-T (1) 1 R553X/G542X (1) 1 621 1 1G-T/621 1 1G-T (1) 1 G542X/M (4) 2 2 CFTR dele 2,3(21kb)/M (1) 1 2 3849 1 10kbC-T/M (2) 1 1 R533X/M (2) 2 N1303K/M (2) 2 895T/M (2) 1 1 E217G/M (1) 1 G551D/M (1) 1 R560T/M (1) 1 2789 1 5G-A/M (1) 1 Total (109) 44 21 10 4 12 18 M, unidentified mutation. Login to comment
86 Kristidis et al. [10] reported that pancreatic insufficiency strongly correlates also with two alleles of DI507, Q493X, G542X, R553X, W1282X, 621 1 1G-T, 1717±1G-A, 556delA, 3659delC, I148T, G480C, V520F and R560T while one or two mutations such as R117H, R334W, A455E, and P574H were correlated with a pancreatic sufficient phenotype. Login to comment
88 An international Cystic Fibrosis Genotype-Phenotype Consortium [25] evaluated DF508 homozygotes and seven of the most common DF508 compound heterozygotes (G542X, R553X, N1303K, W1282X, 1717±1G-A, 621 1 1GT, R117H). Login to comment

[hide] Improved detection of CFTR mutations in Southern C... Hum Mutat. 2001 Oct;18(4):296-307. Wong LJ, Wang J, Zhang YH, Hsu E, Heim RA, Bowman CM, Woo MS
Improved detection of CFTR mutations in Southern California Hispanic CF patients.
Hum Mutat. 2001 Oct;18(4):296-307., [PMID:11668613]

Abstract [show]
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), a common autosomal recessive disease in Caucasians. The broad mutation spectrum varies among different patient groups. Current molecular diagnoses are designed to detect 80-97% of CF chromosomes in Caucasians and Ashkenazi Jews but have a much lower detection rate in Hispanic CF patients. Grebe et al. [1994] reported a 58% detection rate in Hispanic patients. Since then, there has been no large-scale, complete mutational analysis of Hispanic CF patients. In this study, the mutations in 62 Hispanic patients from southern California were investigated. The entire coding and flanking intronic regions of the CFTR gene were analyzed by temporal temperature gradient gel electrophoresis (TTGE) followed by sequencing to identify the mutations. Eleven novel mutations were discovered in this patient group: 3876delA, 406-1G>A, 935delA, 663delT, 3271delGG, 2105-2117del13insAGAAA, 3199del6, Q179K, 2108delA, 3171delC, and 3500-2A>T. Among the mutations, seven were out-of-frame insertions and deletions that result in truncated proteins, two were splice-site mutations, one was an in-frame 6 bp deletion, and one was a missense mutation that involved the non-conservative change of glutamine-179 to lysine. All patients presented severe classical clinical course with pancreatic insufficiency and poor growth, consistent with the nature of truncation mutation. The results indicate that TTGE screening following the analysis of recurrent mutations will substantially improve the mutation detection rate for Hispanic CF patients from southern California.

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94 The next most common mutations were G542X and 3849+10kbC>T, each found in five unrelated patients (4%). Login to comment
95 These frequencies are much higher than those found in Europe and North America where G542X and 3849+10kbC>T mutations account for 2.4 and 0.2% of their patients, respectively. Login to comment
117 Summary of Mutations Found in This Group of Hispanic Patients Exon or Number of Mutation intron chromosomes Frequency % Mutations detected before full gene analysis 91 73.38% 1 F508 10 64 51.6 2 G542X 11 5 4 3 3849+10kb C>T Intron 19 5 4 4 S549N 11 3 2.4 5 I148T 4 2 1.6 6 3120+1G>A 16 2 1.6 7 R334W 7 2 1.6 8 G551D 11 1 0.8 9 N1303K 21 1 0.8 10 W1282X 20 1 0.8 11 R1162X 19 1 0.8 12 G85E 3 1 0.8 13 W1089X 17b 1 0.8 14 Y1092X 17b 1 0.8 15 P205S 6a 1 0.8 Mutations detected by full gene screening 26 20.97% 16 R1066Ca 17b 2 1.6 17 1949del84 13 1 0.8 18 2184delA 13 1 0.8 19 Q98R 4 1 0.8 20 R75X 3 1 0.8 21 G1244E 20 1 0.8 22 3876delA 20 7 5.65 23 935delA 6b 2 1.6 24 406-1G>A Intron 2 2 1.6 25 3271delGG 17a 1 0.8 26 2105-2117del13insAGAAA 13 1 0.8 27 663delT 5 1 0.8 28 3171delC 17a 1 0.8 29 2108delA 13 1 0.8 30 Q179K 5 1 0.8 31 3199del6 17a 1 0.8 32 3500-2 A->T Intron 17b 1 0.8 Total identified 117 (177)b 94.35 (97.5)b Unidentified 7 (3)b 5.65 (2.5)b Total 124 (120)b 100 (100)b a This mutation was also detected by SSCP. Login to comment

[hide] Human genetics: lessons from Quebec populations. Annu Rev Genomics Hum Genet. 2001;2:69-101. Scriver CR
Human genetics: lessons from Quebec populations.
Annu Rev Genomics Hum Genet. 2001;2:69-101., [PMID:11701644]

Abstract [show]
The population of Quebec, Canada (7.3 million) contains approximately 6 million French Canadians; they are the descendants of approximately 8500 permanent French settlers who colonized Nouvelle France between 1608 and 1759. Their well-documented settlements, internal migrations, and natural increase over four centuries in relative isolation (geographic, linguistic, etc.) contain important evidence of social transmission of demographic behavior that contributed to effective family size and population structure. This history is reflected in at least 22 Mendelian diseases, occurring at unusually high prevalence in its subpopulations. Immigration of non-French persons during the past 250 years has given the Quebec population further inhomogeneity, which is apparent in allelic diversity at various loci. The histories of Quebec's subpopulations are, to a great extent, the histories of their alleles. Rare pathogenic alleles with high penetrance and associated haplotypes at 10 loci (CFTR, FAH, HBB, HEXA, LDLR, LPL, PAH, PABP2, PDDR, and SACS) are expressed in probands with cystic fibrosis, tyrosinemia, beta-thalassemia, Tay-Sachs, familial hypercholesterolemia, hyperchylomicronemia, PKU, oculopharyngeal muscular dystrophy, pseudo vitamin D deficiency rickets, and spastic ataxia of Charlevoix-Saguenay, respectively) reveal the interpopulation and intrapopulation genetic diversity of Quebec. Inbreeding does not explain the clustering and prevalence of these genetic diseases; genealogical reconstructions buttressed by molecular evidence point to founder effects and genetic drift in multiple instances. Genealogical estimates of historical meioses and analysis of linkage disequilibrium show that sectors of this young population are suitable for linkage disequilibrium mapping of rare alleles. How the population benefits from what is being learned about its structure and how its uniqueness could facilitate construction of a genomic map of linkage disequilibrium are discussed.

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236 The L206W allele (with a mild phenotypic effect) reflects a particular French Canadian heritage (142), whereas W1282X and G542X are prominent in Ashkenazi Jews (2, 145), which reflects corresponding twentieth century immigrations into Quebec. Login to comment

[hide] Genetic risk factors in infertile men with severe ... Hum Reprod. 2002 Jan;17(1):13-6. Dohle GR, Halley DJ, Van Hemel JO, van den Ouwel AM, Pieters MH, Weber RF, Govaerts LC
Genetic risk factors in infertile men with severe oligozoospermia and azoospermia.
Hum Reprod. 2002 Jan;17(1):13-6., [PMID:11756355]

Abstract [show]
BACKGROUND: Male infertility due to severe oligozoospermia and azoospermia has been associated with a number of genetic risk factors. METHODS: In this study 150 men from couples requesting ICSI were investigated for genetic abnormalities, such as constitutive chromosome abnormalities, microdeletions of the Y chromosome (AZF region) and mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. RESULTS: Genetic analysis identified 16/150 (10.6%) abnormal karyotypes, 8/150 (5.3%) AZFc deletions and 14/150 (9.3%) CFTR gene mutations. An abnormal karyotype was found both in men with oligozoospermia and azoospermia: 9 men had a sex-chromosomal aneuploidy, 6 translocations were identified and one marker chromosome was found. Y chromosomal microdeletions were mainly associated with male infertility, due to testicular insufficiency. All deletions identified comprised the AZFc region, containing the Deleted in Azoospermia (DAZ) gene. CFTR gene mutations were commonly seen in men with congenital absence of the vas deferens, but also in 16% of men with azoospermia without any apparent abnormality of the vas deferens. CONCLUSIONS: A genetic abnormality was identified in 36/150 (24%) men with extreme oligozoospermia and azoospermia. Application of ICSI in these couples can result in offspring with an enhanced risk of unbalanced chromosome complement, male infertility due to the transmission of a Y-chromosomal microdeletion, and cystic fibrosis if both partners are CFTR gene mutation carriers. Genetic testing and counselling is clearly indicated for these couples before ICSI is considered.

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29 Twelve common mutations of the CFTR gene were tested (∆F508, A445E, G542X, 1717-1G→A, R553X, R117H, R1162X, N1303K, W1282X, 3659delC, E60X and S1251N). Login to comment

[hide] ATB(0)/SLC1A5 gene. Fine localisation and exclusio... Eur J Hum Genet. 2001 Nov;9(11):860-6. Larriba S, Sumoy L, Ramos MD, Gimenez J, Estivill X, Casals T, Nunes V
ATB(0)/SLC1A5 gene. Fine localisation and exclusion of association with the intestinal phenotype of cystic fibrosis.
Eur J Hum Genet. 2001 Nov;9(11):860-6., [PMID:11781704]

Abstract [show]
The Na+-dependent amino acid transporter named ATB(0) was previously found to be located in 19q13.3 by fluorescence in situ hybridisation. Genetic heterogeneity in the 19q13.2-13.4 region, syntenic to the Cystic Fibrosis Modulator Locus 1 (CFM1) in mouse, seemed to be associated to the intestinal phenotypic variation of cystic fibrosis (CF). We performed fine chromosomal mapping of ATB(0) on radiation hybrid (RH) panels G3 and TNG. Based on the most accurate location results from TNG-RH panel, mapping analysis evidenced that ATB(0) is localised between STS SHGC-13875 (D19S995) and STS SHGC-6138 in 19q13.3, that corresponds with the immediately telomeric/distal segment of the strongest linkage region within the human CFM1 (hCFM1) syntenic region. Regarding to the genomic structure and exon organisation, our results show that the ATB(0) gene is organised into eight exons. The knowledge of the genomic structure allowed us to perform an exhaustive mutational analysis of the gene. Evaluation of the possible implication of ATB(0) in the intestinal phenotype of CF was performed on the basis of the functional characteristics of the encoded protein, its apparent relevance to meconium ileus (MI) and position in relation to the hCFM1 syntenic region. We have analysed this gene in samples from CF patients with and without MI. Several sequence variations in the ATB(0) gene were identified, although none of them seemed to be related to the intestinal phenotype of CF. Even though no particular allele or haplotype in ATB(0) appears to be associated to CF-MI disease, new SNPs identified should be useful in segregation and linkage disequilibrium analyses in families affected by other disorders caused by the impairment of neutral amino acid transport.

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151 Statistical analysis showed that the higher incidence for P17A and the lower incidence for V512L observed in the general population Table 3 CFTR mutations of the CF patients under study with and without meconium ileus (MI) CF-non MI CF-MI CFTR mutations n CFTR mutations n F508del/R117H 2 F508del/F508del 7 F508del/R334W 3 F508del/L365P 1 F508del/R347P 1 F508del/G542X 1 F508del/621+1G4Ta 1 F508del/621+IG4Ta 1 F508del/M1101K 1 F508del/R1066C 1 F508del/1609delCAa 1 F508del/W1089X 1 F508del/2789+5G4Aa 3 F508del/R1162X 1 F508del/3849+10kbC4T 1 F508del/1609delCAa 1 G542X/G85E 1 F508del/Q1281X 1 G542X/V232D 1 F508del/1811+1.6kbA4G 1 G542X/1811+1.6kb A4Ga 1 F508del/2789+5G4Aa 1 G542X/2789+5G4A 1 F508del/2869insG 1 Q890X/L206W 1 F508del/unknown 1 1811+1.6kbA4G/P205S 1 I507del/I507del 1 R1162X/3272-26A4G 1 G542X/1078delT 1 N1303K/R347H 1 G542X/1811+1.6kbA4Ga 1 N1303K/A1006E+5T 1 S549R/CFTR50kbdel 1 2789+5G4A/405+1G4A 1 R1066C/R1066C 1 W1282X/712-1G4T 1 a CF patient with a sibling presenting identical CFTR genotype and discordance of intestinal phenotype. Login to comment

[hide] Correction of CFTR malfunction and stimulation of ... Hepatology. 2002 Jan;35(1):95-104. Zsembery A, Jessner W, Sitter G, Spirli C, Strazzabosco M, Graf J
Correction of CFTR malfunction and stimulation of Ca-activated Cl channels restore HCO3- secretion in cystic fibrosis bile ductular cells.
Hepatology. 2002 Jan;35(1):95-104., [PMID:11786964]

Abstract [show]
In view of the occurrence of hepatobiliary disorders in cystic fibrosis (CF) this study addresses the role of the cystic fibrosis transmembrane conductance regulator (CFTR) and of Ca(2+)-activated Cl(-) channels in promoting HCO3- secretion in bile ductular cells. Human cholangiocytes were isolated from control livers and from 1 patient with CF (DeltaF508/G542X mutations). Single channel and whole cell currents were analyzed by patch clamp techniques, and HCO3- secretion was determined by fluorometric analysis of the rate of recovery of intracellular pH following alkaline loading. In control cholangiocytes, both cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) catalytic subunit, activated CFTR Cl(-) channels that exhibited a nonrectifying conductance of 8 pS and appeared in clusters. Activation of Cl(-) current by cAMP was associated with an increase in the rate of HCO3- secretion. The basal rate of HCO3- secretion was lower in CF than in control cholangiocytes. In both control and CF cholangiocytes, raising intracellular Ca(2+) concentrations with ionomycin led to a parallel activation of Cl(-) current and HCO3- secretion. Consistent with reports that premature stop codon mutations (class I; e.g., G542X) can be read over by treatment with aminoglycoside antibiotics, exposure of CF cholangiocytes to gentamicin restored activation by cAMP of Cl(-) current and HCO3- secretion. The observation that activation of Ca(2+)-dependent Cl(-) channels can substitute for cystic fibrosis transmembrane conductance regulator (CFTR) in supporting HCO3- secretion and the efficacy of gentamicin in restoring CFTR function and HCO3- secretion in class I mutations are of potential clinical interest.

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1 Human cholangiocytes were isolated from control livers and from 1 patient with CF (⌬F508/G542X mutations). Login to comment
7 Consistent with reports that premature stop codon mutations (class I; e.g., G542X) can be read over by treatment with aminoglycoside antibiotics, exposure of CF cholangiocytes to gentamicin restored activation by cAMP of Cl- current and HCO3 - secretion. Login to comment
26 In addition, the CF cells available to us were heterozygous at the CFTR locus, carrying the ⌬F508 trafficking mutation (class II23) on one allele and the G542X premature stop codon mutation (class I23) on the other. Login to comment
139 Note: (1) Absence of effect of raising intracellular cAMP (column 2) and of the continuous presence of extracellular ATP (column 3); (2) large activation of HCO3 - extrusion by raising intracellular Ca2ϩ concentration with ionomycin (column 4); and (3) unveiling of cAMP-stimulated HCO3 - secretion by restoring translation of CFTR-G542X with gentamycin (column 5). Login to comment
155 This current stimulation was transient and current returned towards control values within 1 minute (data not shown).22,39 Correction of Cl- and HCO3 - Secretory Defect in CFhBDC by Gentamicin Class I mutations of CFTR, which result in premature stop codons (e.g., G542X) can be corrected with certain aminoglycoside antibiotics.24-26 We pretreated CFhBDC with 200 ␮g/mL gentamicin for 18 hours and measured HCO3 - extrusion in the presence of cAMPmix. Login to comment
173 Thus, activation by cAMP of Cl- currents and HCO3 - secretion could both be restored in CF cholangiocytes carrying the premature stop codon mutation (G542X). Login to comment
215 The CFTR-deficient cells used in this study were heterozygous exhibiting the ⌬F508/G542X alleles, mutations belonging to class II and class I groups, respectively. Login to comment
219 We had previously shown that chaperoning with glycerol leads to proper surface membrane targeting of CFTR-⌬F508, which restored cAMP-dependent HCO3 - transport in CF pancreatic ductular cells.9 Unfortunately, no more ⌬F508/G542X cholangiocytes were available and analogous experiments to restore CFTR function in bile ductular cells could not be performed. Login to comment

[hide] Cystic fibrosis mutation testing in Italy. Genet Test. 2001 Fall;5(3):229-33. Bombieri C, Pignatti PF
Cystic fibrosis mutation testing in Italy.
Genet Test. 2001 Fall;5(3):229-33., [PMID:11788089]

Abstract [show]
In Italy, Cystic fibrosis (CF) mutation frequency differences have been observed in different regions. In the northeastern Veneto and Trentino Alto Adige regions, a complete cystic fibrosis transmembrane conductance regulator (CFTR) gene screening in CF patients detected through a newborn screening program has identified about 90% of the mutations. In these two regions, the current detection rate using a CF screening panel containing the 16 most common mutations is 86.6%. CF mutations in some other Italian regions have not been so thoroughly analysed. Available data indicate that a more general national screening panel comprising 31 mutations may detect about 75% of all CF mutations in Italy.

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35 CF MUTATIONS IDENTIFIED IN TWO ITALIAN REGIONS (VENETO AND TRENTINO ALTO ADIGE) Number of alleles Frequency Cumulative Mutation with mutation (%) frequency (%) DF508 107 47.6 47.56 R1162X 22 9.8 57.33 2183 AA ® G 21 9.3 66.67 N1303K 9 4.0 70.67 G542X 6 2.7 73.33 711 1 5 G ® A 6 2.7 76.00 1717-1 G ® A 5 2.2 78.22 G85E 3 1.3 79.56 R553X 3 1.3 80.89 2789 1 5 G ® A 3 1.3 82.22 Q552X 3 1.3 83.56 621 1 1 G ® T 2 0.9 84.44 W1282X 2 0.9 85.33 R347P 1 0.4 85.77 G551D 1 0.4 86.21 3849 1 10 Kb C ® T 1 0.4 86.67a 3132 del TG 2 0.9 87.54 2790-2 A ® G 2 0.9 88.43 457 TAT ® G 1 0.4 88.87 1717-8 G ® A 1 0.4 89.31 R709X 1 0.4 89.75 1898 1 3 A ® G 1 0.4 90.22 Total 203 90.22 Numbers refer to CFTR gene alleles carrying the specified mutation, over total tested alleles (n 5 225) from the affected subjects CF cohort, as indicated in the text (from Bonizzato et al., 1995). Login to comment
38 CF MUTATION PANEL (VENETO AND TRENTINO ALTO ADIGE ITALIAN REGIONS) DF508 R1162X 2183 AA ® G N1303K G542X 711 1 5 G ® A 1717-1 G ® A G85E R553X 2789 1 5 G ® A Q552X 621 1 1 G ® T W1282X R347P G551D 3849 1 10 Kb C ® T Note: Contrary to what is suggested for the U.S. population (Grody et al., 2001), R117H mutation (and its reflex IVS8-5T test) is not included in the panel because it is not commonly found in the Italian CF population (Bonizzato et al., 1995; Estivill et al., 1997; Rendine et al., 1997). Login to comment
44 CF GENE MUTATIONS IN ITALY Number of alleles Frequency Cumulative Mutation screened (%) frequency (%) DF508 3442 51.07 51.07 N1303K 3056 4.84 55.91 G542X 3082 4.83 60.75 2183 AA ® G 2596 2.66 63.41 R1162X 2580 2.42 65.83 1717-1 G ® A 2892 2.11 67.94 W1282X 2600 1.23 69.17 R553X 2882 1.15 70.31 T338I 2306 0.69 71.01 R347P 2642 0.61 71.61 711 1 5 G ® A 2454 0.57 72.18 G85E 1980 0.40 72.59 621 1 1 G ® T 2594 0.39 72.97 R334W 2366 0.30 73.27 R352Q 2112 0.24 73.50 S549N 2118 0.24 73.74 R347H 2184 0.18 73.92 L1077P 1840 0.16 74.09 R1158X 1878 0.16 74.25 541del C 1884 0.16 74.40 R1066H 1918 0.16 74.56 E585X 1922 0.16 74.72 Q552X 2172 0.14 74.86 D1152H 1824 0.11 74.97 2790-2 A ® G 1862 0.11 75.07 3132 del TG 1862 0.11 75.18 3667ins 4 1876 0.11 75.29 DI507 1914 0.10 75.39 1898 1 3 A ® G 1920 0.10 75.50 G1244E 1960 0.10 75.60 1784 del G 2052 0.10 75.69 From Rendine et al. (1997). Login to comment

[hide] Association between genetically determined pancrea... Chest. 2002 Jan;121(1):73-80. Loubieres Y, Grenet D, Simon-Bouy B, Medioni J, Landais P, Ferec C, Stern M
Association between genetically determined pancreatic status and lung disease in adult cystic fibrosis patients.
Chest. 2002 Jan;121(1):73-80., [PMID:11796434]

Abstract [show]
STUDY OBJECTIVES: The association between genotype and phenotype in cystic fibrosis (CF) has been clearly established for pancreatic status, but not for lung disease. DESIGN: Retrospective study. SETTING: A respiratory unit of a teaching hospital. PATIENTS: We studied 51 adult CF patients for whom current data and genotype were available. Thirty-seven patients carried two severe mutations associated with pancreatic insufficiency phenotype (group S). Fourteen patients carried at least one mild (and dominant) mutation associated with pancreatic sufficiency phenotype (group M). MEASUREMENTS: We compared the course of the disease between the two groups, looking for a genotype/phenotype association for lung disease. RESULTS: The mean age of the population was 30 years. Patients with two severe mutations presented more severe disease with earlier onset (1.7 years vs 7.9 years, p = 0.0001). They presented with a more severe respiratory impairment, with a lower mean FEV(1) (29% of predictive value vs 58% of predictive value, p < 0.001); a higher Pseudomonas colonization rate (97% vs 57%, p < 0.01); a more frequent end-stage respiratory insufficiency, defined by a FEV(1) < 30% (73% vs 29%, p < 0.05); and a more marked yearly decline of FEV(1) (3% vs 1.4%, p < 0.001). By multivariate logistic regression analysis, carrying two severe mutations was the only independent predictor of a terminal respiratory insufficiency (relative risk, 6.75; 95% confidence interval, 1.79 to 26.50; p = 0.003). CONCLUSION: This study suggests that pulmonary disease appears to be associated with the severity of CF transmembrane regulator mutations.

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31 The most frequent CF mutations usually found in the French population (⌬F508, ⌬I507, 1717-1G3A, G542X, G551D, R553X, W1282X, N1303K) were analyzed by polymerase chain reaction and allele-specific oligonucleotide with the INNO-LIPA CF2 kit (Innogenetics; Zwijnaarde, Belgium). Login to comment

[hide] Activation of ion secretion via proteinase-activat... Am J Physiol Gastrointest Liver Physiol. 2002 Feb;282(2):G200-10. Mall M, Gonska T, Thomas J, Hirtz S, Schreiber R, Kunzelmann K
Activation of ion secretion via proteinase-activated receptor-2 in human colon.
Am J Physiol Gastrointest Liver Physiol. 2002 Feb;282(2):G200-10., [PMID:11804840]

Abstract [show]
Proteinase-activated receptor (PAR) type 2 (PAR-2) has been shown to mediate ion secretion in cultured epithelial cells and rat jejunum. With the use of a microUssing chamber, we demonstrate the role of PAR-2 for ion transport in native human colonic mucosa obtained from 30 normal individuals and 11 cystic fibrosis (CF) patients. Trypsin induced Cl(-) secretion when added to the basolateral but not luminal side of normal epithelia. Activation of Cl(-) secretion by trypsin was inhibited by indomethacin and was further increased by cAMP in normal tissues but was not present in CF colon, indicating the requirement of luminal CF transmembrane conductance regulator. Effects of trypsin were largely reduced by low Cl(-), by basolateral bumetanide, and in the presence of barium or clotrimazole, but not by tetrodotoxin. Furthermore, trypsin-induced secretion was inhibited by the Ca(2+)-ATPase inhibitor cyclopiazonic acid and in low-Ca(2+) buffer. The effects of trypsin were almost abolished by trypsin inhibitor. Thrombin, an activator of PAR types 1, 3, and 4, had no effects on equivalent short-circuit currents. The presence of PAR-2 in human colon epithelium was confirmed by RT-PCR and additional experiments with PAR-2-activating peptide. PAR-2-mediated intestinal electrolyte secretion by release of mast cell tryptase and potentiation of PAR-2 expression by tumor necrosis factor-alpha may contribute to the hypersecretion observed in inflammatory processes such as chronic inflammatory bowel disease.

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50 Testing of an additional panel of the 19 most prevalent CFTR mutations among the Caucasian population in Europe, including G542X, N1303K, 1717-1 GϾT, W1282X, G551D, R553X, R1162X, R334W, R117H, 621ϩ1GϾT, 3849ϩ10kbCϾT, 3659delC, 1078delT, R347P, A445E, S1251N, ⌬I507, 2183AAϾG, and E60X (ELUCIGENE CF20; AstraZeneca Diagnostics) failed to identify the second disease causing mutation in six CF patients. Login to comment

[hide] Genetic risk factors in chronic pancreatitis. J Gastroenterol. 2002 Jan;37(1):1-9. Teich N, Ockenga J, Keim V, Mossner J
Genetic risk factors in chronic pancreatitis.
J Gastroenterol. 2002 Jan;37(1):1-9., [PMID:11824793]

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70 Approximately 72% of patients with cystic fibrosis are homozygous or compound heterozygous for eight mutations of the CFTR gene on chromosome 7: delta F508, G542X, R553X, W1282X, N1303K, 621 ϩ 1GÆT, 1717-1GÆA, and R117H; whereas the deletion delta F508 alone accounts for about 66% of mutant cystic fibrosis alleles. Login to comment

[hide] Identification of a new cystic fibrosis transmembr... Eur Respir J. 2002 Feb;19(2):374-6. Spitzer E, Staab D, Hanke R, Wahn U, Grosse R
Identification of a new cystic fibrosis transmembrane regulator mutation in a severely affected patient.
Eur Respir J. 2002 Feb;19(2):374-6., [PMID:11866018]

Abstract [show]
By using a combination of multiplex polymerase chain reaction and allele-specific labelled probes, the oligo-ligation assay is designed to detect known cystic fibrosis transmembrane regulator mutations. This study shows that this assay may also be useful to detect new mutations. The second child of a family of Bosnic origin showed all the symptoms of intestinal and pulmonary manifestations of cystic fibrosis. No signal could be obtained for the allele-specific probe 1898+1G>A. This could be explained by a nearby localized sequence change that prevented polymerase chain reaction primers or oligonucleotide probes from binding to the target sequence. Indeed, sequence analysis revealed a new 1894G>T exchange (Glu587Stop). Both parents and the healthy brother carried this mutation. Thus, the index patient was homozygous for 1894G>T, which was inherited from both parents.

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31 Discussion Studies carried out in southern European populations, including the former Yugoslavia, identified DF508, G542X, G551D, 621z1GwT, W1282X and N1303K as the most common CF mutations [6, 7]. Login to comment

[hide] Genetic and clinical features of false-negative in... Acta Paediatr. 2002;91(1):82-7. Padoan R, Genoni S, Moretti E, Seia M, Giunta A, Corbetta C
Genetic and clinical features of false-negative infants in a neonatal screening programme for cystic fibrosis.
Acta Paediatr. 2002;91(1):82-7., [PMID:11883825]

Abstract [show]
A study was performed on the delayed diagnosis of cystic fibrosis (CF) in infants who had false-negative results in a neonatal screening programme. The genetic and clinical features of false-negative infants in this screening programme were assessed together with the efficiency of the screening procedure in the Lombardia region. In total, 774,687 newborns were screened using a two-step immunoreactive trypsinogen (IRT) (in the years 1990-1992), IRT/IRT + delF508 (1993-1998) or IRT/IRT + polymerase chain reaction (PCR) and oligonucleotide ligation assay (OLA) protocol (1998-1999). Out of 196 CF children born in the 10 y period 15 were false negative on screening (7.6%) and molecular analysis showed a high variability in the genotypes. The cystic fibrosis transmembrane regulator (CFTR) gene mutations identified were delF508, D1152H, R1066C, R334W, G542X, N1303K, F1052V, A120T, 3849 + 10kbC --> T, 2789 + 5G --> A, 5T-12TG and the novel mutation D110E. In three patients no mutation was identified after denaturing gradient gel electrophoresis of the majority of CFTR gene exons. Conclusion: The clinical phenotypes of CF children diagnosed by their symptoms at different ages were very mild. None of them presented with a severe lung disease. The majority of them did not seem to have been damaged by the delayed diagnosis. The combination of IRT assay plus genotype analysis (1998-1999) appears to be a more reliable method of detecting CF than IRT measurement alone or combined with only the delF508 mutation.

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8 The cystic brosis transmembrane regulator (CFTR) gene mutations identi ed were delF508, D1152H, R1066C, R334W, G542X, N1303K, F1052V, A120T, 3849 ‡ 10kbC ® T, 2789 ‡ 5G ® A, 5T-12TG and the novel mutation D110E. Login to comment
34 It was initially performed by polyacrylamide gel electrophoretic (PAGE) analysis for the delF508 mutation, and later by polymerase chain reaction (PCR) and oligonucleotide ligation assay (OLA) (31 mutations: G85E, 621 ‡ 1G ® T, R117H, Y122X, 711 ‡ 1G ® T, 1078delT, R347P, R347H, R334W, A455E, 1898 ‡ 1G ® A, 2183-AA ® G, 2789 ‡ 5G ® A, DelF508, I507del, Q493X, V520F, 1717-1G ® A, G542X, G551D, R553X, R560T, S549R, S549N, 3849 ‡ 10kbC ® T, 3849 ‡ 4A ® G, R1162X, 3659delC, W1282X, 3905insT, N1303K) (14). Login to comment
40 Mutation Frequency (%) DelF508 54 N1303K 8 G542X 6.25 1717-1G ® A 2.50 R334W 1.75 2183AA ® G 1.50 R117H, L1077P, W1282X 1.25 D110E, R347P, E585X, 2789 ‡ 5G ® A 0.75 R352Q, R553X, R1066H, D1152H, R1158X, 1782delA, 1898 ‡ 1G ® A, 3659delC 0.50 G85E, R117L, G178R, D579G, H609R, Y1032C, V1153E, R1162X, 621 ‡ 1G ® T, 711 ‡ 1G ® T, 1845delAG o 1846delGA, 2143delT 0.25 Table2.Differencesinthethreestrategiesofneonatalscreening(audit1990-1999). Login to comment
70 Year of birth Patient Sex Age at diagnosis Genotype Sweat test (chloride mEq l¡1 ) 1990 1 BA F 8 mo DF508/2789 ‡ 5G ® A 74, 79 2 LG M 4 y ¡/¡ 84, 83 1991 3 BV F 6 y ¡/¡ a 61, 85, 70 4 CA F 8 y R1066C/D1152H 58, 59 5 CA F 8 y DF508/5T-TG12 65, 67 6 PS M 5 y N1303K/-a 41, 43, 55, 63, 85, 89 1992 7 AE F 1 y R334W/-a 57, 42, 78, 82 8 DA M 4 mo ¡/¡ 85, 101, 143, 9 FA M 1 y ¡/¡ a 70, 75, 98, 114 1993 10 CA F 7 y DF508/5T-TG12 45, 50 1995 11 BM M 3 y DF508/DF508 117, 123 1997 12 DG M 6 mo G542X/D110E 59, 88, 80, 70 13 DE F 2 y D1152H/3849 ‡ 10kbC ® T 31, 35 14 TL M 2 y ¡/¡ a 115, 136 1998 15 CM M 5 mo F1052V/A120T 20, 25 F: female; M: male. Login to comment
80 The CFTR alterations identi ed were D1152H, R1066C, R334W, G542X, N1303K, F1052V, A120T, 3849 ‡ 10kbC ® T, 2789 ‡ 5G ® A, 5T-12TG and the new mutation D110E (19). Login to comment

[hide] The relationship between genotype and exercise tol... Am J Respir Crit Care Med. 2002 Mar 15;165(6):762-5. Selvadurai HC, McKay KO, Blimkie CJ, Cooper PJ, Mellis CM, Van Asperen PP
The relationship between genotype and exercise tolerance in children with cystic fibrosis.
Am J Respir Crit Care Med. 2002 Mar 15;165(6):762-5., 2002-03-15 [PMID:11897641]

Abstract [show]
The relationship between fitness and genotype in children with cystic fibrosis (CF) and at least one copy of the DeltaF508 mutation was examined. Genotype was classified according to the second CF mutation. Fitness was measured by peak aerobic capacity (using a modified Bruce protocol during treadmill exercise) and anaerobic power (using the Wingate test on a cycle ergometer). The class of cystic fibrosis transmembrane regulator proteins (CFTR) mutation was statistically related with aerobic capacity, peak anaerobic power, body mass index, lung function (forced expiratory volume in one second), and disease severity as measured by the Shwachman score. Patients with mutations causing defective CFTR production (Class I) or processing (Class II) had a significantly lower peak aerobic capacity (28.6 +/- 4.2 ml/kg/min and 31.7 +/- 5.4 ml/kg/min, respectively) than those with a mutation conferring defective regulation of CFTR (Class III) (43.9 +/- 6.4 ml/kg/min). The peak anaerobic power in subjects with mutations inducing decreased CFTR conduction (Class IV) or CFTR mRNA (Class V), were significantly higher (11.4 +/- 1.7 and 11.6 +/- 1.5 watts/kg, respectively) than children with Class I (9.7 +/- 1.4 watts/kg), Class II (9.8 +/- 1.4 watts/kg), or Class III (10.5 +/- 1.8 watts/kg) mutations. There were no statistically significant differences in the lung function of patients with the different mutations. These results indicate a relationship between CF genotype and some measures of fitness, the mechanisms of which remain to be determined.

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14 Examples of class I mutations are G542X and W1282X. Login to comment

[hide] DHPLC screening of cystic fibrosis gene mutations. Hum Mutat. 2002 Apr;19(4):374-83. Ravnik-Glavac M, Atkinson A, Glavac D, Dean M
DHPLC screening of cystic fibrosis gene mutations.
Hum Mutat. 2002 Apr;19(4):374-83., [PMID:11933191]

Abstract [show]
Denaturing high performance liquid chromatography (DHPLC) using ion-pairing reverse phase chromatography (IPRPC) columns is a technique for the screening of gene mutations. In order to evaluate the potential utility of this assay method in a clinical laboratory setting, we subjected the PCR products of 73 CF patients known to bear CFTR mutations to this analytic technique. We used thermal denaturation profile parameters specified by the MELT program tool, made available by Stanford University. Using this strategy, we determined an initial analytic sensitivity of 90.4% for any of 73 known CFTR mutations. Most of the mutations not detected by DHPLC under these conditions are alpha-substitutions. This information may eventually help to improve the MELT algorithm. Increasing column denaturation temperatures for one or two degrees above those recommended by the MELT program allowed 100% detection of CFTR mutations tested. By comparing DHPLC methodology used in this study with the recently reported study based on Wavemaker 3.4.4 software (Transgenomic, Omaha, NE) [Le Marechal et al., 2001) and with previous SSCP analysis of CFTR mutations [Ravnik-Glavac et al., 1994] we emphasized differences and similarities in order to refine the DHPLC system and discuss the relationship to the alternative approaches. We conclude that the DHPLC method, under optimized conditions, is highly accurate, rapid, and efficient in detecting mutations in the CFTR gene and may find high utility in screening individuals for CFTR mutations. Hum Mutat 19:374-383, 2002. Published 2002 Wiley-Liss, Inc.

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42 The following mutations have been studied: exon 3: W57G, R74W, R75Q, G85E, 394delTT, 405+ 1G>A; exon 4: E92X, P99L, 441delA, 444delA, 457TAT>G, D110H, R117C, R117H, A120T, 541delC, 544delCA, Q151X, 621+1G>T, 662- 2A>C; exon 7: 1078delT, F331L, R334W, I336K, R347C, R347P, A349V, R352Q, 1221delCT; exon 10: S492F, Q493X, 1609delCA, deltaI507, deltaF508; exon 11: G542X, S549N, G551D, R553X, A559T, R560K, R560T; exon 13: K716X, Q685X, G628R, L719X; exon 17b: H1054D, G1061R, 3320ins5, R1066H, R1066L, R1070Q, 3359delCT, L1077P, H1085R, Y1092X; exon 19: R1162X, 3659delC, 3662delA, 3667del4, 3737delA, I1234V, S1235R, 3849G>A; exon 20: 3860ins31,S1255X,3898insC,3905insT,D1270N, W1282X, Q1291R; and exon 21: N1303H, N1303K, W1316X. Login to comment

[hide] Determination of the relative contribution of thre... Eur J Hum Genet. 2002 Feb;10(2):100-6. Audrezet MP, Chen JM, Le Marechal C, Ruszniewski P, Robaszkiewicz M, Raguenes O, Quere I, Scotet V, Ferec C
Determination of the relative contribution of three genes-the cystic fibrosis transmembrane conductance regulator gene, the cationic trypsinogen gene, and the pancreatic secretory trypsin inhibitor gene-to the etiology of idiopathic chronic pancreatitis.
Eur J Hum Genet. 2002 Feb;10(2):100-6., [PMID:11938439]

Abstract [show]
In the last 5 years, mutations in three genes, the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the cationic trypsinogen (PRSS1) gene, and the pancreatic secretory trypsin inhibitor (PSTI) gene, have been found to be associated with chronic pancreatitis (CP). In this study, using established mutation screening methods, we systematically analysed the entire coding sequences and all exon/intron junctions of the three genes in 39 patients with idiopathic CP (ICP), with a view to evaluating the relative contribution of each gene to the aetiology of the disease. Our results demonstrate that, firstly, 'gain-of-function' mutations in the PRSS1 gene may occasionally be found in an obvious ICP subject. Secondly, presumably 'loss-of-function' mutations in the PSTI gene appear to be frequent, with a detection rate of at least 10% in ICP and, finally, abnormal CFTR alleles are common: at least 20% of patients carried one of the most common CFTR mutations, and about 10% of patients were compound heterozygotes, having at least one 'mild' allele. Thus, in total, about 30% of ICP patients carried at least one abnormal allele in one of the three genes, and this is the most conservative estimate. Moreover, a trans-heterozygous state with sequence variations in the PSTI/CFTR genes was found in three patients. However, an association between the 5T allele in intron 8 of the CFTR gene and ICP remains unproven.

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56 `Gain-of-function' PRSS1 mutations are rare in ICP While PRSS1 mutations are often found in patients with hereditary pancreatitis, they can also be identified in subjects with ICP, albeit with an exceptionally low Table 1 Sequence variations identified in the PRSS1, PSTI, and CFTR genes in 39 patients with ICP CFTR Patient PRSS1 PSTI Mutant PolyT 1 ± a ± ± 7T/7T 2 ± ± F508del/R352Q 9T/7T 3 ± ± F508del/P5L 9T/7T 4 ± ± c.4575+2G4A 9T/7T 5 ± ± ± 7T/7T 6 ± N34Sb ± 7T/7T 7 ± ± ± 7T/5T 8 ± ± F508del/Q1476X 9T/7T 9 ± ± ± 7T/7T 10 ± ± ± 7T/7T 11 ± ± ± 7T/7T 12 ± ± ± 7T/7T 13 ± ± V562I 7T/5T 14 ± ± 2C4A W1282X 7T/5T 15 ± ± IVS3-6T4C 7T/7T 16 R122H ± ± 7T/7T 17 ± ± ± 9T/7T 18 ± ± ± 7T/5T 19 ± ± ± 7T/7T 20 ± N34S/N34S ± 7T/7T 21 ± ± ± 9T/5T 22 ± ± ± 7T/7T 23 ± ± E217G/A1136T 9T/7T 24 ± ± ± 7T/7T 25 ± ± ± NDc 26 ± ± ± ND 27 ± N34S IVS18 ± 20T4C 9T/7T 28 ± ± F508del 9T/7T 29 ± ± ± 7T/7T 30 ± ± N1303K ND 31 ± ± G542X 9T/7T 32 ± ± ± 7T/5T 33 ± ± F508del 9T/7T 34 ± ± 41G4Ad ± 7T/7T 35 ± ± ± 9T/7T 36 ± ± ± 9T/7T 37 ± ± ± 7T/7T 38 ± N34S L967S 7T/7T 39 ± ± ± 7T/5T a Indicates two wild alleles. Login to comment

[hide] Towards the pharmacogenomics of cystic fibrosis. Pharmacogenomics. 2002 Jan;3(1):75-87. Sangiuolo F, D'Apice MR, Bruscia E, Lucidi V, Novelli G
Towards the pharmacogenomics of cystic fibrosis.
Pharmacogenomics. 2002 Jan;3(1):75-87., [PMID:11966405]

Abstract [show]
Cystic fibrosis (CF) is the most common lethal recessive genetic disease affecting children in Europe and the US. CF is a multiorgan disease and may present a variety of clinical symptoms, like chronic obstructive lung disease, exocrine pancreatic insufficiency (PI) and elevated sweat chloride concentration. CF mutations have also been found in other related clinical diseases such as congenital bilateral absence of the vas deferens (CBAVD), disseminated bronchiectasis and chronic pancreatitis. These clinical overlaps pose etiopathogenetic, diagnostic and therapeutic questions. Despite stunning advances in genomic technologies and drug discovery, drug therapy often improves disease symptoms but does not cure the disease. One of the main causes of this failure in CF cure may be attributable to genetic variability and to the scarce knowledge of CF biochemistry. Therefore, knowing the genotype of a patient might help improve drug efficacy, reduce toxicity and suggests innovative genomic-based therapy approaches.

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107 G542X 621 + 1 G→→→→T 3905insT W1282X R553X 1717 - 1 G→→→→A PI Lack of CFTR biosynthesis or defective biosynthesis producing abnormal protein variants. Login to comment
156 G542X, 621+1 G→T, W1282X and R553X belong to the class I group of mutations. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Clin Exp Allergy. 2002 May;32(5):756-61. Eaton TE, Weiner Miller P, Garrett JE, Cutting GR
Cystic fibrosis transmembrane conductance regulator gene mutations: do they play a role in the aetiology of allergic bronchopulmonary aspergillosis?
Clin Exp Allergy. 2002 May;32(5):756-61., [PMID:11994102]

Abstract [show]
BACKGROUND: Previous work suggests that cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations may be implicated in the aetiology of allergic bronchopulmonary aspergilosis (ABPA). OBJECTIVE: To compare the frequency of CF gene mutations in asthmatics with ABPA of varying severity with asthmatics who were skin prick test (SPT)-positive to Aspergillus fumigatus (Af) without evidence of ABPA and asthmatics SPT-negative to Af. METHODS: Thirty-one Caucasian patients with ABPA were identified, together with asthmatics SPT positive to Af without evidence of ABPA (n = 23) and SPT negative to Af (n = 28). Genomic DNA was tested for 16 CF mutations accounting for approximately 85% of CF alleles in Caucasian New Zealanders. RESULTS: Four (12.9%) ABPA patients were found to be carriers of a CF mutation (DeltaF508 n = 3, R117H n = 1), one (4.3%) asthmatic SPT positive to Af without ABPA (DeltaF508), and one (3.6%) asthmatic SPT negative to Af (R117H). All patients with a CF mutation had normal sweat chloride (< 40 mM). There was no significant difference between the frequency of CF mutations in the ABPA patients and asthmatics without ABPA. However, the frequency of CF mutations in the ABPA patients was significantly different (P = 0.0125) to the expected carrier rate in the general population. CONCLUSION: These results lend further support to a possible link between CF mutations and ABPA.

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53 Cystic ®brosis mutation analysis Genomic DNA samples were screened for 16 CF mutations utilizing allelic-speci®c oligonucleotide (ASO) hybridization; ÁF508, ÁI507, R117H, W1282X, 621 IG3T, R334W, R347P, A455E, 1717-IG3A, G542X, 5549N, G551D, R553X, R560T, N1303K and 3849 10KC3T. Login to comment

[hide] Complete screening of the CFTR gene in Argentine c... Clin Genet. 2002 Mar;61(3):207-13. Visich A, Zielenski J, Castanos C, Diez G, Grenoville M, Segal E, Barreiro C, Tsui LC, Chertkoff L
Complete screening of the CFTR gene in Argentine cystic fibrosis patients.
Clin Genet. 2002 Mar;61(3):207-13., [PMID:12000363]

Abstract [show]
In order to establish the nature and the distribution of mutations causing cystic fibrosis (CF) in 220 unrelated Argentine families, the present authors conducted an extensive molecular analysis of the CF transmembrane regulator (CFTR) gene. First, a direct mutation analysis of 13 common mutations was done, enabling the detection of 319 out of 440 CF alleles (72.52%). Then an exhaustive screening of the entire coding region and the adjacent sequences of the CFTR gene was performed in all patients carrying at least one unidentified CF allele using the multiplex heteroduplex analysis assay followed by direct DNA sequencing. Thirty-nine different CF mutations, including five previously undescribed mutations (i.e. L6V, Y362X, 1353insT, 2594delGT and 2686insT) and two novel polymorphisms (i.e. 1170G/C and 3315A/C) were identified. As a result, the overall detection rate increased by up to 83.45%. Besides DeltaF508, only five mutations showed frequencies higher than 1%. In addition, a total of 49% of the mutations were rare because they were found in only one CF family. This wide spectrum of CF mutations is in agreement with the heterogeneous ethnic origin of the Argentine population. The data obtained here may have important consequences for the development of adequate strategies for the molecular diagnosis of CF in Argentina.

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35 Screening for DF508 and 12 other known mutations DF508 and 11 other frequent mutations (i.e. DI507, G551D, R553X, S549N, S549I, R1162X, 1811π1.6KbA»T, G542X, 1717-1G»A, 208 W1282X and N1303K) were detected as previously described (5). Login to comment
56 Frequency of cystic fibrosis transmembrane regulator mutations in the Argentine population: 440 chromosomes analysed Mutation Localization Chromosome Number Percentage DF508 Exon 10 258 58.64 G542X Exon 11 18 4.10 W1282X Exon 20 12 2.73 N1303K Exon 21 12 2.73 R334W Exon 7 5 1.14 1717-1G»A Intron 10 5 1.14 3849π10KbC»T Intron 19 4 0.91 1811π1.6KbA»G Intron 11 4 0.91 IVS8-5T Intron 8 4 0.91 G85E Exon 3 3 0.68 621π1G»T Intron 4 3 0.68 2789π5G»A Intron 14b 3 0.68 DI507 Exon 10 3 0.68 2184delA Exon 13 2 0.45 2566insT Exon 13 2 0.45 2686insT Exon 14a 2 0.45 3659delC Exon 19 2 0.45 R1162X Exon 19 2 0.45 4016insT Exon 21 2 0.45 2789π2insA Intron 14b 2 0.45 L6V Exon 1 1 0.23 297π2A»G Intron 2 1 0.23 W57X Exon 3 1 0.23 R75Q Exon 3 1 0.23 Q220X Exon 6a 1 0.23 Y362X Exon 7 1 0.23 D426C Exon 9 1 0.23 1460delAT Exon 9 1 0.23 1353insT Exon 9 1 0.23 1782delA Exon 11 1 0.23 R553X Exon 11 1 0.23 S549R Exon 11 1 0.23 1898π3A»G Intron 12 1 0.23 2594delGT Exon 13 1 0.23 2183AA»G Exon 13 1 0.23 I1027T Exon 17a 1 0.23 R1066C Exon 17b 1 0.23 G1061R Exon 17b 1 0.23 4005-1G»A Intron 20 1 0.23 Total 367 83.45 209 nificant differences were observed among the compared populations (Table2). Login to comment
83 Only five other mutations (i.e. G542X, W1282X, N1303K, 1717-1G»A and R334W) showed frequencies higher than 1%, while approximately half the mutations (49%) were rare since they were found in only one CF family. Login to comment
99 They have established the group of CF mutations (i.e. DF508, G542X, W1282X, N1303K, 1717-1G»A and R334W) that should be considered in screening programmes based on both IRT and DNA analysis to obtain at least 70% sensitivity. Login to comment

[hide] Cystic fibrosis: a worldwide analysis of CFTR muta... Hum Mutat. 2002 Jun;19(6):575-606. Bobadilla JL, Macek M Jr, Fine JP, Farrell PM
Cystic fibrosis: a worldwide analysis of CFTR mutations--correlation with incidence data and application to screening.
Hum Mutat. 2002 Jun;19(6):575-606., [PMID:12007216]

Abstract [show]
Although there have been numerous reports from around the world of mutations in the gene of chromosome 7 known as CFTR (cystic fibrosis transmembrane conductance regulator), little attention has been given to integrating these mutant alleles into a global understanding of the population molecular genetics associated with cystic fibrosis (CF). We determined the distribution of CFTR mutations in as many regions throughout the world as possible in an effort designed to: 1) increase our understanding of ancestry-genotype relationships, 2) compare mutational arrays with disease incidence, and 3) gain insight for decisions regarding screening program enhancement through CFTR multi-mutational analyses. Information on all mutations that have been published since the identification and cloning of the CFTR gene's most common allele, DeltaF508 (or F508del), was reviewed and integrated into a centralized database. The data were then sorted and regional CFTR arrays were determined using mutations that appeared in a given region with a frequency of 0.5% or greater. Final analyses were based on 72,431 CF chromosomes, using data compiled from over 100 original papers, and over 80 regions from around the world, including all nations where CF has been studied using analytical molecular genetics. Initial results confirmed wide mutational heterogeneity throughout the world; however, characterization of the most common mutations across most populations was possible. We also examined CF incidence, DeltaF508 frequency, and regional mutational heterogeneity in a subset of populations. Data for these analyses were filtered for reliability and methodological strength before being incorporated into the final analysis. Statistical assessment of these variables revealed that there is a significant positive correlation between DeltaF508 frequency and the CF incidence levels of regional populations. Regional analyses were also performed to search for trends in the distribution of CFTR mutations across migrant and related populations; this led to clarification of ancestry-genotype patterns that can be used to design CFTR multi-mutation panels for CF screening programs. From comprehensive assessment of these data, we offer recommendations that multiple CFTR alleles should eventually be included to increase the sensitivity of newborn screening programs employing two-tier testing with trypsinogen and DNA analysis.

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75 This includes the analyses of Lac St. Jean-Quebec-Toronto,∆F508 in South America, and G542X in Brittany and Southern France. Login to comment
101 Mutations that fall into this category include: 1) G542X, of single origin, associated with the ancient Phoenicians [Loirat et al., 1997], 2) N1303K, also of single origin, and linked to ancient Mediterranean populations, and 3) G551D, also of single origin [Cashman et al., 1995], having been associated with the ancient Celtic tribes [Macek et al., 1991]. Login to comment
109 Mutational Arrays, Detection Rates and Methods by Region* Estimated Projected detection of Number of Number of Country/ allele two CFTR mutations chromosomes Region Mutation array detectiona mutationsb includedc (max/min)d Reference Europe Albania ∆F508 (72.4%) C276X (0.7%) 74.5 55.5 4 270/146 CFGAC [1994]; Macek et al. G85E (0.7%) R1070Q (0.7%) [2002] Austria ∆F508 (62.9%) 457TAT→G (1.2%) 76.6 58.7 11 1516/580 Estiville et al. [1997]; Dörk et al. (total) G542X (3.3%) 2183AA→G (0.7%) [2000]; Macek et al. [2002] CFTRdele2,3 (2.1%) N1303K (0.6%) R1162X (1.9%) I148T (0.5%) R553X (1.7%) R117H (0.5%) G551D (1.2%) Austria ∆F508 (74.6%) 2183AA→G (2.4%) 95.3 90.8 8 126 Stuhrmann et al. [1997] (tyrol) R1162X (8.7%) G551D (1.6%) G542X (2.4%) R347P (1.6%) 2789+5G→A (2.4%) Q39X (1.6%) Belarus ∆F508 (61.2%) R553X (0.5%) 75.2 56.6 9 278/188 Dörk et al. [2000]; Macek et al. G542X (4.5%) R334W (0.5%) [2002] CFTRdele2,3 (3.3%) R347P (0.5%) N1303K (3.2%) S549N (0.5%) W1282X (1.0%) Belgium ∆F508 (75.1%) 622-1A→C (0.5%) 100.0 100.0 27 1504/522 Cuppens et al. [1993]; Mercier et G542X (3.5%) G458V (0.5%) al. [1993]; CFGAC [1994]; N1303K (2.7%) 1898+G→C (0.5%) Estivill et al.[1997] R553X (1.7%) G970R (0.5%) 1717-1G→A (1.6%) 4218insT (0.5%) E60X (1.6%) 394delTT (0.5%) W1282X (1.4%) K830X (0.5%) 2183A→G+2184delA (1.2%) E822K (0.5%) W401X (1.0%) 3272-1G→A (0.5%) A455E (1.0%) S1161R (0.5%) 3272-26A→G (1.0%) R1162X (0.5%) S1251N (1.0%) 3750delAG (0.5%) S1235R (0.8%) S1255P (0.5%) ∆I507 (0.6%) Bulgaria ∆F508 (63.6%) R75Q (1.0%) 93.0 86.5 21 948/432 Angelicheva et al. [1997]; (total) N1303K (5.6%) 2183AA→G (0.9%) Estivill et al. [1997]; Macek G542X (3.9%) G1244V+S912L (0.9%) et al. [2002] R347P (2.2%) G85E (0.9%) 1677delTA (2.1%) 2184insA (0.9%) R1070Q (1.8%) L88X+G1069R (0.8%) Q220X (1.2%) 2789+5G→A (0.8%) 3849+10KbC→T (1.1%) G1244E (0.8%) W1282X (1.0%) 1717-1G→A (0.8%) 2176insC (1.0%) Y919C (0.7%) G1069R (1.0%) WORLDWIDEANALYSISOFCFTRMUTATIONS581 Bulgaria 1) DF508 4) 1677delTA - - 6 13 Angelicheva et al. [1997] (ethnic 2) R347P 5) Q493R Turks) 3) G542X 6) L571S - - 1 30 Angelicheva et al. [1997] Bulgaria 1) DF508 (100.0%) (Gypsy) Croatia ∆F508 (64.5%) G551D (1.1%) 72.5 52.6 5 276 Macek et al. [2002] G542X (3.3%) 3849+10KbC→T (0.7%) N1303K (2.9%) Czech ∆F508 (70.0%) 1898+1G→T (2.0%) 89.6 80.3 10 2196/628 CFGAC [1994]; Estiville et al. Republic CFTRdele2,3 (5.5%) 2143delT (1.2%) [1997]; Dörk et al. [2000]; G551D (3.8%) R347P (0.8%) Macek et al. [2002] N1303K (2.9%) 3849+10KbC→T (0.6%) G542X (2.2%) W1282X (0.6%) Denmark ∆F508 (87.5%) G542X (0.7%) 92.3 85.2 6 1888/678 CFGAC [1994]; Schwartz et al. (excluding 394delTT (1.8%) 621+1G→T (0.6%) [1994]; Estiville et al. [1997] Faroe) N1303K (1.1%) 3659delC (0.6%) Estonia ∆F508 (51.7%) R117C (1.7%) 80.2 64.3 10 165/80 Estivill et al. [1997]; Klaassen et 394delTT (13.3%) E217G (1.7%) al. [1998]; Macek et al. S1235R (3.3%) R1066H (1.7%) [2002] 359insT (1.7%) 3659delC (1.7%) I1005R (1.7%) S1169X (1.7%) Finland ∆F508 (46.2%) G542X (1.9%) 78.8 62.1 4 132/52 CFGAC [1994]; Kere et al. 394delTT (28.8%) 3372delA (1.9%) [1994]; Estivill et al. [1997] France ∆F508 (67.7%) 2789+5G→T (0.79%) 79.7 63.6 12 17854/7420 Chevalier-Porst et al. [1994]; (total) G542X (2.94%) 2184delA+2183A→G (0.77%) Estivill et al. [1997]; Claustres et al. [2000]; Guilloud-Bataille N1303K (1.83%) G551D (0.74%) et al. [2000] 1717-1G→A (1.35%) 1078delT (0.63%) W1282X (0.91%) ∆I507 (0.62%) R553X (0.86%) Y122K (0.59%) France ∆F508 (75.8%) R297Q (0.8%) 98.7 97.4 18 599/365 Férec et al. [1992]; Scotet et al. (Brittany) 1078delT (4.0%) R347H (0.8%) [2000] G551D (3.6%) I1234V (0.8%) N1303K (3.0%) R553X (0.8%) R117H (1.7%) 2789+5G→A (0.8%) 3272-26A→G (1.3%) 4005+1G→A (0.7%) G542X (1.1%) 621+1G→T (0.6%) 1717-1G→A (1.0%) ∆I507 (0.6%) G1249R (0.8%) W846X (0.5%) France ∆F508 (70.0%) N1303K (0.8%) 90.4 81.7 16 250 Claustres et al. [1993] (southern) G542X (6.4%) 3737delA (0.8%) 1717-1G→A (1.6%) R1162X (0.8%) L206W (1.2%) Y1092X (0.8%) R334W (1.2%) S945L (0.8%) ∆I507 (1.2%) K710X (0.8%) 2184delA (1.2%) 1078delT (0.8%) R1158X (1.2%) Y122X (0.8%) (Continued) BOBADILLAETAL. Login to comment
110 Germany ∆F508 (71.8%) 1789+5G→A (0.9%) 87.6 76.7 17 5662/1316 Dörk et al. [1992]; Dörk et al. R553X (2.0%) 3272-26A→G (0.9%) [1994]; Tümmler et al. [1996]; N1303K (1.8%) W1282X (0.7%) Estivill et al. [1997]; Dörk et G542X (1.2%) 2143delT (0.7%) al. [2000] R347P (1.2%) 1078delT (0.6%) CFTRdele2,3 (1.2%) 2183AA→G (0.6%) 3849+10KbC→T (1.0%) 2184insA (0.6%) G551D (0.9% 3659delC (0.6%) 1717-1G→A (0.9%) Greece ∆F508 (52.9%) 3272-26A→G (0.8%) 82.2 67.6 22 2097/718 Kanavakis et al. [1995]; Estivill 621+1G→T (5.0%) R1070Q (0.8%) et al. [1997]; Tzetis et al. G542X (4.1%) W496X (0.7%) [1997]; Macek et al. [2002] N1303K (3.3%) 621+3A→G (0.7%) 2183AA→G (1.8%) ∆I507 (0.7%) 2789+5G→A (1.7%) W1282X (0.7%) E822X (1.6%) 574delA (0.7%) R117H (1.2%) 1677delTA (0.7%) R334W (1.1%) A46D (0.6%) R1158X (1.0%) 3120+1G→A (0.6%) G85E (1.0%) G551D (0.5%) Hungary ∆F508 (54.9%) W1282X (1.8%) 68.3 46.6 9 1133/976 CFGAC [1994]; Estivill et al. 1717-1G→A (1.9%) G542X (1.7%) [1997]; Macek et al. [2002] R553X (2.1%) N1303K (1.3%) Y1092X (1.8%) G551D (1.0%) S1196X (1.8%) Ireland ∆F508 (70.4%) G542X (1.0%) 82.1 67.4 7 801/509 CFGAC [1994]; Estivill et al. G551D (5.7%) 621+1G→T (0.8%) [1994] R117H (2.4%) 1717-1G→A (0.6%) R560T (1.2%) Italy ∆F508 (50.9%) ∆I507 (0.65%) 60.3 36.4 9 3524 Estivill et al. [1997] (total) G542X (3.1%) W1282X (0.62%) 1717-1G→A (1.6%) Y122K (0.59%) N1303K (1.4%) G551D (0.53%) R553X (0.94%) Italy ∆F508 (47.6%) R553X (1.3%) 87.1 75.9 15 225 Bonizzato et al. [1995] (Northeast) R1162X (9.8%) 2789+G→A (1.3%) 2183AA→G (9.3%) Q552X (1.3%) N1303K (4.0%) 621+1G→T (0.9%) G542X (2.7%) W1282X (0.9%) 711+5G→A (2.7%) 3132delTG (0.9%) 1717-1G→A (2.2%) 2790-2A→G (0.9%) G85E (1.3%) TABLE 1. Continued. Estimated Projected detection of Number of Number of Country/ allele two CFTR mutations chromosomes Region Mutation array detectiona mutationsb includedc (max/min)d Reference WORLDWIDEANALYSISOFCFTRMUTATIONS583 Italy ∆F508 (56.4%) 711+1G→T (1.3%) 85.7 73.4 13 660/396 Castaldo et al. [1996]; Castaldo (southern) N1303K (6.8%) G1244E (1.3%) et al. [1999] G542X (5.7%) R1185X (1.3%) W1282X (3.8%) L1065P (1.3%) 1717-1G→A (2.3%) R553X (1.1%) 2183AA→G (1.9%) I148T (0.7%) 4016insT (1.8%) Latvia 1) DF508 (58.3%) 4) CFTRdele2,3 (2.8%) - - 6 36 Dörk et al. [2000]; Macek et al. 2) 3849+10KbC®T (8.3%) 5) W1282X (2.8%) [2002] 3) N1303K (5.6%) 6) 394delTT (2.8%) Lithuania ∆F508 (31.0%) N1303K (2.0%) 39.0 15.2 4 94 Dörk et al. [2000]; Macek et al. R553X (4.0%) CFTRdele2,3 (2.0%) [2002] Macedonia ∆F508 (54.3%) 711+3A→G (1.0%) 69.2 47.9 12 559/226 Petreska et al. [1998]; Dörk et G542X (4.2%) 3849G→A (1.0%) al. [2000]; Macek et al. N1303K (2.0%) 2184insA (0.9%) [2002] CFTRdele2,3 (1.3%) 457TAT→G (0.7%) 621+1G→T (1.3%) V139E (0.7%) 611-1G→T (1.2%) 1811+1G→C (0.6%) Netherlands ∆F508 (74.2%) R1162X (0.9%) 86.8 75.3 9 3167/1442 Gan et al. [1995]; Estiville et al. A455E (4.7%) S1251N (0.9%) [1997]; Collee et al. [1998] G542X (1.8%) N1303K (0.9%) 1717-1G→A (1.5%) W1282X (0.7%) R553X (1.2%) Norway ∆F508 (60.2%) G551D (1.2%) 69.8 48.7 6 410/242 Schwartz et al. [1994]; Estivill 394delTT (4.2%) G542X (0.6%) et al. [1997] R117H (3.0%) N1303K (0.6%) Poland ∆F508 (57.1%) CFTRdele2,3 (1.8%) 73.5 54.0 11 4046/1726 CFGAC [1994]; Estivill et al. 3849+10Kb C→T (2.7%) R560T (1.5%) [1997]; Dörk et al [2000]; G542X (2.6%) W1282X (0.7%) Macek et al. [2002] 1717-1G→A (2.4%) ∆I507 (0.5%) R553X (1.9%) G551D (0.5%) N1303K (1.8%) Portugal ∆F508 (44.7%) R334W (0.7%) 49.7 24.7 5 739/454 CFGAC [1994]; Estivill et al. G542X (1.6%) N1303K (0.7%) [1997] R1066C (2.0%) Romania ∆F508 (36.6%) G542X (1.4%) 51.5 26.5 11 224/74 CFGAC [1994]; Estivill et al. 2043delG (2.0%) R553X (1.4%) [1997]; Popa et al. [1997]; W1282X (1.7%) G576X (1.4%) Macek et al. [2002] 1717-2A→G (1.4%) 1898+1G→A (1.4%) I148T (1.4%) 2183AA→G (1.4%) 621+1G→T (1.4%) Russia ∆F508 (54.4%) 552insA (0.9%) 70.7 50.0 12 5073/2562 CFGAC [1994]; Estivill et al. CFTRdele2,3 (5.0%) G542X (0.9%) [1997]; Dörk et al. [2000]; R553X (3.5%) R334W (0.9%) Macek et al. [2002] 2183AA→G (1.3%) 1677delTA (0.8%) W1282X (1.0%) Y122X (0.5%) 394delTT (1.0%) 1367del5 (0.5%) (Continued) BOBADILLAETAL. Login to comment
111 Slovakia ∆F508 (57.3%) CFTRdele2,3 (1.2%) 82.7 68.4 14 908/254 CFGAC [1994]; Estivill et al. G542X (6.8%) 3849+10KbC→T (1.0%) [1997]; Dörk et al. [2000]; R553X (4.0%) S42F (0.9%) Macek et al. [2002] N1303K (3.4%) R75X (0.9%) 2143delT (1.8%) G85E (0.9%) R347P (1.4%) 605insT (0.9%) W1282X (1.3%) 1898+1G→A (0.9%) Slovenia ∆F508 (57.8%) R347P (1.1%) 79.7 63.5 16 455/132 CFGAC [1994]; Dörk et al. 2789+5G→A (4.1%) S4X (0.8%) [2000]; Macek et al. [2002] R1162X (3.2%) 457TAT→G (0.8%) G542X (1.9%) D192G (0.8%) Q552X (1.5%) R553X (0.8%) Q685X (1.5%) A559T (0.8%) 3905insT (1.5%) 2907delTT (0.8%) CFTRdele2,3 (1.5%) 3667ins4 (0.8%) Spain ∆F508 (52.7%) G85E (0.8%) 80.2 64.3 21 3608/1356 Chillón et al. [1994]; Casals et G542X (8.0%) R1066C (0.8%) al. [1997]; Estivill et al. [1997] N1303K (2.5%) 2789+5G→A (0.7%) 3601-111G→C (2.0%) 2869insG (0.7%) 1811+1.6Kb A→G (1.7%) ∆I507 (0.6%) R1162X (1.6%) W1282X (0.6%) 711+1G→T (1.3%) L206W (0.5%) R334W (1.2%) R709X (0.5%) Q890X (1.0%) K710X (0.5%) 1609delCA (1.0%) 3272-26A→G (0.5%) 712-1G→T (1.0%) Sweden ∆F508 (66.6%) E60X (0.6%) 85.9 73.8 10 1357/662 Schwartz et al. [1994]; Estivill et 394delTT (7.3%) Y109C (0.6%) al. [1997]; Schaedel et al. 3659delC (5.4%) R117H (0.6%) [1999] 175insT (2.4%) R117C (0.6%) T338I (1.2%) G542X (0.6%) Switzerland ∆F508 (57.2%) K1200E (2.1%) 91.3 83.4 9 1268/1173 Estivill et al. [1997]; R553X (14.0%) N1303K (1.2%) Hergersberg et al. [1997] 3905insT (9.8%) W1282X (1.1%) 1717-1G→A (2.7%) R347P (0.6%) G542X (2.6%) Ukraine ∆F508 (65.2%) CFTRdele2,3 (1.1%) 74.6 55.7 6 1055/580 Estivill et al. [1997]; Dörk et al. R553X (3.6%) G551D (1.8%) [2000]; Macek et al. [2002] N1303K (2.4%) W1282X (0.5%) United ∆F508 (75.3%) 621+1G→T (0.93%) 81.6 66.6 5 19622/9815 Schwartz et al. [1995b]; Kingdom G551D (3.1%) 1717-1G→A (0.57%) Estivill et al. [1997] (total) G542X (1.7%) TABLE 1. Continued. Estimated Projected detection of Number of Number of Country/ allele two CFTR mutations chromosomes Region Mutation array detectiona mutationsb includedc (max/min)d Reference WORLDWIDEANALYSISOFCFTRMUTATIONS585 United ∆F508 (56.6%) 621+1G→T (1.8%) 69.1 47.7 7 456 CFGAC [1994] Kingdom G551D (3.7%) R117H (1.5%) (N. Ireland) R560T (2.6%) ∆I507 (0.9%) G542X (2.0%) United ∆F508 (19.2%) 621+2T→C (3.8%) 84.4 71.2 11 52 Malone et al. [1998] Kingdom Y569D (15.4%) 2184insA (3.8%) (Pakistani) Q98X (11.5%) R560S (1.9%) 1525-1G→A (9.6%) 1898+1G→T (1.9%) 296+12T→C (7.7%) R709X (1.9%) 1161delC (7.7%) United ∆F508 (71.3%) 1717-1G→A (1.0%) 86.4 74.6 9 1236/730 Shrimpton et al. [1991]; Kingdom G551D (5.5%) 621+1G→T (0.6%) Gilfillan et al. [1998] (Scotland) G542X (4.0%) ∆I507 (0.6%) R117H (1.4%) R560T (0.6%) P67L (1.4%) United ∆F508 (71.6%) 1717-1G→A (1.1%) 98.7 97.4 17 183 Cheadle et al. [1993] Kingdom 621+1G→T (6.6%) 3659delC (0.5%) (Wales) 1898+1G→A (5.5%) R117H (0.5%) G542X (2.2%) N1303K (0.5%) G551D (2.2%) E60X (0.5%) 1078delT (2.2%) S549N (0.5%) R1283M (1.6%) 3849+10KbC→T (0.5%) R553X (1.1%) 4016insT (0.5%) ∆I507 (1.1%) Yugoslavia ∆F508 (68.9%) 3849G→A (1.0%) 82.2 67.6 11 709/398 Dabovic et al. [1992]; Estivill et G542X (4.0%) N1303K (0.8%) al. [1997]; Macek et al. R1162C (3.0%) 525delT (0.5%) (submitted for publication) 457TAT→G (1.0%) 621+1G→T (0.5%) I148T (1.0%) G551D (0.5%) Q552X (1.0%) Middle East/Africa Algeria 1) DF508 (20.0%) 4) 1812-1G®A (5.0%) - - 5 20 Loumi et al. [1999] 2) N1303K (20.0%) 5) V754M (5.0%) 3) 711+1G®T (10.0%) Jewish W1282X (48.0%) 3849+10KbC→T (6.0%) 95.0 90.3 6 261 Kerem et al. [1995] (Ashkenazi) ∆F508 (28.0%) N1303K (3.0%) G542X (9.0%) 1717-1G→A (1.0%) Jewish 1) N1303K - - 1 6 Kerem et al. [1995] (Egypt) Jewish 1) Q359K/T360K - - 1 8 Kerem et al. [1995] (Georgia) Jewish 1) DF508 2) 405+1G®A - - 2 11 Kerem et al. [1995] (Libya) Jewish 1) DF508 (72.0%) 3) D1152H (6.0%) - - 3 33 Kerem et al. [1995] (Morocco) 2) S549R (6.0%) Jewish ∆F508 (35.0%) W1282X (2.0%) 43.0 18.5 4 51 Shoshani et al. [1992] (Sepharadim) G542X (4.0%) S549I (2.0%) (Continued) BOBADILLAETAL. Login to comment
112 Jewish 1) 405+1G®A (48.0%) 3) W1282X (17.0%) - - 4 23 Kerem et al. [1995] (Tunisia) 2) DF508 (31.0%) 4) 3849+10KbC®T (4.0%) Jewish 1) G85E 4) G542X - - 6 10 Kerem et al. [1995] (Turkey) 2) DF508 5) 3849+10KbC®T 3) W1282X 6) W1089X Jewish (Yemen) None - - 0 5 Kerem et al. [1995] Lebanon 1) DF508 (35.0%) 6) 4096-28G®A (2.5%) - - 9 40 Desgeorges et al. [1997] 2) W1282X (20.0%) 7) 2789+5G®A (2.5%) 3) 4010del4 (10.0%) 8) M952I (2.5%) 4) N1303K (10.0%) 9) E672del (2.5%) 5) S4X (5.0%) Reunion ∆F508 (52.0%) 1717-1G→A (0.7%) 90.4 81.7 9 138 Cartault et al. [1996] Island Y122X (24.0%) G542X (0.7%) 3120+1G→A (8.0%) A309G (0.7%) A455E (2.2%) 2789+5G→A (0.7%) G551D (1.4%) Saudi North: 3) H139L - - North 1 49 families El-Harith et al. [1997]; Arabia 1) 1548delG 4) L1177X Central 3 Kambouris et al. [1997]; Central: 5) DF508 South 4 Banjar et al. [1999] 1)I1234V 6) 3120+1G®A West 9 2)1548delG 7) 425del42 East 6 3)DF508 8) R553X South: 9) N1303K 1) I1234V East: 2) 1548delG 1) 3120+1G®A 3) 711+1G®T 2) H139L 4) 3120+1G®A 3) 1548delG West: 4) DF508 1) I1234V 5) S549R 2) G115X 6) N1303K Tunisia ∆F508 (17.6%) G85E (2.6%) 58.7 34.5 11 78 Messaoud et al. [1996] G542X (8.9%) W1282X (2.6%) 711+1G→T (7.7%) Y122X (1.3%) N1303K (6.4%) T665S (1.3%) 2766del8NT (6.4%) R47W+D1270N (1.3%) R1066C (2.6%) Turkeye ∆F508 (24.5%) 1066L (1.3%) 80.6 65.0 36 1067/670 Yilmaz et al. [1995]; Estivill et al. 1677delTA (4.1%) E822X (1.3%) [1997]; Onay et al. [1998]; 2789+5G→A (3.9%) 2183+5G→A+2184insA (1.3%) Macek et al. [2002] 2181delA (3.8%) D110H (0.8%) R347H (3.6%) P1013L (0.8%) N1303K (2.9%) 3172delAC (0.8%) 621+1G→T (2.6%) 1259insA (0.8%) G542X (2.6%) M1028I (0.8%) TABLE 1. Continued. Estimated Projected detection of Number of Number of Country/ allele two CFTR mutations chromosomes Region Mutation array detectiona mutationsb includedc (max/min)d Reference WORLDWIDEANALYSISOFCFTRMUTATIONS587 E92K (2.6%) 4005+1G→A (0.7%) A96E (2.6%) W1282X (0.7%) M152V (2.6%) I148T (0.6%) 2183AA→G (2.5%) R1162X (0.6%) 296+9A→T (1.6%) D1152H (0.6%) 2043delG (1.4%) W1098X (0.6%) E92X (1.4%) E831X (0.6%) K68N (1.4%) W496X (0.6%) G85E (1.3%) F1052V (0.5%) R1158X (1.3%) L571S (0.5%) United Arab S549R (61.5%) ∆F508 (26.9%) 88.4 78.1 2 86/52 Frossard et al. [1988]; Emirates Frossard et al. [1999] North/Central/South Americas Argentina ∆F508 (58.6%) N1303K (1.8%) 69.1 47.7 5 326/228 CFGAC [1994]; Chertkoff et al. W1282X (3.9%) 1717-1G→A (0.9%) [1997] G542X (3.9%) Brazilf ∆F508 (47.7%) W1282X (1.3%) 66.8 44.6 10 820/500 CFGAC [1994]; Cabello et al. (total) G542X (7.2%) G85E (1.3%) [1999]; Raskin et al. [1999]; R1162X (2.5%) R553X (0.7%) Bernardino et al. [2000] R334W (2.5%) L206W (0.6%) N1303K (2.4%) 2347delG (0.6%) South East: >∆F508, G542X South: >N1303K Brazil ∆F508 (31.7%) N1303K (2.5%) 42.5 18.1 3 120 Parizotto and Bertuzzo [1997] (Sao Paulo) G542X (8.3%) Canada ∆F508 (59.0%) G542X (0.5%) 98.5 97.0 13 381/200 Rozen et al. [1992]; (Lac St. Jean) 621+1G→T (24.3%) N1303K (0.5%) De Braekeleer et al. [1998] A445E (8.2%) Q890X (0.5%) Y1092X (1.2%) S489X (0.5) 711+1G→T (1.0%) R117C (0.5%) I148T (1.0%) R1158 (0.5%) G85E (0.8%) Canada ∆F508 (71.4%) ∆I507 (1.3%) 90.9 82.6 7 77 Rozen et al. [1992] (Quebec City) 711+1G→T (9.1%) Y1092X (1.3%) 621+1G→T (5.2%) N1303K (1.3%) A455E (1.3%) Canada ∆F508 (70.9%) W1282X (0.9%) 82.0 67.2 10 632 Kristidis et al. [1992] (Toronto) G551D (3.1%) R117H (0.9%) G542X (2.2%) 1717-1G→A (0.6%) 621+1G→T (1.3%) R560T (0.6%) N1303K (0.9%) ∆I507 (0.6%) Chile ∆F508 (29.2%) R553X (4.2%) 33.4 11.2 2 72 Rios et al. [1994] Columbia 1) DF508 (35.4%) 3) N1303K (2.1%) - - 4 48 Restrepo et al. [2000] 2) G542X (6.3%) 4) W1282X (2.1%) Ecuador 1) DF508 (25%) - - 1 20 Paz-y-Mino et al. [1999] (Continued) BOBADILLAETAL. Login to comment
113 Mexico ∆F508 (41.6%) G551S (0.5%) 75.5 57.0 35 374/194 Orozco et al.[1993]; Villalobos- G542X (5.6%) 1078delT (0.5%) Torres et al. [1997]; Liang et al. ∆I507 (2.5%) Y1092X (0.5%) [1998]; Orozco et al. [2000] S549N (1.9%) R117H (0.5%) N1303K (1.7%) G85E (0.5%) R75X (1.5%) 1716G→A (0.5%) 406-1G→A (1.5%) W1204X (0.5%) I148T (1.5%) W1098C (0.5%) 3849+10KbC→T (1.5%) 846delT (0.5%) 621+1G→T (1.2%) P750L (0.5%) 2055del9→A (1.0%) V754M (0.5%) 935delA (1.0%) R75Q (0.5%) I506T (1.0) W1096X (0.5%) 3199del6 (1.0%) L558S (0.5%) 2183AA→G (1.0%) 4160insGGGG (0.5%) G551D (0.5%) 297-1G→A (0.5%) R553X (0.5%) H199Y (0.5%) 1924del7 (0.5%) United States ∆F508 (68.6%) R553X (0.9%) 79.7 63.5 10 25048 Cystic Fibrosis Foundation (total) G542X (2.4%) 621+1G→T (0.9%) [1998] G551D (2.1%) 1717-1G→A (0.7%) W1282X (1.4%) 3849+10KbC→T (0.7%) N1303K (1.3%) R117H (0.7%) United States ∆F508 (48.0%) S1255X (1.4%) 77.3 59.8 16 160/148 Carles et al. [1996]; Macek et al. (African 3120+1G→A (12.2%) 444delA (0.7%) [1997]; Dörk et al. [1998]; American) 2307insA (2.0%) R334W (0.7%) Friedman et al. [1998] A559T (2.0%) ∆I507 (0.7%) R553X (2.0%) 1717-1G→A (0.7%) ∆F311 (2.0%) G542X (0.7%) G480C (1.4%) S549N (0.7%) 405+3A→C (1.4%) G551D (0.7%) United States 1) L1093P - - 1 2 Yee et al. [2000] (Cherokee) United States Non-French: French: Non- Non- Non- Non- Bayleran et al. [1996] (Maine) ∆F508 (82.0%) ∆F508 (58%) French: French: French: French: G542X (2.6%) 711+1G→T (8.3%) 95.3 90.8 11 191 G551D (2.6%) I148T (4.2%) French: French: French: French: N1303K (2.1%) A455E (4.2%) 80.3 64.5 8 72 R560T (1.0%) 1717-1G→A (1.4%) Total: 621+1G→T (1.0%) G85E (1.4%) 263 711+1G→T (1.0%) 621+1G→T (1.4%) R117H (1.0%) Y1092X (1.4%) 1717-1G→A (1.0%) G85E (0.5%) W1282X (0.5%) TABLE 1. Continued. Estimated Projected detection of Number of Number of Country/ allele two CFTR mutations chromosomes Region Mutation array detectiona mutationsb includedc (max/min)d Reference WORLDWIDEANALYSISOFCFTRMUTATIONS589 United States ∆F508 (46.0%) R334W (1.6%) 58.5 34.2 7 129 Grebe et al. [1994] (SW Hispanic) G542X (5.4%) W1282X (0.8%) 3849+10KbC→T (2.3%) R553X (0.8%) R1162X (1.6%) United States 1) R1162X - - 3 17 Mercier et al. [1992] (SW Native 2) D648V American) 3) G542X United States 1) R1162X 3) G542X - - 4 16 Mercier et al. [1994] (Zuni Pueblo) 2) 3849+10KbC®T 4) D648V Venezuela ∆F508 (29.6%) G542X (3.7%) 33.3 11.1 2 54 Restrepo et al. [2000] Other Regions Australia ∆F508 (76.9%) 621+1G→T (1.1%) 88.7 78.7 8 761/464 CFGAC [1994] G551D (4.5%) N1303K (0.9%) G542X (2.8%) W1282X (0.6%) R553X (1.3%) R117H (0.6%) East Asia 1) 1898+1G®T 2) 1898+5G®T - - 2 28 Suwanjutha et al. [1998] Hutterite 1) M1101K (69.0%) 2) DF508 (31.0%) - - 2 32 Zielenski et al. [1993] Brethren New Zealand ∆F508 (78.0%) N1303K (1.9%) 87.4 76.4 5 636 CFGAC [1994] G551D (4.4%) 621+1G→T (1.1%) G542X (2.0%) *This table presents the mutation panels for all regions investigated in this study. Login to comment
155 G542X is most common in the Mediterranean regions of Europe and Africa. Login to comment
159 This gradient of decreasing G542X prevalence can be seen on both the international and intranational levels as the distance from the Mediterranean Sea increases. Login to comment
163 G542X has been implicated as a mutation that was introduced into the Mediterranean region by the migration of Phoenicians [Loirat et al., 1997]. Login to comment
164 Across Europe, G542X is found in significantly higher proportions in countries, and in regions of countries, which border the Mediterranean Sea (Table 1). Login to comment
165 Not surprisingly, G542X has its highest rates of prevalence in the south of Spain and in the north of Africa (South Spain, 14.4%; Tunisia, 8.9%). Login to comment
168 When one studies CF chromosomes in those populations of the New World that were heavily influenced by such countries as Spain and Italy, the G542X mutation frequencies are virtually identical. Login to comment
169 The mutation N1303K is another CFTR allele that has a similar distribution patterns as G542X, and may also have been introduced via the Mediterranean route (Table 1). Login to comment
193 The top 10 list for the United States (Table 1) includes five CFTR alleles found in populations with distinct ethnic ancestries, i.e., G542X, G551D, W1282X, N1303K, and 3849+10KbC→T. Login to comment
213 Ideal Recommended CFTR Mutation Screening Panel for 2001 Neonatal Screening in the USA* Location Estimated Mutation in CFTRa percentageb Reason for inclusion DF508 Exon 10 68.6% CFF registry, >1%, Pan-European G542X Exon 11 2.4% CFF registry, >1%, Mediterranean G551D Exon 11 2.1% CFF registry, >1%, Celtic W1282X Exon 20 1.4% CFF registry, >1%, Ashkenazi Jew N1303K Exon 21 1.3% CFF registry, >1%, Mediterranean R553X Exon 11 0.9% CFF registry, >0.5%, Hispanic 621+1G®T Intron 4 0.9% CFF registry, >0.5%, multi-ethnic 1717-1G®A Intron 10 0.7% CFF registry, >0.5%, Italian 3849+10KbC®T Intron 19 0.7% CFF registry, >0.5%, Hispanic R117Hc Exon 4 0.7% CFF registry, >0.5% 1898+1G→T Intron 12 0.4% CFF registry, >0.1%, East Asian DI507 Exon 10 0.3% CFF registry, >0.1%, Hispanic 2789+5G®A Intron 14b 0.3% CFF registry, >0.1% G85E Exon 3 0.3% CFF registry, >0.1% R347P Exon 7 0.2% CFF registry, >0.1% R334W Exon 7 0.2% CFF registry, >0.1%, multi-ethnic R1162X Exon 19 0.2% CFF registry, >0.1%, multi-ethnic R560T Exon 11 0.2% CFF registry, >0.1% 3659delC Exon 19 0.2% CFF registry, >0.1% A455E Exon 9 0.2% CFF registry, >0.1% 2184delA Exon 13 0.1% CFF registry, >0.1% S549N Exon 11 0.1% CFF registry, >0.1%, multi-ethnic 711+1G®T Intron 5 0.1% CFF registry, >0.1% R75X Exon 3 0.2% Hispanic 406-1G→A Intron 3 0.2% Hispanic I148T Exon 4 0.2% Hispanic, French 2055del9→A Exon 13 0.1% Hispanic 935delA Exon 6b 0.1% Hispanic I506T Exon 10 0.1% Hispanic 3199del6 Exon 17a 0.1% Hispanic 2183AA→G Exon 13 0.1% Hispanic 3120+1G®A Intron 16 1.5% African American, Arabian 2307insA Exon 13 0.2% African American A559T Exon 11 0.2% African American ∆F311 Exon 7 0.2% African American G480C Exon 10 0.2% African American 405+3A→C Intron 3 0.2% African American S1255X Exon 20 0.2% African American L1093P Exon 17b Undetermined Native American D648V Exon 13 Undetermined Native American I1234V Exon 19 Undetermined Arabian linkage S549R Exon 11 Undetermined Arabian linkage 1898+5G→T Intron 12 Undetermined East Asian linkage CFTRdele2,3 Exons 2,3 Undetermined Eastern European linkage (Slavic) Y1092X Exon 17b Undetermined French linkage 394delTT Exon 3 Undetermined Nordic linkage Y569D Exon 12 Undetermined Pakistani linkage 3905insT Exon 20 Undetermined Swiss linkage (also: Amish, Acadian, Mennonite) 1898+1G®A Intron 12 Undetermined Welsh linkage M1101k Exon 17b Undetermined Hutterite ancestry *This table presents the top 50 mutations in the USA based on the Cystic Fibrosis Foundation CF Registry data from 1997 [Cystic Fibrosis Foundation, 1998], and data generated during our investigation. Login to comment

[hide] Prenatal detection of cystic fibrosis by ultrasono... J Med Genet. 2002 Jun;39(6):443-8. Scotet V, De Braekeleer M, Audrezet MP, Quere I, Mercier B, Dugueperoux I, Andrieux J, Blayau M, Ferec C
Prenatal detection of cystic fibrosis by ultrasonography: a retrospective study of more than 346 000 pregnancies.
J Med Genet. 2002 Jun;39(6):443-8., [PMID:12070257]

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196 The heterozygous fetuses carried a severe molecular abnormality (∆F508 (n=9) or G542X (n=1)), except one who carried a mild mutation (R347H). Login to comment
202 Therefore, the second mutation Table 1 Incidence of cystic fibrosis and CF heterozygosity among fetuses with echogenic bowel in Brittany, France (1991-2000) Fetuses with echogenic bowel 142 Affected fetuses Number 14 Incidence 1/10 Genotypes ∆F508/∆F508 9 ∆F508/4005+1G→A 1 ∆F508/3129del4 1 ∆F508/Q220X 1 ∆F508/W1282X 1 ∆F508/1717-1G→A 1 CF incidence in the general population during the present study 1/2987 Risk of CF: echogenic bowel fetuses/general population 294 Heterozygous fetuses Number 11 Incidence 1/13 Mutations ∆F508 9 G542X 1 R347H 1 CF heterozygosity in the general population during the present study 1/28 Risk of CF heterozygosity: echogenic bowel fetuses/general population 2.2 Letter www.jmedgenet.com will be identified in 88.5% of the 22.6% of fetuses for which the first analysis identified only one mutation (that is, in 20.0% of all CF fetuses). Login to comment
241 However, they based their comparison on an expected carrier rate in the general population which appears to be overestimated.1 The CFTR mutations identified in fetuses with echogenic bowel that have been reported so far are associated with pancreatic insufficiency (for example, ∆F508, G542X, G551D, Table 2 Ability of the ultrasound examination to detect cystic fibrosis Cystic fibrosis TotalYes No Utrasound examination Abnormal 14 128 142 Normal 112 346 300 346 412 Total 126 346 428 346 554 2183AA→G, ∆F311).9 13 27 43 To our knowledge, only one mutation associated with a mild phenotype (R117H)13 and one mild complex CFTR allele (D443Y-G576A-R668C)44 have been identified in two of the CF affected fetuses. Login to comment

[hide] Development and evaluation of a PCR-based, line pr... Clin Chem. 2002 Jul;48(7):1121-3. Wang X, Myers A, Saiki RK, Cutting GR
Development and evaluation of a PCR-based, line probe assay for the detection of 58 alleles in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.
Clin Chem. 2002 Jul;48(7):1121-3., [PMID:12089190]

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68 Amplicon Size, bp Mutations (polymorphisms) Exon 13 598 2307 insA Intron 8, exon 09 548 A455E, 5T (7/9 T polymorphism) Exon 10 482 G480C, ⌬I507, ⌬F508 (F508C, I507V, I506V polymorphisms) Intron 10, exon 11 433 1717-1G3A, G542X, G551D, R553X, A559T, R560T Exon 19 420 R1162X, 3659delC Exon 21 397 N1303K Exon 20 359 S1255X, W1282X Exon 07 328 1078delT, R334W, R347P Exon 04, intron 4 288 R117H, 621ϩ1G3T Intron 14b 248 2789ϩ5G3A Intron 19 237 3849ϩ10kbC3T Exon 03 210 G85E, 405ϩ3A3C Intron 5 166 711ϩ1G3T Intron 16 139 3120ϩ1G3A Clinical Chemistry 48, No. Login to comment
88 The genotypes of each sample are as follows: lane 1, ϩ/ϩ (ϩ is the wild type); lane 2, 5T, R117H/3659delC; lane 3, G542X/ϩ; lane 4, I506V/ϩ; lane 5, I507V/ϩ; lane 6, F508C/⌬F508; lane 7, G85E/⌬F508; lane 8, 405ϩ3A3C/3120ϩ1G3C; lane 9, R117H/ϩ; lane 10, 621ϩ1G3T/⌬F508; lane 11, 711ϩ1G3T/⌬F508; lane 12, 1078delT/ϩ; lane 13, R334W/⌬F508; lane 14, R347P/⌬F508; lane 15, A455E/ϩ; lane 16, G480C/⌬F508; lane 17, ⌬I507/ϩ; lane 18, ⌬F508/ϩ; lane 19, 1717-1G3A/ϩ; lane 20, G542X/ϩ; lane 21, G551D/⌬F508; lane 22, R553X/ϩ; lane 23, R560T/⌬F508; lane 24, G551D/A559T; lane 25, 2307insA/ϩ; lane 26, 2789ϩ5G3A/⌬F508; lane 27, 3120ϩ1G3A/⌬F508; lane 28, R1162X/R1162X; lane 29, 3659delC/⌬F508; lane 30, 3849ϩ10kbC3T/⌬F508; lane 31, S1255X/⌬F508; lane 32, W1282X/G542X; lane 33, N1303K/ϩ. Login to comment

[hide] Predicting the risk of cystic fibrosis with abnorm... Am J Med Genet. 2002 Jun 15;110(2):109-15. Muller F, Simon-Bouy B, Girodon E, Monnier N, Malinge MC, Serre JL
Predicting the risk of cystic fibrosis with abnormal ultrasound signs of fetal bowel: results of a French molecular collaborative study based on 641 prospective cases.
Am J Med Genet. 2002 Jun 15;110(2):109-15., 2002-06-15 [PMID:12116247]

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Hyperechogenic fetal bowel is prenatally detected by ultrasound during the second trimester of pregnancy in 0.1-1.8% of fetuses. It has been described as a normal variant but has often been associated with severe diseases, notably cystic fibrosis (CF). The aim of our study was to determine the risk of CF in a prospective study of 641 fetuses with ultrasonographically abnormal fetal bowel and the residual risk when only one mutation is detected in the fetus. Fetal cells and/or parental blood cells were screened for CFTR mutations. Two screening steps were used, the first covering the mutations most frequently observed in French CF patients (mutation detection rate of 70-90%) and, when a CF mutation was detected, a DGGE-sequencing strategy. We observed a 3.1% risk of CF when a digestive tract anomaly was prenatally observed at routine ultrasound examination. The risk was higher when hyperechogenicity was associated with bowel dilatation (5/29; 17%) or with the absence of gall bladder (2/8; 25%). The residual risk of CF was 11% when only one CF mutation was detected by the first screening step, thereby justifying in-depth screening. Mutations associated with severe CF (DeltaF508 mutation) were more frequently observed in these ultrasonographically and prenatally detected CF cases. However, the frequency of heterozygous cases was that observed in the normal population, which demonstrates that heterozygous carriers of CF mutations are not at increased risk for hyperechogenic bowel. In conclusion, fetal bowel anomalies indicate a risk of severe cystic fibrosis and justify careful CFTR molecular analysis.

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46 Depending on the laboratory, the methods most frequently used were reverse dot blot with the Inno-Lipa CF2 kit (Murex) (eight mutations detected: DF508, DI507, G542X, G551D, R553X, 1717-1G ! Login to comment
48 A, G542X, G551D, R553X, R560T, S549R, S549N, 3849 þ 10kbC ! Login to comment
64 Of them, 14 were homozygous for DF508 and three were compound heterozygous for DF508 and another mutation (W1282X, G542X, 1078delT). Login to comment
73 Fetuses Carrying Two CFTR Mutations Cases CFTR Gene Mutations Ultrasound Findings Outcome 1-9 DF508/DF508 Hyperechogenic bowel TOP 10,11 DF508/DF508 Hyperechogenic bowel þ dilated loop TOP 12 DF508/DF508 Hyperechogenic bowel þ dilated loop þ gall bladder not seen TOP 13 DF508/DF508 Hyperechogenic bowel þ gall bladder not seen TOP 14 DF508/DF508 Intestinal dilated loops (absent at 22 wks) Birth, CF-affected, meconium ileus at birth 15 DF508/W1282X Hyperechogenic bowel (absent at 22 wks) TOP 16 DF508/G542X Hyperechogenic bowel þ dilated loop TOP 17 DF508/1078delT Hyperechogenic bowel þ dilated loop (absent at 22 wks) Birth, CF-affected,* meconium ileus at birth 18 DF508/O220X Hyperechogenic bowel þ dilated loop (present at 33 wks) Birth, CF-affected,* meconium ileus at birth 19 1078delT/394delTT Hyperechogenic bowel TOP 20** CFTRdele19/CFTRdele19 Hyperechogenic bowel (present at 33 wks) Birth, CF-affected, absence of meconium ileus at birth 21 W846X/G576A-R668C Hyperechogenic bowel Birth, potential absence of vas deferens TOP ¼ termination of pregnancy; Wks ¼ weeks of amenorrhea. Login to comment
103 Heterozygous Cystic Fibrosis Cases With Abnormal Fetal Bowel at Ultrasound Examination Cases CFTR Gene mutations Ultrasound findings Outcome 22-27 DF508/X Hyperechogenic bowel Birth, thriving 28-29 DF508/X Hyperechogenic bowel Premature birth (32 wks), thriving 30 DF508/X Hyperechogenic bowel TOP cardiomegaly þ pulmonary hypoplasia 31 DF508/X Hyperechogenic bowel Lost to follow-up 32 DF508/X Hyperechogenic bowel þ short femur Died day 2 after birth, fetal distress 33 DF508/X Intestinal dilated loops Birth, thriving 34 DF508/X Hyperechogenic bowel þ fetal hydrops Birth, parvovirus-affected, thriving 35 DF508/X Intra-abdominal calcifications Birth, thriving 36 G542X/X Hyperechogenic bowel þ polyhydramnios Birth, thriving 37 R117H/X Hyperechogenic bowel Birth, thriving 38 A534E/X Hyperechogenic bowel Birth, thriving 39 D836Y/X Dilated loop (small bowel) þ polyhydramnios Birth, small bowel atresia, operated, not CF-affected In the present study, most CF cases with intestinal anomalies (15/20) were observed during the second trimester of pregnancy, because in France all pregnant women undergo ultrasound examinations at 11, 22, and 33 weeks. Login to comment
110 It has also been demonstrated that the risk of meconium ileus is higher among children with the G542X mutation [Hamosh et al., 1992] and one of the 20 CF cases was a DF508/G542X compound heterozygote. Login to comment

[hide] Hyperechogenic bowel loops and meconium ileus in a... Prenat Diagn. 2002 Jul;22(7):636-7. Orgad S, Berkenstadt M, Achiron R, Yahav Y, Gazit E, Barkai G, Loewenthal R
Hyperechogenic bowel loops and meconium ileus in a fetus carrying the D1152H and G542X cystic fibrosis CFTR mutations.
Prenat Diagn. 2002 Jul;22(7):636-7., [PMID:12124706]

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20 Hyperechogenic bowel loops and meconium ileus in a fetus carrying the D1152H and G542X cystic fibrosis CFTR mutations In the Jewish cystic fibrosis (CF) patient population, 12 mutations account for more than 91% of the CF chromosomes. Login to comment
21 Ashkenazi Jews were tested in the past only for F508del, W1282X, G542X, N1303K and 3849+10KbC>T of the cystic fibrosis transmembrane conductance regulator (CFTR) gene (Kerem et al., 1995, 1997; Abeliovich et al., 1992, 1996). Login to comment
32 Since the US finding was compatible with CF, DNA from the woman was analysed and was found to carry the G542X CFTR mutation. Login to comment

[hide] Genotype-phenotype correlation in cystic fibrosis:... Am J Med Genet. 2002 Jul 22;111(1):88-95. Salvatore F, Scudiero O, Castaldo G
Genotype-phenotype correlation in cystic fibrosis: the role of modifier genes.
Am J Med Genet. 2002 Jul 22;111(1):88-95., 2002-07-22 [PMID:12124743]

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More than 1,000 mutations have been identified in the cystic fibrosis (CF) transmembrane regulator (CFTR) disease gene. The impact of these mutations on the protein and the wide spectrum of CF phenotypes prompted a series of Genotype-Phenotype correlation studies. The CFTR genotype is invariably correlated with pancreatic status-in about 85% of cases with pancreatic insufficiency and in about 15% of cases with pancreatic sufficiency. The correlations between the CFTR genotype and pulmonary, liver, and gastrointestinal expression are debatable. The heterogeneous phenotype in CF patients bearing the same genotype or homozygotes for nonsense mutations implicated environmental and/or genetic factors in the disease. However, the discordant phenotype observed in CF siblings argued against a major role of environmental factors and suggested that genes other than CFTR modulate the CF phenotype. A locus that modulates gastrointestinal expression was identified in mice and subsequently in humans. By analyzing nine CF patients discordant for meconium ileus we were able to show that this locus had a dominant effect. Moreover, in a collaborative study we found a higher rate of polymorphisms in beta-defensin genes 1 and 2 in CF patients and in controls. In another multicenter study mutations in alpha-1 antitrypsin (A1AT) and mannose binding lectin genes were found to be independent risk factors for liver disease in CF patients. The body of evidence available suggests that the variegated CF phenotype results from complex interactions between numerous gene products.

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25 Several mutations of this group have a frequency of > 2% among CF chromosomes within most populations studied, e.g., W1282X [Shoshani et al., 1992], R553X, and G542X [Casals et al., 1993]. Login to comment
65 Secondly, pulmonary expression was heterogeneous in CF patient homozygotes for nonsense mutations, although CFTR mRNA was not detected at the pulmonary level in these patients [Hamosh et al., 1991]; for example, a mild pulmonary expression has been reported in several patient homozygotes for nonsense mutations [Cutting et al., 1990b; Cuppens et al., 1990], such as R553X [Castaldo et al., 1996] and G542X [Castaldo et al., 1997]. Login to comment
87 Similarly, we described a CF patient homozygous for the G542X mutation who had a very severe liver phenotype, unlike the six previously reported cases with this CFTR genotype, who were free of liver involvement [Castaldo et al., 1997]. Login to comment

[hide] Screening for cystic fibrosis in newborn infants: ... J Med Screen. 2002;9(2):60-3. Corbetta C, Seia M, Bassotti A, Ambrosioni A, Giunta A, Padoan R
Screening for cystic fibrosis in newborn infants: results of a pilot programme based on a two tier protocol (IRT/DNA/IRT) in the Italian population.
J Med Screen. 2002;9(2):60-3., [PMID:12133923]

Abstract [show]
OBJECTIVE: To assess the performance of a two tier neonatal screening programme (IRT/DNA/IRT) for cystic fibrosis, based on immunoreactive trypsinogen (IRT) followed by direct cystic fibrosis transmembrane conductance regulator (CFTR) gene analysis (based on a panel of up to 31 mutations) in hypertrypsinaemic newborn infants and to compare it with a previous screening protocol. SETTING: The study comprised all the newborn infants in the period 1 October 1998 to 31 December 1999 in the Lombardia region, north western Italy. METHODS: The screening strategy consisted of an immunoreactive trypsinogen assay from dried blood spots, a polymerase chain reaction (PCR) followed by an oligonucleotide ligation assay (PCR-OLA), and a sequence code separation. RESULTS: 104 609 newborn infants were screened. 1457 hypertrypsinaemic infants (1.39%) were analysed with the PCR-OLA assay. 18 newborn homozygotes or compound heterozygotes for CFTR mutations were identified and referred to the cystic fibrosis (CF) centre at a mean age of 3 weeks. 125 infants presenting only one mutation were recalled for a sweat test: a diagnosis of CF was made in 13 infants, and parents of 112 neonates identified as carriers (1:13) received genetic counselling. The remaining 1314 hypertrypsinaemic newborn infants were recalled for IRT retesting and 177 were referred for a sweat test because the second IRT measurement was above the cut off value. Among this group a further two infants were diagnosed with CF (1.1%) leading to a CF prevalence of 1:3170. CONCLUSIONS: This strategy resulted in an early and accurate diagnosis of CF. The IRT/DNA/IRT protocol with an OLA assay was shown to be useful in an Italian population with a genetic heterogeneity, leading to the identification of 94% of infants with CF.

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266 Mutations identified by the assay are G85E, 621+1G→T, R117H, Y122X, 711+1G→T, 1078delT, R347P, R347H, R334W, A455E, 1898+1G→A, 2183-AA→G, 2789+5G→A, delF508, I507del, Q493X, V520F, 1717-1G→A, G542X, G551D, R553X, R560T, S549R, S549N, 3849+10kbC→T, 3849+4A→G, R1162X, 3659delC, W1282X, 3905insT, and N1303K. Login to comment
280 Genotypes are shown in table 1: 18% of our patients were delF508 homozygotes, 15% were homozygotes for other CFTR alleles (N1303K, 2183AA→G, 1717-1G→A, R334W, G542X), 21% were compound heterozygotes, and a further 43% were heterozygotes. Login to comment
285 The CFTR mutations identified and their frequencies among carriers were as follows: delF508 (72 chromosomes, 64.2%), N1303K (12, 10.7%), R117H (9, 8%), G542X (7, 6.25%), R347H, R1162X, 2789+5G→A (2 alleles each, 1.8%), 1898+1G→A, 1717-1G→A, W1282X, 2183-AA→G, 621+1G→T, and 3849+10kbC→T (1, 0.9%). Login to comment
310 Since 1998, in our CF centre, an expanded DNA CFTR gene analysis and repeat sweat test after 6-12 months of life have been performed in hypertrypsinaemic Table 1 Genotypes of 33 patients with CF identified in the 15 month period Two CFTR mutations identified (18 patients) by PCR/OLA: ∆F508/∆F508 6 N1303K/N1303K 2 ∆F508/N1303K 3 R334W/R334W 1 ∆F508/G542X 2 G542X/G542X 1 ∆F508/3659delC 1 2183AA→G/ 2183AA→G 1 ∆F508/R1162X 1 One CFTR mutation identified (13 patients) by PCR/OLA: ∆F508/D1152H* 1 ∆F508/Y1032C* 1 ∆F508/R1066H* 1 ∆F508/UN 6 ∆F508/R1066C* 1 W1282X/L1077P* 1 ∆F508/D579G* 1 G85E/UN 1 No CFTR mutation identified (two patients) by PCR/OLA: 711+3A→G*/UN 1 D110E*/D110E* 1 *CFTR alleles identified by analysis by denaturing gradient gel electrophoresis and sequencing. Login to comment

[hide] Urogenital abnormalities in male children with cys... Arch Dis Child. 2002 Aug;87(2):135-8. Blau H, Freud E, Mussaffi H, Werner M, Konen O, Rathaus V
Urogenital abnormalities in male children with cystic fibrosis.
Arch Dis Child. 2002 Aug;87(2):135-8., [PMID:12138064]

Abstract [show]
BACKGROUND: Congenital bilateral absence of the vas deferens (CBAVD) is presumed to occur prenatally and is present in over 99% of adult males with cystic fibrosis (CF). AIMS: To describe ultrasonic features in male children with CF. We aimed to describe urogenital anomalies, comparing pancreatic sufficient and insufficient CF patients. METHODS: Pelvic and scrotal ultrasonography were performed in 12 boys with CF aged 2-12 years and 16 age matched healthy controls. RESULTS: Nine patients had pancreatic insufficiency (PI): seven had two severe mutations and two had unknown mutations. Three boys were pancreatic sufficient (PS), two with splicing mutations (5T and 3849+10kb C-T respectively) and borderline sweat tests. Seminal vesicles were visualised in 5/12 patients and 8/16 controls, compared to non-visualisation reported in all adults with CBAVD. Testicular microlithiasis was found in 4/18 PI, 0/6 PS, and 0/32 control testes, compared to 0.6-1.4% in healthy males and 15% in CF adults; 7/18 PI, 4/6 PS, and 0/32 control testes were smaller than predicted for age. The epididymal head was non-homogeneous with cysts, hypo-, or hyper-echogenicity in 5/18 PI, 1/6 PS, and 0/32 control testes. CONCLUSIONS: Genital abnormalities may occur early in CF, but are less common than described in adults. They are found more often in pancreatic insufficient than in pancreatic sufficient CF patients. However, a positive finding, if present, may aid in the diagnosis of the latter. A larger longitudinal study is recommended to better define the onset and progression of urogenital abnormalities.

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264 Seven of these were homozygous or compound heterozygotes for the genotypes ∆F508, W1282X, G542X, S549R, and 405+1G→A. Login to comment
294 The vas deferens appeared similar to Table 1 Genotype and clinical features in pancreatic insufficient boys Pt no. Age (y) Sweat chloride CFTR mutations FEV1 Wt Ht Sh. score 1 2 69 W1282X/405+1G→A - 3 3 77 2 3 100 W1282X/W1282X 98% 50 75 92 3 5 78 Unknown 94% 50 10 92 4 6 75 Unknown 86% 50 25 85 5 7 76 W1282X/G542X 105% 95 40 90 6 8 99 W1282X/∆F508 112% 85 50 98 7 9 90 S549R/S549R 77% 25 10 70 8 10 84 W1282X/∆F508 102% 25 20 89 9 10 97 W1282X/∆F508 87% 50 50 98 FEV1, forced expiratory volume in 1 second; Wt , weight, percentile for age; Ht, height, percentile for age; Sh. score, Shwachman score. Login to comment

[hide] Analysis by mass spectrometry of 100 cystic fibros... Hum Reprod. 2002 Aug;17(8):2066-72. Wang Z, Milunsky J, Yamin M, Maher T, Oates R, Milunsky A
Analysis by mass spectrometry of 100 cystic fibrosis gene mutations in 92 patients with congenital bilateral absence of the vas deferens.
Hum Reprod. 2002 Aug;17(8):2066-72., [PMID:12151438]

Abstract [show]
BACKGROUND: Limited mutation analysis for congenital bilateral absence of the vas deferens (CBAVD) has revealed only a minority of men in whom two distinct mutations were detected. We aimed to determine whether a more extensive mutation analysis would be of benefit in genetic counselling and prenatal diagnosis. METHODS: We studied a cohort of 92 men with CBAVD using mass spectrometry and primer oligonucleotide base extension to analyse an approximately hierarchical set of the most common 100 CF mutations. RESULTS: Analysis of 100 CF mutations identified 33/92 (35.9%) patients with two mutations and 29/92 (31.5%) with one mutation, compound heterozygosity accounting for 94% (31/33) of those with two mutations. This panel detected 12.0% more CBAVD men with at least one mutation and identified a second mutation in >50% of those considered to be heterozygotes under the two routine 25 mutation panel analyses. CONCLUSION: Compound heterozygosity of severe/mild mutations accounted for the vast majority of the CBAVD patients with two mutations, and underscores the value of a more extensive CF mutation panel for men with CBAVD. The CF100 panel enables higher carrier detection rates especially for men with CBAVD, their partners, partners of known CF carriers, and those with 'mild' CF with rarer mutations.

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20 Given the frequency of CF mutations, especially in the Caucasian population ( in 25), and the common request by CBAVD men to sire their own offspring by using surgical Table I. The 100 most common cystic fibrosis mutations listed by exon Mutationa Exonb Frequency (%)c G85E 3 0.1 394delTT 3 Swedish E60X 3 Belgium R75X 3 405ϩ1G→A Int 3 R117H 4 0.30 Y122X 4 French 457TAT→G 4 Austria I148T 4 Canada (French Canadian) 574delA 4 444delA 4 R117L 4 621ϩ1G→T Int 4 0.72 711ϩ1G→T Int 5 Ͼ0.1 712-1G→T Int 5 711ϩ5G→A Int 5 Italy (Caucasian) L206W 6a R347P 7 0.24 1078delT 7 Ͼ0.1 R334W 7 Ͼ0.1 1154InsTC 7 T338I 7 Italy R347H 7 Turkey Q359K/T360K 7 Israel (Georgian Jews) I336K 7 R352Q 7 G330X 7 S364P 7 A455E 9 0.20 I507 10 0.21 F508 10 66.02 1609delCA 10 Spain (Caucasian) V520F 10 Q493X 10 C524X 10 G480C 10 Q493R 10 1717-1G→A Int 10 0.58 R553X 11 0.73 G551D 11 1.64 G542X 11 2.42 R560T 11 Ͼ0.1 S549N 11 Q552X 11 Italy S549I 11 Israel (Arabs) A559T 11 African American R553G 11 R560K 11 1812-1G→A Int 11 A561E 12 E585X 12 Y563D 12 Y563N 12 1898ϩ1G→A Int 12 0.22 1898ϩ1G→C Int 12 2183AA→G 13 Italian 2184delA 13 Ͻ0.1 K710X 13 2143delT 13 Moscow (Russian) 2184InsA 13 1949del84 13 Spain (Spanish) 2176InsC 13 2043delG 13 2307insA 13 2789ϩ5G→A Int 14b Ͼ0.1 2869insG 15 S945L 15 Q890X 15 3120G→A 16 2067 Table I. continued Mutationa Exonb Frequency (%)c 3120ϩ1G→A Int 16 African American 3272-26A→G Int 17a R1066C 17b Portugal (Portugese) L1077P 17b R1070Q 17b Bulgarian W1089X 17b M1101K 17b Canada (Hutterite) R1070P 17b R1162X 19 0.29 3659delC 19 Ͼ0.1 3849G→A 19 3662delA 19 3791delC 19 3821delT 19 Russian Q1238X 19 S1235R 19 France, South S1196X 19 K1177R 19 3849ϩ10kbC→T Int 19 0.24 3849ϩ4A→G Int 19 W1282X 20 1.22 S1251N 20 Dutch, Belgian 3905insT 20 Swiss, Acadian, Amish G1244E 20 R1283M 20 Welsh W1282R 20 D1270N 20 S1255X 20 African American 4005ϩ1G→A Int 20 N1303K 21 1.34 W1316X 21 aMutations were chosen according to their frequencies (Cystic Fibrosis Genetic Analysis Consortium, 1994; Zielenski and Tsui, 1995; Estivill et al., 1997). Login to comment
34 The mutations in the 25 mutation panel were: ∆F508, G542X, N1303K, G551D, W1282X, 1717-1G→A, R553X, 621ϩ1G→T, R1162X, 2183AA→G, R117H, ∆I507, R560T, 3849ϩ10kbC→T, S549N, S549I, S549R, R1283M, R1283K, R553G, R560K, R117L, 1774delCT, 1811ϩ1G→C, and 4006-61del14. Login to comment
35 ACMG 25 mutation panel (ACMG25): The following mutations are the recommended core mutations for general population CF carrier screening by American College of Medical Genetics (ACMG) (Grody, et al 2001): ∆F508, G542X, N1303K, G551D, W1282X, 1717-1G→A, R553X, 621ϩ1G→T, R1162X, R117H, ∆I507, 1898ϩ1G→A, G85E, R347P, A455E, R560T, R334W, 3849ϩ10kbC→T, 3659delC, 1078delT, 2789ϩ5G→A, 711ϩ1G→T, 2184delA, 3120ϩ1G→A and I148T. Login to comment
86 CFTR mutations in 92 men with congenital bilateral absence of vas deferens Mutations CFTR mutation panels CF25 CF25 ϩ 5T ACMG25 ACMG25 ϩ 5T CF100 Mutations detected in ∆F508 39 39 39 39 39 CF25 mutation panel R117H 4 4 4 4 4 W1282X 4 4 4 4 4 G551D 3 3 3 3 3 G542X 1 1 1 1 1 N1303K 1 1 1 1 1 IVS8-polyT IVS8-5T 33 33 33 Additional mutations L206W 3 detected not in CF25 D1270N 2 mutation panel 1154InsTC 1 3272-26A→G 1 A455E 1 1 1 R334W 1 1 1 Q890X 1 Total 14 52 85 54 87 95 respectively, in the total number of patients with at least one mutation. Login to comment
91 CFTR genotypes in 92 men with congenital bilateral absence of vas deferens Genotypesa CFTR mutation panelsb CF25 CF25 ϩ 5T ACMG25 ACMG25 ϩ 5T CF100 Two mutations ∆F508/5T 16 16 16 W1282X/5T 4 4 4 ∆F508/R117Hc 3 3 3 3 3 G542X/5T 1 1 1 G551D/5T 1 1 1 ∆F508/L206W 2 ∆F508/A455E 1 1 1 ∆F508/3272-26A→G 1 Q890X/5T 1 L206W/5T 1 D1270N/D1270N 1 5T/5T 1 1 1 Sub-total 3 26 4 27 33 One mutation ∆F508/ϩ 36 20 35 19 16 5T/ϩ 9 9 7 G551D/ϩ 3 2 3 2 2 G542X/ϩ 1 1 R117H/ϩ 1 1 1 1 1 N1303K/ϩ 1 1 1 1 1 W1282X/ϩ 4 4 R334W/ϩ 1 1 1 1154InsTC/ϩ 1 Sub-total 46 33 46 33 29 Total (%) 49 (53.3) 59 (64.1) 50 (54.3) 60 (65.2) 62 (67.4) No mutation (%) 43 (46.7) 33 (35.9) 42 (45.7) 32 (34.8) 30 (32.6) aMutations L206W, 3272-26A→G, Q890X, D1270N, 1154InsTC and 5T are not in either CF25 and ACMG25 panels, while A455E and R334W are not in CF25, but are part of ACMG25 panel. Login to comment
93 bThe increase in the number of patients with two mutations under CF25ϩ5T, ACMG25ϩ5T and CF100, compared with CF25 and ACMG25, was due to identification of a second mutation and reflected by the reduction of the number of patients with one mutation (e.g. ∆F508/ϩ, G551D/ϩ, G542X/ϩ, W1282X/ϩ). Login to comment

[hide] A phase II, double-blind, randomized, placebo-cont... Hum Gene Ther. 2002 Jul 20;13(11):1349-59. Wagner JA, Nepomuceno IB, Messner AH, Moran ML, Batson EP, Dimiceli S, Brown BW, Desch JK, Norbash AM, Conrad CK, Guggino WB, Flotte TR, Wine JJ, Carter BJ, Reynolds TC, Moss RB, Gardner P
A phase II, double-blind, randomized, placebo-controlled clinical trial of tgAAVCF using maxillary sinus delivery in patients with cystic fibrosis with antrostomies.
Hum Gene Ther. 2002 Jul 20;13(11):1349-59., 2002-07-20 [PMID:12162817]

Abstract [show]
tgAAVCF, an adeno-associated cystic fibrosis transmembrane conductance regulator (CFTR) viral vector/gene construct, was administered to 23 patients in a Phase II, double-blind, randomized, placebo-controlled clinical trial. For each patient, a dose of 100,000 replication units of tgAAVCF was administered to one maxillary sinus, while the contralateral maxillary sinus received a placebo treatment, thereby establishing an inpatient control. Neither the primary efficacy endpoint, defined as the rate of relapse of clinically defined, endoscopically diagnosed recurrent sinusitis, nor several secondary endpoints (sinus transepithelial potential difference [TEPD], histopathology, sinus fluid interleukin [IL]-8 measurements) achieved statistical significance when comparing treated to control sinuses within patients. One secondary endpoint, measurements of the anti-inflammatory cytokine IL-10 in sinus fluid, was significantly (p < 0.03) increased in the tgAAVCF-treated sinus relative to the placebo-treated sinus at day 90 after vector instillation. The tgAAVCF administration was well tolerated, without adverse respiratory events, and there was no evidence of enhanced inflammation in sinus histopathology or alterations in serum-neutralizing antibody titer to adeno-associated virus (AAV) capsid protein after vector administration. In summary, this Phase II trial confirms the safety of tgAAVCF but provides little support of its efficacy in the within-patient controlled sinus study. Various potentially confounding factors are discussed.

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157 Of the 23 treated patients, 11 were homozygous for DF508, 3 were DF508 heterozygouswith an unidentifiedallele, 2 were DF508 heterozygous with G542X, 6 were DF508 heterozygous with another allele (one each of 3849110KB, 3905 insert T, 62111, G85E, R334W, and W1282X) and 1 patient was G542X heterozygous with an unidentified allele. Login to comment

[hide] Variant cystic fibrosis phenotypes in the absence ... N Engl J Med. 2002 Aug 8;347(6):401-7. Groman JD, Meyer ME, Wilmott RW, Zeitlin PL, Cutting GR
Variant cystic fibrosis phenotypes in the absence of CFTR mutations.
N Engl J Med. 2002 Aug 8;347(6):401-7., 2002-08-08 [PMID:12167682]

Abstract [show]
BACKGROUND: Cystic fibrosis is a life-limiting autosomal recessive disorder with a highly variable clinical presentation. The classic form involves characteristic findings in the respiratory tract, gastrointestinal tract, male reproductive tract, and sweat glands and is caused by loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR ) gene. Nonclassic forms of cystic fibrosis have been associated with mutations that reduce but do not eliminate the function of the CFTR protein. We assessed whether alteration in CFTR function is responsible for the entire spectrum of variant cystic fibrosis phenotypes. METHODS: Extensive genetic analysis of the CFTR gene was performed in 74 patients with nonclassic cystic fibrosis who had been referred by 34 medical centers. We evaluated two families that each included a proband without identified mutations and a sibling with nonclassic cystic fibrosis to determine whether there was linkage to the CFTR locus and to measure the extent of CFTR function in the sweat gland and nasal epithelium. RESULTS: Of the 74 patients studied, 29 had two mutations in the CFTR gene, 15 had one mutation, and 30 had no mutations. A final genotype of two mutations was more common among patients who had been referred after screening for common cystic fibrosis-causing mutations identified one mutation than among those who had been referred after screening had identified no such mutations (26 of 34 patients vs. 3 of 40 patients, P<0.001). Comparison of clinical features and sweat chloride concentrations revealed no significant differences among patients with two, one, or no CFTR mutations. Haplotype analysis in the two families revealed no linkage to CFTR. Although each of the affected siblings had elevated sweat chloride concentrations, measurements of cyclic AMP-mediated ion and fluid transport in the sweat gland and nasal epithelium demonstrated the presence of functional CFTR. CONCLUSIONS: Factors other than mutations in the CFTR gene can produce phenotypes clinically indistinguishable from nonclassic cystic fibrosis caused by CFTR dysfunction.

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71 MUTATION IDENTIFIED BY SCREENING FOR COMMON MUTATIONS MUTATION IDENTIFIED BY DNA SEQUENCING NO. OF PATIENTS ∆F508 5T* 3 ∆F508 D1152H 2 ∆F508 2789+2insA 2 ∆F508 R117C 2 ∆F508 D110H 1 ∆F508 2789+5G→A 1 ∆F508 P205S 1 ∆F508 L967S 1 ∆F508 I1027T 1 ∆F508 L206W 1 ∆F508 T1053I and 5T 1 ∆F508 V920M and 5T 1 ∆F508 R1070W 1 ∆F508 D579G 1 ∆F508 P67L 1 ∆F508 2811G→T†‡ 1 G85E F191V† 1 R117H G103X and 5T 1 I148T I556V 1 G542X R1162L 1 W1282X D1152H 1 None L138ins and 3272-26 A→G 1 None G463D† and 5T 1 None F693L and 5T 1 ∆F508 None 6 G551D None 1 W1282X None 1 None 5T 4 None 2307insA 1 None L997F 1 None V520I 1 None None 30 in Subject II-2 in Family 1. Login to comment

[hide] Spatial and temporal distribution of cystic fibros... Hum Genet. 2002 Sep;111(3):247-54. Epub 2002 Aug 1. Scotet V, Gillet D, Dugueperoux I, Audrezet MP, Bellis G, Garnier B, Roussey M, Rault G, Parent P, De Braekeleer M, Ferec C
Spatial and temporal distribution of cystic fibrosis and of its mutations in Brittany, France: a retrospective study from 1960.
Hum Genet. 2002 Sep;111(3):247-54. Epub 2002 Aug 1., [PMID:12215837]

Abstract [show]
Cystic fibrosis (CF) is the most common severe inherited disorder that affects children in Caucasian populations. The aim of this study was to define the spatial and temporal distribution of CF and its mutations in Brittany (western France) where the frequency of the disease is high. We retrospectively registered all CF patients born in Brittany since 1960 by cross-checking various data sources (e.g. medical care centres, genetics laboratories, hospital archives). Councils were contacted so that the place of residence of patients at birth could be determined. Moreover, the spectrum of CF transmembrane conductance regulator (CFTR) mutations and their spatial distribution across Brittany were determined. A total of 520 patients was registered in this study. The incidence of CF was assessed according to administrative (department, district) and diocesan divisions of Brittany and its evolution analysed over four decades. The incidence of CF was 1/2630, with a west/east gradient that was confirmed over time (Finistere: 1/2071 vs Ille-et-Vilaine: 1/3286). At present, the incidence of CF is decreasing, mainly as a result of prenatal diagnosis. An excellent mutation detection rate of 99.7% was obtained. Western Brittany presented a specific spectrum of mutations: 1078delT (9.4% of mutated alleles in the diocese of Cornouaille), G551D (7.7% in the diocese of Leon), 4005+1G-->A (2.9% in Cornouaille) and W846X (1.5% in western Brittany). On the other hand, the eastern region showed a spectrum more similar to the overall picture in France as a whole. This study enabled a precise measurement of the incidence of CF in Brittany to be obtained. The high frequency of the CFTR mutated alleles may result from founder effects and genetic drifts. Moreover, the study brings together the regional specificities of the CFTR gene and highlights disparities that exist in this part of France, both in incidence and in mutation distribution. These are attributable to different degrees of isolation and of population movements between the eastern and western parts of the region. Given that this is the first time that such a detailed study of the CFTR gene has been performed on a large population, this heightened knowledge of the epidemiology of CF in Brittany should provide a basis for the improvement of diagnostic strategies and refinement of genetic counselling.

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118 His genotype was ∆F508/∆F508 Mutation Exon Basse-Bretagne Haute-Bretagne Brittanya ∆F508 10 446 75.6% 224 73.7% 672 75.0% 1078delT 7 31 5.3% 3 1.0% 34 3.8% G551D 11 21 3.6% 12 3.9% 33 3.7% N1303K 21 3 0.5% 9 3.0% 12 1.3% W846X 14a 9 1.5% 1 0.3% 10 1.1% 2789+5G→A 14b 3 0.5% 6 2.0% 9 1.0% 1717-1G→A 11 5 0.8% 3 1.0% 8 0.9% Y1092X 17b 1 0.2% 6 2.0% 7 0.8% 4005+1G→A 20 6 1.0% 1 0.3% 7 0.8% E60X 3 3 0.5% 3 1.0% 6 0.7% 621+1G→T 4 3 0.5% 3 1.0% 6 0.7% R347H 7 6 1.0% 0 0.0% 6 0.7% S492F 10 2 0.3% 3 1.0% 5 0.6% G542X 11 4 0.7% 1 0.3% 5 0.6% 3272-26A→G 17b 2 0.3% 3 1.0% 5 0.6% R117H 4 3 0.5% 1 0.3% 4 0.4% G91R 3 3 0.5% 0 0.0% 3 0.3% ∆I507 10 1 0.2% 2 0.7% 3 0.3% R553X 11 3 0.5% 0 0.0% 3 0.3% W1282X 20 2 0.3% 1 0.3% 3 0.3% A72D 3 0 0.0% 2 0.7% 2 0.2% G85E 3 0 0.0% 2 0.7% 2 0.2% F311L 7 0 0.0% 2 0.7% 2 0.2% 1221delCT 7 2 0.3% 0 0.0% 2 0.2% R560K 11 0 0.0% 2 0.7% 2 0.2% 2622+1G→A 13 2 0.3% 0 0.0% 2 0.2% S945L 15 0 0.0% 2 0.7% 2 0.2% I1234V 19 2 0.3% 0 0.0% 2 0.2% G1249R 20 2 0.3% 0 0.0% 2 0.2% 3905insT 20 2 0.3% 0 0.0% 2 0.2% Unidentified - 3 0.5% 0 0.0% 3 0.3% Total - 590 65.7% 304 34.3% 896 100% IVS17bTA, IVS17bCA) of Irish, Scottish, English, Breton and Czech subjects who were carriers of this mutation, and showed that all these alleles carried a unique haplotype (16-7-17), testifying to the Celtic origin of this mutation (Cashman et al. 1995). Login to comment
161 The G542X mutation, which is the most frequent in France after the ∆F508 deletion (2.9%), is solely present on 0.6% of the mutated alleles in the population studied here. Login to comment

[hide] Aminoglycoside suppression of a premature stop mut... J Mol Med (Berl). 2002 Sep;80(9):595-604. Epub 2002 Jul 3. Du M, Jones JR, Lanier J, Keeling KM, Lindsey JR, Tousson A, Bebok Z, Whitsett JA, Dey CR, Colledge WH, Evans MJ, Sorscher EJ, Bedwell DM
Aminoglycoside suppression of a premature stop mutation in a Cftr-/- mouse carrying a human CFTR-G542X transgene.
J Mol Med (Berl). 2002 Sep;80(9):595-604. Epub 2002 Jul 3., [PMID:12226741]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Since approximately 5% of all mutant CF alleles are stop mutations, it can be calculated that approximately 10% of CF patients carry a premature stop mutation in at least one copy of the CFTR gene. Certain ethnic groups, such as the Ashkenazi Jewish population, carry a much higher percentage of CF stop mutations. Consequently, a therapeutic strategy aimed at suppressing this class of mutation would be highly desirable for the treatment of this common genetic disease. We have shown previously that aminoglycoside antibiotics can suppress premature stop mutations in the CFTR gene in a bronchial epithelial cell line [Nat Med (1997) 3:1280]. To address whether aminoglycosides can suppress a CFTR premature stop mutation in an animal model, we constructed a transgenic mouse with a null mutation in the endogenous CFTR locus (Cftr-/-) that also expressed a human CFTR-G542X cDNA under control of the intestinal fatty acid binding protein promoter. We then investigated whether the daily administration of the aminoglycoside antibiotics gentamicin or tobramycin could restore the expression of a detectable level of CFTR protein. Immunofluorescence staining of intestinal tissues from Cftr-/- hCFTR-G542X mice revealed that gentamicin treatment resulted in the appearance of hCFTR protein at the apical surface of the glands of treated mice. Weaker staining was also observed in the intestinal glands following tobramycin treatment. Short-circuit current measurements made on intestinal tissues from these mice demonstrated that a significant number of positive cAMP-stimulated transepithelial chloride current measurements could be observed following gentamicin treatment (P=0.008) and a near significant number following tobramycin treatment (P=0.052). When taken together, these results indicate that gentamicin, and to a lesser extent tobramycin, can restore the synthesis of functional hCFTR protein by suppressing the hCFTR-G542X premature stop mutation in vivo.

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1 M. Du · J. Lanier · K.M. Keeling · D.M. Bedwell (✉) Department of Microbiology, 1530 Third Avenue, South, The University of Alabama at Birmingham, Birmingham, AL 35294-2170, USA e-mail: dbedwell@uab.edu Tel.: +1-205-9346593, Fax: +1-205-9755482 J.R. Jones · D.M. Bedwell Department of Human Genetics, University of Alabama at Birmingham, Birmingham, Alabama, USA J.R. Lindsey Department of Genomics and Pathobiology, University of Alabama at Birmingham, Birmingham, Alabama, USA Z. Bebök · E.J. Sorscher Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama, USA M. Du · J.R. Jones · J. Lanier · K.M. Keeling · J.R. Lindsey A. Tousson · Z. Bebök · E.J. Sorscher · D.M. Bedwell Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, USA J.A. Whitsett · C.R. Dey Division of Pulmonary Biology, Children`s Hospital Medical Center, Cincinnati, Ohio, USA W.H. Colledge Department of Physiology, University of Cambridge, Cambridge, UK M.J. Evans Cardiff School of Biosciences, Cardiff University, Cardiff, UK Present address: J.R. Jones, Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, USA J Mol Med (2002) 80:595-604 DOI 10.1007/s00109-002-0363-1 O R I G I N A L A RT I C L E Ming Du · Julie R. Jones · Jessica Lanier Kim M. Keeling · J. Russell Lindsey Albert Tousson · Zsuzsa Bebök · Jeffrey A. Whitsett Chitta R. Dey · William H. Colledge Martin J. Evans · Eric J. Sorscher · David M. Bedwell Aminoglycoside suppression of a premature stop mutation in a Cftr-/- mouse carrying a human CFTR-G542X transgene Received: 1 February 2002 / Accepted: 17 May 2002 / Published online: 3 July 2002 (c) Springer-Verlag 2002 MING DU received her Ph.D. degree in Biochemistry at Chiba University, Japan. Login to comment
11 To address whether aminoglycosides can suppress a CFTR premature stop mutation in an animal model, we constructed a transgenic mouse with a null mutation in the endogenous CFTR locus (Cftr-/-) that also expressed a human CFTR-G542X cDNA under control of the intestinal fatty acid binding protein promoter. Login to comment
13 Immunofluorescence staining of intestinal tissues from Cftr-/- hCFTR-G542X mice revealed that gentamicin treatment resulted in the appearance of hCFTR protein at the apical surface of the glands of treated mice. Login to comment
16 When taken together, these results indicate that gentamicin, and to a lesser extent tobramycin, can restore the synthesis of functional hCFTR protein by suppressing the hCFTR-G542X premature stop mutation in vivo. Login to comment
17 Keywords Aminoglycoside · Cystic fibrosis · CFTR-G542X transgene · Premature stop mutation Introduction Cystic fibrosis (CF) is the most prevalent autosomal recessive disorder in the Caucasian population, affecting 1 in 2,500 newborns. Login to comment
32 To do this, a transgenic mouse expressing a hCFTR cDNA containing the G542X stop mutation under control of the rat intestinal fatty-acid binding protein (FABP) promoter was constructed. Login to comment
33 We then examined the ability of aminoglycosides to induce hCFTR expression in Cftr-/- hCFTR-G542X mice. Login to comment
35 These results provide evidence that gentamicin (and to a lesser extent, tobramycin) can suppress the hCFTR-G542X mutation in vivo. Login to comment
36 Materials and methods Plasmid construction The plasmid containing hCFTR-G542X under control of the rat FABP promoter was derived from a FABP-hCFTR-WT plasmid [14]. Login to comment
37 The G542X premature stop mutation was introduced by the direct exchange of a 3043 bp BspE1/NcoI fragment from pDB436, yielding the FABP-CFTR-G542X plasmid pDB488. Login to comment
38 In addition to the G542X mutation, this plasmid contains an additional SalI restriction site that was introduced at codon 764 of the CFTR cDNA. Login to comment
40 Generation of transgenic mice A 1.2 kb DNA fragment containing the rat intestinal fatty-acid binding protein (FABP) promoter (kindly provided by Jeffrey Gordon, Washington University, St. Louis) was used to direct expression of the human CFTR cDNA containing the G542X mutation to the intestinal epithelial cells of mice using methods previously described [14]. Login to comment
41 The chimeric FABP-hCFTR-G542X gene construct was microinjected into fertilized oocytes. Login to comment
43 The FABP-hCFTR-G542X transgene was detected by Southern analysis in founder mice using a 2.54 kb BamHI fragment from the hCFTR gene as a probe. Login to comment
45 Animals that expressed the FABP-hCFTR-G542X transgene were then bred with heterozygous Cftr+/Cftrtm1Cam mice (hereafter referred to as Cftr+/- mice). Login to comment
48 Animals that contained single copies of both the Cftrtm1Cam allele and the FABP-hCFTR-G542X transgene were interbred until the NeoR marker was eliminated and the progeny were homozygous for the FABP-hCFTR-G542X transgene. Login to comment
49 Cftr+/- mice that were homozygous for the FABP-hCFTR-G542X transgene were then intercrossed to generate the desired Cftr-/- progeny carrying the transgene. Login to comment
53 Amplification of the FABP-hCFTR-G542X transgene DNA was carried out using the primers DB985 (5'-CAAGATAGAA AGAGGACAGT TGTT-3') and DB986 (5'-TTGAGGGTTG ACATAGGTGC TTGAA-3'). Login to comment
54 This primer pair generated a 1557 bp fragment containing both the G542X mutation and the new SalI site. Login to comment
57 mRNA expression in intestinal tissue To determine whether the FABP-CFTR-G542X transgene was expressed, mRNA was isolated from intestinal tissues of FABP-hCFTR-G542X or FABP-hCFTR-WT transgenic mice. Login to comment
59 The hCFTR-G542X PCR product was digested with the restriction enzyme SalI to demonstrate that the predicted 954 and 603 bp fragments were produced. Login to comment
63 Aminoglycoside treatment Aminoglycoside treatment of homozygous Cftr-/- mice carrying the FABP-hCFTR-G542X transgene was initiated 16 days after birth (1 week before weaning). Login to comment
78 In control experiments, glibenclamide was found to block the forskolin-activated Isc in Cftr+/- mice carrying the hCFTR-G542X transgene. Login to comment
93 In vitro transcription/translation A modified form of a readthrough reporter system previously used to monitor the suppression of stop mutations by aminoglycosides [10] was used to determine whether the aminoglycosides gentamicin and tobramycin could suppress the CFTR-G542X mutation in an in vitro rabbit reticulocyte lysate translation system (Promega TNT system). Login to comment
96 In this study, the readthrough cassette contained the CFTR-G542X premature stop mutation and six upstream and downstream codons from the hCFTR gene. Login to comment
100 Efficient translation termination at the G542X stop mutation produced a 25 kDa protein, while suppression of the stop codon resulted in the synthesis of a 35 kDa protein. Login to comment
102 Suppression of the G542X mutation was expressed as percent readthrough, which was calculated as the [35 kDa protein / (25 kDa + 35 kDa proteins)] × 100. Login to comment
103 Results Genetic characterization of a Cftr-/- mouse expressing the hCFTR-G542X transgene The objective of this study was to determine whether the aminoglycosides gentamicin or tobramycin can suppress a hCFTR premature stop mutation in an animal model. Login to comment
105 As a result, we bred FABP-hCFTR-G542X mice with Cftr+/- mice that carried the HPRT gene inserted in exon 10 of the mouse Cftr gene [4, 15]. Login to comment
106 Male and female Cftr+/- mice that were homozygous for the FABP-hCFTR-G542X transgene were produced and used as breeders for all subsequent experiments. Login to comment
107 From these breeder pairs, offspring homozygous for both the Cftrtm1Cam allele and the FABP-hCFTR-G542X transgene were obtained at a frequency of 25%. Login to comment
108 To confirm the presence of the FABP-hCFTR-G542X transgene in these animals, a PCR reaction with hCFTR-specific primers was carried out using genomic DNA isolated from both the FABP-hCFTR-G542X and FABP-hCFTR-WT transgenic mice. Login to comment
110 As a unique indentifier, the hCFTR-G542X transgene also contained a new SalI restriction site polymorphism that does not alter the amino acid sequence of the hCFTR protein. Login to comment
111 Digestion of the PCR products with SalI produced the expected restriction fragments of 954 and 603 bp from the PCR product from the hCFTR-G542X mouse, but not from the hCFTR-WT mouse. Login to comment
112 This result confirmed the presence of the FABP-hCFTR-G542X transgene. Login to comment
113 We next analyzed the expression of the FABP-hCFTR-G542X transgene. Login to comment
114 Since the FABP promoter is expressed predominantly in intestine [14], mRNA was isolated from intestinal tissues from either hCFTR-G542X or hCFTR-WT transgenic mice. Login to comment
116 As can be seen in Fig. 1, a 1,557 bp fragment could be detected in both the hCFTR-G542X and hCFTR-WT transgenic mice. Login to comment
117 In addition, digestion of the cDNA fragment generated by RT-PCR with SalI site again confirmed that a SalI site was present exclusively in the mouse line carrying the hCFTR-G542X transgene. Login to comment
118 These results confirmed that the FABP-hCFTR-G542X construct is expressed in intestinal tissue from the FABP-hCFTR-G542X mice. Login to comment
119 Pathologic characterization of Cftr-/- mice expressing the hCFTR-G542X transgene Mice homozygous for both the Cftr-/- allele and the FABP-hCFTR-G542X transgene were generally smaller than their heterozygous littermates, as observed in various Cftr-/- mouse models [19]. Login to comment
121 While the survival of the Cftr-/- hCFTR-G542X mice was generally high during the first few weeks after birth, we found that most of these animals died in the days following weaning. Login to comment
123 To determine the primary cause of death of the Cftr-/- hCFTR-G542X mice, a comprehensive histopathologic examination was carried out on several animals. Login to comment
124 In particular, our attention focused on comparing healthy and sick Cftr-/- hCFTR-G542X mice in the days following weaning. Login to comment
127 Some of these findings are il- 598 Fig. 1A, B Generation of Cftr-/- FABP-hCFTR-G542X mice. Login to comment
128 A Schematic diagram of the FABP-hCFTR-G542X construct. Login to comment
129 B PCR from genomic DNA showing the presence of the hCFTR-G542X transgene (left) and an RT-PCR showing that the hCFTR-G542X transgene is expressed in intestinal tissues (right). Login to comment
131 A similar analysis of Cftr+/+ FABP-hCFTR-G542X littermates found that all tissues were normal. Login to comment
132 These results confirmed that the cftr-/- FABP-hCFTR-G542X mice, like other Cftr-/-mouse models, frequently experience intestinal blockage. Login to comment
133 However, little or no intestinal pathology was observed in Cftr-/- hCFTR-G542X mice that were not ill, other than a minimal retention of mucus in the crypts of the small and large intestine (data not shown). Login to comment
135 In preliminary experiments, we found that the subcutaneous administration of gentamicin at a similar dose was well tolerated in the FABP-hCFTR-G542X mice. Login to comment
139 Effect of aminoglycoside treatment on the survival of Cftr-/- hCFTR-G542X mice We first asked whether the subcutaneous administration of the aminoglycosides increased the survival of Cftr-/- mice carrying the hCFTR-G542X transgene. Login to comment
141 As a control for any change in survival of these animals caused by antibacterial effects rather than suppression of the hCFTR-G542X stop mutation, we also treated a control group of mice 599 Fig. 2 Histopathology observed in the jejunum of: A a Cftr+/+ mouse carrying the hCFTR-G542X transgene, and B an ill Cftr-/- mouse carrying the hCFTR-G542X transgene. Login to comment
146 Groups of both treated and untreated Cftr-/- hCFTR-G542X mice had a high rate of survival up to 20 days after birth (Fig. 4). Login to comment
154 hCFTR activity is detected in Cftr-/- hCFTR-G542X mice following aminoglycoside treatment The CFTR protein is a cAMP-activated chloride channel that facilitates transepithelial chloride conductance following its activation by cAMP agonists. Login to comment
156 In intestinal tissue from untreated Cftr+/- hCFTR-G542X mice, we observed a strong increase in transepithelial chloride current that ranged from 6.2 to 26.6 µA/cm2 following forskolin addition, demonstrating the presence of endogenous CFTR activity in these heterozygous animals. Login to comment
157 When in600 Fig. 4 Survival of homozygous Cftr-/- hCFTR-G542X mice. Login to comment
160 A Tracing from the ileum of a heterozygous Cftr +/- mouse carrying the hCFTR-G542X (without aminoglycoside treatment). Login to comment
161 B Tracing from the ileum of a homozygous Cftr-/- hCFTR-G542X mouse (without aminoglycoside treatment). Login to comment
162 C Tracing from the ileum of a homozygous Cftr-/- hCFTR-G542X (following gentamicin treatment). Login to comment
163 D Tracing from the ileum of a homozygous Cftr-/- hCFTR-G542X (following tobramycin treatment). Login to comment
167 The "n" value represents the total number of samples assayed under each condition testinal tissue from untreated Cftr-/- hCFTR-G542X mice was examined following forskolin treatment, no change in the transepithelial chloride current was detected in 13 independent sample s (Figs. Login to comment
171 In Cftr-/- hCFTR-G542X mice treated with gentamicin, we observed a modest increase in Isc upon forskolin addition in 6 of 15 samples. Login to comment
174 In cftr-/- hCFTR-G542X mice treated with tobramycin, we observed an Isc increase in 3 of 11 samples following forskolin addition, which approached statistical significance (P=0.052). Login to comment
176 These results suggest that both gentamicin and tobramycin can restore a low level of hCFTR activity in Cftr-/- hCFTR-G542X mice. Login to comment
177 hCFTR protein can be detected in intestinal glands following aminoglycoside treatment Since our functional analysis detected the presence of cAMP-activated Isc currents in intestinal tissues of Cftr-/- hCFTR-G542X mice following aminoglycoside treatment, we next asked whether the hCFTR protein could be detected. Login to comment
179 In gentamicin-treated Cftr -/- hCFTR-G542X 601 Fig. 7 CFTR immunofluorescence in intestinal tissues. Login to comment
180 Samples from the duodenum of homozygous Cftr-/- hCFTR-G542X mice (harvested from untreated, gentamicin-treated, or tobramycin-treated animals) were incubated with either preimmune serum or CFTR-NBD1 serum. Login to comment
181 Similar samples from heterozygous Cftr+/- animals that lacked the hCFTR-G542X transgene were also examined following gentamicin treatment. Login to comment
186 Furthermore, a positive signal was not observed in tissues from gentamicin-treated Cftr -/- hCFTR-G542X mice when a pre-immune serum was used, or when the hCFTR-specific antiserum was used on samples from mice that had not been treated with gentamicin. Login to comment
187 We also confirmed that a specific signal was not observed in Cftr+/- mice that did not carry the hCFTR-G542X transgene following gentamicin treatment. Login to comment
189 When taken together with the functional data presented above, these results indicate that treatment of Cftr-/- hCFTR-G542X mice with either gentamicin or tobramycin can promote the synthesis of full-length, functional CFTR that localizes to the apical epithelium of the submucosal and mucosal glands of this transgenic mouse. Login to comment
193 To more directly determine the ability of gentamicin and tobramycin to suppress the hCFTR-G542X mutation, we constructed a readthrough reporter construct for use in an in vitro translation system that contains the hCFTR-G542X stop mutation and six flanking codons upstream and downstream of the premature stop mutation (Fig. 8). Login to comment
205 B Example of gentamicin- and tobramycin-mediated suppression of the hCFTR-G542X stop mutation. Login to comment
206 C Quantitation of data shown in B full-length CFTR from a transgene containing the hCFTR-G542X premature stop mutation in vivo. Login to comment
208 To optimize our chance of detecting the appearance of hCFTR protein produced by suppression of the hCFTR-G542X mutation, we administered 34 µg/g gentamicin by subcutaneous injection a once daily. Login to comment
214 Our results also demonstrate for the first time that tobramycin is capable of weakly suppressing the hCFTR-G542X stop mutation in vivo. Login to comment
215 The hCFTR-G542X stop mutation is a UGAG tetranucleotide termination signal. Login to comment
219 Both gentamicin and tobramycin were found to suppress the hCFTR-G542X stop mutation, although gentamicin appeared to mediate this effect much more efficiently than tobramycin. Login to comment
237 In the present study, we used a similar FABP-hCFTR construct to show that aminoglycosides can suppress the hCFTR-G542X mutation. Login to comment
238 Significantly, we found that gentamicin treatment of the FABP-hCFTR-G542X mouse resulted in the appearance of CFTR in the mucosal and submucosal glands, but not in the crypts of Lieberkuhn or the intestinal villi. Login to comment

[hide] Demographics of the UK cystic fibrosis population:... Eur J Hum Genet. 2002 Oct;10(10):583-90. McCormick J, Green MW, Mehta G, Culross F, Mehta A
Demographics of the UK cystic fibrosis population: implications for neonatal screening.
Eur J Hum Genet. 2002 Oct;10(10):583-90., [PMID:12357328]

Abstract [show]
The objective was to determine the composition of the Cystic Fibrosis (CF) Population attending specialist UK CF centres in terms of age, gender, age at diagnosis, genotype and ethnicity. With the planned introduction of the national CF screening programme in the UK, cystic fibrosis transmembrane regulator (CFTR) mutations were compared between different ethnic groups enabling a UK-specific frequency of mutations to be defined. Data were analysed from the patient biographies held in the UK CF Database (see www.cystic-fibrosis.org.uk). The currently registered population of 5,274 CF patients is 96.3% Caucasian with a male preponderance that significantly increases with age. The majority of the 196 non-Caucasian CF patients are from the Indian Subcontinent (ISC), of which one in 84 UK CF patients are of Pakistani origin. The commonest CFTR mutation, deltaF508, is found in 74.1% of all CF chromosomes. In the Caucasian CF population, 57.5% are deltaF508 homozygotes but the UK ISC CF population with only 24.7%, has significantly fewer deltaF508 homozygotes patients (95% confidence interval (CI) 0.2-0.4). The distribution of Caucasian patients with deltaF508/deltaF508, deltaF508/Other and Other/Other does not fit the expected distribution with a Hardy-Weinberg model unless those patients without a detected mutation are excluded (P<0.001). The UK CF Database has shown the UK CF population to have distinct characteristics separate from the North American and European CF Registries. The ISC group contains many mutations not recognised by current genetic analysis, and one in four ISC patients have no CFTR mutations identified. The CFTR analysis proposed for the screening programme would detect 96% of patients registered in the database, but is unlikely to achieve the desired >80% detection rates in the ethnic minority groups. Screen-positive, non-Caucasian infants without an identifiable CFTR mutation should be referred for a sweat test and genetic counselling when serum trypsinogen concentrations remain elevated after birth.

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79 It is envisaged that the proposed screening programme will be based on a three-stage protocol.6 In Table 3 Genotypes of the UK CF Caucasian and ISC populations Percentage of Percentage of genotyped UK CF genotyped UK CF Caucasian population ISC population Genotype n=4753 (%) n=78 (%) DF508/DF508 57.5 24.7 DF508/Unknown 11.5 3.5 DF508/G551D 5.1 0.0 DF508/G542X 2.8 0.0 Unknown/Unknown 2.7 27.1 DF508/621+1G?T 2.0 1.2 DF508/R117H 2.0 0.0 DF508/1898+1G?A 1.0 0.0 DF508/1717-G?A 0.9 0.0 DF508/N1303K 0.8 0.0 DF508 DI507 0.8 0.0 DF508/R553X 0.6 0.0 DF508/R560T 0.6 0.0 DF508/Q493X 0.5 0.0 G551D/Unknown 0.4 0.0 Other/Other 2.8 15.3* DF508/Other 6.7 0.0 Y569D/Y569D 0.0 8.2 L218X/L218X 0.0 3.5 1161delC/1161delC 0.0 3.5 R709X/V456A 0.0 2.4 G542X/G542X 0.4 2.4 Other/Unknown 1.0 3.5 The shaded areas represent the commonest genotypes in the ISC population. Login to comment
85 Table 4 The commonest CFTR mutations in the UK Genotypes UK CF population Genotyped UK Caucasian CF Genotyped UK CF ISC (n=9866 chromosomes) population (n=9506 chromosomes) population (n=156 chromosomes) CFTR mutation gene frequency per 1000 genes gene frequency per 1000 genes gene frequency per 1000 genes DF508 741.0 752.0 294.9 G551D 33.7 34.3 12.8 G542X 18.5 18.4 25.6 R117H 12.5 12.7 0.0 621+1G?T 12.7 12.7 6.4 1717-1G?A 5.8 5.8 0.0 1898+1G?A 5.7 5.9 0.0 N1303K 5.6 5.4 0.0 DI507 4.8 5.0 0.0 R560T 4.2 4.3 0.0 R553X 3.3 3.4 0.0 1154insTC 3.2 3.3 0.0 Q493X 2.8 2.9 0.0 3659delC 2.8 2.9 0.0 E60X 2.4 2.4 0.0 W1282X 2.7 2.7 0.0 P67L 2.1 2.1 0.0 G85E 2.1 2.0 0.0 V520F 1.6 1.7 0.0 1078delT 1.3 1.4 0.0 Y569D 1.5 0.0 96.2 L218X 0.6 0.0 38.5 1161delC 0.7 0.1 38.5 R1162X 0.9 0.6 19.2 R709X 0.4 0.2 12.8 3849+10kbC?T 1.2 0.8 19.2 S549R* 0.6 0.0 0.0 *S549R mutations appear in the non-Caucasian but not the ISC subgroup. Login to comment
96 There are similar frequencies for the more common mutations between our UK Caucasian CF data (Table 4) and the Schwarz data (respectively, G551D 3.43% and 3.08%; G542X 1.84% and 1.68%; 621+1G?A 1.27% and 0.93%; 1717-1G?A 0.58% and 0.57%). Login to comment
97 In North America, DF508 accounts for 71.2%, with G542X (2.4%), G551D (2.4%), W1282X (1.4%), N1303K (1.3%) and R553X (0.9%).8 Genotype frequencies in CF have previously been shown to fit a Hardy - Weinberg model in a smaller regional UK study.9 In the current study, we find that the genotype frequencies only satisfy the Hardy-Weinberg equilibrium provided we exclude those without an identified CFTR mutation in the Other/Other category. Login to comment
101 When compared with a European CFTR geographic distribution,10 the UK CF patients possess a greater proportion of DF508, G551D and 621+1G?T mutations, and a smaller proportion of G542X, N1303K, W1282X and R1162X mutations. Login to comment
102 In France, the five most common genotypes were DF508/DF508 (47.8%), DF508 /G542X (3.4%), DF508/N1303K (2.7%), DF508 /1717-1G?A (2.1%) and DF508/2789+5G?A (1.5%) (Desgeorges M, personal communication) which is different to the commonest genotypes found in the UK population (Table 3). Login to comment
103 N1303K and G542X occur at a frequency of around 5% in Italy.11 In Germany, a study of 658 CF families revealed mutation frequencies of R553X (1.8%), N1303K (1.3%), G542X (1.1%), G551D (0.8%) and R347P (0.8%).12 The frequency of CFTR mutations recorded for just over 1000 patients for the Irish CF Database include G551D in 7%, R117H in 2% and DF508 in 72% of patients.13 In the white South African population, a paper based on 192 patients found that DF508 accounts for 76% of the mutations with 3272-26A?G (4%), 394delTT (3.6%) and G542X (1.3%) the other most common mutations.14 It is suggested that the 3272-26A?G and 394delTT mutations are more common due to a founder effect in white South Africans of European descent. Login to comment

[hide] Correction of G551D-CFTR transport defect in epith... Br J Pharmacol. 2002 Oct;137(4):504-12. Zegarra-Moran O, Romio L, Folli C, Caci E, Becq F, Vierfond JM, Mettey Y, Cabrini G, Fanen P, Galietta LJ
Correction of G551D-CFTR transport defect in epithelial monolayers by genistein but not by CPX or MPB-07.
Br J Pharmacol. 2002 Oct;137(4):504-12., [PMID:12359632]

Abstract [show]
1. This study compares the effect of three chemically unrelated cystic fibrosis transmembrane conductance regulator (CFTR) activators on epithelial cell monolayers expressing the G551D-CFTR mutant. 2. We measured Cl(-) transport as the amplitude of short-circuit current in response to the membrane permeable cAMP analogue 8-(4-chlorophenylthio)adenosine-3'-5'-cyclic monophosphate (CPT-cAMP) alone or in combination with a CFTR opener. The correction of G551D-CFTR defect was quantified by comparison with maximal activity elicited in cells expressing wild type CFTR. To this end we used Fisher rat thyroid (FRT) cells transfected with wild type or G551D CFTR, and primary cultures of human nasal epithelial cells. 3. In both types of epithelia, cAMP caused activation of Cl(-) transport that was inhibited by glibenclamide and not by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid. After normalising for CFTR expression, the response of FRT-G551D epithelia was 1% that of wild type monolayers. 4. Addition of genistein (10-200 micro M), but not of 8-cyclopentyl-1,3-dipropylxanthine (CPX, 1-100 micro M) or of the benzo[c]quinolizinium MPB-07 (10-200 micro M) to FRT-G551D epithelia pre-treated with cAMP, stimulated a sustained current that at maximal genistein concentration corresponded to 30% of the response of wild type epithelia. 5. The genistein dose-response curve was bell-shaped due to inhibitory activity at the highest concentrations. The dose-dependence in G551D cells was shifted with respect to wild type CFTR so that higher genistein concentrations were required to observe activation and inhibition, respectively. 6. On human nasal epithelia the correction of G551D-CFTR defective conductance obtained with genistein was 20% that of wild type. The impressive effect of genistein suggests that it might correct the Cl(-) transport defect on G551D patients.

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70 The second mutation is presently unknown, but is not one of the 15 most frequent mutations found in the CF patients of Northeast Italy, namely F508del, I507del, R1162X, 2183AA4G, N1303K, 3849+10KbC4T, G542X, 1717-1G4A, R553X, Q552X, G85E, 711+5G4A, 3132delTG, 2789+5G4A, W1282X. Login to comment

[hide] Standards and guidelines for CFTR mutation testing... Genet Med. 2002 Sep-Oct;4(5):379-91. Richards CS, Bradley LA, Amos J, Allitto B, Grody WW, Maddalena A, McGinnis MJ, Prior TW, Popovich BW, Watson MS, Palomaki GE
Standards and guidelines for CFTR mutation testing.
Genet Med. 2002 Sep-Oct;4(5):379-91., [PMID:12394352]

Abstract [show]
One mission of the ACMG Laboratory Quality Assurance (QA) Committee is to develop standards and guidelines for clinical genetics laboratories, including cytogenetics, biochemical, and molecular genetics specialties. This document was developed under the auspices of the Molecular Subcommittee of the Laboratory QA Committee by the Cystic Fibrosis (CF) Working Group. It was placed on the "fast track" to address the preanalytical, analytical, and postanalytical quality assurance practices of laboratories currently providing testing for CF. Due to the anticipated impact of the ACMG recommendation statement endorsing carrier testing of reproductive couples, it was viewed that CF testing would increase in volume and that the number of laboratories offering CF testing would also likely increase. Therefore, this document was drafted with the premise of providing useful information gained by experienced laboratory directors who have provided such testing for many years. In many instances, "tips" are given. However, these guidelines are not to be interpreted as restrictive or the only approach but to provide a helpful guide. Certainly, appropriately trained and credentialed laboratory directors have flexibility to utilize various testing platforms and design testing strategies with considerable latitude. We felt that it was essential to include technique-specific guidelines of several current technologies commonly used in laboratories providing CF testing, since three of the four technologies discussed are available commercially and are widely utilized. We take the view that these technologies will change, and thus this document will change with future review.

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50 Depending upon the ethnic group, these mutation frequencies may be difficult to obtain (Table 1).5-7 CF 2.8.1 The most common mutations in the Ashkenazi Jewish population have been described.8-10 These include W1282X, ⌬F508, G542X, N1303K, and 3849 ϩ 10 kbCϾT. Login to comment
307 ⌬F508 R553X R1162X 2184delA 3120ϩ1GϾA ⌬I507 G542X G551D W1282X N1303K 621ϩ1GϾT R117H 1717-1GϾA A455E R560T G85E R334W R347P 711ϩ1GϾT 1898ϩ1GϾA 1078delT 3849ϩ10kbCϾT 2789ϩ5GϾA 3659delC I148T CF 3.3.2 Inclusion of the common R117H mutation in the test panel screens for CBAVD as well as for CF: The phenotypic consequences of the R117H mutation are modulated in cis by the 5/7/9T polypyrimidine tract in intron 8 such that R117H/7T is associated with CBAVD and R117H/5T is associated with CF.34 Moreover, the 5T allele is associated as a trans mutation in CBAVD.35 It is recommended that the 5/7/9T variant be excluded from the routine carrier screen but tested as a reflex for carriers shown to be heterozygous for the R117H mutation. Login to comment

[hide] Splice mutation 1811+1.6kbA>G causes severe cystic... J Med Genet. 2002 Nov;39(11):e73. Reboul MP, Bieth E, Fayon M, Biteau N, Barbier R, Dromer C, Desgeorges M, Claustres M, Bremont F, Lacombe D, Iron A
Splice mutation 1811+1.6kbA>G causes severe cystic fibrosis with pancreatic insufficiency: report of 11 compound heterozygous and two homozygous patients.
J Med Genet. 2002 Nov;39(11):e73., [PMID:12414835]

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127 Genotype-phenotype data are available especially for common genotypes,2 but information remains scarce or lacking for genotypes of compound heterozygotes with a rare mutation and homozygotes for a rare mutation.3 Among rare CFTR gene mutations, 1811+1.6kbA>G is practically absent in CF patients from other parts of France, but it is not rare in patients genotyped in the south west of France, occurring in fourth place after F508del, G542X, and N1303K. Login to comment
140 RESULTS The results of genotyping the 13 CF patients carrying the mutation 1811+1.6kbA>G were: two homozygotes for 1811+1.6kbA>G/1811+1.6kbA>G and 11 compound heterozygotes for 1811+1.6kbA>G and for another CFTR mutation, that is, F508del (n=6), N1303K (n=2), G542X (n=1), 2183 AA>G (n=1), and W1063X (n=1). Login to comment
148 Key points • The splice mutation 1811+1.6kbA>G of the CFTR gene is the fourth most frequent mutation (after F508del, G542X, and N1303K) in CF patients from the south west of France. Login to comment
157 This is the case for nonsense and frameshift mutations always associated with severe CF with PI: they come into class I (for instance, G542X) when they lead to an absence of functional CFTR and into the "new" class V (for instance, Q1412X) when they cause the presence of a functional but unstable CFTR at the apical membrane. Login to comment

[hide] Cystic fibrosis and Chiari type I malformation: au... Pediatr Dev Pathol. 2003 Jan-Feb;6(1):88-93. Epub 2002 Nov 6. Rakheja D, Xu Y, Burns DK, Veltkamp DL, Margraf LR
Cystic fibrosis and Chiari type I malformation: autopsy study of two infants with a rare association.
Pediatr Dev Pathol. 2003 Jan-Feb;6(1):88-93. Epub 2002 Nov 6., [PMID:12415481]

Abstract [show]
Cystic fibrosis (CF), an epithelial cell transport disorder caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, is not generally associated with malformations of the central nervous system (CNS). This report describes two African-American children who died at less than 2 years of age with known Chiari I malformations and were found, unexpectedly at autopsy, to have the classic pancreatic and respiratory changes of CF. Both patients had suffered from failure to thrive that had been attributed to their CNS malformations. One child also had recurrent pneumonia and died with Pseudomonas sepsis. Mutational analysis for > 70 common CFTR mutations identified a single delta F508 mutation in one patient and a single 3120+1G to A mutation in the other. Their second CFTR mutations were not identified. The association of CF with Chiari I malformation is not likely to be purely coincidental, as the probability of such an occurrence in African-Americans is greater than one in 7,500,000 patients. It is possible that the CFTR gene may play a previously unrecognized role in CNS development. Alternatively, this CNS abnormality may be acquired due to the metabolic and electrolyte imbalances that characteristically occur in CF.

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101 In two patients, the second mutations could not be identified, while the remaining two patients showed G542X and W1282X as their second mutation, respectively. Login to comment

[hide] Survey of CF mutations in the clinical laboratory. BMC Clin Pathol. 2002 Nov 19;2(1):4. Huber K, Mirkovic B, Nersesian R, Myers A, Saiki R, Bauer K
Survey of CF mutations in the clinical laboratory.
BMC Clin Pathol. 2002 Nov 19;2(1):4., 2002-11-19 [PMID:12437773]

Abstract [show]
BACKGROUND: Since it is impossible to sequence the complete CFTR gene routinely, clinical laboratories must rely on test systems that screen for a panel of the most frequent mutations causing disease in a high percentage of patients. Thus, in a cohort of 257 persons that were referred to our laboratory for analysis of CF gene mutations, reverse line probe assays for the most common CF mutations were performed. These techniques were evaluated as routine first-line analyses of the CFTR gene status. METHODS: DNA from whole blood specimens was extracted and subjected to PCR amplification of 9 exons and 6 introns of the CFTR gene. The resulting amplicons were hybridised to probes for CF mutations and polymorphisms, immobilised on membranes supplied by Roche Molecular Systems, Inc. and Innogenetics, Inc. Denaturing gradient gel electrophoresis and sequencing of suspicious fragments indicating mutations were done with CF exon and intron specific primers. RESULTS: Of the 257 persons tested over the last three years (referrals based on 1) clinical symptoms typical for/indicative of CF, 2) indication for in vitro fertilisation, and 3) gene status determination because of anticipated parenthood and partners or relatives affected by CF), the reverse line blots detected heterozygote or homozygote mutations in the CFTR gene in 68 persons (26%). Eighty-three percent of those affected were heterozygous (47 persons) or homozygous (10 persons) for the DeltaF508 allele. The only other CF-alleles that we found with these tests were the G542X allele (3 persons), the G551D allele (3 persons), the 3849+10kb C-T allele (2 persons) the R117H allele (2 persons) and the 621+1G-T allele (1 person).Of the fifteen IVS8-5T-polymorphisms detected in intron 8, seven (47%) were found in males referred to us from IVF clinics. These seven 5T-alleles were all coupled with a heterozygous DeltaF508 allele, they make up 35% of the males with fertility problems (20 men) referred to us. CONCLUSIONS: In summary, the frequency of CF chromosomes in the cohort examined with these tests was 26%, with the DeltaF508 allele affecting 83% of the CF chromosomes. It is a substantial improvement for routine CF diagnostics to have available a test system for 30 mutations plus the polypyrimidine length variants in intron 8. Our results show that this test system allows a routine first-line analyses of the CFTR gene status.

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8 The only other CF-alleles that we found with these tests were the G542X allele (3 persons), the G551D allele (3 persons), the 3849+10kb C-T allele (2 persons) the R117H allele (2 persons) and the 621+1G-T allele (1 person). Login to comment
36 F508C, I507V, I506V polymorphism exon 11 1717-1G → A, G542X, S549N, G551D, R553X, R560T exon 20 W1282X exon 21 N1303K intron 19 3849+10kb C → T Innogenetics assay: exon 3 394delTT, G85E, E60X exon/intron 4 621+1G-T, R117H exon 7 1078delT, R347P, R334W exon 13 2143delT, 2183AA-G, 2184delA exon 19 R1162X, 3659delC intron 5 711+5G-A intron8/exon 9 A455E,, 5T,7T,9T intron 14b 2789+5G-A intron 19 3849+10kb C-T Table 2: Genotypes of patients with mutations, final results Group 1) (patients with symptoms typical for/indicative of CF) No. Login to comment
38 IRT, normal sweat test f 0 7T/9T DF508/3849+10kb C-T x 2 CF, substantiation f 0 9T/9T 621+1G-T/621+1G-T 3 CF, substantiation f 1 9T/9T DF508/DF508 x 4 CF, substantiation f 5 9T/9T DF508/DF508 x x x 5 CF, substantiation f 7 9T/9T DF508/G542X x x 6 CF, substantiation, rec. diarrhoe, pancreas insufficiency, pos. sweat test f 8 9T/9T DF508/DF508 x 7 CF, substantiation f 12 9T/9T DF508/DF508 x 8 CF, substantiation f 13 9T/9T DF508/DF508 x 9 f 13 7T/9T DF508/WT 10 CF, substantiation f 16 9T/9T DF508/G542X 11 indicative linkage analysis f 22 7T/9T DF508/WT x 12 f 24 7T/9T DF508/WT x 13 bronchiectasis, bronchopulmonal infections since infancy f 28 7T/9T DF508/3849+10kbC-T x 14 pos. sweat test f 28 9T/9T DF508/WT x 15 typical clinic, pos. sweat test f 31 7T/9T DF508/WT x x 16 f 32 7T/7T 3849+10kb C-T/WT 17 pulmonal course typical of CF f 32 7T/9T DF508/WT x x x 18 f 34 7T/7T G551D/WT x x 19 f 41 7T/7T DF508/WT 20 CF, substantiation f 56 7T/9T DF508/3849+10kb C-T x 21 22 CF, substantiation m 0 9T/9T DF508/DF508 x 23 m 1 7T/9T DF508/WT x 24 impaired lung function, intestinal complications m 3 7T/9T DF508/WT x x 25 CF, substantiation m 5 9T/9T DF508/DF508 26 m 12 7T/7T G551D/WT x x 27 CF, substantiation m 17 9T/9T DF508/DF508 28 m 18 7T/7T R117H/WT&1466delAATT/1466delAATT 1466delAATT x 29 pos sweat test m 20 7T/9T DF508/WT 30 CF, substantiation m 25 9T/9T DF508/DF508 31 . Login to comment
40 infect., azoospermia, pancreatitis m 31 9T/9T DF508/WT 35 CF, substantiation m 33 9T/9T DF508/DF508 x 36 m 33 7T/9T DF508/WT 37 m 33 7T/9T DF508/WT 38 m 38 7T/9T R117H/G542X Group 2a) (Patients from IVF clinics) No. Login to comment
41 : Diagnosis Sex Age Intron 8 16 mut. 29 mut.b seq.c DGGEd 39 m 24 7T/9T WT 40 m 25 9T/9T WT 41 m 28 5T/9T DF508/WT x 42 m 28 5T/9T DF508/WT 43 m 29 5T/9T DF508/WT x x x 44 m 30 7T/7T WT x 45 m 31 5T/9T DF508/WT x x x 46 m 31 7T/7T WT x 47 m 31 7T/9T WT x 48 m 33 7T/9T DF508/WT x 49 m 34 7T/7T WT x 50 m 34 9T/9T DF508/WT x 51 m 35 7T/9T G542X/WT 52 m 36 5T/9T DF508/WT ents, respectively) had given informed consent for genetic analysis as required by the Austrian law. Login to comment
50 : Diagnosis Sex Age Intron 8 16 mut. 29 mut.b seq.c DGGEd 59 f 42 7T/7T 3849+10kb C-T/WT x 60 f 15 7T/9T DF508/WT x 61 f 20 7T/9T DF508/WT x 62 f 23 7T/9T DF508/WT x 63 f 25 7T/9T DF508/WT x x x 64 f 26 7T/9T DF508/WT 65 f 32 7T/9T DF508/WT x 66 f 40 7T/9T DF508/WT x x 67 f 65 9T/9T DF508/WT 68 f 30 7T/9T DF508/WT x 69 m 14 9T/9T DF508/WT x 70 m 16 7T/9T DF508/WT 71 m 25 7T/9T DF508/WT x x x 72 m 28 5T/9T DF508/WT x 73 m 32 7T/9T DF508/WT x x 74 m 45 7T/9T DF508/WT x x 75 m 48 7T/9T DF508/WT x 76 m 69 7T/9T DF508/WT 77 m 30 7T/9T G542X/WT x x 78 m 15 7T/7T G551D/WT x Details for diagnoses, number of mutations analysed, methods used, and other specifics for individuals with found mutations within the three groups are shown. Login to comment
95 Among 114 children < 18 yrs in group 1), we found 9 patients to be homozygote for ∆F508, two compound heterozygote for ∆F508/G542X, one compound heterozygote for ∆F508/3849+10kbC-T, five heterozygote for ∆F508, one G551D/WT, one R117H/WT, one homozygote for 621+1G-T, and one girl with 5T/7T alleles in intron 8 (total of 18% with mutations). Login to comment
96 Twenty-two percent of the adults in group 1) had CFTR mutations, namely two ∆F508/∆F508, thirteen ∆F508/ WT, one compound for R117H/WT and 1466delAATT (frameshift mutation in exon 9), one R117H/G542X, one G551D/WT, one 3849+10kb C-T/WT, one compound heterozygote for ∆F508/1248+1 A → G (splice mutation in intron 7), and two individuals with ∆F508/3849+10kb C-T. Login to comment
102 Nine of these men (45%) had normal alleles (and a benign thymidine polymorphism in intron 8) at the loci analysed, seven men had a genotype of 5T/9T with ∆F508/WT, one showed 7T/9T with ∆F508/ WT, one had 9T/9T with ∆F508/WT, and one had 7T/9T with G542X/WT. Login to comment
106 Of 54 individuals in group 3, tested because their partners or relatives had CFTR mutations, 37% had mutated alleles: seventeen persons had the genotype ∆F508/WT, one had G551D/WT, one had 3849+10kb C-T, and one person had G542X/WT (table 2). Login to comment
116 In comparison to CF mutation-frequencies in some European countries, the CF alleles we found (>2%) with our tests show the following distribution in our cohort: ∆F508: 83% (Romania:27%, Switzerland: 43%, Denmark: 87,2% [19]); G542X: 4,4% (France: 3,1%, Italy: 4,8%, Spain: 7,7%); G551D: 4,4% (UK: 3,1%, Czechia: 4,0%, Ireland: 6,9%), 3849+10kbC-T: 2,9% (Germany: 1,2%, Poland: 2,6%, Latvia: 12,5%), R117H: 2,9% (Greece: 1,2%, Ireland: 2,0%, Norway: 3,0%) Because we participate in the European Quality assessment trial for Cystic Fibrosis, we could evaluate the quality of the Roche Amplicor CF test in this regard as well. Login to comment

[hide] Highest heterogeneity for cystic fibrosis: 36 muta... Am J Med Genet. 2002 Dec 1;113(3):250-7. Kilinc MO, Ninis VN, Dagli E, Demirkol M, Ozkinay F, Arikan Z, Cogulu O, Huner G, Karakoc F, Tolun A
Highest heterogeneity for cystic fibrosis: 36 mutations account for 75% of all CF chromosomes in Turkish patients.
Am J Med Genet. 2002 Dec 1;113(3):250-7., 2002-12-01 [PMID:12439892]

Abstract [show]
We analyzed the CFTR locus in 83 Turkish cystic fibrosis patients to identify mutations, haplotypes, and the carrier frequency in the population. We detected 36 different mutations in 125 (75%) of the total 166 CF chromosomes. Seven novel mutations were identified: four missense (K68E, Q493P, E608G, and V1147I), two splice-site (406 -3T > C and 3849 +5G > A), and one deletion (CFTRdele17b,18). The data showed that the Turkish population has the highest genetic heterogeneity at the CFTR locus reported so far. The results of this thorough molecular analysis at the CFTR locus of a population not of European descent shows that CF is not uncommon in all such populations. The large number of mutations present, as well as the high heterogeneity in haplotypes associated with the mutations suggests that most of the mutations have persisted for a long time in the population. Consistently, the carrier frequency is assessed to be high, indicating that the disease in the population is ancient.

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80 Haplotypes Associated With the Mutations Identified in 83 Turkish CF Patients* Mutation Total number of alleles Number of alleles Number of patients Haplotypes Homo Hetero DF508 39 (23.5) 6 7 23 M 28 13 1 0 1 6 7 23 M 30 13 1 0 1 6 9 23 M 31 13 1 0 1 6 7 23 M 31 13 11 4 3 6 7 23 M 7 17 2 0 2 6 7 16 M 31 13 3 1 1 6 7 17 M 31 13 17 5 7 6 7 17 M 32 13 3 1 1 1677delTA 12 (7.2) 7 7 16 V 30 13 12 5 2 2183AA > G 7 (4.2) 7 7 16 M 30 13 1 0 1 7 9 16 M 31 13 4 2 0 7 7 16 M 32 13 2 1 0 G542X 6 (3.6) 6 7 23 M 32 13 6 3 0 F1052V 5 (3.0) 6 7 17 M 7 13 4 1 2 7 5 17 M 7 17 1 0 1 W1282X 5 (3.0) 7 7 17 M 7 17 4 1 2 7 7 17 M 7 18 1 0 1 E92K 4 (2.4) 7 7 16 V 46 13 3 1 1 7 7 17 V 46 13 1 0 1 1525 À 1G > A 4 (2.4) 7 7 17 M 7 17 4 2 0 2789 þ 5G > A 4 (2.4) 7 9 17 M 7 17 3 1 1 7 5 17 M 7 17 1 0 1 N1303K 4 (2.4) 7 7 23 M 31 13 2 0 2 6 7 22 M 30 13 1 0 1 6 7 23 M 30 13 1 0 1 A46D 3 (1.8) 6 9 23 M 31 13 1 0 1 6 7 23 M 31 13 2 1 0 2184insA 3 (1.8) 7 5 17 V 30 13 1 0 1 7 7 16 V 30 13 2 0 2 R1070Q 3 (1.8) 7 7 16 M 31 13 1 0 1 7 7 17 M 31 13 2 0 2 Q493Pa 2 (1.2) 6/7 5 16 M 46 13 2 1 0 3849 þ 5G > Aa 2 (1.2) 7 7 16 M 31 13 2 1 0 CFTRdele17b,18a 2 (1.2) 6 9 16 V - - 2 1 0 K68Ea 1 (0.6) 6 9 17 M 7 13 1 0 1 R74W 1 (0.6) 6 7 16 M 32 16 1 0 1 306delTAGA 1 (0.6) 7 7 16 M 7 17 1 0 1 D110H 1 (0.6) 7 9 16 V 30 13 1 0 1 I125T 1 (0.6) 6 7 23 V 7 16 1 0 1 406 À 3T > Ca 1 (0.6) 7 7 16 V 33 17 1 0 1 I148T 1 (0.6) 6/7 7 16/17 M 7 17/23 1 0 1 621 þ 1G > T 1 (0.6) 6 7 21 V 31 13 1 0 1 R347P 1 (0.6) 7 9 17 V 30 13 1 0 1 S466X 1 (0.6) 7 7 23 M 33 13 1 0 1 L571S 1 (0.6) 7 7 16 V 29 13 1 0 1 1717 À 1G > A 1 (0.6) 7 9 17 M 7 16 1 0 1 E608Ga 1 (0.6) 7 9 16 M/V 29/31 13 1 0 1 2043delG 1 (0.6) 7 9 17 M 7 17 1 0 1 P1013L 1 (0.6) 6 5 16 M 21 18 1 0 1 R1066L 1 (0.6) 7 7 17 M 7 13 1 0 1 3129del4 1 (0.6) 7 7 16 V 29 13 1 0 1 V1147Ia 1 (0.6) 6 7 17 M 33 17 1 0 1 S1235R 1 (0.6) 6 7 17 M 39 13 1 0 1 CFTRdele2,3 1 (0.6) 7 7 16 V 33 13 1 0 1 Total 125 (75) 125 32 61 *The order of the polymorphisms is IVS6GATT, Tn, IVS8CA, M470V, IVS17BTA and IVS17BCA. Login to comment
159 All of the mutations that are also frequent in other populations, with the exception of G542X and 1525 À 1G > A, were found to be associated with more than one haplotype, indicating that these mutations have persisted for a long time in our population. Login to comment

[hide] Genes in the vicinity of CFTR modulate the cystic ... Hum Genet. 2003 Jan;112(1):1-11. Epub 2002 Oct 3. Mekus F, Laabs U, Veeze H, Tummler B
Genes in the vicinity of CFTR modulate the cystic fibrosis phenotype in highly concordant or discordant F508del homozygous sib pairs.
Hum Genet. 2003 Jan;112(1):1-11. Epub 2002 Oct 3., [PMID:12483292]

Abstract [show]
Cystic fibrosis (CF) is the most common severe autosomal recessive disease among Caucasians and is caused by lesions within the cystic fibrosis transmembrane conductance regulator ( CFTR) gene. The variability of CF disease severity suggests the effect of modifying factors. Thirty-four highly concordant and highly discordant F508del homozygous sib pairs, who have been selected out of a group of 114 pairs for extreme disease phenotypes by nutritional and pulmonary status, were typed at single nucleotide polymorphisms (SNPs) and short tandem repeat polymorphisms (STRPs) in the 24-cM CFTR-spanning region between D7S525 and D7S495. Allele frequencies differed significantly at D7S495, located within a 21-cM distance 3' of CFTR, comparing concordant mildly affected, concordant severely affected and discordant sib pairs, as judged by hypothesis-free permutation analysis by Monte Carlo simulation. A rare haplotype of two SNPs within the leptin gene promotor was found exclusively among the concordant mildly affected pairs. All concordant sib pairs shared the paternal F508del chromosome between CFTR and D7S495, whilst the cohort of discordant sib pairs inherited equal proportions of recombined and non-recombined parental chromosomes. We conclude that disease manifestation in CF is modulated by loci in the partially imprinted region 3' of CFTR that determine stature, food intake and energy homeostasis, such as the Silver-Russel-Syndrome candidate gene region and LEP.

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11 The frequent CFTR mutations, F508del, N1303K, and G542X, are associated with several intragenic STRP haplotypes, but occur on the same flanking SNP haplotype (Dörk et al. 1992; Sereth et al. 1993; Morral et al. 1993; Cuppens et al. 1994; Morral et al. 1996). Login to comment

[hide] A clinical perspective of cystic fibrosis and new ... Am J Med Genet A. 2003 Jan 30;116A(3):262-7. Kulczycki LL, Kostuch M, Bellanti JA
A clinical perspective of cystic fibrosis and new genetic findings: relationship of CFTR mutations to genotype-phenotype manifestations.
Am J Med Genet A. 2003 Jan 30;116A(3):262-7., 2003-01-30 [PMID:12503104]

Abstract [show]
The present report describes several aspects of the relationship of mutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene to phenotype expression of the disease including several clinical vignettes from the authors' experience. The genotype-phenotype relationships in CF are complex, and are affected by many factors, including pollution, smoking, bacterial infection, malnutrition, and certain therapeutic agents. The number of CFTR mutations is growing continuously and rapidly, and more than 1,000 mutations have been discovered so far. From a genetic point of view, the deltaF508 mutation is not only the most frequently encountered but also the most severe genetic lesion for homozygotes. The great clinical variability observed in patients with CF, particularly the severity of lung disease, involvement of the pancreas, and male infertility, are beginning to be better understood through the knowledge, although incomplete, of CFTR mutations and their phenotype expressions. This knowledge has had very significant research and clinical applications in all dimensions of the CF problem. It has not only contributed to the enhancement of better diagnosis and clinical management, but it also has opened new and unanticipated lines of investigation and research.

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12 The frequency of other mutations, for example R553X, G542X, and 1717-1(G-A), is estimated to range from 1-3% [Cystic Fibrosis Genetic Analysis Consortium, 1990, 1994]. Login to comment
84 At the age of 60, genetic testing indicated two mutations H1282X (severe) and A 445E (mild), confirming the CF diagnosis as a compound heterzygote with normal alleles for D F508, -G551D, -R553X, -G542X, and N1303K [Kulczycki et al., 1998]. Login to comment

[hide] Cystic fibrosis transmembrane regulator gene mutat... J Trop Pediatr. 2002 Dec;48(6):348-50. Eskandarani HA
Cystic fibrosis transmembrane regulator gene mutations in Bahrain.
J Trop Pediatr. 2002 Dec;48(6):348-50., [PMID:12521276]

Abstract [show]
A genotypic study was undertaken to characterize the cystic fibrosis transmembrane regulator gene mutations (CFTR) in the Bahraini cystic fibrosis (CF) population using a polymerase chain reaction-based direct gene test to search for 15 common CF mutations amongst Arabs. During the period October 2000 to May 2001, 19 patients (12 males and seven females; aged at time of study between 4 months and 14 years with a mean age of 5.4 +/- 4.3 years) from 13 families were recruited in the study. Patients were diagnosed as having CF, based on a typical clinical picture and sweat chloride levels > 60 mmol/l and were screened for CFTR mutations. The rate of consanguinity among the families was 77 per cent. Eight mutations were detected in 21 of the 26 alleles examined. The overall detection rate was approximately 81 per cent. The allele frequency of the eight mutations was estimated to be approximately 73 per cent. There was no specific phenotypic pattern that correlated with a specific genotype. All families except two were of Bahraini origin. Of the eight mutations detected, four were common among Bahrainis (2043delG > 548A --> T > 4041C --> G = deltaF508, in order of decreasing frequency), accounting for 66 per cent of the Bahraini CF alleles. However, we also detected four different heterozygous mutations, namely: 1161delC, 1756G -->T, 3120 + 1G --> A, and 3661A --> T, accounting for 16 per cent of the Bahraini CF alleles.

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25 Isolation and PCR amplification of genomic DNA Genomic DNA was extracted from leucocytes according to standard procedures.10 PCR amplification of DNA was performed by preparation of a 50-µl reaction mixture that contained appropriate primers using standard protocols.4 Mutation analysis All patients were screened for 15 common mutations amongst Arabs by restriction enzyme digestion analysis with appropriate enzymes according to specific protocols4,5 and/or using the amplification refractory mutation system (ARMS-PCR) technique.11 These mutations were: 406-2A→G (intron 3), 425del42 (exon 4), 475G→T (exon 4), 548A→T (exon 4), 1161delC (exon 7), 1548delG (exon10), F508 (exon 10), G542X (exon 11), 2043delG (exon 13), 3120+1G→A (intron 16), 3661A→T (exon 19), 3849+10KbC→T (intron 19), I1234V (exon 19), W1282X (exon 20), and N1303K (exon 21). Login to comment

[hide] Extensive sequencing of the cystic fibrosis transm... Genet Med. 2003 Jan-Feb;5(1):9-14. Strom CM, Huang D, Chen C, Buller A, Peng M, Quan F, Redman J, Sun W
Extensive sequencing of the cystic fibrosis transmembrane regulator gene: assay validation and unexpected benefits of developing a comprehensive test.
Genet Med. 2003 Jan-Feb;5(1):9-14., [PMID:12544470]

Abstract [show]
PURPOSE: To develop a sequencing assay for the gene to identify mutations in patients with cystic fibrosis (CF). METHODS: An automated assay format was developed to sequence all exons and splice junctional sequences, the promotor region, and parts of introns 11 and 19. RESULTS: After validating the assay using 20 known samples, DNA of seven patients, four of whom were heterozygous for a known CF mutation, was sequenced. Known CF mutations were detected in seven of the eight chromosomes, and a novel missense mutation was detected in the eighth. In addition, this assay allowed 14 ambiguous results obtained using the Roche CF gold strips to be resolved. Three false-positive diagnoses were prevented; a different mutation at the same codon was identified in two patients and confirmation was provided in the remaining nine cases. CONCLUSIONS: Sequencing of the gene provides important information for CF patients and is a valuable adjunct to a carrier screening program to resolve ambiguities in panel testing.

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54 Table 2 Mutant samples used for validation of sequencing assay Mutation expected wt/wt (3 patients) delta F508/wt (2 patients) R117H/wt (3 patients) 2789 ϩ 5 G 3 A/2789 ϩ 5 G 3 A (both parents confirmed carriers) R117H/delta F508 (2 patients) delta F508/I148T delta F508/R1066C delta F508/3848 ϩ 10 kb C 3 T delta F508/G542X R117H/I148T (2 patients) 2307 delA/N1303K deltaF 508/711 ϩ 1 G 3 T deltaF 508/1898 ϩ 1 G 3 A G551D/N1303K 2789 ϩ 5G3A. Login to comment

[hide] [National program for neonatal screening for cysti... J Gynecol Obstet Biol Reprod (Paris). 2003 Feb;32(1 Suppl):1S56-60. Navarro J, Grosskopf C, Vidailhet M, Briard ML, Farriaux JP
[National program for neonatal screening for cystic fibrosis: implementation and preliminary results].
J Gynecol Obstet Biol Reprod (Paris). 2003 Feb;32(1 Suppl):1S56-60., [PMID:12592165]

Abstract [show]
Neonatal screening for cystic fibrosis was decided by the national medical authorities after a common investigation conducted by the French association ADPHE and national health insurance fund. Based on therapeutic progress and the proposed method using determination of blood immunoreactive trypsin then study of the main CF mutations, there is strong hope of effective CF detection and clinical benefit for the patients.

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46 3 Les mutations étudiées sont : 1717-1G > A - G542X - W 1282 X - N 1303 K - DF 508 (M) - 3849 + 10kbC > T - 621+1 G > T - R553X - G 551D, R117H, R1162X - R 334W - A455E - 2183 AA > G - 3659delC-- 1078 delT - D1507 - R347P - S 1251N, E60X, 2789+5G > A - 394del T - G 85 E - 1811+1.6 - Y122X - 711+1G > T - W 846 X - Y 1092 - 3272-26A > G. Login to comment

[hide] Modulation of Ca2+-activated Cl- secretion by baso... Pediatr Res. 2003 Apr;53(4):608-18. Epub 2003 Feb 5. Mall M, Gonska T, Thomas J, Schreiber R, Seydewitz HH, Kuehr J, Brandis M, Kunzelmann K
Modulation of Ca2+-activated Cl- secretion by basolateral K+ channels in human normal and cystic fibrosis airway epithelia.
Pediatr Res. 2003 Apr;53(4):608-18. Epub 2003 Feb 5., [PMID:12612194]

Abstract [show]
Human airway epithelia express Ca2+-activated Cl- channels (CaCC) that are activated by extracellular nucleotides (ATP and UTP). CaCC is preserved and seems to be up-regulated in the airways of cystic fibrosis (CF) patients. In the present study, we examined the role of basolateral K+ channels in CaCC-mediated Cl- secretion in native nasal tissues from normal individuals and CF patients by measuring ion transport in perfused micro Ussing chambers. In the presence of amiloride, UTP-mediated peak secretory responses were increased in CF compared with normal nasal tissues. Activation of the cAMP pathway further increased CaCC-mediated secretion in CF but not in normal nasal mucosa. CaCC-dependent ion transport was inhibited by the chromanol 293B, an inhibitor of cAMP-activated hKvLQT1 K+ channels, and by clotrimazole, an inhibitor of Ca2+-activated hSK4 K+ channels. The K+ channel opener 1-ethyl-2-benzimidazolinone further increased CaCC-mediated Cl- secretion in normal and CF tissues. Expression of hSK4 as well as hCACC-2 and hCACC-3 but not hCACC-1 was demonstrated by reverse transcriptase PCR on native nasal tissues. We conclude that Ca2+-activated Cl- secretion in native human airway epithelia requires activation of Ca2+-dependent basolateral K+ channels (hSK4). Co-activation of hKvLQT1 improves CaCC-mediated Cl- secretion in native CF airway epithelia, and may have a therapeutic effect in the treatment of CF lung disease.

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32 CF patients were genotyped for the following common CFTR mutations: ⌬F508, R553X, N1303 K, G542X, G551D, and R347P. Login to comment
33 The following genotypes were identified: ⌬F508/ ⌬F508 (n ϭ 15); ⌬F508/G542X (n ϭ 1); ⌬F508/G551D (n ϭ 1); ⌬F508/- (n ϭ 2); -/- (n ϭ 8) (- ϭ mutation not identified). Login to comment

[hide] Cystic fibrosis mutation frequencies in an Irish p... Clin Genet. 2003 Feb;63(2):121-5. Devaney J, Glennon M, Farrell G, Ruttledge M, Smith T, Houghton JA, Maher M
Cystic fibrosis mutation frequencies in an Irish population.
Clin Genet. 2003 Feb;63(2):121-5., [PMID:12630958]

Abstract [show]
The incidence of cystic fibrosis (CF) at birth in Ireland is 1/1461. Neonate CF genetic testing is not routinely performed in Ireland. Currently, screening is only carried out where there is clinical evidence or a family history to suggest disease. Here we report the frequencies of common CF mutations occurring in an Irish population composed of samples collected from western, mid-western and southern regions of Ireland. Rarer CF mutations were also identified in a selected number of CF patients. In addition, a number of polymorphisms were identified, some of which are reported to be functionally and phenotypically important.

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17 Eight common mutations were screened using polymerase chain reaction-restriction enzyme analysis (PCR-REA): R117H, 1717±1G > A, DI507, DF508, G542X, G551D, R553X and R560T. Login to comment
26 PCR-REA An in-house PCR-REA procedure was used to screen for the eight common mutations (R117H, 1717±1G > A, DI507, DF508, G542X, G551D, R553X and R560T). Login to comment
65 Frequency of common CF mutations Mutation Numberof chromosomes Frequency (%) R117H 25 2.70 1717^1G >A 20 2.16 DI507 4 0.43 DF508 658 70.97 G542X 4 0.43 G551D 70 7.55 R553X 2 0.22 R560T 4 0.43 Total 788 85 Frequencypercentages areadjustedtorepresent 85%. Login to comment
74 The G542X mutation (0.43%) was found at a lower frequency than the UK (15) (1.68%), Ireland (11) (1%) and Northern Ireland (13) (2.2%). Login to comment

[hide] Longitudinal follow-up of exocrine pancreatic func... J Pediatr Gastroenterol Nutr. 2003 Apr;36(4):474-8. Walkowiak J, Nousia-Arvanitakis S, Agguridaki C, Fotoulaki M, Strzykala K, Balassopoulou A, Witt M, Herzig KH
Longitudinal follow-up of exocrine pancreatic function in pancreatic sufficient cystic fibrosis patients using the fecal elastase-1 test.
J Pediatr Gastroenterol Nutr. 2003 Apr;36(4):474-8., [PMID:12658038]

Abstract [show]
BACKGROUND: A progressive decline in pancreatic function is possible in cystic fibrosis (CF) patients with exocrine pancreatic sufficiency. The secretin-cholecystokinin test is invasive and not acceptable as a repeatable procedure for children. Steatorrhea, conversely, has low sensitivity. Therefore, the aim of the present study was to evaluate the usefulness of the noninvasive fecal elastase-1 (E1) test for the longitudinal assessment of exocrine pancreatic function (EPF) in pancreatic-sufficient (PS) CF patients. METHODS: One hundred eighty-four CF patients were included in the study. In all subjects, E1 concentrations and fecal fat excretion were measured. PS patients were followed for 5 years. RESULTS: At the beginning of the study, 35 (19.0%) CF patients were PS, and 32 (17.4%) had normal E1 concentrations. Longitudinal measurements of E1 concentrations in PS patients with CF demonstrated stable enzyme output in 27 and gradual decrease in 8. The decrease was rapid in five infant patients and gradual in three older patients. The decrease of E1 concentrations preceded the appearance of steatorrhea in all eight subjects. CONCLUSIONS: The decline of EPF in patients with CF appears more frequently during the first months and years of life. However, late PS to pancreatic-insufficient (PI) conversion is also possible. The appearance of maldigestion is preceded by the decrease of fecal E1 concentration. Thus, the fecal E1 test is a helpful screening tool for the longitudinal assessment of declining EPF in PS patients with CF to demonstrate pancreatic deterioration. In suspected patients, fecal fat excretion should be assessed.

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51 RESULTS Among the patients studied, the following mutations of the CFTR gene were present (n): ⌬F508 (223), 621+G-T (10), N1303K (9), 3849+10kbC-T (6), G542X (5), CFTRdele2,3(21kB) (4), E822X (4), 1717-1G-A (3), E836X (3), G1069-L88X (2), R533X (1), G85E (1), 1677delTA (1), G1069R (1), 1525-1G-A (1), and 2789+5G-A (1). Login to comment

[hide] Clinical characteristics and genotype analysis of ... Clin Otolaryngol Allied Sci. 2003 Apr;28(2):125-32. Cimmino M, Cavaliere M, Nardone M, Plantulli A, Orefice A, Esposito V, Raia V
Clinical characteristics and genotype analysis of patients with cystic fibrosis and nasal polyposis.
Clin Otolaryngol Allied Sci. 2003 Apr;28(2):125-32., [PMID:12680831]

Abstract [show]
The prevalence of nasal polyps in a group of paediatric patients with cystic fibrosis was prospectively studied in comparison with a control group with cystic fibrosis but without polyps. Clinical variables, including pulmonary function tests, skin testing and mucociliary transport, were carried out in both groups, as well as genotype analysis. Endoscopic intranasal evaluation identified polyps in 29 of 89 patients (33%). Statistical analysis revealed that patients with nasal polyposis had better pulmonary function, a higher rate of Pseudomonas aeruginosa colonization, more hospitalizations, and more prevalence of allergy to Aspergillus fumigatus than did the comparison group. We found no statistically different genotype distribution between the polyposis and the control group. However, it can be emphasized that the prevalence of the compound heterozygous genotype is higher in the nasal polyposis group than in controls. Our observations suggest that other genetic and environmental factors could play an important role in the development of nasal polyposis.

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47 Analysis of mutations in the CFTR gene as tested by the multiplex polymerase chain reaction (PCR), followed by the reverse dot-blot technique, which searches for 29 of the most frequent mutations (DF508, N1303K, G542X, W1282X, 1717±1 G-A, R553X, 2183 AA-G, DI507, G551D, R560T, 3849 10kbC > T, R1162X, 3659delC, 3905insT, G85E, 621 1GT, R117H, R347P, R334W, A455E, 2789 5GA, Q552X, S1251N, 3905insT, 394delTT, E60X, 2143delT, 2184delA, 711 5G > A), and by ASO dot-blot for the following mutations: I148T, R1158X, 4016 1T, G1244E G >A.26 Statistical analysis was performed using multivariate analysis, by forward stepwise comparison; it was done to ®nd out which of the examined characteristics could be associated (P < 0.01) to nasal polyposis. Login to comment
91 Frequency (percentage) of compound heterozygous genotypes by nasal polyposis status in patients with cystic fibrosis Group with nasal polyposis (n 15) Comparison group (n 20) DF508/G542X 2 (13.33) 2 (10) DF508/N1303K 2 (13.33) 4 (20) DF508/AA2183G 1 (6.67) 0 DF508/IG1717 1 (6.67) 1 (5) DF508/W1282X 1 (6.67) 1 (5) DF508/unspecified 8 (53.33) 12 (60) their study group included patients with nasal polyposis that required surgery. Login to comment
117 Several authors9,16,17 reported that certain CFTR mutations (i.e. G551D, G542X, W1282X) are associated with severe phenotypes, particularly pancreatic insuf®ciency. Login to comment

[hide] Spatial patterns of cystic fibrosis mutation spect... Eur J Hum Genet. 2003 May;11(5):385-94. Lao O, Andres AM, Mateu E, Bertranpetit J, Calafell F
Spatial patterns of cystic fibrosis mutation spectra in European populations.
Eur J Hum Genet. 2003 May;11(5):385-94., [PMID:12734544]

Abstract [show]
Cystic fibrosis (CF) is the most frequent severe recessive disorder in European populations. We have analyzed its mutation frequency spectrum in 94 European, North African and SW Asian populations taken from the literature. Most major mutations as well as the incidence of CF mutations showed clinals patterns as demonstrated by autocorrelogram analysis. More importantly, measures of mutation diversity did also show clinal patterns, with mutation spectra being more diverse in southern than in northern Europe. This increased diversity would imply roughly a three-fold long-term effective population size in southern than in northern Europe. Distances were computed among populations based on their CF mutation frequencies and compared with distances based on other genic regions. CF-based distances correlated with mtDNA but not with Y-chromosome-based distances, which may be a consequence of the relatively homogeneous CF mutation frequencies in European populations.

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52 As for the direction of the clines, F508del peaks in NW Europe and declines towards SE Europe, G542X declines from SW to NE Europe, G551D is almost restricted to NW Europe, and N1303 K and W1282X show gradients from SW Asia and SE Europe towards NW Europe. Login to comment
66 The correlations with Y-chromosome-based Table 1 94 middle Eastern, North African and European populations used in the analysis Population 2N F508del G542X G551D N1303K W1282X Rare Other Unknown Hmax Ymax Incidence Referencesa Austria 592 0.660 0.022 0.012 0.005 0.002 0.064 0.019 0.216 0.562 0.96 49 Belgium 646 0.752 0.025 0.002 0.028 0.012 0.053 0.046 0.082 0.430 0.56 50 Bulgaria 208 0.654 0.034 0.000 0.067 0.000 0.096 0.067 0.082 0.563 0.97 51 Crete 26 0.462 0.077 0.000 0.038 0.000 0.231 0.038 0.154 0.785 2.87 Czech Republic 584 0.697 0.021 0.034 0.026 0.005 0.051 0.021 0.146 0.512 0.78 Denmark 678 0.872 0.006 0.001 0.010 0.001 0.034 0.035 0.040 0.239 0.23 0.000210 Great Britain 0.000414b North England 4111 0.772 0.008 0.023 0.005 0.001 0.032 0.011 0.148 0.403 0.50 Scotland 1167 0.751 0.033 0.061 0.003 0.003 0.033 0.023 0.093 0.430 0.56 0.000504 South England 3679 0.769 0.020 0.029 0.005 0.002 0.032 0.009 0.133 0.407 0.51 Wales 372 0.659 0.024 0.030 0.005 0.000 0.134 0.065 0.083 0.557 0.94 Estonia 25 0.640 0.000 0.000 0.000 0.000 0.160 0.080 0.120 0.577 1.02 Former Yugoslavia 203 0.700 0.030 0.000 0.010 0.005 0.039 0.069 0.148 0.506 0.76 Finland 52 0.462 0.019 0.000 0.000 0.000 0.288 0.019 0.212 0.713 1.90 France 0.000232 Alsace 126 0.595 0.024 0.000 0.016 0.008 0.040 0.008 0.310 0.646 1.38 Aquitaine 116 0.612 0.034 0.000 0.017 0.000 0.043 0.009 0.284 0.626 1.26 Auvergne 102 0.725 0.039 0.000 0.029 0.010 0.020 0.000 0.176 0.474 0.67 Burgundy 168 0.702 0.024 0.000 0.006 0.000 0.060 0.006 0.202 0.507 0.77 Brittany 582 0.744 0.009 0.024 0.017 0.003 0.064 0.002 0.137 0.444 0.60 0.000343 Centre 218 0.716 0.050 0.000 0.023 0.000 0.023 0.000 0.188 0.486 0.71 Champagne 182 0.665 0.049 0.000 0.016 0.000 0.055 0.005 0.209 0.556 0.94 Franche-Comt ´ e 118 0.746 0.085 0.000 0.085 0.025 0.059 0.000 0.000 0.431 0.56 Languedoc 90 0.700 0.022 0.011 0.033 0.000 0.044 0.000 0.189 0.511 0.78 Llimousin 44 0.545 0.023 0.000 0.068 0.000 0.023 0.023 0.318 0.705 1.83 Loire Valley 308 0.737 0.006 0.019 0.013 0.003 0.032 0.000 0.188 0.457 0.63 Lorraine 286 0.717 0.031 0.000 0.000 0.000 0.042 0.000 0.210 0.486 0.70 Lower Normandie 174 0.644 0.017 0.023 0.017 0.000 0.069 0.000 0.230 0.585 1.06 Midi-Pyr ´ en ´ ees 114 0.649 0.035 0.000 0.018 0.009 0.018 0.000 0.272 0.580 1.03 Nord 468 0.660 0.019 0.004 0.015 0.002 0.053 0.006 0.239 0.563 0.97 Paris Region 830 0.643 0.027 0.007 0.010 0.012 0.035 0.000 0.266 0.585 1.06 Picardie 200 0.650 0.040 0.000 0.040 0.010 0.080 0.000 0.180 0.574 1.01 Poitou 100 0.770 0.030 0.000 0.020 0.000 0.020 0.000 0.160 0.408 0.51 Provence- Cote d`Azur 178 0.674 0.028 0.000 0.051 0.017 0.028 0.006 0.197 0.544 0.89 Rhone-Alpes 668 0.668 0.036 0.001 0.027 0.009 0.018 0.009 0.232 0.552 0.92 Upper Normandie 248 0.645 0.020 0.008 0.012 0.004 0.048 0.004 0.258 0.584 1.05 Germany Baden-W ¨ urttemberg 59 0.763 0.000 0.000 0.034 0.000 0.051 0.102 0.051 0.412 0.52 Bavaria 177 0.740 0.017 0.017 0.000 0.000 0.040 0.011 0.175 0.453 0.62 Berlinc 132 0.773 0.015 0.000 0.023 0.000 0.038 0.015 0.136 0.403 0.50 Bremend 74 0.689 0.014 0.014 0.000 0.000 0.054 0.014 0.216 0.528 0.84 Lower Saxony 198 0.803 0.005 0.005 0.015 0.000 0.015 0.030 0.126 0.355 0.41 North-Rhine/ Westphalia 174 0.736 0.006 0.006 0.000 0.006 0.069 0.034 0.144 0.458 0.63 Saxonye 83 0.639 0.012 0.012 0.024 0.000 0.036 0.036 0.241 0.594 1.10 Rhineland-Palatinaf 59 0.525 0.017 0.000 0.051 0.000 0.085 0.068 0.254 0.721 1.99 Greece Ipiros/Ionian Islands 46 0.609 0.000 0.000 0.043 0.000 0.087 0.043 0.217 0.632 1.30 Peloponese/Attica 89 0.573 0.000 0.022 0.045 0.000 0.146 0.045 0.169 0.667 1.52 Thesalia/Macedonia/ Thrace 61 0.672 0.066 0.000 0.033 0.000 0.033 0.082 0.115 0.543 0.89 Hungary 57 0.439 0.018 0.000 0.018 0.018 0.070 0.018 0.421 0.811 3.43 Italy Abruzzo 66 0.500 0.061 0.000 0.091 0.076 0.030 0.000 0.242 0.739 2.19 Basilicata 75 0.467 0.107 0.000 0.067 0.027 0.067 0.013 0.253 0.769 2.61 Calabria 149 0.430 0.034 0.000 0.047 0.020 0.054 0.034 0.383 0.813 3.46 distances were similar (r ¼ 0.147, P ¼ 0.116 and after controlling for geographical distance r ¼ 0.054, P ¼ 0.296). Login to comment
68 Table 1 (continued) Population 2N F508del G542X G551D N1303K W1282X Rare Other Unknown Hmax Ymax Incidence Referencesa Campania 223 0.610 0.040 0.000 0.067 0.018 0.040 0.004 0.220 0.623 1.25 Emilia-Romagna 242 0.541 0.058 0.000 0.025 0.008 0.050 0.000 0.318 0.704 1.82 0.000170 Friuli 24 0.375 0.125 0.000 0.042 0.042 0.083 0.083 0.250 0.855 4.85 Lazio 236 0.462 0.030 0.000 0.093 0.013 0.034 0.013 0.356 0.778 2.75 Liguria 44 0.591 0.114 0.000 0.023 0.000 0.045 0.000 0.227 0.646 1.38 Lombardia 399 0.499 0.038 0.000 0.038 0.010 0.090 0.050 0.276 0.743 2.24 Marche 144 0.389 0.056 0.000 0.083 0.014 0.063 0.007 0.389 0.841 4.29 Molise 27 0.481 0.037 0.000 0.074 0.000 0.037 0.000 0.370 0.775 2.70 Piemonte 117 0.675 0.034 0.000 0.000 0.000 0.043 0.017 0.231 0.544 0.89 Puglia 245 0.543 0.053 0.000 0.073 0.000 0.041 0.012 0.278 0.698 1.77 Sardegna 141 0.582 0.057 0.000 0.028 0.000 0.028 0.142 0.163 0.641 1.35 Sicilia 387 0.525 0.062 0.000 0.034 0.023 0.067 0.021 0.269 0.719 1.97 Toscana 191 0.508 0.042 0.000 0.037 0.010 0.031 0.005 0.366 0.740 2.21 Trentino 113 0.513 0.027 0.009 0.009 0.009 0.204 0.053 0.177 0.718 1.96 Umbria 37 0.676 0.081 0.000 0.027 0.000 0.027 0.000 0.189 0.545 0.90 Veneto 552 0.449 0.014 0.000 0.031 0.000 0.188 0.033 0.284 0.785 2.87 0.000370 Ireland Republic of Ireland 509 0.727 0.010 0.069 0.004 0.000 0.037 0.014 0.139 0.467 0.65 0.000684 Northern Ireland 876 0.619 0.021 0.045 0.001 0.000 0.063 0.047 0.205 0.612 1.19 0.000553 52 Israel 367 0.322 0.054 0.000 0.030 0.362 0.065 0.082 0.084 0.754 2.39 0.000304 Lebanon 40 0.350 0.000 0.000 0.100 0.200 0.025 0.225 0.100 0.794 3.04 0.000390 53 Netherlands 1442 0.744 0.013 0.001 0.009 0.007 0.072 0.019 0.135 0.444 0.60 0.000252 Norway 168 0.667 0.006 0.012 0.006 0.000 0.071 0.000 0.238 0.555 0.93 0.000152 Poland 444 0.662 0.023 0.007 0.020 0.002 0.043 0.020 0.223 0.560 0.96 Portugal Faro/Beja 25 0.680 0.000 0.000 0.000 0.000 0.040 0.000 0.280 0.547 0.90 Lisboag 100 0.480 0.030 0.000 0.000 0.000 0.080 0.060 0.350 0.767 2.57 Setubal/Evora 33 0.485 0.000 0.000 0.000 0.000 0.121 0.091 0.303 0.767 2.57 Russia 445 0.618 0.007 0.002 0.004 0.004 0.031 0.031 0.301 0.617 1.22 0.000051 Slovakia 254 0.559 0.075 0.000 0.035 0.016 0.075 0.016 0.224 0.680 1.62 Spain Andalucı´a 314 0.538 0.086 0.013 0.013 0.013 0.083 0.096 0.159 0.694 1.73 Arago´n 65 0.523 0.031 0.000 0.015 0.000 0.123 0.138 0.169 0.708 1.86 Castilla la Mancha 69 0.478 0.058 0.000 0.043 0.000 0.014 0.029 0.377 0.771 2.63 Paı´s Valencia` 125 0.464 0.104 0.000 0.056 0.000 0.096 0.040 0.240 0.771 2.63 Castilla Leo´n/ La Rioja 187 0.604 0.048 0.000 0.011 0.000 0.102 0.107 0.128 0.623 1.24 54 Catalonia 109 0.642 0.055 0.000 0.037 0.009 0.083 0.064 0.110 0.582 1.05 0.000187 Extremadura 63 0.460 0.048 0.000 0.016 0.000 0.079 0.127 0.270 0.776 2.72 Galicia 93 0.624 0.097 0.000 0.011 0.000 0.161 0.075 0.032 0.596 1.11 Madrid 51 0.510 0.059 0.020 0.039 0.000 0.059 0.020 0.294 0.742 2.23 Murcia 40 0.250 0.125 0.000 0.025 0.025 0.175 0.200 0.200 0.889 6.74 Basque Country 31 0.710 0.000 0.000 0.000 0.000 0.065 0.097 0.129 0.497 0.74 Sweden 165 0.733 0.006 0.000 0.000 0.000 0.103 0.085 0.073 0.448 0.60 0.000130 Switzerland 95 0.432 0.032 0.000 0.011 0.000 0.263 0.168 0.095 0.732 2.11 Tunisia 78 0.179 0.090 0.000 0.064 0.026 0.128 0.115 0.397 0.941 14.51 55 Turkey 263 0.274 0.038 0.000 0.042 0.004 0.087 0.114 0.441 0.907 8.44 56 Ukraine 396 0.543 0.000 0.005 0.000 0.000 0.018 0.000 0.434 0.706 1.84 57 Total 29131 0.674 0.025 0.015 0.017 0.009 0.053 0.024 0.182 0.586 1.06 N, sample size (in number of CF chromosomes); F508del, G542X, G551D, 1303K, and W1282X, relative frequencies of the main mutations; rare, relative frequency of mutations not listed in Table 2 of reference 58; other, relative frequency of mutations listed in Table 2 of reference 58. unknown, fraction of chromosomes asociated to disease bearing unidentified mutations. Login to comment
86 However, the effects of the two parameters are not equivalent: Figure 1 Geographical distribution (a) and spatial autocorrelogram (b) of F508del mutation in 94 middle Eastern, North African, and European populations. The X-axis represents geographic distance between samples; the Y-axis represents Moran`s index; a single asterisk (n) denotes Po0.05; double asterisks (nn) denote Po0.01. Figure 2 Geographical distribution (a) and spatial autocorrelogram (b) of the G542X mutation in 94 middle Eastern, North African, and European populations. The X-axis represents geographic distance between samples; the Y-axis represents Moran`s index; a single asterisk (n) denotes Po0.05; double asterisks (nn) denote Po0.01. for any Ne, doubling it would double y; for a typical CF allele frequency f0 ¼ 0.02, doubling it would mean just a 2% reduction in y. Login to comment

[hide] Analysis of cystic fibrosis transmembrane conducta... Am J Med Genet A. 2003 Jul 1;120A(1):72-6. Timmreck LS, Gray MR, Handelin B, Allito B, Rohlfs E, Davis AJ, Gidwani G, Reindollar RH
Analysis of cystic fibrosis transmembrane conductance regulator gene mutations in patients with congenital absence of the uterus and vagina.
Am J Med Genet A. 2003 Jul 1;120A(1):72-6., 2003-07-01 [PMID:12794695]

Abstract [show]
The relationship between cystic fibrosis transmembrane conductance regulator gene (CFTR) mutations and congenital absence of the uterus and vagina (CAUV) was examined. CFTR mutations have previously been associated with congenital bilateral absence of the vas deferens (CBAVD). CBAVD is caused by a disruption in the vas deferens, a Wolffian duct derivative. Because the embryologic development of the Mullerian ducts directly depends on the prior normal development of the Wolffian ducts, the same gene products may be necessary for normal embryologic development of both ductal systems. This study evaluated the role of CFTR mutations in the development of CAUV. DNA samples from 25 patients with CAUV were tested for the presence of 33 of the most common CFTR mutations. Protein-coding DNA fragments from the CFTR gene were amplified in vitro by the polymerase chain reaction (PCR) and analyzed for mutations using allele-specific oligonucleotide (ASO) probes. Two patients were heterozygous for CFTR mutations. One was heterozygous for the W1282X mutation and the other was heterozygous for the DeltaF508 mutation. The incidence of the 33 CFTR mutations found in the patients with CAUV (8%) was twice that found in the general population (4%), but much less than the incidence of CFTR mutations in men with CBAVD (80%). This data suggests that it is unlikely for CFTR mutations to cause CAUV in females as they cause CBAVD in some males. Furthermore, the data suggest that CAUV in females may be the same disorder as CBAVD in males who do not have CFTR mutations.

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82 CFTR Gene Mutations Tested DF508 R334W Y1092X 5T variant Y122X R347H G542X S549R 3,849 þ 4 G551D 3,849 þ 10 kb 2,789 þ 5 W1282X R553X 711 þ 1 3,905 þ T 621 þ 1 1,898 þ 1 N1303K 1,717À1 R1162X R117H 1078dT A455E D1507 Q493X 218dA R347P V520F G85E R560T S549N 3659dC Wolffian duct must occur at a time when the Mu¨llerian duct is no longer dependent on the Wolffian duct for development. Login to comment

[hide] Comparison of the CFTR mutation spectrum in three ... Hum Mutat. 2003 Jul;22(1):105. Scotet V, Barton DE, Watson JB, Audrezet MP, McDevitt T, McQuaid S, Shortt C, De Braekeleer M, Ferec C, Le Marechal C
Comparison of the CFTR mutation spectrum in three cohorts of patients of Celtic origin from Brittany (France) and Ireland.
Hum Mutat. 2003 Jul;22(1):105., [PMID:12815607]

Abstract [show]
This study aims to compare the spectrum of the mutations identified in the gene responsible for cystic fibrosis in three cohorts of patients of Celtic origin from Brittany and Ireland. It included 389 patients from Brittany, 631 from Dublin and 139 from Cork. The CFTR gene analysis relied on the detection of the most common mutations, followed by a complete gene scanning using DGGE or D-HPLC. High mutation detection rates were obtained in each cohort: 99.6%, 96.8%, and 96.0% respectively. A high frequency of the c.1652_1655 del3 mutation (F508del: 74.8% to 81.3%) and of the "Celtic" mutation (c.1784G>A (G551D): 3.7% to 9.7%) was observed in each population. Apart from this, the mutation spectrums differed. In Brittany, the most common abnormalities were: c.1078delT (3.6%), c.4041C>G (N1303K: 1.4%), c.2670G>A (W846X(2): 1.0%) and c.1717-1G>A (1.0%), whereas in the cohort of Dublin, the main mutations were: c.482G>A (R117H: 3.0%), c.1811G>C (R560T: 2.4%) and c.621+1G>T (1.7%). Finally, in the Cork area, only the c.482G>A mutation (R117H) reached a frequency of 1%. Two previously-unreported mutations were identified in the Dublin cohort: c.2623-2A>G and c.3446T>G (M1105R). This collaborative study highlights the similarities of the CFTR alleles in the Breton and Irish populations, but also the disparities that exist between these populations, despite their common origin. Each population has its own history, with its mixture of founder effects and genetic drifts, which are at the origin of the current mutation distribution. The molecular study of the CFTR gene provides new tools for retracing European populations' histories.

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19 This spectrum is noteworthy because it includes a main mutation, accounting for 70% of the mutated alleles world-wide (the deletion F508del), four other mutations observed with a frequency over 1% (G542X: 2.4%, G551D: 1.6%, N1303K: 1.3%, W1282X: 1.2%) and a multitude of private abnormalities (Tsui, 2003). Login to comment
44 Firstly, the National Centre for Medical Genetics, Dublin performed an analysis of the most common CFTR mutations, using the ARMS test (Ferrie et al., 1992), which enables the detection of the following mutations: F508del, R117H, I507del, G542X, G551D, R560T, N1303K, R352Q, 1717-1G>A and 621+1G>T. Login to comment
64 Spectrum of the CFTR Mutations Identified in the Cohorts from Brittany, Dublin Centre, and Cork Area Nucleotide Amino acid change * change Exon Number Frequency Number Frequency Number Frequency 211delG 2 1 0.1% 310G>T E60X 3 5 0.6% 4 0.3% 347C>A A72D 3 1 0.1% 368G>A W79X 3 1 0.1% 386G>A G85E 3 2 0.3% 3 0.2% 403G>A G91R 3 2 0.3% 482G>A R117H 4 4 0.5% 38 3.0% 4 1.4% 498T>A Y122X 4 1 0.1% 574delA 4 1 0.1% 577G>A G149R 4 1 0.1% 621+1G>T int 4 5 0.6% 21 1.7% 790C>T Q220X 6a 1 0.1% 875+1G>C int 6a 1 0.4% 905delG 6b 1 0.1% 1065C>G F311L 7 2 0.3% 1078delT 7 28 3.6% 1132C>T R334W 7 1 0.1% 1172G>A R347H 7 5 0.6% 1172G>T R347L 7 1 0.1% 1172G>C R347P 7 1 0.1% 1187G>A R352Q 7 3 0.2% 2 0.7% 1208A>G Q359R 7 1 0.1% 1154insTC 7 2 0.2% 1221delCT 7 2 0.3% 1248+1G>A int 7 1 0.1% 1249-27delTA int 7 1 0.4% 1334G>A W401X 8 1 0.1% 1461ins4 9 5 0.4% 1471delA 9 2 0.2% 1607C>T S492F 10 2 0.3% 1609C>T Q493X 10 1 0.1% 1648_1653delATC I507del 10 3 0.4% 10 0.8% 1 0.4% 1652_1655del 3 bp F508del 10 582 74.8% 966 76.5% 226 81.3% 1690G>T V520F 10 4 0.3% 1717-1G>A int 10 8 1.0% 9 0.7% 1756G>T G542X 11 5 0.6% 8 0.6% 1779T>G S549R 11 1 0.1% 1784G>A G551D 11 29 3.7% 82 6.5% 27 9.7% 1789C>G R553G 11 1 0.1% 1789C>T R553X 11 3 0.4% 1 0.1% 1806delA 11 1 0.1% 1811G>A R560K 11 2 0.3% 1811G>C R560T 11 30 2.4% 2 0.7% 1819T>A Y563N 12 1 0.1% 1853C>A P574H 12 1 0.1% 1898+1G>A int 12 1 0.1% 2184delA 13 1 0.1% 1 0.1% 2184insA 13 1 0.1% 2622+1G>A int 13 1 0.1% 2 0.2% 2622+1G>T int 13 1 0.1% 2623-2A>G ** int 13 1 0.1% 2670G>A W846X2 14a 8 1.0% 2752-1G>T int 14a 1 0.1% 2752-26A>G int 14a 2 0.2% 2789+5G>A int 14b 6 0.8% 2966C>T S945L 15 2 0.3% 3007delG 15 4 0.3% 3040G>C G970R 15 1 0.1% 3062C>T S977F 16 1 0.1% 3120+1G>A int 16 1 0.1% 3272-26A>G int 17a 4 0.5% 2 0.2% 2 0.7% 3320dupli(CTATG) 17b 1 0.1% 3329G>A R1066H 17b 1 0.1% 3340C>T R1070W 17b 1 0.1% 3408C>A Y1092X 17b 7 0.9% 3442G>T E1104X 17b 1 0.1% 3446T>G ** M1105R 17b 1 0.1% 3586G>C D1152H 18 1 0.1% 3601-17T>C + 1367delC int 18 + 9 1 0.1% 3616C>T R1162X 19 1 0.1% 2 0.2% 3659delC 19 2 0.2% 3832A>G I1234V 19 2 0.3% 3849+4A>G int 19 1 0.1% 3849+10kbC>T int 19 3 0.2% 3877G>A G1249R 20 1 0.1% 3884G>A S1251N 20 1 0.1% 3898insC 20 1 0.1% 3905insT 20 2 0.3% 3978G>A W1282X 20 3 0.4% 4005+1G>A int 20 6 0.8% 4016insT 21 1 0.1% 4041C>G N1303K 21 11 1.4% 5 0.4% 4136T>C L1335P 22 1 0.1% 1 0.4% 4279insA 23 1 0.1% Unidentified Unidentified - 3 0.4% 41 3.2% 11 4.0% Total 778 100.0% 1262 100.0% 278 100.0% * All nucleotide changes correspond to cDNA numbering. Login to comment
76 Number Frequency Number Frequency 1652_1655del 3 bp F508del 384 75.6% 196 73.1% 582 74.8% 1784G>A G551D 17 3.3% 12 4.5% 29 3.7% 1078delT 25 4.9% 3 1.1% 28 3.6% 4041C>G N1303K 3 0.6% 8 3.0% 11 1.4% 2670G>A W846X2 7 1.4% 1 0.4% 8 1.0% 1717-1G>A 5 1.0% 3 1.1% 8 1.0% 3408C>A Y1092X 1 0.2% 6 2.2% 7 0.9% 2789+5G>A 2 0.4% 4 1.5% 6 0.8% 4005+1G>A 5 1.0% 1 0.4% 6 0.8% 310G>T E60X 3 0.6% 2 0.7% 5 0.6% 621+1G>T 2 0.4% 3 1.1% 5 0.6% 1172G>A R347H 5 1.0% 5 0.6% 1756G>T G542X 4 0.8% 1 0.4% 5 0.6% 482G>A R117H 3 0.6% 1 0.4% 4 0.5% 3272-26A>G 2 0.4% 2 0.7% 4 0.5% 1648_1653delATC I507del 1 0.2% 2 0.7% 3 0.4% 1789C>T R553X 3 0.6% 3 0.4% 3978G>A W1282X 2 0.4% 1 0.4% 3 0.4% Unidentified Unidentified 3 0.6% 3 0.4% Total Total 508 100.0% 268 100.0% 778 100.0% Basse-Bretagne Haute-Bretagne Brittany * Amino acid change Nucleotide change Table 3: Distribution of the Main CFTR Nutations Observed in the Irish Cohorts (Dublin and Cork) The 62 mutations detected in Brittany combined to give 81 different genotypes in CF patients. Login to comment
98 Number Frequency Number Frequency 1652_1655del 3 bp F508del 966 76.5% 226 81.3% 1192 77.4% 1784G>A G551D 82 6.5% 27 9.7% 109 7.1% 482G>A R117H 38 3.0% 4 1.4% 42 2.7% 1811G>C R560T 30 2.4% 2 0.7% 32 2.1% 621+1G>T 21 1.7% 21 1.4% 1648_1653delATC I507del 10 0.8% 1 0.4% 11 0.7% 1717-1G>A 9 0.7% 9 0.6% 1756G>T G542X 8 0.6% 8 0.5% 1187G>A R352Q 3 0.2% 2 0.7% 5 0.3% 1461ins4 5 0.4% 5 0.3% 4041C>G N1303K 5 0.4% 5 0.3% 310G>T E60X 4 0.3% 4 0.3% 1690G>T V520F 4 0.3% 4 0.3% 3007delG 4 0.3% 4 0.3% 3272-26A>G 2 0.2% 2 0.7% 4 0.3% 386G>A G85E 3 0.2% 3 0.2% 3849+10kbC>T 3 0.2% 3 0.2% Unidentified Unidentified 41 3.2% 11 4.0% 52 3.4% Total Total 1262 100.0% 278 100.0% 1540 100.0% Dublin cohort Cork cohort Ireland Amino acid change Nucleotide change We noted similar high frequencies of the F508del and G551D mutations in the three cohorts studied. Login to comment

[hide] Abnormal passive chloride absorption in cystic fib... J Clin Invest. 2003 Jul;112(1):118-25. Russo MA, Hogenauer C, Coates SW Jr, Santa Ana CA, Porter JL, Rosenblatt RL, Emmett M, Fordtran JS
Abnormal passive chloride absorption in cystic fibrosis jejunum functionally opposes the classic chloride secretory defect.
J Clin Invest. 2003 Jul;112(1):118-25., [PMID:12840066]

Abstract [show]
Due to genetic defects in apical membrane chloride channels, the cystic fibrosis (CF) intestine does not secrete chloride normally. Depressed chloride secretion leaves CF intestinal absorptive processes unopposed, which results in net fluid hyperabsorption, dehydration of intestinal contents, and a propensity to inspissated intestinal obstruction. This theory is based primarily on in vitro studies of jejunal mucosa. To determine if CF patients actually hyperabsorb fluid in vivo, we measured electrolyte and water absorption during steady-state perfusion of the jejunum. As expected, chloride secretion was abnormally low in CF, but surprisingly, there was no net hyperabsorption of sodium or water during perfusion of a balanced electrolyte solution. This suggested that fluid absorption processes are reduced in CF jejunum, and further studies revealed that this was due to a marked depression of passive chloride absorption. Although Na+-glucose cotransport was normal in the CF jejunum, absence of passive chloride absorption completely blocked glucose-stimulated net sodium absorption and reduced glucose-stimulated water absorption 66%. This chloride absorptive abnormality acts in physiological opposition to the classic chloride secretory defect in the CF intestine. By increasing the fluidity of intraluminal contents, absence of passive chloride absorption may reduce the incidence and severity of intestinal disease in patients with CF.

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58 The Journal of Clinical Investigation | July 2003 | Volume 112 | Number 1 119 Table 1 Demographics and CFTR mutation analysis Patient Sex, age CFTR mutation analysis Meconium BMIA FVC FEV1 Work/school Number of Experiments ileus (% pred) (% pred) statusB hospital admissionsC conductedD 1 F, 33 ∆F508/1898 + 1G-A No 23 112 106 1 1 b, c, d, e 2 M, 28 ∆F508/∆F508 Yes 18 35 23 2 2 b, c, d, e 3 F, 21 W1282X/W1282X Yes 22 77 76 1 1 a, b, d 4 M, 26 G551D/G542X No 23 68 65 1 1 b 5 M, 20 ∆F508/∆F508 Yes 19 106 99 1 1 b 6 M, 24 ∆F508/∆F508 Yes 18 69 58 1 1 b 7 M, 34 ∆F508/∆F508 No 22 64 45 1 0 a, b, d 8 F, 20 ∆F508/∆F508 No 21 81 91 1 2 a, c, d, e 9 M, 28 ∆F508/∆F508 No 18 83 67 1 1 a, d 10 F, 38 ∆F508/∆F508 No 22 58 39 3 4E a 11 M, 28 ∆F508/∆F508 Yes 18 55 44 1 1 c, e 12 M, 27 NegativeF/∆F508 No 22 48 44 2 2 c, e AHealthy adult body mass index: 18.5-24.9 kg/m2. Login to comment

[hide] Molecular consequences of cystic fibrosis transmem... Gut. 2003 Aug;52(8):1159-64. Ahmed N, Corey M, Forstner G, Zielenski J, Tsui LC, Ellis L, Tullis E, Durie P
Molecular consequences of cystic fibrosis transmembrane regulator (CFTR) gene mutations in the exocrine pancreas.
Gut. 2003 Aug;52(8):1159-64., [PMID:12865275]

Abstract [show]
BACKGROUND AND AIMS: We tested the hypothesis that the actual or predicted consequences of mutations in the cystic fibrosis transmembrane regulator gene correlate with the pancreatic phenotype and with measures of quantitative exocrine pancreatic function. METHODS: We assessed 742 patients with cystic fibrosis for whom genotype and clinical data were available. At diagnosis, 610 were pancreatic insufficient, 110 were pancreatic sufficient, and 22 pancreatic sufficient patients progressed to pancreatic insufficiency after diagnosis. RESULTS: We identified mutations on both alleles in 633 patients (85.3%), on one allele in 95 (12.8%), and on neither allele in 14 (1.9%). Seventy six different mutations were identified. The most common mutation was DeltaF508 (71.3%) followed by G551D (2.9%), G542X (2.3%), 621+1G-->T (1.2%), and W1282X (1.2%). Patients were categorized into five classes according to the predicted functional consequences of each mutation. Over 95% of patients with severe class I, II, and III mutations were pancreatic insufficient or progressed to pancreatic insufficiency. In contrast, patients with mild class IV and V mutations were consistently pancreatic sufficient. In all but four cases each genotype correlated exclusively with the pancreatic phenotype. Quantitative data of acinar and ductular secretion were available in 93 patients. Patients with mutations belonging to classes I, II, and III had greatly reduced acinar and ductular function compared with those with class IV or V mutations. CONCLUSION: The predicted or known functional consequences of specific mutant alleles correlate with the severity of pancreatic disease in cystic fibrosis.

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130 The most common mutation was ∆F508 (71.3%) followed by G551D (2.9%), G542X (2.3%), 621+1G→T (1.2%), and W1282X (1.2%). Login to comment
279 For example, a patient carrying a class I mutation (for example, G542X) in combination with ∆F508 (class II) was classified as class I although the genotype is in fact a compound heterozygote of class I/II. Login to comment
293 The most common mutations were: ∆F508 (in 943 chromosomes, 71.2%), G551D (39, 2.9%), G542X (31, 2.3%), 621+1G→T, W1282X (16, 1.2%), and R117H (11, 0.8%). Login to comment
309 Table 2 Genotype classification according to the functional consequences of CFTR gene mutations Pancreatic status Class I Class II Class III Class IV Class V PS F1 , 875+1G→C(2) F, F (1) F, G551D (1) F, R117H (11) F,3849+10kbC→T (5) F, G85E2 (1) F, R347H (3) F,3272-26A→G (4) F, S1251N (2) F,A445E (3) F, D614G (1) F,P574H (2) F, R347P (1) F,3120G>A (1) R117H,R117H (1) F, 5T (8) F, L1335P (1) F,2789+5G→A (1) F,P67L (1) F,R347P/R347H (1) F,V232D(2) R334W, R334W(1) PS→PI F,3659delC (1) F,F (15) F,G551D (1) F, I1234V (1) F,2184insA (1) F,R560T (1) PI F, G542X (27) F,F (365) F, G551D (28) F, 621+1G→T (13) F, R560T (7) F,R553X (7) F, N1303K (9) F, R1162X (6) F,L1077P (2) F, 3659delC (5) F, I48T (1) F, 1717-1G→A (5) F,A559T (1) F, W1282X (5) F, G85E2 (2) F, 711+1G→T (5) G551D,G551D(1) F,2184delA(4) F,H199R (1) W1282X,W1282X (4) F,I1072T(1) F,Y1092X (3) F,S549 (R75Q) (1) F,556delA (3) F, Q493X (3) F,4016InsT (3) F, 3120+1G→A (2) F, G551D/R553X (2) F,Q814X(2) F,1154insTC (2) F,441delA (1) F, 4326delTC (1) F,Q552X(1) F,3007delG (1) F,2184insA (1) F, 4010del4 (1) F,3905insT (1) F,1078delT(1) F,E1104X (1) F,3876delA (1) F,4374+1G→T (1) F,E585X (1) F, E60X (1) CFTR, cystic fibrosis transmembrane regulator; PI, pancreatic insufficiency; PS, pancreatic sufficiency. Login to comment
322 The four next most common mutations reported in this study (G551D, G542X, 621+1G→T, and W1282X) each accounted for Table 3 Pancreatic ductular and acinar secretion classified according to the functional consequences of CFTR gene mutations Functional class n Ductular function Acinar function Fluid† (ml/kg/h) Bicarbonate‡ (mmol/kg/h) Chloride‡ (mmol/kg/h) Trypsin* (U/kg/h) Colipase* (U/kg/h) Total lipase* (U/kg/h) Class I 7 2.3 (1.5) 0.03 (0.02) 0.15 (0.14) 19 (42) 142 (201) 290 (388) Class II 33 2.9 (2.4) 0.04 (0.04) 0.21 (0.33) 31 (63) 111 (197) 202 (276) Class III 6 1.1 (0.87) 0.02 (0.02) 0.12 (0.07) 109 (256) 235 (483) 608 (1280) Class IV 14 4.0 (2.8) 0.19 (0.2) 0.32 (0.12) 1032 (768) 9840 (10698) 12563 (10461) Class V 10 4.8 (3.2) 0.12 (0.07) 0.36 (0.23) 1535 (1406) 12161 (13550) 16449 (15482) Control 21 10.0 (3.9) 0.57 (0.22) 0.62 (0.3) 2291 (836) 13036 (5958) 19767 (8623) Values are mean (SD). Login to comment

[hide] Detection of cystic fibrosis mutations by peptide ... Clin Chem. 2003 Aug;49(8):1318-30. Malehorn DE, Telmer CA, McEwen SB, An J, Kinsey AD, Retchless AC, Mason C, Vieta WM, Jarvik JW
Detection of cystic fibrosis mutations by peptide mass signature genotyping.
Clin Chem. 2003 Aug;49(8):1318-30., [PMID:12881448]

Abstract [show]
BACKGROUND: The diversity of genetic mutations and polymorphisms calls for the development of practical detection methods capable of assessing more than one patient/one nucleotide position per analysis. METHODS: We developed a new method, based on peptide mass signature genotyping (PMSG), for the detection of DNA mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Exons of the gene were amplified, cloned, and expressed in Escherichia coli as peptide fusions, in natural as well as unnatural reading frames. Peptide analytes were purified by immobilized metal affinity chromatography and analyzed by matrix-assisted, laser desorption/ionization time-of-flight mass spectrometry. Synthetic and natural DNA samples with the 25 mutations recommended for CFTR carrier screening (Grody et al. Genet Med 2001;3:149-54) were assessed using the PMSG test for the CFTR gene. RESULTS: Peptide analytes ranged from 6278 to 17 454 Da and varied 30-fold in expression; highly expressing peptides were observed by electron microscopy to accumulate as inclusion bodies. Peptides were reliably recovered from whole-cell lysates by a simple purification method. CFTR mutations caused detectable changes in resulting mass spectrometric profiles, which were >95% reliably detected in blinded testing of replicate synthetic heterozygous DNA samples. Mutation detection was possible with both sample pooling and multiplexing. The PMSG CFTR test was used to determine compound heterozygous mutations in DNA samples from cystic fibrosis patients, which were confirmed by direct DNA sequencing. CONCLUSIONS: The PMSG test of the CFTR gene demonstrates unique capabilities for determining the sequence status of a DNA target by sensitively monitoring the mass of peptides, natural or unnatural, generated from that target.

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133 In the case of exon 11, a second analyte (exon 11.2) was designed in an alternative forward reading frame to detect the 1717-1 GϾA mutation, but it also scanned the remainder of the exon and registered its own characteristic mass shifts resulting from G542X and R553X mutations. Login to comment
138 ⌬b 3 R Y 9863.78 G85E SerϾPhe 9923.90 Y 60.12 4.1 R N 7047.69 R117H AlaϾVal 7075.76 N 28.07 4.2 R Y 11161.32 lI48T AsnϾSer 11134.32 Y -27.00 621ϩ1 GϾT TyrϾTAA 6513.09 N -4648.23 5 R Y 11081.45 711ϩ1 GϾT ThrϾAsn 11094.48 Y 13.03 7.1 R N 7383.08 1078⌬T frameshift 9201.10 Y 1818.02 7 R Y 12233.9 R334W ArgϾGln 12205.87 Y -28.03 R347P ArgϾGly 12134.79 Y -99.11 9 F Y 14049.68 A455E AlaϾGlu 14107.74 Y 58.06 10.2 R Y 10525.57 ⌬I507 ⌬ Asp 10410.50 Y -115.07 ⌬F508 ⌬ Asp & LysϾAsn 10396.43 Y -129.14 11.2 F Y 11173.32 1717-1 GϾA GlyϾArg 11272.46 Y 99.14 G542X TrpϾLeu 11100.27 Y -73.05 G551D no change 11173.32 Y 0.00 R553X ThrϾMet 11203.42 Y 30.10 R560T no change 11173.32 Y 0.00 11 F N 8465.27 1717-1 GϾA no change 8465.27 N 0.00 G542X GlyϾTGA 6584.17 N -1881.10 G551D GlyϾAsp 8523.33 N 58.06 R553X ArgϾTGA 7541.18 N -924.09 R560T ArgϾThr 8410.21 N -55.06 12 F Y 10372.51 1898ϩ1 GϾA GlyϾAsp 10430.57 Y 58.06 13.2A R Y 10103.23 2184⌬A frameshift 8726.91 N -1376.32 14B R Y 9291.17 2789ϩ5 GϾA LeuϾPhe 9325.21 Y 34.04 16 F N 9398.67 3120ϩ1 GϾA ValϾIle 9412.72 N 14.05 19 F Y 17455.96 R1162X ArgϾTGA 6280.13 N -11175.83 3659⌬C frameshift 9650.06 N -7805.90 19i F Y 9699.9 3849ϩ10kB CϾT ArgϾTGA 7131.04 N -2568.86 20 F N 11125.48 W1282X TrpϾTGA 9370.40 N -1755.08 21 F Y 11183.44 N1303K AsnϾLys 11197.54 Y 14.10 a Denotes the directionality of exonic sequence when expressed as peptide. Login to comment
181 The heterozygous mutations depicted are as follows: (A), exon 3 wt/G85E; (B), exon 4.1 wt/R117H; (C), exon 4.2 wt/I148T; (D), exon 4.2 wt/621 ؉ 1G>T; (E), exon 5 wt/711 ؉ 1G>T; (F), exon 7.1 wt/1078⌬T; (G), exon 7 wt/R334W; (H), exon 7 wt/R347P; (I), exon 9 wt/A455E; (J), exon 10.2 wt/⌬I507; (K), exon 10.2 wt/⌬F508; (L), exon 11.2 wt/1717-1G>A; (M), exon 11 wt/G542X; (N), exon 11 wt/G551D; (O), exon 11 wt/R553X; (P), exon 11 wt/R560T; (Q), exon 12 wt/1898 ؉ 1G>A; (R), exon 13.2A wt/2184⌬A; (S), exon 14B wt/2789 ؉ 5G>A; (T), exon 16 wt/3120 ؉ 1G>A; (U), exon 19 wt/R1162X; (V), exon 19 wt/3659⌬C; (W), intron 19 wt/3849 ؉ 10kbC>T; (X), exon 20 wt/W1282X; (Y), exon 21 wt/N1303K. typical yield of purified protein was 1-30 ␮g/test well, depending on the analyte species. Login to comment
227 The remaining two compound heterozygotes (P175 and P176) had both mutations in exon 11; the detection of apparently wild-type analyte in these samples is attributable to translational suppression of the G542X mutation. Login to comment
228 The G542X and R553X mutations registered as mass shifts in both the exon 11 and exon 11.2 analytes, as predicted. Login to comment
229 Mutations predicted on the basis of their peptide mass Table 2. Summary of PMSG screening of putative compound heterozygous patient samples.a Exon Sample P154 P156 P158 P164 P165 P166 P168 P169 P175 P176 3.1 9871 9868 9872 9867 9863 9861 9866 9867 9861 9868 4.1 7054 7052 7057 7049 7048 7039 7048 7044 7047 7046 4.2 11172 11164 11175 11164 11157 11166 11159 11158 11163 11156 5 11096 11084 11098 11088 11088 11071 11084 11079 11076 11085 7.1 7386 7392 7382 7390 7382 7383 7379 7380 7387 7386 7 12232 12229 12234 12231 12237 12238 12239 12239 12240 12238 9 14064 14060 14065 14056 14062 14045 14050 14049 NAb 14051 10.2 10534 10531 10542 10533 No peakc 10525 10528 10527 10527 10524 Mutant 10404 10399 10409 10401 10400 10396 10398 10397 11.2 11186 11180 11182 11182 11179 11168 11175 11178 11075 11179 Mutant 11112 11205 11209 11105 11106 11 8477 8470 8477 8469 8467 8459 8468 8465 8465 ‫؍‬ supd 8459 ‫؍‬ supd Mutant 6591 8420 8427 7541 7539 8409 & 6581 8403 & 6576 12 10382 10376 10394 10379 10385 10365 10370 10370 10378 10366 13.2A 10103 10104 10103 10104 10105 10099 10099 10100 10098 10100 Mutant 8723 14B 9299 9294 9306 9300 9293 9283 9289 9291 9295 9294 16 9414 9403 9408 9402 9409 9391 9400 9396 9398 9396 19 17486 17476 17478 17481 17452 17447 17472 17453 17461 17448 Intron 19 9712 9709 9708 9709 9714 9696 9697 9704 9702 9700 20 11138 11128 11138 11135 11131 11117 NA 11122 11120 11116 Mutant 9372 21 11191 11189 11190 11187 11185 11181 11183 11185 11187 11183 Sequence result ⌬F508 ⌬F508 ⌬F508 ⌬F508 ⌬F508 ⌬F508 ⌬F508 ⌬F508 G542X G542X G542X W1282X R560T G551D ⌬F508 2183AAϾG R553X R553X R560T R560T a Shaded boxes highlight test analytes revealing evidence of mutation. Login to comment
234 d sup, slight wild-type mass analyte attributable to translational suppression of G542X mutation. Login to comment

[hide] HFE alleles in an Irish cystic fibrosis population... Genet Test. 2003 Summer;7(2):155-8. Devaney J, Maher M, Smith T, Houghton JA, Glennon M
HFE alleles in an Irish cystic fibrosis population.
Genet Test. 2003 Summer;7(2):155-8., [PMID:12885340]

Abstract [show]
The variable clinical manifestations of cystic fibrosis (CF) suggest the influence of modifier genes. Genetic and environmental factors that determine whether an individual will develop associated complications are still being determined. It has been proposed that the gene for hemochromatosis, HFE, may be a modifier locus for CF disease phenotype. Recent research has suggested a relationship between mutations to the HFE gene and the development of meconium ileus (MI) and liver disease in CF. This study aims to expand our knowledge of the HFE mutations C282Y and H63D carrier rate in an Irish population of CF allele carriers. PCR restriction enzyme analysis was performed on blood samples from CF patients to identify the C282Y and H63D mutations. HFE status of CF allele carriers and CF patients (Delta F508) homozygotes with and without meconium ileus was determined. The carrier frequency for C282Y was 30.8% for the Delta F508 homozygote MI positive group, as compared to 12.5% for the non-Delta F508 MI positive group but did not reach statistical significance (p = 0.27). Interestingly, no Delta F508 homozygote patients were homozygous for the C282Y mutation.

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57 Our non-DF508 CF patients were screened for the R117H, 1717-1G R A, DI507, G542X, G551D, R553X, R560K, and R560T CFTR mutations prior to inclusion in this study; it is interesting to note that the G542X and G551D alleles have positive and negative associations, respectively, with MI development (Schwarz et al., 1995; Feingold and Gailloud-Bataille, 1999). Login to comment

[hide] Mutation analysis of the cystic fibrosis transmemb... Eur J Hum Genet. 2003 Sep;11(9):687-92. Perri F, Piepoli A, Stanziale P, Merla A, Zelante L, Andriulli A
Mutation analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the cationic trypsinogen (PRSS1) gene, and the serine protease inhibitor, Kazal type 1 (SPINK1) gene in patients with alcoholic chronic pancreatitis.
Eur J Hum Genet. 2003 Sep;11(9):687-92., [PMID:12939655]

Abstract [show]
Susceptibility to alcoholic chronic pancreatitis (ACP) could be genetically determined. Mutations in cationic trypsinogen (PRSS1), cystic fibrosis transmembrane conductance regulator (CFTR), and serine protease inhibitor, Kazal type 1 (SPINK1) genes have been variably associated with both the hereditary and the idiopathic form of chronic pancreatitis (CP). Our aim was to analyze the three genes in ACP patients. Mutational screening was performed in 45 unrelated ACP patients and 34 patients with alcoholic liver disease (ALD). No mutation of PRSS1 was found in ACP and ALD patients. Three mutations of CFTR were detected in four ACP patients with a prevalence (8.9%) not significantly different from that observed (3.0%) in ALD patients and from that expected (3.2%) in our geographical area. Neither compound heterozygotes for CFTR nor trans-heterozygotes for CFTR/SPINK1 were found. One ACP patient (2.2%) was found to carry the most common mutation (N34S) of SPINK1 compared to none of the ALD patients (P=NS). In five other patients (two with ACP and three with ALD) other rare variants, including P55S, were found. In contrast with the hereditary and the idiopathic forms of CP, in which mutations of PRSS1, CFTR, and SPINK1 genes may occur, ACP is still a "gene(s)-orphan" disease. The supposed genetic susceptibility to ACP relies on other yet unknown gene(s) which could affect the alcohol metabolism or modulate the pancreatic inflammatory response to alcohol abuse.

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33 Mutation screening of the CFTR gene The 31 most frequent mutations (F508del, I507del, G551D, G542X, N1303K, 1717-1G4A, W1282X, R553X, R347P, R347H, R334W, 3849+10kb C4T, R117H, 621+1G4T, A455E, S549N, R560T, S549R, V520F, Q493X, 3849+ 4A4G, 1078delT, R1162X, 3659delC, 3905insT, Y122X, 2183delAA4G, 2789+5G4A, 1898+1G4A, 711+1G4T, and G85E) were examined with the polymerase chain reaction (PCR) followed by an oligonucleotide ligation assay (OLA, Applied Biosystems, Foster City, CA, USA) and finally a sequence-coded separation. Login to comment
67 Our working hypothesis was that the pancreatic damage due to high alcohol intake could be due to either an abnormal allele in one of the three genes or a combination of multiple mutations occurring in the two alleles of the same gene (compound heterozygote) or different genes (trans- Table 1 Demographic and clinical data ACP All CFTR Mut SPINK1 Mut Mut negative N 45 4 3 38 Sex (M/F) 44/1 4/0 3/0 37/1 Age (years) 3875 3673 3774 3875 Age at onset (years) 3075 2976 3075 3276 Symptoms` duration (years) 872 773 773 673 Alcohol (g/day) 160743 130720 133723 166744 Smokers 26 3 2 21 ALD N 34 1 3 30 Sex (M/F) 28/6 1/0 3/0 24/6 Age (years) 4476 46 3977 4476 Age at onset (years) 3677 35 33711 3776 Symptoms` duration (years) 873 11 774 673 Alcohol (g/day) 140741 140 133723 140743 Smokers 19 1 1 17 Table 2 Sequence variations identified in the PRSS1, CFTR, and SPINK1 genes in 45 ACP patients CFTR Patients n PRSS1 Mutant Poly T SPINK1 1 1 Fa F508del NDb F 2 1 F G542X 7/9 F 3 1 F 711+1G>T 7/7 F 4 1 F 711+1G>T 7/7 F 5 1 F F 7/7 P55S 6 1 F F 5/7 IVS2-23A>T 7 1 F F 7/9 N34S 8 1 F F 7/7 ND 9-10 2 F F 5/7 F 11-37 27 F F 7/7 F 38-45 8 F F 7/9 F a indicates two wild alleles. Login to comment

[hide] The phenotypic consequences of CFTR mutations. Ann Hum Genet. 2003 Sep;67(Pt 5):471-85. Rowntree RK, Harris A
The phenotypic consequences of CFTR mutations.
Ann Hum Genet. 2003 Sep;67(Pt 5):471-85., [PMID:12940920]

Abstract [show]
Cystic fibrosis is a common autosomal recessive disorder that primarily affects the epithelial cells in the intestine, respiratory system, pancreas, gall bladder and sweat glands. Over one thousand mutations have currently been identified in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene that are associated with CF disease. There have been many studies on the correlation of the CFTR genotype and CF disease phenotype; however, this relationship is still not well understood. A connection between CFTR genotype and disease manifested in the pancreas has been well described, but pulmonary disease appears to be highly variable even between individuals with the same genotype. This review describes the current classification of CFTR mutation classes and resulting CF disease phenotypes. Complex disease alleles and modifier genes are discussed along with alternative disorders, such as disseminated bronchiectasis and pancreatitis, which are also thought to result from CFTR mutations.

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31 The majority of the other CFTR mutations are very rare with only four other mutations (G542X, N1303K, G551D and W1282X) having overall frequencies above 1%. Login to comment
47 These mutations, the most common being G542X, prevent the synthesis of a stable protein or result in the production of a truncated protein due to the creation of a premature termination codon. Login to comment
56 Alternatively, recent studies showed that premature stop codons, such as G542X and R553X, were suppressed by the addition of aminoglycoside antibiotics (e.g. gentamicin or G418) that are known to stimulate the suppression of stop codons in various organisms by near-cognate mis-pairing of an aminoacyl-tRNA with the premature stop codon (Howard et al. 1996). Login to comment

[hide] A haplotype-based molecular analysis of CFTR mutat... Hum Mol Genet. 2003 Sep 15;12(18):2321-32. Lee JH, Choi JH, Namkung W, Hanrahan JW, Chang J, Song SY, Park SW, Kim DS, Yoon JH, Suh Y, Jang IJ, Nam JH, Kim SJ, Cho MO, Lee JE, Kim KH, Lee MG
A haplotype-based molecular analysis of CFTR mutations associated with respiratory and pancreatic diseases.
Hum Mol Genet. 2003 Sep 15;12(18):2321-32., 2003-09-15 [PMID:12952861]

Abstract [show]
Aberrant membrane transport caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is associated with a wide spectrum of respiratory and digestive diseases as well as cystic fibrosis. Using a gene scanning method, we found 11 polymorphisms and mutations of the CFTR gene in the Korean population. Individual variants at these sites were analyzed by conventional DNA screening in 117 control and 75 patients having bronchiectasis or chronic pancreatitis. In a haplotype determination based on a Bayesian algorithm, 15 haplotypes were assembled in the 192 individuals tested. Several haplotypes, especially with Q1352H, IVS8 T5, and E217G, were found to have disease associations in a case-control study. Notably, a common polymorphism of M470V appears to affect the intensity of the disease association. Among the two haplotypes having IVS8 T5, the T5-V470 haplotype showed higher disease association than the T5-M470 haplotype. In addition, a Q1352H mutation found in a V470 background showed the strongest disease association. The physiological significances of the identified mutations were rigorously analyzed. Non-synonymous E217G and Q1352H mutations in the M470 background caused a 60-80% reduction in CFTR-dependent Cl(-) currents and HCO3(-) -transport activities. Surprisingly, the additional M470V polymorphic variant with the Q1352H mutation completely abolished CFTR-dependent anion transport activities. These findings provide the first evidence on the importance of CFTR mutations in the Asian population. Importantly, the results also reveal that interactions between multiple genetic variants in cis affect the final function of the gene products.

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15 Several mutations of CFTR, such as F508del, G542X and N1303K, are associated with severe CF phenotypes and display high disease penetrance. Login to comment
74 CFTR genetic variants analyzed in this study Variations found by TDGS Most common worldwide disease-causing mutations Reported disease-associated microsatellite À8G/C (50 UTR)a R117H (exon 4) T5-7,9 (IVS 8) (16) I125T (exon 4)b 621 þ 1G > T (intron 4) E217G (exon 6a)b F508del (exon 10) 1059C > T (exon 7, A309)a 1717-1G > A (intron 10) M470V (exon 10)b G542X (exon 11) I556V (exon 11)b G551D (exon 11) 2694T/G (exon 14a, T854)b R553X (exon 11) Q1352H (exon 22)b R1162X (exon 19) R1453W (exon 24)b W1282X (exon 20) N1303K (exon 21) Mutation names and nucleotide numbers are presented according to the Cystic Fibrosis Genetic Analysis Consortium (CFGAC; www.genet.sickkids.on.ca/). Login to comment
160 However, over 95% of the three most common CF-causing mutations, F508del, G542X and N1303K, arise from this haplotype. Login to comment

[hide] CFTR genotypes in patients with normal or borderli... Hum Mutat. 2003 Oct;22(4):340. Feldmann D, Couderc R, Audrezet MP, Ferec C, Bienvenu T, Desgeorges M, Claustres M, Mittre H, Blayau M, Bozon D, Malinge MC, Monnier N, Bonnefont JP, Iron A, Bieth E, Dumur V, Clavel C, Cazeneuve C, Girodon E
CFTR genotypes in patients with normal or borderline sweat chloride levels.
Hum Mutat. 2003 Oct;22(4):340., [PMID:12955726]

Abstract [show]
In recent years, some patients bearing "atypical" forms of cystic fibrosis (CF) with normal sweat chloride concentrations have been described. To identify the spectrum of mutant combinations causing such atypical CF, we collected the results of CFTR (ABCC7) mutation analysis from 15 laboratories. Thirty patients with one or more typical symptoms of the disease associated with normal or borderline sweat chloride levels and bearing two CFTR mutations were selected. Phenotypes and genotypes of these 30 patients are described. A total of 18 different CFTR mutations were observed in the 60 chromosomes analysed. F508del was present in 31.6 % of the mutated chromosomes and 3849+10kbC>T in 13.3 %. R117H, D1152H, L206W, 3272-26A>G, S1235R, G149R, R1070W, S945L, and the poly-T tract variation commonly called IVS8-5T were also observed. The relative frequency of CFTR mutations clearly differed from that observed in typical CF patients or in CBAVD patients with the same ethnic origin. A mild genotype with one or two mild or variable mutations was observed in all the patients. These findings improve our understanding of the distribution of CFTR alleles in CF with normal or borderline sweat chloride concentrations and will facilitate the development of more sensitive CFTR mutation screening.

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44 Table 1 : Genotypes and Phenotypes of Patients with Normal or BordIerline Sweat Tests Patient Age at diagnosis (years) CFTR GENOTYPE* Allele 1 Allele 2 SWEAT CL- MEAN (MMOL/L) PHENOTYPE 1 0.2 F508del G149R 38 P+PI, neonatal hypertrypsinemia, 2 0.3 G551D R117H-7T 31 neonatal hypertrypsinemia 3 0.4 F508del R1070W 30.5 neonatal hypertrypsinemia 4 0.4 F508del R117H-7T 52 P 5 0.6 F508del 3849+10kbC>T 48 P 6 0.11 F508del S945L 58 P+PI 7 1 F508del 5T 40 P+CBAVD 8 2 F508del L206W 53 P 9 2 W1282X 5T 42.5 P 10 5 F508del 3849+10kbC>T 55.5 P 11 5 F508del L206W 55 P 12 5 G91R 5T 47.5 P 13 6 G551D S1235R+5T 49.5 P, neonatal hypertrypsinemia 14 7 F508del 3849+10kb 50 P, nasal popyposis 15 13 F508del R117H-7T 58 P, nasal polyposis 16 18 F508del 5T 60.5 P 17 20 G542X 3849+10kbC>T 52 P+PI 18 21 I507del 3849+10kbC>T 54 P, bronchiectasis 19 30 R347P 3849+10kbC>T 43 P, Pseudomonas colonisation 20 30 I507del L206W 57.5 CBAVD, chronic cough 21 31 F508del R117H-7T 60 CBAVD 22 32 G542X 3849+10kbC>T 30 P, Pseudomonas colonisation 23 34 F508del 3272-26A>G 64 P, CBAVD 24 37 R1070Q D1152H 56 CBAVD, bronchectasis 25 46 F508del D1152H 43 P 26 55 F508del D1152H 48 P, Pseudomonas colonisation 27 56 I507del S1235R 53 P 28 >18 F508del D1152H 60 P+PI 29 >20 F508del 3849+10kbC>T 18 P, bronchiectasis 30 >20 F508del 3272-26A>G 61 P *All mutations are named in accordance with the numbering used in the CFTR Mutation Database: http://www.genet.sickkids.on.ca/cftr/. Login to comment

[hide] Liver cirrhosis and portal hypertension in cystic ... Eur J Gastroenterol Hepatol. 2003 Oct;15(10):1073-8. Efrati O, Barak A, Modan-Moses D, Augarten A, Vilozni D, Katznelson D, Szeinberg A, Yahav J, Bujanover Y
Liver cirrhosis and portal hypertension in cystic fibrosis.
Eur J Gastroenterol Hepatol. 2003 Oct;15(10):1073-8., [PMID:14501614]

Abstract [show]
OBJECTIVES: Liver disease is the second cause of death in cystic fibrosis. The most deleterious complication of liver disease is portal hypertension, which has an estimated prevalence of up to 8%. Portal hypertension may manifest itself by splenomegaly, hypersplenism, gastro-oesophageal bleeding and ascites. The aim of our study was to determine the prevalence, risk factors and invasive management of portal hypertension at our centre. METHODS: One hundred and fifty patients with cystic fibrosis were followed up between 1975 and 2000 in the national cystic fibrosis centre in Israel. Forty patients (27%) had liver disease. All underwent clinical evaluation and laboratory and imaging studies. RESULTS: Portal hypertension was diagnosed in 10 patients (7%), of whom eight were male. The mean age at diagnosis was 11 years (range, 4-17 years). All had severe mutations of the cystic fibrosis transmembrane conductance regulator gene (the CFTR gene), pancreatic insufficiency, meconium ileus or distal intestinal obstruction syndrome and variceal bleeding. Seven patients underwent sclerotherapy to control acute bleeding. Four underwent portosystemic shunting (functioning up to 37 years). Two patients with severe lung and liver disease underwent transjugular intrahepatic portosystemic shunting, which provided bleeding control, but both died while waiting for lung/liver transplantation. One patient underwent liver transplantation due to liver failure and still had good liver and lung function 10 years later. CONCLUSIONS: Portal hypertension is more common among Israeli patients with cystic fibrosis. The unique genetic composition of our population may explain this phenomenon. Risk factors include male gender, pancreatic insufficiency, severe CFTR mutations, meconium ileus and meconium ileus equivalent. Sclerotherapy is the main option to control oesophageal variceal bleeding, while portosystemic shunts offer a prolonged alternative treatment for refractory bleeding. A transjugular intrahepatic portosystemic shunt and liver transplantation may also be effective, but further research is required in order to establish their role.

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92 Unauthorized reproduction of this article is prohibited. Table 2 Details of the 10 cystic fibrosis patients with portal hypertension Pulmonary function test Symptoms Liver Patient of portal synthetic Procedures number Sex Mutation FEV1 FVC hypertension function (year) 1 M W1282X/W1282X 50% 55% H,V Abnormal Splenectomy & splenorenal shunt (`63) 2 F A¨ F508/G542X 75% 90% H,V Abnormal Portacaval end to side shunt (`77) 3 M G542X/W1282X 82% 92% H,V Abnormal Liver transplantation (`90) 4 M A¨ F508/W1282X 65% 80% H,V,A Abnormal Sclerotherapy & portacaval shunt (`98) 5 M G542X/W1282X 30% 40% H,V,A Abnormal Sclerotherapy & TIPSS (x2): right hepatic to left portal shunt (`99) 6 M A¨ F508/G542X 70% 80% H,V Abnormal Sclerotherapy & portosystemic shunt: superior mesenteric vein to left renal vein (`99) 7 M A¨ F508/A¨ F508 22% 25% H,V,A Abnormal Recurrent sclerotherapy (x5) & TIPSS 8 F W1282X/N1303K 75% 85% H,V,A Abnormal Sclerotherapy 9 M G542X/G542X 65% 70% H,V,A Abnormal Sclerotherapy 10 M A¨ F508/A¨ F508 55% 85% H,V Borderline Sclerotherapy M, male; F, female; FEV1, forced expiratory volume in 1s; FVC, forced vital capacity; H, hypersplenism; V, varices; A, ascites; TIPSS, transjugular intrahepatic portosystemic shunt. Login to comment

[hide] Gentamicin-induced correction of CFTR function in ... N Engl J Med. 2003 Oct 9;349(15):1433-41. Wilschanski M, Yahav Y, Yaacov Y, Blau H, Bentur L, Rivlin J, Aviram M, Bdolah-Abram T, Bebok Z, Shushi L, Kerem B, Kerem E
Gentamicin-induced correction of CFTR function in patients with cystic fibrosis and CFTR stop mutations.
N Engl J Med. 2003 Oct 9;349(15):1433-41., 2003-10-09 [PMID:14534336]

Abstract [show]
BACKGROUND: Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene containing a premature termination signal cause a deficiency or absence of functional chloride-channel activity. Aminoglycoside antibiotics can suppress premature termination codons, thus permitting translation to continue to the normal end of the transcript. We assessed whether topical administration of gentamicin to the nasal epithelium of patients with cystic fibrosis could result in the expression of functional CFTR channels. METHODS: In a double-blind, placebo-controlled, crossover trial, patients with stop mutations in CFTR or patients homozygous for the DeltaF508 mutation received two drops containing gentamicin (0.3 percent, or 3 mg per milliliter) or placebo in each nostril three times daily for two consecutive periods of 14 days. Nasal potential difference was measured at base line and after each treatment period. Nasal epithelial cells were obtained before and after gentamicin treatment from patients carrying stop mutations, and the C-terminal of surface CFTR was stained. RESULTS: Gentamicin treatment caused a significant reduction in basal potential difference in the 19 patients carrying stop mutations (from -45+/-8 to -34+/-11 mV, P=0.005) and a significant response to chloride-free isoproterenol solution (from 0+/-3.6 to -5+/-2.7 mV, P<0.001). This effect of gentamicin on nasal potential difference occurred both in patients who were homozygous for stop mutations and in those who were heterozygous, but not in patients who were homozygous for DeltaF508. After gentamicin treatment, a significant increase in peripheral and surface staining for CFTR was observed in the nasal epithelial cells of patients carrying stop mutations. CONCLUSIONS: In patients with cystic fibrosis who have premature stop codons, gentamicin can cause translational "read through," resulting in the expression of full-length CFTR protein at the apical cell membrane, and thus can correct the typical electrophysiological abnormalities caused by CFTR dysfunction.

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15 Howardetal.demonstratedthattwoCFTR-associated stop mutations could be suppressed by treating cells with low doses of an aminoglycoside antibiotic.12 Bedwell et al. demonstrated that after incubation of bronchial epithelial cell line IB3-1, whichcarriesaW1282XmutationofCFTR,withami- noglycosides, cyclic AMP (cAMP)-activated chloride conductance and the expression of functional CFTR were restored to the apical membrane.13 Recently, Zsembery et al. isolated cholangiocytes from the liver of a patient carrying the G542X mutation of CFTR and incubated them with gentamicin, resulting in the expression of cAMP-activated chloride transport.14 Thus, in vitro, gentamicin obviated the effect of stop-codon mutations on the transcription and translation of CFTR. This effect has subsequently been demonstrated in a number of models of other diseases caused by stop mutations,includingmusculardystrophy,15 Hurler`ssyndrome,16 cystinosis,17 late infantile neuronal ceroid lipofuscinosis,18 and disorders involving the p53 gene.19 In a previous open pilot study, we found that topical application of gentamicin drops to the nose augmented chloride transport in epithelial cells of nine patients with cystic fibrosis who had at least one W1282X allele.20 Subsequently, Clancy et al.,21 in an open study, administered gentamicin intravenously to five patients who were heterozygous for stop mutations and found that four of the patients had hyperpolarization of the nasal potential differ- enceaftertheadministrationofisoproterenol,indicating that chloride transport was induced across the apical surface. Login to comment
62 Of the 24 study patients,11carriedtwostopmutations:6werehomo- zygous for W1282X, 3 were compound heterozygous for W1282X/G542X, and 2 were compound heterozygous for W1282X/3849+10KbC˚T. Login to comment
129 In vitro studies using quantitative immunohistochemistry have shown that after incubation with an aminoglycoside, cells from patients with cystic fibrosis have as much as 25 percent (in those with the R553X mutation) to 35 percent (in those with the G542X mutation) of the concentration of full-length CFTR observed in cells transfected with a wild-type CFTR complementary DNA.12,13 This in- creaseinfunctionalCFTRmightexceedthethresh- old required for normally functioning respiratory epithelial cells and might thus have corrected cell-membrane function in our patients. Login to comment

[hide] Pathophysiology and management of pulmonary infect... Am J Respir Crit Care Med. 2003 Oct 15;168(8):918-51. Gibson RL, Burns JL, Ramsey BW
Pathophysiology and management of pulmonary infections in cystic fibrosis.
Am J Respir Crit Care Med. 2003 Oct 15;168(8):918-51., 2003-10-15 [PMID:14555458]

Abstract [show]
This comprehensive State of the Art review summarizes the current published knowledge base regarding the pathophysiology and microbiology of pulmonary disease in cystic fibrosis (CF). The molecular basis of CF lung disease including the impact of defective cystic fibrosis transmembrane regulator (CFTR) protein function on airway physiology, mucociliary clearance, and establishment of Pseudomonas aeruginosa infection is described. An extensive review of the microbiology of CF lung disease with particular reference to infection with P. aeruginosa is provided. Other pathogens commonly associated with CF lung disease including Staphylococcal aureus, Burkholderia cepacia, Stenotrophomonas maltophilia, Achromobacter xylosoxidans and atypical mycobacteria are also described. Clinical presentation and assessment of CF lung disease including diagnostic microbiology and other measures of pulmonary health are reviewed. Current recommendations for management of CF lung disease are provided. An extensive review of antipseudomonal therapies in the settings of treatment for early P. aeruginosa infection, maintenance for patients with chronic P. aeruginosa infection, and treatment of exacerbation in pulmonary symptoms, as well as antibiotic therapies for other CF respiratory pathogens, are included. In addition, the article discusses infection control policies, therapies to optimize airway clearance and reduce inflammation, and potential future therapies.

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77 Class 1 mutations, exemplified by G542X, contain premature stop mutations that create truncated mRNA. Login to comment

[hide] Mutations of the CFTR gene in pancreatic disease. Pancreas. 2003 Nov;27(4):332-6. Pezzilli R, Morselli-Labate AM, Mantovani V, Romboli E, Selva P, Migliori M, Corinaldesi R, Gullo L
Mutations of the CFTR gene in pancreatic disease.
Pancreas. 2003 Nov;27(4):332-6., [PMID:14576497]

Abstract [show]
INTRODUCTION: An association has been found between CFTR gene mutations and chronic pancreatitis; however, there is a lack of information about the frequency of CFTR gene mutations in acute pancreatitis and in pancreatic cancer. AIM: To prospectively evaluate the prevalence of CFTR gene mutations in acute pancreatitis, chronic pancreatitis, and pancreatic cancer. METHODOLOGY: Ninety-eight consecutive patients were studied and divided into 3 groups: 34 patients with acute pancreatitis, 46 patients with chronic pancreatitis, and 18 patients with pancreatic cancer. The mutation analysis of the CFTR gene was carried out using diagnostic commercial kits for the simultaneous detection of 29 mutations and Tn polymorphism. RESULTS: Among the 98 patients studied, 12 (12.2%) had CFTR gene mutations: 2 of the 34 patients (5.9%) with acute pancreatitis, 9 of the 46 (19.6%) with chronic pancreatitis, and 1 of the 18 (5.6%) with pancreatic cancer. All the mutations were found in heterozygosis (2 DeltaF508, 1 W1282X, and 9 T5 allele). CONCLUSION: Our prospective study adds further information about the frequency of CFTR mutations in patients with a single episode of acute pancreatitis. Furthermore, our results suggest an association of CFTR gene mutations with chronic alcoholic pancreatitis and emphasize the need for a multicenter study, possibly multinational, to conclusively establish the role of CFTR mutations as a genetic susceptibility factor for this disease.

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59 The 29 Mutations and the Tn Polymorphism Which Can Be Detected by INNO-LiPA Assays Mutation Exon/Intron (i) E60X, G85E, 394delTT 3 621 + 1G > T, R117H (i) 4, 4 711 + 5G > A (i) 5 1078delT, R347P, R334W 7 A455E, Tn (i) 8, 9 ⌬F508, ⌬I507 10 G542X, 1717-1 G > A, G551D, R553X, R560T, Q552X (i) 10, 11 2183AA > G, 2184del A, 2143delT 13 2789 + 5G > A (i) 14b R1162X, 3659delC 19 3849 + 10kbC > T (i) 19 3905insT, W1282X, S1251N 20 N1303K 21 Group 3: pancreatic cancer CFTR gene mutations were identified only in 1 of the 18 patients (5.6%) with this cancer. Login to comment

[hide] Isolated idiopathic chronic pancreatitis associate... Gastroenterol Clin Biol. 2003 Aug-Sep;27(8-9):821-4. Reboul MP, Laharie D, Amouretti M, Lacombe D, Iron A
Isolated idiopathic chronic pancreatitis associated with a compound heterozygosity for two mutations of the CFTR gene.
Gastroenterol Clin Biol. 2003 Aug-Sep;27(8-9):821-4., [PMID:14586256]

Abstract [show]
We report the case of a patient suffering from idiopathic chronic pancreatitis (ICP) and compound heterozygous for mutations G542X and S1235R of the cystic fibrosis transmembrane regulator (CFTR) gene. The patient had normal sweat test and no other clinical sign usually linked with a typical or moderate pathology (bronchiectasis, nasal polyposis, congenital absence of the vas deferens) of the CFTR gene. G542X is a severe mutation, which is usually found in classical cystic fibrosis when associated with other severe mutations. S1235R is a quite rare abnormality recently reported as being potentially pathogenic when combined in trans with a second CF mutation. Our case is quite similar to the only other six patients in the literature in whom only the pancreas is affected and who bear a rare mutation with moderate effect. The history and the clinical features of our patient indicate an unambiguous isolated ICP in which the presence of the S1235R mutation--in trans with regard to G542X--is likely responsible for the ICP phenotype. This case could throw light on some of the as yet poorly known abnormalities of the CFTR gene in the ICP phenotype.

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1 SUMMARY We report the case of a patient suffering from idiopathic chronic pancreatitis (ICP) and compound heterozygous for mutations G542X and S1235R of the cystic fibrosis transmembrane regulator (CFTR) gene. Login to comment
3 G542X is a severe mutation, which is usually found in classical cystic fibrosis when associated with other severe mutations. Login to comment
6 The history and the clinical features of our patient indicate an unambiguous isolated ICP in which the presence of the S1235R mutation - in trans with regard to G542X - is likely responsible for the ICP phenotype. Login to comment
8 RE´SUME´ Pancréatite chronique idiopathique isolée associée à une hétérozygotie composite pour deux mutations du gène CFTR Marie-Pierre REBOUL, David LAHARIE, Michel AMOURETTI, Didier LACOMBE, Albert IRON (Gastroenterol Clin Biol 2003;27:821-824) Nous rapportons le cas d`un malade souffrant de pancréatite chronique idiopathique (PCI) hétérozygote composite pour les mutations G542X et S1235R du gène CFTR. Login to comment
10 G542X est une mutation sévère rencontrée assez fréquemment - en association avec d`autres mutations sévères - dans des formes de mucoviscidose classique. Login to comment
13 L`histoire et le tableau clinique de la maladie chez notre malade évoquent de manière complète et sans ambiguïté une PCI isolée dans laquelle la présence de la mutation S1235R - en position trans par rapport à G542X - est très vraisemblablement responsable du phénotype de pancréatite chronique idiopathique. Login to comment
23 He had an original compound heterozygosity for the severe G542X mutation and the mild S1235R mutation. Login to comment
44 This revealed the G542X mutation with a heterozygous status. Login to comment
67 In five patients (no 5, 9, 10, 11, 15) who bear a frequent mutation well known for its severity (F508del or G542X), the involvement of the ICP phenotype could lie in their "second" missense mutation, i.e. L997F in exon 17b (Patient no 5), I1027T in exon 17a (Patient no 9), D1152H in exon 18 (Patients no 10 and 11) and S1235R in exon 19 (Patient no 15); and the presence of 2 of these 4 missense mutations in patient no 7 could actually strengthen this hypothesis but to date little is known about the possible impact of his 5T allele on the phenotype (possible sterility). Login to comment
75 It is a rare moderate mutation recently studied by Cuppens`s team [16] who showed that the deleterious character Fig. 2 - Pedigree of the family (limited to the subjects investigated) with mention of the haplotypes IVS8CA-TGm-Tn-M470V-IVS17bTA-IVS17bCA and the presence or absence (-) of the 2 familial mutations G542X (exon 10) and S1235R (exon 19). Login to comment
76 ↑ proband Arbre généalogique de la famille (limité aux sujets analysés) avec mention des haplotypes IVS8CA-TGm-Tn-M470V-IVS17bTA-IVS17bCA et de la présence ou de l`absence (-) des 2 mutations familiales G542X (exon 10) et S1235R (exon 19). Login to comment
80 Patient CFTR no PolyT genotype Sex genotype Age (years) Sweat chloride (mmol/L) Anamnestic features known to be associated with atypical CF Reference 1 F508del/R117H 9T/7T M 45 29 CBAVD [4] 2 N1303K/R117H 9T/7T F n.a. 37 bronchiectasis, sinusitis, positive NPD [5] 3 R1162X/2789+5G>A 7T/7T F n.a. 108 chronic cough [5] 4 I336K/R75Q 7T/7T F 26 26 nasal polyposis [7] 5 F508del/L997F 9T/7T M 17 24 none [11] 6 3849+10kbC>T/3878delG 7T/7T M 14 n.a. none [11] 7 S1235R/L997F 5T/7T M 27 25 none [11] 8 F508del/R117H n.a. M 45 29 CBAVD, smooth P. aeruginosa [12] 9 F508del/I1027T n.a. F 32 59 none [12] 10 F508del/D1152H n.a. M 8 62 none [12] 11 F508del/D1152H n.a. F 15 32 none [12] 12 F508del/P574H n.a. F 26 81 sinus surgery, S. aureus, S. maltophilia [12] 13 F508del/3120G>A n.a. F 40 n.a. n.a. [12] 14 F508del/G1069R n.a. M 16 n.a. n.a. [12] 15 G542X/S1235R 7T/7T M 35 15 none [this study] n.a.: not available. Login to comment

[hide] High allelic heterogeneity between Afro-Brazilians... Genet Test. 2003 Fall;7(3):213-8. Raskin S, Pereira L, Reis F, Rosario NA, Ludwig N, Valentim L, Phillips JA 3rd, Allito B, Heim RA, Sugarman EA, Probst CM, Faucz F, Culpi L
High allelic heterogeneity between Afro-Brazilians and Euro-Brazilians impacts cystic fibrosis genetic testing.
Genet Test. 2003 Fall;7(3):213-8., [PMID:14641997]

Abstract [show]
Cystic fibrosis (CF) is an autosomal recessive disease caused by at least 1,000 different mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). To determine the frequency of 70 common worldwide CFTR mutations in 155 Euro-Brazilian CF patients and in 38 Afro-Brazilian CF patients, we used direct PCR amplification of DNA from a total of 386 chromosomes from CF patients born in three different states of Brazil. The results show that screening for seventy mutations accounts for 81% of the CF alleles in Euro-Brazilians, but only 21% in the Afro-Brazilian group. We found 21 different mutations in Euro-Brazilians and only 7 mutations in Afro-Brazilians. The frequency of mutations and the number of different mutations detected in Euro-Brazilians are different from Northern European and North American populations, but similar to Southern European populations; in Afro-Brazilians, the mix of CF-mutations is different from those reported in Afro-American CF patients. We also found significant differences in detection rates between Euro-Brazilian (75%) and Afro-Brazilian CF patients (21%) living in the same state, Minas Gerais. These results, therefore, have implications for the use of DNA-based tests for risk assessment in heterogeneous populations like the Brazilians. Further studies are needed to identify the remaining CF mutations in the different populations and regions of Brazil.

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11 Although the major mutation causing CF accounts for 66% of mutant chromosomes screened worldwide, at least 1,000 sequence alterations associated with the disease have been identified in the CFTR gene during the past years, and their frequencies vary between populations (Tsui, 1990, 1992; Cystic Fibrosis Genetic AnalysisConsortium,1994, 1999).Previously, we have shown allelic heterogeneity in Brazilian CF patients of European origin by screening for DF508 and another four common worldwide mutations (G542X, N1303K, G551D, and R553X). Login to comment
54 Only three mutations have an overall frequency higher than 4%-DF508, G542X, and R1162X-and a fourth, 312011G R A, has a frequency of 2.6% in the Afro-Brazilian group. Login to comment
61 In this study, we also found that only four among 70 CFTR mutations included in the screening panel were present in CF patients born in the three different states of Brazil and in the two population subgroups (Euro- and Afro-Brazilians) studied; that is, DF508, G542X, R1162X, and G85E. Login to comment
63 FREQUENCIES OF 70 CFTR MUTATIONS IN DIFFERENT STATES OF BRAZIL, BY CONTINENTA L GROUP CFTR mutations SC PR MG detected n n n n % n % N % DF508 53 39 54 146 47.1 8 10.5 154 39.9 G542X 6 9 8 23 7.4 1 1.3 24 6.2 R1162X 9 2 4 15 4.8 2 2.6 17 4.4 N1303K 5 5 0 10 3.2 0 0 10 2.6 R334W 5 1 4 10 3.2 0 0 10 2.6 G85E 2 2 4 8 2.6 1 1.3 9 2.3 1717-1G®A 1 3 2 6 1.9 0 0 6 1.6 W1282X 4 1 1 6 1.9 0 0 6 1.6 3849110kbC®T 1 3 1 5 1.6 0 0 5 1.3 R553X 0 2 0 2 0.7 0 0 2 0.5 1812-1G®A 0 1 3 4 1.3 1 1.3 5 1.3 2183AA®G 2 1 0 3 1.0 0 0 3 0.8 312011G®A 0 0 2 2 0.7 2 2.6 4 1.0 Y1092X 0 1 1 2 0.7 1 1.3 3 0.8 G551D 0 0 0 0 0 0 0 0 0 W1089X 0 0 1 1 0.3 0 0 1 0.3 6211G®T 0 1 0 1 0.3 0 0 1 0.3 Q1238X 0 1 0 1 0.3 0 0 1 0.3 711-1G®T 0 1 0 1 0.3 0 0 1 0.3 R347P 1 0 0 1 0.3 0 0 1 0.3 189811G®A 1 0 0 1 0.3 0 0 1 0.3 I507 0 0 1 1 0.3 0 0 1 0.3 Subtotal 91 73 86 250 80.7 16 21.1 266 68.9 Alleles with CFTR 5 27 28 60 19.4 60 79.0 120 31.1 mutations not detected Total 96 100 114 310 100.0 76 100.0 386 100.0 Detection rate (%) 94.8 73.0 75.4 250 80.7 16 21.1 266 68.9 The following 70 CFTR mutations were selected and tested on the basis of frequency in various populations, known association with CF, or predicted deleterious effect on the CFTR protein product; DF508, G542X, N1303K, G551D, R553X, DI507, A455E, A559T, C524X, D1270N, E60X, G178R, G330X, G85E, 2307insA, I148T, K710X, P574H, Q1238X, Q493X, Q890X, R1158X, R1162X, R117H, R334W, R347H, R347P 2307insA, I148T, K710X, P574H, Q1238X, Q493X, Q890X, R1158X, R1162X, R117H, R334W, R347H, R347P 2307insA, 1148T, K710X, P574H, Q1238X, Q493X, Q890X, R1158X, R1162X, R117H, R334W, R347H, R347P, R352Q, R560T, S1196X, S1255X, S364P, S549N, S549R, V520F, W1089X, W1282X, W1310X, W1316X, Y1092X, Y122X, Y563D, 1078delT,1677delTA,1717-1G-A,1812-1G-A,1898 1 1G-A, 2043delG,2183delAA-G, 2184delA, 2789 1 5G-A, 2869insG, 2909delT, 3120 1 1G-A, 3120G-A, 3358delAC, 3659delC, 3662delA, 3750delAG, 3791delC, 3821delT, 3849 1 10KbC-T, 3849 1 4A-G, 3905insT, 405 1 1G-A, 444delA, 556delA, 574delA, 621 1 1G-T, and 711 1 1G-T. aSC, Santa Catarina State; PR, Parana State; MG, Minas Gerais State; n, number of chromosomes. Login to comment
69 G542X Our data show that G542X is the second most common CF mutation in two of the three Euro-Brazilian CF states studied (PR and MG), with an overall frequencyof 6.2% (Table 1). Login to comment
70 The presence of G542X in the Afro-Brazilian group was, as for DF508, likely the result of Caucasian admixture, in agreement with North American data (Macek et al., 1997). Login to comment
101 The presence in Afro-Brazilians of at least four CF alleles that are common in Caucasians (i.e., DF508, G542X, G85E, R1162X) indicates that the incidence of CF in Afro-Brazilians is due, at least in part, to genetic admixture. Login to comment

[hide] Emerging drug treatments for cystic fibrosis. Expert Opin Emerg Drugs. 2003 Nov;8(2):523-35. Zeitlin PL
Emerging drug treatments for cystic fibrosis.
Expert Opin Emerg Drugs. 2003 Nov;8(2):523-35., [PMID:14662004]

Abstract [show]
Cystic fibrosis (CF) is one of the most common life-shortening inherited disorders. Mutations in the cystic fibrosis transmembrane regulator (CFTR) gene disrupt the localisation and function of the cAMP-mediated chloride channel. Most of the morbidity and mortality arise from the lung disease which is characterised by excessive inflammation and chronic infection. Research into the mechanisms of wild-type and mutant CFTR biogenesis suggest that multiple drug targets can be identified. This review explores the current understanding of the nature of the different mutant CFTR forms and the potential for repair of the chloride channel defect. High-throughput screening, pharmacogenomics and proteomics bring recent technological advances to the field.

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57 Examples in this class include G542X, W1282X, R553X and 621 + 1 G→T. Login to comment
88 Class of mutation Molecular mechanism Pancreatic status (if known) Examples 1 No CFTR protein synthesis PI W1282X, G542X, R553X, 621 + 1 G→T, 1717-1 G→A, 3905insT, 394delTT 2 Abnormal CFTR processing and trafficking PI ∆F508, N1303K, P574H 3 Defective CFTR regulation (normal trafficking) PI G551D, G551S, G1349D, S1255P 4 Decreased CFTR chloride conductance PS R117H, R334W, R347P, P547H 5 Reduced synthesis and trafficking of normal CFTR PS A455E, 3849 + 10kb C→T, (5T) 6A Reduced apical stability PI S1455X, Q1412S, 4326delTC, 4279insA 6B Defective regulation of other ion channels PI G551D Note that the G551D is placed in Class 3 for defective regulation and Class 6B for defective regulation of the outwardly rectifying chloride channel. Login to comment

[hide] Cytokine secretion by cystic fibrosis airway epith... Am J Respir Crit Care Med. 2004 Mar 1;169(5):645-53. Epub 2003 Dec 11. Becker MN, Sauer MS, Muhlebach MS, Hirsh AJ, Wu Q, Verghese MW, Randell SH
Cytokine secretion by cystic fibrosis airway epithelial cells.
Am J Respir Crit Care Med. 2004 Mar 1;169(5):645-53. Epub 2003 Dec 11., 2004-03-01 [PMID:14670800]

Abstract [show]
It is controversial whether mutations in cystic fibrosis transmembrane conductance regulator intrinsically dysregulate inflammation. We characterized passage 2 human tracheobronchial epithelial cell cultures morphologically and physiologically and determined whether cytokine production or nuclear factor-kappaB activation was systematically altered in cystic fibrosis (CF) cells. Non-CF and CF cells originating from a total of 33 and 25 lungs, respectively, were available for culture on plastic or at an air-liquid interface until well differentiated. Forskolin-stimulated short-circuit currents were present in representative polarized non-CF cultures and were absent in CF cultures, whereas uridine 5'-triphosphate-stimulated currents were present in both. Constitutive or interleukin (IL)-1beta-induced IL-8 or IL-6 secretion or nuclear factor-kappaB activity was not significantly different between non-CF and CF cells. The cytokines regulated upon activation, normal T cell expressed and secreted (RANTES) and IL-10 were not detectable. Stimulation with tumor necrosis factor-alpha or a synthetic toll-like receptor 2 agonist or variable doses and times of Staphylococcus aureus culture filtrate revealed a single dose- and time-dependent difference in IL-8 production by CF cells. Interestingly, although IL-8 secretion after stimulation with Pseudomonas aeruginosa filtrates was not greater in CF cells in the absence of human serum, it was variably greater in its presence. Thus, although exaggerated responses may develop under certain conditions, our results do not support an overall intrinsically hyperinflammatory phenotype in CF cells.

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49 3 14 NTD 51 F 3 14 30 F Not genotyped 3 15 TD 48 F 3 15 22 F ⌬F508/⌬F508 3, 4 16 NTD 45 F 3 16 42 M 2789ϩ5GϾA/N1303K 4 17 NTD 28 M 4 17 32 F ⌬F508/⌬F508 4 18 NTD 46 M 4 18 27 F ⌬F508/G542X 4 19 TD 20 M 4 19 21 F ⌬F508/G85E 4 20 PF 63 M 4 20 26 F N1303K/? Login to comment

[hide] Prenatal screening for cystic fibrosis: past, pres... Expert Rev Mol Diagn. 2004 Jan;4(1):49-62. Richards CS, Grody WW
Prenatal screening for cystic fibrosis: past, present and future.
Expert Rev Mol Diagn. 2004 Jan;4(1):49-62., [PMID:14711349]

Abstract [show]
Prenatal screening for cystic fibrosis is reviewed. The disease, gene involved, molecular basis of disease, genotype/phenotype correlations and pilot trials are discussed, as well as historical perspectives, background and American College of Medical Genetics/American College of Obstetricians and Gynecologists recommendations. A number of complex challenges to the implementation of cystic fibrosis screening exist, including mutation testing of the cystic fibrosis transmembrane conductance regulator gene (CFTR), as well as laboratory and clinical issues. Current technologies for CFTR testing include reverse dot blots, amplification refractory mutation detection systems, oligonucleotide ligation assays, the Invader assay and NanoChip system. Emerging technologies are also considered, as well as quality assurance measures including analytical and clinical validation, reporting, residual risk calculations and prenatal diagnosis. An even greater challenge is clinical implementation, which focuses upon education and communication, choosing models, reporting, counseling and prenatal diagnosis.

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37 The second most common mutation in the general Caucasian population is G542X (2.4%) [5], and there are another 4-5 above the 1% level. Login to comment

[hide] CFTR gene and cystic fibrosis. J Gastroenterol Hepatol. 2004 Feb;19(2):228. Gaskin KJ
CFTR gene and cystic fibrosis.
J Gastroenterol Hepatol. 2004 Feb;19(2):228., [PMID:14731137]

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23 Contributed by Kevin J Gaskin Department of Gastroenterology and James Fairfax Institute of Pediatric Nutrition,The Children`s Hospital,Westmead, NSW 2145, Australia Table 1 Classification of cystic fibrosis transmembrane conductance regulator (CFTR) mutations Type Description Example I CFTR mRNA or protein not formed G542X II CFTR trafficking defect, and protein fails to locate in cell membrane DF508 III Regulation defect. CFTR inserts into cell membrane but no response to cAMP G551D IV Channel defect. CFTR inserts into cell membrane but function is reduced R117H V Synthesis defect. CFTR inserts into membrane and functions normally, but the amount of CFTR synthesized is reduced from normal Login to comment

[hide] Neonatal screening for cystic fibrosis: France ris... J Inherit Metab Dis. 2003;26(8):729-44. Farriaux JP, Vidailhet M, Briard ML, Belot V, Dhondt JL
Neonatal screening for cystic fibrosis: France rises to the challenge.
J Inherit Metab Dis. 2003;26(8):729-44., [PMID:14739679]

Abstract [show]
This paper describes the adjustments to the French neonatal screening programme required by the introduction of systematic screening for cystic fibrosis (CF), taking into account both the legal and statutory framework and the lessons of a pilot study carried out 10 years ago. The French association for the screening and prevention of infant handicaps (AFDPHE) has been mandated by its regulatory agencies to organize screening for CF in France (metropolitan and overseas territories). During the year 2001, expert groups (Technical Aspects, Information, Ethics and Genetics, Criteria for CF Centres, Protocol for the Care of a Newborn with CF) issued recommendations for the establishment of a national programme that would guarantee efficiency and adequate patient care from the time of diagnosis onward. The programme is based on a strategy combining immunoreactive trypsin (IRT) assay and the analysis of DNA mutations in dried blood samples obtained at 3 days of age. When an elevated IRT value is found, DNA analysis is performed on the same sample. Owing to the relative regional heterogeneity existing in France, 30 selected mutations are used, which provide 85% coverage. The Ethics and Genetics Committee recommended that, in order to avoid arousing anxiety by a recall, informed consent, according to the French legislation on bioethics, should be obtained for all neonates at birth by having the parents sign directly on the sampling paper. Information brochures for parents and health professionals have been designed. A new organization of patient care, involving the creation of CF centres recognized by the Ministry of Health, has been decided; all children diagnosed are to be referred to such centres, where they can be well cared for by a trained staff with sufficient means. The programme was implemented region by region in France, from the beginning of the year 2002 to early 2003. The expert groups still meet periodically to evaluate the implementation of the programme and to check that the terms of the agreement between the AFDPHE and the Social Security Agency are complied with.

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114 In its present version, the kit allows screening for 20 CFTR gene mutations (F508del, G542X, N1303K, 1717-1G>A, G551D, W1282X, R553X, I507del, 1078delT, 2183AA>G, 3849 þ 10kbC>T, R1162X, 621 þ 1G>T, R334W, R347P, 3659delC, R117H, S1251N, E60X, A455E) in one workday; moreover, it does not require any speci'c equipment. Login to comment

[hide] Cystic fibrosis in Uruguay. Genet Mol Res. 2002 Mar 31;1(1):32-8. Luzardo G, Aznarez I, Crispino B, Mimbacas A, Martinez L, Poggio R, Zielenski J, Tsui LC, Cardoso H
Cystic fibrosis in Uruguay.
Genet Mol Res. 2002 Mar 31;1(1):32-8., [PMID:14963811]

Abstract [show]
We conducted clinical and genetic analyses of 52 cystic fibrosis (CF) patients in Uruguay, which is about half of the known affected individuals in the country. A relatively high proportion had a mild presentation, characterized by pancreatic sufficiency (28%), a strong pulmonary component (97%), and borderline sweat electrolyte measurements (25%). Mutational analysis of CF chromosomes demonstrated a relatively low incidence of the DeltaF508 allele (40%) and a large number of other cystic fibrosis conductance regulator mutations, with an overall detection rate of about 71%. Fifteen different mutations were detected in our patients: DeltaF508, G542X, R1162X, G85E, N1303K, R334W, R75Q, R74W, D1270N, W1282X, DeltaI507, 2789+5G-->A, R1066C, -816C/T, R553X, as well as RNA splicing variant IVS8-5T. This group of Uruguayan CF patients has some characteristics in common with other populations of similar origin (Hispanics), as well as some unique characteristics.

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36 The CFTR mutations were detected by using one or more of the following methods: a) Reverse hybridization technique for eight mutations frequent in Europe (∆F508, G542X, N1303K, 1717-1G→A, W1282X, G551D, R553X and ∆I507), using a commercial kit from Inno Lipa CF2, Innogenetics, Belgium. Login to comment
42 RESULTS Genetic analysis led to the detection of 15 different mutations: ∆F508, G542X, R1162X, G85E, N1303K, R334W, R75Q, R74W, D1270N, W1282X, ∆I507, 2789+5G→A, R1066C, R553X and -816C/T. Login to comment
47 Mutation Cumulative (%)%N ∆F508 G542X R1162X G85E N1303K R334W R75Q Other mutations* Unknown 42 6 3 3 3 2 2 13 30 40.4 5.7 2.9 2.9 2.9 1.9 1.9 12.5 28.9 40.4 46.1 49.0 51.9 54.9 56.7 58.6 71.1 99.9 *R74W, D1270N, W1282X, ∆I507, 2789+5G→A, R1066C, -816C/T, R553X, 5T (3 cases associated to other mutations, 2 cases without known second mutation). Login to comment
49 The most prevalent mutation, ∆F508, was found in 42/104 CF chromosomes, with the second most common allele being the G542X (6/104). Login to comment
59 Genotypes N Percent ∆F508/∆F508 ∆F508/R1162X ∆F508/G85E ∆F508/G542X ∆F508/5T ∆F508/R334W ∆F508/1303X ∆F508/R1066C ∆F508/Unknown ∆I507/2789+G-A R74W/D1270N N1303K/G542X N1303K/R553K -816C-T/5T 5T/Unknown G542X/Unknown R75Q/Unknown W1282X/Unknown Unknown/Unknown 8 3 3 3 2 2 1 1 11 1 1 1 1 1 2 2 2 1 6 15.4 5.8 5.8 5.8 3.9 3.9 1.9 1.9 21.2 1.9 1.9 1.9 1.9 1.9 3.9 3.9 3.9 1.9 11.5 All individuals had pulmonary symptoms.All those carrying the ∆F508/∆F508 genotype had pancreatic insufficiency. Login to comment
89 We have also observed differences in the distribution and frequencies of non-∆F508 mutations between Uruguayans and patients from other LatinAmerican countries, in particular compared to theArgentinean population.AmongArgentine CF patients, seven mutations (∆F508, G542X, W1282X, N1303K, 17171G→A, R553X, R1162X) constituted 67.5% of the observed alleles (Chertkoff et al., 1997), while in our population 15 mutations corresponded to a similar cumulative percentage (71%). Login to comment
90 There is an agreement between the most common Uruguayan CFTR mutations (∆F508, G542X, R1162X, N1303K, R334W, W1282X and R553X) and those reported in the geographical regions from where most Uruguayans`ancestors originated, namely, Spain, the Canary Islands, Italy and the Basque regions. Login to comment
98 In U.S. CF patients, only three mutations (∆F508, G542X, G551D) have gene frequencies higher than 2%, and altogether represent 72.5% of the CF chromosomes with a ∆F508 frequency of 68% (Cystic Fibrosis Foundation, 1997). Login to comment
99 TheArgentine study shows similar proportions, with three mutations with a gene frequency exceeding 2% and covering 64% of the total number of chromosomes (∆F508 = 57.0%, G542X = 3.94%, W1282X = 3.07%; Chertkoff et al., 1997). Login to comment

[hide] Improved detection of cystic fibrosis mutations in... Hum Reprod. 2004 Mar;19(3):540-6. Epub 2004 Jan 29. Danziger KL, Black LD, Keiles SB, Kammesheidt A, Turek PJ
Improved detection of cystic fibrosis mutations in infertility patients with DNA sequence analysis.
Hum Reprod. 2004 Mar;19(3):540-6. Epub 2004 Jan 29., [PMID:14998948]

Abstract [show]
BACKGROUND: Accurate determination of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is critical for genetic counselling and treatment of obstructive azoospermia. Of concern is that detection rates with routine CFTR mutation panels vary widely depending on patient ancestry; and such panels have limited value for azoospermic patients, who are more likely to carry rare mutations. An alternative approach offers comprehensive, CFTR mutation analysis by a DNA sequence method. We investigated whether this method could improve CFTR detection rates in men with obstructive azoospermia in a prospective study of men with obstructive azoospermia and their partners who were referred for genetic counselling and testing at one of two institutions. METHODS: Sixteen patients with congenital absence of the vas deferens (CAVD, n = 14) or idiopathic obstructive azoospermia (n = 2) were studied. DNA from all patients was analysed for mutations by the DNA sequence method. In addition to this method, six men underwent CFTR analysis by a common 25 or 31 mutation panel coupled with poly T analysis. In 10 subjects, common mutation panel findings were inferred from DNA sequence method results. RESULTS: Overall, 12/16 (75%) azoospermic patients had one or more CFTR mutations and/or 5T alleles, including 12 mutations in 10 patients (two compound heterozygotes) and seven 5T alleles in six patients (one homozygote). The sequence method detected all mutations and three variants of unknown significance. By comparison, the common mutation panels detected only 3/12 mutations (25%) and 0/3 variants. CONCLUSION: The DNA sequence method detects more CFTR mutations than common mutation panels. Given the serious, clinical consequences of transmitting such mutations, this study underscores the importance of accurate, CFTR mutation detection in men with obstructive azoospermia and their partners.

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59 Polyacrylamide gels were analysed for the presence of mutations following staining in ethidium bromide (EtBr) and image capture under UV using the Gel Doc 1000 system Table I. List of CFTR mutations included in common mutation panels American College of Medical Genetics CF panel (25 mutations) DF508 G542X G551D R117H W1282X N1303K R1162X 3849+10kbC®T DI507 R553X 1717-1G®A 621+1G®T R560T 3659delC 3120+1G®A I148T G85E R334W A455E 1898+1G®A 2148delA 711+1G®T 2789+5G®A R347P 1078delT Six additional mutations and one polymorphism in UCSF panel (31 mutations) Y1092X R347H 3849+4 Q493X 3905insT S549N F508C (polymorphism) (BioRad). Login to comment

[hide] Direct visualization of cystic fibrosis transmembr... Clin Chem. 2004 May;50(5):836-45. Epub 2004 Mar 9. Strom CM, Clark DD, Hantash FM, Rea L, Anderson B, Maul D, Huang D, Traul D, Chen Tubman C, Garcia R, Hess PP, Wang H, Crossley B, Woodruff E, Chen R, Killeen M, Sun W, Beer J, Avens H, Polisky B, Jenison RD
Direct visualization of cystic fibrosis transmembrane regulator mutations in the clinical laboratory setting.
Clin Chem. 2004 May;50(5):836-45. Epub 2004 Mar 9., [PMID:15010427]

Abstract [show]
BACKGROUND: The recommendation for population- based cystic fibrosis (CF) carrier screening by the American College of Medical Genetics for the 25 most prevalent mutations and 6 polymorphisms in the CF transmembrane regulatory gene has greatly increased clinical laboratory test volumes. We describe the development and technical validation of a DNA chip in a 96-well format to allow for high-throughput genotype analysis. METHODS: The CF Portrait chip contains an 8 x 8 array of capture probes and controls to detect all requisite alleles. Single-tube multiplex PCR with 15 biotin-labeled primer pairs was used to amplify sequences containing all single-nucleotide polymorphisms to be interrogated. Detection of a thin-film signal created by hybridization of multiplex PCR-amplified DNA to complementary capture probes was performed with an automated image analysis instrument, NucleoSight. Allele classification, data formatting, and uploading to a laboratory information system were fully automated. RESULTS: The described platform correctly classified all mutations and polymorphisms and can screen approximately 1300 patient samples in a 10-h shift. Final validation was performed by two separate 1000-sample comparisons with Roche CF Gold line probe strips and the Applera CF OLA, Ver 3.0. The CF Portrait Biochip made no errors during this validation, whereas the Applera assay made seven miscalls of the IVS-8 5T/7T/9T polymorphism CONCLUSIONS: The CF Portrait platform is an automated, high-throughput, DNA chip-based assay capable of accurately classifying all CF mutations in the recommended screening panel, including the IVS-8 5T/7T/9T polymorphism.

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178 The optimal spotting conditions for each probe are indicated by the boxes around spots in C. wild-type controls and heterozygotes for each ACMG mutation and polymorphism, DNA from 12 compound heterozygotes (⌬F508/1898 ϩ 1GϾA, 711 ϩ 1GϾT/⌬F508, G85E/621 ϩ 1GϾT, 3659delC/⌬F508, 3120 ϩ 1GϾA/ 621 ϩ 1GϾT, R347P/G551D, A455E/⌬F508, R560T/ dF508, R553X/⌬F508, 621 ϩ 1GϾT/⌬F508, 621 ϩ 1GϾT/ 711 ϩ 1GϾT, R117H/⌬F508, and I506V/⌬F508) and DNA from 4 homozygous patients (⌬F508 and 2789 ϩ 5GϾA, 3849 ϩ 10kbCϾT, and G542X) was used in validation experiments. Login to comment
199 In this series, there were 17 ⌬F508 heterozygous patient samples, 1 ⌬F508 homozygous sample, 2 R117H heterozygous samples, and 1 heterozygous patient sample each for I148T, G542X, R553X, R347P, and 2789 ϩ 5GϾA, for a total of 26 mutant alleles. Additional mutant alleles detected in the control samples included three fixed control samples (⌬F508 homozygous, 5T/WT, 3659delC/⌬F508) on every plate and two heterozygous samples (R560T and 1078delT) and one heterozygous sample each for R334W, A455E, R347P, R117H, ⌬I507, I507V, G551D, and 1717-1GϾA as rotating controls. Login to comment
203 In this comparison, there were 19 ⌬F508 heterozygous patient samples, 3 I148T heterozygous samples, 3 R117H heterozygous and 1 R117H homozygous samples, 2 W1282X heterozygous samples, and 1 heterozygous patient sample each for G551D, R553X, R1162X, and 3849 ϩ 10kBCϾT, for a total of 36 mutant alleles. Additional mutant alleles detected for this study included fixed controls ⌬F508 homozygous, 5T/WT, and a N1303K heterozygous sample on all plates, and one heterozygous sample each for R560T, G542X, R553X, W1282X, 2184delA, G85E, I148T, 621 ϩ 1GϾT, R334W, R117H, 1078delT, and 1717-1GϾA as rotating controls. Login to comment

[hide] Detection of five common CFTR mutations by rapid-c... Clin Chem. 2004 Apr;50(4):773-5. Dempsey E, Barton DE, Ryan F
Detection of five common CFTR mutations by rapid-cycle real-time amplification refractory mutation system PCR.
Clin Chem. 2004 Apr;50(4):773-5., [PMID:15044340]

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4 The five mutations [and the percentages of Irish (3) and worldwide (2) cases] are F508del (77.4%, 66.0%), G551D (7.1%, 1.6%), R117H (2.7%, 0.3%), 621ϩ1 GϾT (1.4%, 0.7%), and G542X (0.5%, 2.4%). Login to comment
5 Four of these fall into the severe class of mutations in which the mRNA is incorrectly spliced (621ϩ1 GϾT), or in which the protein is not synthesized (G542X) or is blocked during processing (F508del), or its regulation is blocked (G551D). Login to comment
23 The second reaction detects the 621ϩ1 GϾT and G542X mutations (1.9% frequency in Ireland, 3.1% worldwide). Login to comment
28 The CF11ARMS-A (5Ј-TAT- GATTACATTAGAAGGAAGATGTGCCTTT-F-3Ј) and CF11ARMS-P (5Ј-LCRed705-AATTCAGATTGAGCAT- ACT-P-3Ј) hybridization probes detect both the G551D and G542X ARMS products. Login to comment

[hide] Role of CFTR mutations in adult bronchiectasis. Thorax. 2004 Apr;59(4):357-8. King PT, Freezer NJ, Holmes PW, Holdsworth SR, Forshaw K, Sart DD
Role of CFTR mutations in adult bronchiectasis.
Thorax. 2004 Apr;59(4):357-8., [PMID:15047968]

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230 The patients were screened for the 10 most common mutations in the local population (DF508, D1507, V520F, G542X, G551D, R553X, R117H, 621+1GRT, A455E and N1303K) responsible for 82% of cases of CF and the 5T mutation by previously published methods.7 8 Ethical approval for the project was obtained from the ethics committee at MMC. Login to comment

[hide] Molecular analysis using DHPLC of cystic fibrosis:... BMC Med Genet. 2004 Apr 14;5:8. D'Apice MR, Gambardella S, Bengala M, Russo S, Nardone AM, Lucidi V, Sangiuolo F, Novelli G
Molecular analysis using DHPLC of cystic fibrosis: increase of the mutation detection rate among the affected population in Central Italy.
BMC Med Genet. 2004 Apr 14;5:8., 2004-04-14 [PMID:15084222]

Abstract [show]
BACKGROUND: Cystic fibrosis (CF) is a multisystem disorder characterised by mutations of the CFTR gene, which encodes for an important component in the coordination of electrolyte movement across of epithelial cell membranes. Symptoms are pulmonary disease, pancreatic exocrine insufficiency, male infertility and elevated sweat concentrations. The CFTR gene has numerous mutations (>1000) and functionally important polymorphisms (>200). Early identification is important to provide appropriate therapeutic interventions, prognostic and genetic counselling and to ensure access to specialised medical services. However, molecular diagnosis by direct mutation screening has proved difficult in certain ethnic groups due to allelic heterogeneity and variable frequency of causative mutations. METHODS: We applied a gene scanning approach using DHPLC system for analysing specifically all CFTR exons and characterise sequence variations in a subgroup of CF Italian patients from the Lazio region (Central Italy) characterised by an extensive allelic heterogeneity. RESULTS: We have identified a total of 36 different mutations representing 88% of the CF chromosomes. Among these are two novel CFTR mutations, including one missense (H199R) and one microdeletion (4167delCTAAGCC). CONCLUSION: Using this approach, we were able to increase our standard power rate of mutation detection of about 11% (77% vs. 88%).

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77 Finally, in a single CF patient, we detected two different mutations [G542X (1756 G to T) and S549R (1779 T to G)] in the same exon 11-derived DNA amplicon, which showed a peculiar DHPLC pattern, different from that observed when only one of these mutations was present (data not show). Login to comment
89 Table 1: Primers and DHPLC (oven temperature, gradient) analysis conditions for 6b and 9 exons of the CFTR gene exon Primer 5' → 3' Amplicon length Oven temp (°C) % B buffer start/end 6b F - CAGAGATCAGAGAGCTGGG 323 56 55/63 R - GAGGTGGAAGTCTACCATGA 9 F - GGGATTTGGGGAATTATTTG 279 55 54/62 R - TCTCCAAAAATACCTTCCAG Table 2: CF mutations identified in cohort of 290 patients from the Central Italy Mutation Nucleotide change Exon/intron N % Method delF508 1652delCTT 10 328 56.36 INNO-LiPA, DHPLC N1303K 4041 C to G 21 51 8.76 INNO-LiPA, DHPLC G542X 1756 G to T 11 42 7.21 INNO-LiPA, DHPLC W1282X 3978 G to A 20 15 2.60 INNO-LiPA, DHPLC S549R 1779 T to G 11 8 1.37 DHPLC 621+1G-T 621+1 G to T Intron 4 7 1.20 INNO-LiPA, DHPLC 1717-1G-A 1717-1 G to A Intron 10 5 0.86 INNO-LiPA, DHPLC G85E 386 G to A 3 4 0.69 INNO-LiPA, DHPLC R553X 1789 C to T 11 4 0.69 INNO-LiPA, DHPLC H139R 548 A to G 6a 3 0.51 DHPLC R347P 1172 G to C 7 3 0.51 INNO-LiPA, DHPLC L1065P 3326 T to C 17b 3 0.51 DHPLC L1077P 3362 T to C 17b 3 0.51 DHPLC S4X 143 C to A 1 2 0.34 DHPLC D110H 460 G to C 4 2 0.34 DHPLC R334W 1132 C to T 7 2 0.34 INNO-LiPA, DHPLC M348K 1175 T to A 7 2 0.34 DHPLC 1259insA 1259 ins A 8 2 0.34 DHPLC S549N 1778 G to A 11 2 0.34 DHPLC L558S 1805 T to C 11 2 0.34 DHPLC 2183+AA-G 2183 A to G and 2184 del A 13 2 0.34 INNO-LiPA, DHPLC 2789+5G-A 2789+5 G to A Intron 14b 2 0.34 INNO-LiPA, DHPLC R1066C 3328 C to T 17b 2 0.34 DHPLC 3667ins4 3667insTCAA 19 2 0.34 DHPLC S42F 257 C to T 2 2 0.34 DHPLC R117L 482 G to T 4 1 0.17 DHPLC H199R 728 A to G 6a 1 0.17 DHPLC R334L 1133 G to T 7 1 0.17 DHPLC T338I 1145 C to T 7 1 0.17 DHPLC G551D 1784 G to A 11 1 0.17 INNO-LiPA, DHPLC Q552X 1786 C to T 11 1 0.17 INNO-LiPA, DHPLC D614G 1973 A to G 13 1 0.17 DHPLC A1006E 3149 C to A 17a 1 0.17 DHPLC 4016insT 4016 ins T 21 1 0.17 DHPLC 4040delA 4040 del A 21 1 0.17 DHPLC 4167del7 4167 delCTAAGCC 22 1 0.17 DHPLC Detected 511 88.10 Unknown 69 11.90 Total 580 100.00 N = number of CF chromosomes; % = frequency. Login to comment

[hide] A finger sweat chloride test for the detection of ... Pancreas. 2004 Apr;28(3):e80-5. Naruse S, Ishiguro H, Suzuki Y, Fujiki K, Ko SB, Mizuno N, Takemura T, Yamamoto A, Yoshikawa T, Jin C, Suzuki R, Kitagawa M, Tsuda T, Kondo T, Hayakawa T
A finger sweat chloride test for the detection of a high-risk group of chronic pancreatitis.
Pancreas. 2004 Apr;28(3):e80-5., [PMID:15084988]

Abstract [show]
OBJECTIVES: Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene are associated with chronic pancreatitis in Caucasians. We developed a simple method for measuring finger sweat chloride concentration to test whether CFTR dysfunction underlies chronic pancreatitis in Japan where cystic fibrosis (CF) is rare. METHODS: We studied 25 patients with chronic (21 alcoholic and 4 idiopathic) pancreatitis and 25 healthy volunteers. Sweat chloride concentrations were measured by a finger sweat chloride test. We analyzed DNA for 20 common CFTR mutations in Europeans, 9 CF-causing mutations in Japanese, and 2 polymorphic loci, a poly-T tract and (TG) repeats, at intron 8. RESULTS: Thirteen patients (52%) had sweat chloride levels >60 mmol/L, a level consistent with CF, while only 4 (16%) healthy subjects exceeded this level. The 29 CF mutations and the 5T allele were detected in neither the patients nor controls. The (TG) 12 allele was common in both the patients (58%) and controls (48%). The (TG) 12/12 genotype was common in alcoholic pancreatitis (29%) compared with the (TG) 11/11 (10%). Patients with the (TG) 12/12 genotype had significantly higher sweat chloride concentrations than the controls. CONCLUSION: CFTR dysfunction as evidenced by a finger sweat chloride test is present in about half of Japanese patients with chronic pancreatitis, suggesting that this test may be useful for detecting the high-risk group. A higher proportion of the (TG) 12 allele may be a genetic background for elevated sweat chloride concentrations in Japanese patients.

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50 The DNA samples were analyzed using an amplification refractory mutation system kit for 20 common major CFTR mutations (E60X, R117H, R334W, R347P, A455E, ⌬I507, ⌬F508, G542X, G551D, R553X, 621+1G>T, 1078delT, R1162X, S1251N, W1282X, N1303K, 1717-1G>A, 2183AA>G, 3659delC, 3849+10kbC>T) (Elucigene CF 20, AstraZeneca Diagnostics, Abingdon, UK) following the standard procedures recommended by the manufacturer. Login to comment

[hide] Genetic evidence for CFTR dysfunction in Japanese:... J Med Genet. 2004 May;41(5):e55. Fujiki K, Ishiguro H, Ko SB, Mizuno N, Suzuki Y, Takemura T, Yamamoto A, Yoshikawa T, Kitagawa M, Hayakawa T, Sakai Y, Takayama T, Saito M, Kondo T, Naruse S
Genetic evidence for CFTR dysfunction in Japanese: background for chronic pancreatitis.
J Med Genet. 2004 May;41(5):e55., [PMID:15121783]

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218 The 20 most common CF mutations (E60X, R117H, R334W, R347P, A455E, DI507, DF508, G542X, G551D, R553X, 621+1GRT, 1078delT, R1162X, S1251N, W1282X, N1303K, 1717-1GRA, 2183AARG, 3659delC, and 3849+10kbCRT) were tested by an Elucigene CF20 kit (AstraZeneca Diagnostics, Abingdon, Oxfordshire, UK). Login to comment

[hide] Bayesian risk assessment for autosomal recessive d... J Med Genet. 2004 May;41(5):e70. Ogino S, Wilson RB, Grody WW
Bayesian risk assessment for autosomal recessive diseases: fetal echogenic bowel with one or no detectable CFTR mutation.
J Med Genet. 2004 May;41(5):e70., [PMID:15121798]

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185 If a relative of parent A or parent B is affected or an obligate carrier, this table can still be applied when neither that relative nor any other family member has been tested. Table 3 Summary of carrier frequencies for cystic fibrosis, overall mutation detection rates by the ACMG 25 mutation panel, and frequencies of major mutations for each major ethnic group (adapted from Richards et al. and Bobadilla et al.)4 18 Ethnic group Cystic fibrosis carrier frequency Overall mutation detection rate by ACMG CFTR 25 mutation panel (%) Frequency DF508 among all disease alleles (%) Other major mutations (%)* Non-Hispanic 1/25 90 70 G542X 2.4 Caucasian G551D 2.1 W1282X 1.4 N1303K 1.3 Ashkenazi Jewish 1/25 97 30 W1282X 48 G542X 9.0 3849+10kbCRT 6.0 N1303K 3.0 1717-1GRA 1.0 African-American 1/65 69 48 3120+1GRA 12 2307insA 2.0 A559T 2.0 R553X 2.0 DF311 2.0 G480C 1.4 405+3ARC 1.4 S1255X 1.4 Hispanic American 1/46 57 46 G542X 5.4 3849+10kbCRT 2.3 R1162X 1.6 R334W 1.6 Asian American 1/90 ? Login to comment

[hide] Bronchiectasis in adult patients: an expression of... Clin Genet. 2004 Jun;65(6):490-5. Casals T, De-Gracia J, Gallego M, Dorca J, Rodriguez-Sanchon B, Ramos MD, Gimenez J, Cistero-Bahima A, Olveira C, Estivill X
Bronchiectasis in adult patients: an expression of heterozygosity for CFTR gene mutations?
Clin Genet. 2004 Jun;65(6):490-5., [PMID:15151509]

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While all patients with cystic fibrosis (CF) have mutations in both CFTR alleles, often only one CFTR change is detected in patients with other lung disorders. The aim of this study was to investigate whether heterozygosity for CFTR mutations could be a determinant risk factor in the development of bronchiectasis in adult patients. We have performed the CFTR gene analysis in a cohort of 55 bronchiectasis adult patients with unknown etiology. The 5T variant (TG)m and the M470V polymorphisms were also analyzed. A general population in which the same molecular analysis was previously performed was used as the control group. The mutational spectrum of patients was also compared with that found in our CF population. CFTR mutations/variants were found in 20 patients (36%), 14 with only one mutant gene (25%). All six patients colonized by Staphylococcus aureus presented with at least one CFTR change (p = 0.001). No statistical significance was observed between patients with and without mutations for other clinical features. The 5T variant was found in four patients. Additionally, 90% of patients with mutations had the more functional M470 allele (p < 0.001). These results suggest the involvement of the CFTR gene in bronchiectasis of unknown etiology in adult patients.

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57 One stop mutation (G542X) and one splice-site mutation (2789þ 5G> A) were detected in two other patients. Login to comment
81 Table2.ClinicalfeaturesandCFTRgenotypesfoundin20adultpatientswithbronchiectasis SampleSex/age Ageonset (years) FEV1/FVC (%predicted) Bacterial colonization Sweattest (mEq/l) Lobes affected Clinical features FirstCFTR change Second CFTRchangeM470V 1M/41520/43P30a >4-F508delL997FM/V 2F/231785/89P,S46a >4SN,ABPA,PNF508del-M/M 3F/24160/74P,S49a >4SN,PNF508del-M/V 4M/55-87/84S32a 2-F508del-M/V 5c F/372991/93S41a >4PNF508del-M/V 6F/333286/84No51a 2-G542X-M/M 7F/306101/112No56a >4-2789þ5G>A5T-12TGM/V 8F/3815106/104No29a 2OtitisS1235R-M/V 9F/34Birth75/100H20a >4SNV562L5T-11TGM/V 10d F/36530/51P20a >4SN,PNG1237S-M/V 11d M/401473/92H26a 3SN,PN,OZG1237S-M/V 12F/23541/47S23a >4HemoptysisR347HR75QV/V 13F/68548/52No34a >4PNY1014C5T-12TGV/V 14M/643088/84H39a 2-R75Q-M/V 15M/40Childhood56/79No33b >4SN,asthmaV754M-M/M 16M/474594/108No19a 2SN,PNQ179K-M/V 17M/23Childhood38/34No28a 2SN,PN5T-12TG5T-11TGM/V 18F/695068/89S52a 4DiabetesG576A,R668C-M/V 19F/47Childhood16/18P64b >4-G576A,R668C-M/V 20F/38672/88No39b >4SN,ABPA,asthma1716G/A-M/M M,male;F,female;FEV1,forcedexpiratoryvolumein1s(%ofpredictedvalueforheight);FVC,forcedvitalcapacity(%ofpredictedvalueforheight);P,Pseudomonas aeruginosa;H,Haemophilusinfluenza;S,Staphylococcusaureus;SN,sinusitis;ABPA,allergicbronchopulmonaryaspergillosis;PN,pneumonia;OZ,oligozoospermia. Login to comment
94 Among the common mutations reported in Spanish CF families (19), only three were detected in the bronchiectasis group (F508del, G542X, 2789 þ 5G> A). Login to comment

[hide] Population-based newborn screening for genetic dis... Pediatrics. 2004 Jun;113(6):1573-81. Comeau AM, Parad RB, Dorkin HL, Dovey M, Gerstle R, Haver K, Lapey A, O'Sullivan BP, Waltz DA, Zwerdling RG, Eaton RB
Population-based newborn screening for genetic disorders when multiple mutation DNA testing is incorporated: a cystic fibrosis newborn screening model demonstrating increased sensitivity but more carrier detections.
Pediatrics. 2004 Jun;113(6):1573-81., [PMID:15173476]

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OBJECTIVES: Newborn screening for cystic fibrosis (CF) provides a model to investigate the implications of applying multiple-mutation DNA testing in screening for any disorder in a pediatric population-based setting, where detection of affected infants is desired and identification of unaffected carriers is not. Widely applied 2-tiered CF newborn screening strategies first test for elevated immunoreactive trypsinogen (IRT) with subsequent analysis for a single CFTR mutation (DeltaF508), systematically missing CF-affected infants with any of the >1000 less common or population-specific mutations. Comparison of CF newborn screening algorithms that incorporate single- and multiple-mutation testing may offer insights into strategies that maximize the public health value of screening for CF and other genetic disorders. The objective of this study was to evaluate technical feasibility and practical implications of 2-tiered CF newborn screening that uses testing for multiple mutations (multiple-CFTR-mutation testing). METHODS: We implemented statewide CF newborn screening using a 2-tiered algorithm: all specimens were assayed for IRT; those with elevated IRT then had multiple-CFTR-mutation testing. Infants who screened positive by detection of 1 or 2 mutations or extremely elevated IRT (>99.8%; failsafe protocol) were then referred for definitive diagnosis by sweat testing. We compared the number of sweat-test referrals using single- with multiple-CFTR-mutation testing. Initial physician assessments and diagnostic outcomes of these screened-positive infants and any affected infants missed by the screen were analyzed. We evaluated compliance with our screening and follow-up protocols. All Massachusetts delivery units, the Newborn Screening Program, pediatric health care providers who evaluate and refer screened-positive infants, and the 5 Massachusetts CF Centers and their affiliated genetic services participated. A 4-year cohort of 323 506 infants who were born in Massachusetts between February 1, 1999, and February 1, 2003, and screened for CF at approximately 2 days of age was studied. RESULTS: A total of 110 of 112 CF-affected infants screened (negative predictive value: 99.99%) were detected with IRT/multiple-CFTR-mutation screening; 2 false-negative screens did not show elevated IRT. A total of 107 (97%) of the 110 had 1 or 2 mutations detected by the multiple- CFTR-mutation screen, and 3 had positive screens on the basis of the failsafe protocol. In contrast, had we used single-mutation testing, only 96 (87%) of the 110 would have had 1 or 2 mutations detectable by single-mutation screen, 8 would have had positive screens on the basis of the failsafe protocol, and an additional 6 infants would have had false-negative screens. Among 110 CF-affected screened-positive infants, a likely "genetic diagnosis" was made by the multiple-CFTR-mutation screen in 82 (75%) versus 55 (50%) with DeltaF508 alone. Increased sensitivity from multiple-CFTR-mutation testing yielded 274 (26%) more referrals for sweat testing and carrier identifications than testing with DeltaF508 alone. CONCLUSIONS: Use of multiple-CFTR-mutation testing improved sensitivity and postscreening prediction of CF at the cost of increased referrals and carrier identification.

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79 The 16-mutation panel included ⌬F508, R117H, G551D, G542X, W1282X, N1303K, R334W, 621 ϩ 1GϾT, R553X, ⌬I507, 1717-1GϾA, R347P, R560T, 3849 ϩ 10kbCϾT, A455E, and S549N. Login to comment
122 False-Negative Results One of the 2 infants who were not detected by the Ͼ95th daily percentile screen had an IRT value at the 93.9th percentile and meconium ileus (positive sweat test; G542X/unknown); the other infant missed by the screen had an IRT value at the 84th percentile and presented at 2 months with failure to thrive and upper respiratory tract infection (positive sweat test; ⌬F508/R117H). Login to comment
126 The 15th infant`s IRT did not prompt a DNA assay; subsequent diagnostic testing revealed no ⌬F508 mutation but detected another (G542X) that is included in our multiple-CFTR-mutation testing panel. Login to comment
159 Genotypes and Frequencies Observed in 112 CF-Affected Infants First Mutation Second Mutation N ⌬F508 ⌬F508 55 ⌬F508 R117H 7* ⌬F508 G551D 4 ⌬F508 N1303K 3 ⌬F508 W1282X 3 ⌬F508 G542X 2 ⌬F508 1898 ϩ 1 G Ͼ A 2 G85E R117C 2 ⌬F508 1717-GϾA 1 ⌬F508 3849 ϩ 10kbC Ͼ T 1 ⌬F508 R1066C 1 ⌬F508 Y1092X 1 ⌬F508 L206W 1 ⌬F508 R560T 1 ⌬F508 1152H 1 ⌬F508 621 ϩ 1G Ͼ T 1 R117H G551D 1 R117H G85E 1 G551D 2789 ϩ 5GϾA 1 G551D R117C 1 G85E 711 ϩ 1GϾT 1 W1282X 3849 ϩ 10kbCϾT 1 R553X 2183AAϾG 1 A455E S549R 1 ⌬F508 Unknown† 13 N1303K Unknown 2 G542X Unknown 1 Unknown Unknown 2 * Includes 1 of the false-negative screens. Login to comment

[hide] The role of CFTR and SPINK-1 mutations in pancreat... AIDS. 2004 Jul 23;18(11):1521-7. Felley C, Morris MA, Wonkam A, Hirschel B, Flepp M, Wolf K, Furrer H, Battegay M, Bernasconi E, Telenti A, Frossard JL
The role of CFTR and SPINK-1 mutations in pancreatic disorders in HIV-positive patients: a case-control study.
AIDS. 2004 Jul 23;18(11):1521-7., 2004-07-23 [PMID:15238770]

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OBJECTIVE: Pancreatic disorders in HIV-positive patients are frequent. CFTR and SPINK-1 mutations have been reported to increase the risk of pancreatitis, but no data are available in HIV-positive patients. This study will evaluate the frequency of CFTR mutations and SPINK-1 polymorphisms in HIV-positive patients with clinical pancreatitis or asymptomatic elevation of serum pancreatic enzymes. METHOD: Cases (patients with hyperamylasemia) were identified during a toxicity study conducted in August 1999 among 1152 participants of the Swiss HIV Cohort Study. We designed a case-control study in which each case was matched one to one to an HIV-infected control according to sex, age, CD4 cell count, viraemia and medication use. CFTR mutations and SPINK-1 polymorphisms were studied using polymerase chain reaction techniques. RESULTS: Fifty-one HIV-positive patients with hyperamylasemia were detected among 1152 participants in the toxicity study (4.4%). There were 13 carriers of CFTR and SPINK-1 mutations (12.7%). Amylase levels were 316 +/- 130 U/l for the group with mutations, and 135 +/- 18 U/l for non-carriers (P = 0.79). However, among patients with hyperamylasemia, those with CFTR or SPINK-1 mutations had 648 +/- 216 U/l amylase levels compared with 232 +/- 28 U/l for those without (P = 0.025). Ten patients had acute pancreatitis, four of whom had CFTR mutations or SPINK-1 polymorphisms (40%) compared with seven of the control patients (14%) (P = 0.01). CONCLUSION: CFTR mutations and SPINK-1 polymorphisms are frequent among HIV-positive patients suffering from acute pancreatitis. These mutations may increase the susceptibility to pancreatitis when exposed to environmental risk factors.

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42 Samples were tested: (i) for 20 common CFTR mutations (delF508, 621+1G.T, G542X, 3849+10kbC.T, N1303K, 3659delC, 1717-1G.A, 1078delT, W1282X, R347P, G551D, A455E, R553X, S1251N, R1162X, delF507, R334W, 2183AA.G, R117H, and E60X; Elucigene CF20; Orchid Biosciences, Abingdon, UK); (ii) for the CFTR IVS8 5T variant (Elucigene CF Poly-T; Orchid); and (iii) for the SPINK-1 N34S polymorphism, by poly- Copyright (c) Lippincott Williams & Wilkins. Login to comment

[hide] The puzzle of genes and environmental risk factors... AIDS. 2004 Jul 23;18(11):1591-3. Ockenga J
The puzzle of genes and environmental risk factors for disease susceptibility: putting the pieces together.
AIDS. 2004 Jul 23;18(11):1591-3., 2004-07-23 [PMID:15238778]

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11 Approximately 72% of cystic fibrosis patients are homozygous or compound heterozygous for eight mutations of the CFTR regulator gene on chromosome 7: delta F508, G542X, R553X, W1282X, N1303K, 621 + 1G!T, 1717-1G!A, R117H [3]; whereas the deletion delta F508 alone accounts for approximately 66% of mutant cystic fibrosis alleles. Login to comment

[hide] Cystic fibrosis as a cause of infertility. Reprod Biol. 2004 Jul;4(2):119-29. Jarzabek K, Zbucka M, Pepinski W, Szamatowicz J, Domitrz J, Janica J, Wolczynski S, Szamatowicz M
Cystic fibrosis as a cause of infertility.
Reprod Biol. 2004 Jul;4(2):119-29., [PMID:15297887]

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Cystic fibrosis (CF) is one of the autosomal recessive diseases, caused by mutations in a gene known as cystic fibrosis transmembrane regulator (CFTR). The majority of adult males with CF (99%) is characterized by congenital bilateral absence of vas deferens (CBAVD). CBAVD is encountered in 1-2% of infertile males without CF. Females with CF are found to be less fertile than normal healthy women. In females with CF, delayed puberty and amenorrhoea are common due to malnutrition. CFTR mutations are also associated with congenital absence of the uterus and vagina (CAUV). The National Institutes of Health recommend genetic counseling for any couple seeking assisted reproductive techniques with a CF male or obstructive azoospermia which is positive for a CF mutation.

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58 CFTR screening includes the most frequent CFTR mutations, for example in the German population: ΔF508, R347P, G542X, S549I, N, R (A→C), G551D, R553X, N1303K, 3849+10kbC→T [11]. Login to comment

[hide] Characterization of cystic fibrosis conductance tr... Hum Reprod. 2004 Nov;19(11):2502-8. Epub 2004 Aug 27. Grangeia A, Niel F, Carvalho F, Fernandes S, Ardalan A, Girodon E, Silva J, Ferras L, Sousa M, Barros A
Characterization of cystic fibrosis conductance transmembrane regulator gene mutations and IVS8 poly(T) variants in Portuguese patients with congenital absence of the vas deferens.
Hum Reprod. 2004 Nov;19(11):2502-8. Epub 2004 Aug 27., [PMID:15333598]

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BACKGROUND: Cystic fibrosis conductance transmembrane regulator (CFTR) gene mutations and IVS8 poly(T) variants in Portuguese patients with bilateral (CBAVD) and unilateral (CUAVD) congenital absence of the vas deferens remain to be evaluated. METHODS: Patient screening was carried out by PCR, denaturing gradient gel electrophoresis and DNA sequencing. RESULTS: CFTR mutations were found in 18 out of 31 (58.1%) CBAVD and in three of four (75%) CUAVD patients. The most frequent mutations were F508del and R334W in CBAVD and G542X in CUAVD, with the allelic frequencies of R334W (6.5%) and G542X (25%) being particular to the Portuguese population. The 5T allelic frequency was 3.5% in the fertile male population, 25% in CUAVD and 27.4% in CBAVD patients. The combined frequency of mutations (CFTR+5T) was increased in CBAVD to 22 out of 31 (71%). The frequency of CFTR mutations was compared with that of patients with secondary obstructive azoospermia (OAZ; one out of 16, 6.3%) and non-obstructive azoospermia (NOAZ; two out of 22, 9.1%) with conserved spermatogenesis, which were similar to the general population. However, whereas the 5T allelic frequency in OAZ was similar to that of the general population (3.1%), it was increased in NOAZ cases (14.3%). CONCLUSIONS: Data confirm that CFTR+5T mutations represent the most common genetic abnormality in CAVD, and suggest that cases of NOAZ may be associated with the 5T allele.

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5 The most frequent mutations were F508del and R334W in CBAVD and G542X in CUAVD, with the allelic frequencies of R334W (6.5%) and G542X (25%) being particular to the Portuguese population. Login to comment
98 The association of CFTR mutations with the 5T allele also did not increase the number of patients with at least one mutation detected. Overall, in the four CUAVD patients, the 5T allele and the G542X mutation were the most frequent mutations found, which accounted for two of the eight (25%) total alleles. Login to comment
106 G/- 1 - 1 7/9 5T/5T 2 - - 5/5 (£2) 5T/- 2 - - 5/7 (£2) CUAVD F508del/- 1 - 1 7/9 G542X/- 2 - 2 5/9 (£2) OAZ F508del/- 1 - 1 7/9 5T/- 1 - - 5/7 NOAZ-HP 3659delC/- 1 - 1 7/7 F508del/- 1 - 1 5/9 5T/5T 1 - - 5/5 5T/- 4 - - 5/7 (£4) Severe mutations are in bold. Login to comment
135 This frequency was due to the allelic frequency of G542X (25%) in Portuguese CUAVD males, which is higher than in other countries where it was also found (7-17%), France (Je´ze´quel et al., 2000) and Spain (Casals et al., 2000). Login to comment
150 The increased allelic frequency of R334W in CBAVD (6.5%) and of G542X in CUAVD (25%) appears particular to the Portuguese population. Login to comment

[hide] Familial concordance of phenotype and microbial va... Pediatr Pulmonol. 2004 Oct;38(4):292-7. Picard E, Aviram M, Yahav Y, Rivlin J, Blau H, Bentur L, Avital A, Villa Y, Schwartz S, Kerem B, Kerem E
Familial concordance of phenotype and microbial variation among siblings with CF.
Pediatr Pulmonol. 2004 Oct;38(4):292-7., [PMID:15334505]

Abstract [show]
The clinical spectrum of cystic fibrosis (CF) is influenced by the cystic fibrosis transmembrane conductance regulator (CFTR) genotype. However, variable courses of the disease were demonstrated among patients with identical genotypes. Since siblings share identical CFTR mutations and environmental factors, they can serve as a model to assess the effect of modifier genes on disease expression, and also to evaluate cross-infection. The aim of this study was to compare disease expression among siblings with CF. All sibling pairs treated at 7 CF centers in Israel were included in the study. Data were collected from patients' medical charts. Fifty families with at least 2 siblings were identified. As expected, the second-born sibling was diagnosed at an earlier age compared to the first-born. The mode of CF presentation at diagnosis showed significant familial concordance. In the families where the first sibling presented with gastrointestinal manifestations, 79% of the second siblings also presented with gastrointestinal manifestations. When gastrointestinal manifestations were absent in the first sibling, only 12% of the second siblings presented with gastrointestinal manifestations (P < 0.0001). Likewise, when the first sibling presented with respiratory symptoms, 60% of the second siblings presented with the similar symptoms. However, when the first sibling presented without respiratory symptoms, only 12% of the second siblings presented with respiratory symptoms (P < 0.001). Meconium ileus (MI) was present in 20 patients (21%). In 10 families where the first-born sibling had MI, 8 (80%) of the subsequent siblings had MI. On the other hand, in the 39 families where the first-born sibling did not have MI, only 2 (5%) subsequent siblings had MI (P < 0.001). Pancreatic insufficiency (PI) also had high familial concordance (P < 0.0001). Percentile growth for weights and heights and lung function (FVC, FEV(1), and FEF(25-75)) at ages 7 and 10 years were similar between siblings. P. aeruginosa grew from sputum in 89% of our study patients. When P. aeruginosa was isolated from the first-born patient, 91% of the second siblings were also positive for P. aeruginosa, whereas when the initial sibling was not a carrier of P. aeruginosa, only 50% of subsequent siblings were positive (P < 0.0001). This familial concordance was not observed for S. aureus. By contrast, the age of first isolation of P. aeruginosa and S. aureus was significantly earlier in the second sibling than in the first for the two bacteria: 10.3 +/- 5.1 vs. 7.3 +/- 5.2 years (P < 0.05) for P. aeruginosa, and 11.5 +/- 5.4 years vs. 6.8 +/- 5.1 years (P < 0.05) for S. aureus. CF siblings tend to share similar phenotypes that are not mutation-dependent. The lack of variability between siblings in mode of initial CF presentation, rates of MI, pulmonary function, and nutritional status supports the role of modifier genes in the determination of these factors.

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66 The following genotypes were found among the families: DF508/DF508 (7), W1282X/W1282X (4), N1303K/N1303K (3), DF508/ W1282X (3), W1282X/N1303K (2), DF508/G542X (2), DF508/3849þ10 kb C ! Login to comment
69 T (1), G542X/G542X (1), G85E/G85E (1), 3849þ10 kb C ! Login to comment
71 A (1), S549R/S549R (1), Q359K/Q360K (1), DF508/Unknown (4), W1282X/Unknown (3), G542X/Unknown (2), G85E/Unknown (1), and Q359K/ Unknown (1). Login to comment

[hide] Cystic fibrosis screening: lessons learned from th... Genet Med. 2004 May-Jun;6(3):136-40. Strom CM, Crossley B, Redman JB, Buller A, Quan F, Peng M, McGinnis M, Sun W
Cystic fibrosis screening: lessons learned from the first 320,000 patients.
Genet Med. 2004 May-Jun;6(3):136-40., [PMID:15354331]

Abstract [show]
PURPOSE: To examine the data from > 335,000 Cystic fibrosis (CF) tests to detect unsuspected findings and obtain clinical data when indicated to optimize genetic counseling. METHODS: A proprietary database containing 335,204 consecutive CF DNA tests and 445 CF prenatal diagnostic tests was queried. Clinical information was obtained for prenatal and selected nonprenatal cases by telephone contact with physician offices. RESULTS: The mutation 1078delT was found in much lower frequency than expected with rates of only 1:55,867 tests and 0.06% of CF mutations. This level is below the threshold set by the American College of Medical Genetics. Homozygosity was observed for 2789+5G>A in a 29-year-old women and compound heterozygosity with delta F408 in a 40-year-old woman with isolated chronic sinusitis. Many patients elected prenatal diagnosis when not at a 1:4 risk due to echogenic bowel or IVS-8 5T issues. CONCLUSIONS: With the exception of 1078delT, all CF mutations in the ACMG panel were detected with a frequency of > 0.1% of CF chromosomes. When ACMG guidelines are strictly adhered to, population-based CF carrier screening will accurately identify couples at risk for having children with CF.

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47 Frequency, all tests Frequency, CF mutations (%) delta F508 7610 1:44 75% R117H/7T or 9T 1030 1:325 NAb R117H/5T 103 1:3,254 0.51c W1282X 529 1:625 5.2 G542X 382 1:909 3.8 G551D 278 1:1,250 2.7 N1303K 201 1:1,668 2.0 3849ϩ10kb CϾT 167 1:2,007 1.6 1717-1 GϾA 102 1:3,286 1.0 R553X 102 1:3,286 1.0 621ϩ1 GϾT 98 1:3,420 0.97 2789ϩ5 GϾA 82 1:4,087 0.80 3120ϩ1 GϾA 73 1:4,591 0.72 R1162X 54 1:6,207 0.53 R334W 54 1:6,207 0.53 685E 52 1:6,446 0.51 R560T 52 1:6,446 0.51 Delta I507 51 1:6,572 0.50 711ϩ1 GϾT 40 1:8,380 0.39 1898ϩ1 GϾA 37 1:9,059 0.36 3659 del C 36 1:9,311 0.36 A455E 34 1:9,858 0.33 R347P 33 1:10,158 0.32 2184 del A 14 1:23,943 0.14 1078 del T 6 1:55,867 0.06 a I148T has been eliminated from these data. Login to comment

[hide] Cystic fibrosis population carrier screening: 2004... Genet Med. 2004 Sep-Oct;6(5):387-91. Watson MS, Cutting GR, Desnick RJ, Driscoll DA, Klinger K, Mennuti M, Palomaki GE, Popovich BW, Pratt VM, Rohlfs EM, Strom CM, Richards CS, Witt DR, Grody WW
Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel.
Genet Med. 2004 Sep-Oct;6(5):387-91., [PMID:15371902]

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70 It has been ar- Table 1 CFTR mutation frequency among individuals with clinically diagnosed cystic fibrosis by racial/ethnic group and in a pan-ethnic U.S. population CFTR mutation Mutation frequency among individuals with clinically diagnosed cystic fibrosis (%) Non-Hispanic Caucasian Hispanic Caucasian African American Asian American Ashkenazi Jewish Pan-Ethnic Population5 delF508 72.42 54.38 44.07 38.95 31.41 66.31 G542X 2.28 5.10 1.45 0.00 7.55 2.64 W1282X 1.50 0.63 0.24 0.00 45.92 2.20 G551D 2.25 0.56 1.21 3.15 0.22 1.93 621ϩ1GϾT 1.57 0.26 1.11 0.00 0.00 1.30 N1303K 1.27 1.66 0.35 0.76 2.78 1.27 R553X 0.87 2.81 2.32 0.76 0.00 1.21 dell507 0.88 0.68 1.87 0.00 0.22 0.90 3849ϩ10kbCϾT 0.58 1.57 0.17 5.31 4.77 0.85 3120ϩ1GϾT 0.08 0.16 9.57 0.00 0.10 0.86 R117H 0.70 0.11 0.06 0.00 0.00 0.54 1717-1GϾT 0.48 0.27 0.37 0.00 0.67 0.44 2789ϩ5GϾA 0.48 0.16 0.00 0.00 0.10 0.38 R347P 0.45 0.16 0.06 0.00 0.00 0.36 711ϩ1GϾT 0.43 0.23 0.00 0.00 0.10 0.35 R334W 0.14 1.78 0.49 0.00 0.00 0.37 R560T 0.38 0.00 0.17 0.00 0.00 0.30 R1162X 0.23 0.58 0.66 0.00 0.00 0.30 3569delC 0.34 0.13 0.06 0.00 0.00 0.28 A455E 0.34 0.05 0.00 0.00 0.00 0.26 G85E 0.29 0.23 0.12 0.00 0.00 0.26 2184delA 0.17 0.16 0.05 0.00 0.10 0.15 1898ϩ1GϾA 0.16 0.05 0.06 0.00 0.10 0.13 l148T 0.09 0.09 0.05 0.00 0.10 0.08 1078delT 0.02 0.09 0.00 0.00 0.00 0.03 Total 88.40 71.90 64.51 48.93 94.14 84.00 gued that a laboratory is obligated to report any and all information that is gleaned from a test system, however, there is no regulatory requirement and practice varies. Login to comment

[hide] CFTR mutation distribution among U.S. Hispanic and... Genet Med. 2004 Sep-Oct;6(5):392-9. Sugarman EA, Rohlfs EM, Silverman LM, Allitto BA
CFTR mutation distribution among U.S. Hispanic and African American individuals: evaluation in cystic fibrosis patient and carrier screening populations.
Genet Med. 2004 Sep-Oct;6(5):392-9., [PMID:15371903]

Abstract [show]
PURPOSE: We reviewed CFTR mutation distribution among Hispanic and African American individuals referred for CF carrier screening and compared mutation frequencies to those derived from CF patient samples. METHODS: Results from CFTR mutation analyses received from January 2001 through September 2003, were analyzed for four populations: Hispanic individuals with a CF diagnosis (n = 159) or carrier screening indication (n = 15,333) and African American individuals with a CF diagnosis (n = 108) or carrier screening indication (n = 8,973). All samples were tested for the same 87 mutation panel. RESULTS: In the Hispanic population, 42 mutations were identified: 30 in the patient population (77.5% detection rate) and 33 among carrier screening referrals. Five mutations not included in the ACMG/ACOG carrier screening panel (3876delA, W1089X, R1066C, S549N, 1949del84) accounted for 7.55% detection in patients and 5.58% among carriers. Among African American referrals, 33 different mutations were identified: 21 in the patient population (74.4% detection) and 23 in the carrier screening population. Together, A559T and 711+5G>A were observed at a detection rate of 3.71% in CF patients and 6.38% in carriers. The mutation distribution seen in both the carrier screening populations reflected an increased frequency of mutations with variable expression such as D1152H, R117H, and L206W. CONCLUSIONS: A detailed analysis of CFTR mutation distribution in the Hispanic and African American patient and carrier screening populations demonstrates that a diverse group of mutations is most appropriate for diagnostic and carrier screening in these populations. To best serve the increasingly diverse U.S. population, ethnic-specific mutations should be included in mutation panels.

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35 87 mutation panel The following mutations were included in the panel: ⌬F508, ⌬F311, ⌬I507, A455E, A559T, C524X, D1152H, D1270N, E60X, G178R, G330X, G480C, G542X, G551D, G85E, G91R, I148T, K710X, L206W, M1101K, N1303K, P574H, Q1238X, Q359K/T360K, Q493X, Q552X, Q890X, R1066C, R1158X, R1162X, R117C, R117H, R1283M, R334W, R347H, R347P, R352Q, R553X, R560T, S1196X, S1251N, S1255X, S364P, S549I, S549N, S549R, T338I, V520F, W1089X, W1282X, Y1092X, Y563D, 1078delT, 1161delC, 1609delCA, 1677delTA, 1717-1GϾA, 1812-1GϾA, 1898ϩ1GϾA, 1898ϩ5GϾT, 1949del84, 2043delG, 2143delT, 2183delAAϾG, 2184delA, 2307insA, 2789ϩ5GϾA, 2869insG, 3120ϩ1GϾA, 3120GϾA, 3659delC, 3662delA, 3791delC, 3821delT, 3849ϩ10kbCϾT, 3849ϩ4AϾG, 3905insT, 394delTT, 405ϩ1GϾA, 405ϩ3AϾC, 444delA, 574delA, 621ϩ1GϾT, 711ϩ1GϾT, 711ϩ5GϾA, 712-1GϾT, 3876delA CFTR mutation analysis Genomic DNA was extracted from peripheral blood lymphocytes, buccal cell swabs, or bloodspots by Qiagen QIAmp 96 DNA Blood Kit. Specimens were tested for 87 mutations by a pooled allele-specific oligonucleotide (ASO) hybridization method as previously described.16,17 Two multiplex chain reactions (PCR) were used to amplify 19 regions of the CFTR gene. Login to comment
56 Among 318 CF patient chromosomes, 30 mutations were identified with ⌬F508, G542X, R334W, 3120ϩ1GϾA, W1089X, 3876delA, and R1066C representing 52.52% of the total. Login to comment
63 The most prevalent mutations were as follows: ⌬F508, D1152H, R117H, G542X, L206W, I148T (3199del6 status unknown), ⌬I507, R1066C, R553X, 3849ϩ10kbCϾT, and R334W representing 83.72% of the total identified. Login to comment
69 With the exception of W1089X, the next 6 most frequent mutations in the patient population (G542X, R334W, 3120ϩ1GϾA, 3876delA, W1089X, and R1066C) were all seen in the carrier population at frequencies of 1.4% to 4.2%. Login to comment
86 An additional 4 mutations (G551D, 1717-1GϾA, G542X, 711ϩ5GϾA) were detected twice (0.93% each), whereas 12 other mutations were identified on one chromosome each. Login to comment
108 In the current study, 42 different mutations were identified among the Hispanic individuals (patients and carriers) tested and the most common mutations included those previously reported to be common among Hispanics, 3876delA,32 W1089X,17 as well as mutations considered frequent in African Americans (3120ϩ1GϾA)19 and panethnic (e.g., G542X, ⌬I507) populations.33 Although regional variation in overall detection rates may occur, these data provide general guidance when developing a panethnic mutation panel and information useful for genetic counseling purposes. Login to comment

[hide] Clinical sensitivity of prenatal screening for cys... Genet Med. 2004 Sep-Oct;6(5):405-14. Palomaki GE, FitzSimmons SC, Haddow JE
Clinical sensitivity of prenatal screening for cystic fibrosis via CFTR carrier testing in a United States panethnic population.
Genet Med. 2004 Sep-Oct;6(5):405-14., [PMID:15371905]

Abstract [show]
PURPOSE: To estimate CFTR mutation frequencies, clinical sensitivities (proportions of carrier couples or affected fetuses detected), and birth prevalence estimates for broad racial/ethnic groups and for a panethnic U.S. population. METHODS: Published sources of information were identified, corrected when appropriate, and summarized. Combining racial/ethnic-specific mutation frequencies and birth prevalence estimates allowed the computation of panethnic estimates. RESULTS: Two of the 25 recommended mutations do not meet the 0.1% threshold in a panethnic population set by the American College of Medical Genetics. The clinical sensitivities are estimated to be 71.9%, 51.7%, 41.6%, 88.6%, and 23.4% for non-Hispanic Caucasians, Hispanic Caucasian, African American, Ashkenazi Jewish Caucasian, and Asian American couples, respectively. Birth prevalence estimates are 1:2,500, 1:13,500, 1:15,100, 1:2,270, and 1:35,100, whereas the number of couples needed to screen to detect an affected fetus are about 3,200, 26,120; 36,040; 2,600, and 129,600, respectively, for the same racial/ethnic groups. CONCLUSIONS: Overall, the panethnic estimates for CFTR mutation frequencies are similar to those for non-Hispanic Caucasians. However, large differences in both clinical sensitivity and birth prevalence exist between the broad racial/ethnic groups examined. Whether and how the differences in the numbers of couples needed to screen to detect an affected fetus are to be included in prenatal screening for cystic fibrosis needs to be more explicitly addressed.

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32 Data from the International Cystic Fibrosis Consortium were taken from Table 1 of its publication.4 Data from the Cystic Fibrosis Foundation National Patient Registry were taken from the year 1999 and stratified according to whether or not the patient was seen Table 1 CFTR mutation frequencies among Hispanic Caucasians with cystic fibrosis within the recommended minimum testing panel Ordera Mutation Mutation frequency (%) CF Consortiumb CF Foundationc Average Cumulative 1 delF508 45.51 63.25 54.38 54.38 2 G542X 5.11 5.09 5.10 59.48 8 delI507 0.59 5.02 2.81 62.29 22 R334W 2.25 1.31 1.78 64.07 6 N1303K 1.65 1.67 1.66 65.73 10 3849 ϩ 10kbC Ͼ T 1.60 1.53 1.57 67.30 7 R553X 0.63 0.73 0.68 67.98 5 W1282X 0.53 0.73 0.63 68.61 19 R1162X 0.57 0.58 0.58 69.19 3 G551D 0.31 0.80 0.56 69.75 12 1717 - 1G Ͼ T 0.10 0.44 0.27 70.02 4 621 ϩ 1G Ͼ T 0.00 0.51 0.26 70.28 14 711 ϩ 1G Ͼ T 0.10 0.36 0.23 70.51 18 G85E 0.10 0.36 0.23 70.74 11 2789 ϩ 5G Ͼ A 0.10 0.22 0.16 70.90 13 R347P 0.10 0.22 0.16 71.06 20 2184delA 0.10 0.22 0.16 71.22 24 3120 ϩ 1G Ͼ T 0.10 0.22 0.16 71.38 17 3569delC 0.10 0.15 0.13 71.51 9 R117H 0.00 0.22 0.11 71.62 23 I148T 0.10 0.07 0.09 71.71 25 1078delT 0.10 0.07 0.09 71.80 16 A455E 0.10 0.00 0.05 71.85 21 1898 ϩ 1G Ͼ A 0.10 0.00 0.05 71.90 15 R560T 0.00 0.00 0.00 71.90 All 25 59.95 83.77 71.90 a The order is based on that found for non-Hispanic Caucasians.3 b Based on between 178 and 958 chromosomes (International Cystic Fibrosis Genetic Analysis Consortium.4 c Based on 1374 chromosomes from clinically diagnosed persons registered in the Cystic Fibrosis Foundation National Patient Registry. Login to comment
80 The larger data- Table 2 CFTR mutation frequencies among African American individuals with cystic fibrosis within the recommended minimum testing panel Ordera Mutation Mutation frequency (%) CF Consortiumb CF Foundationc Average Cumulative 1 delF508 35.50 52.63 44.07 44.07 24 3120 ϩ 1G Ͼ T 12.50 6.64 9.57 53.64 8 delI507 0.74 3.89 2.32 55.96 7 R553X 2.37 1.37 1.87 57.83 2 G542X 1.18 1.72 1.45 59.28 3 G551D 0.59 1.83 1.21 60.49 4 621 ϩ 1G Ͼ T 1.18 1.03 1.11 61.60 19 R1162X 0.74 0.57 0.66 62.26 22 R334W 0.74 0.23 0.49 62.75 12 1717 - 1G Ͼ T 0.74 0.00 0.37 63.12 6 N1303K 0.00 0.69 0.35 63.47 5 W1282X 0.00 0.47 0.24 63.71 10 3849 ϩ 10kbC Ͼ T 0.00 0.34 0.17 63.88 15 R560T 0.00 0.34 0.17 64.05 18 G85E 0.00 0.23 0.12 64.17 9 R117H 0.00 0.11 0.06 64.23 13 R347P 0.00 0.11 0.06 64.29 17 3569delC 0.00 0.11 0.06 64.35 21 1898 ϩ 1G Ͼ A 0.00 0.11 0.06 64.41 20 2184delA 0.10 0.00 0.05 64.46 23 I148T 0.10 0.00 0.05 64.51 11 2789 ϩ 5G Ͼ A 0.00 0.00 0.00 64.51 14 711 ϩ 1G Ͼ T 0.00 0.00 0.00 64.51 16 A455E 0.00 0.00 0.00 64.51 25 1078delT 0.00 0.00 0.00 64.51 All 25 56.46 72.42 64.51 a The order is based on that found for non-Hispanic Caucasians.3 b Based on between 79 and 169 chromosomes reported by the International Cystic Fibrosis Genetic Analysis Consortium.4 c Based on 874 chromosomes from clinically diagnosed persons registered in the Cystic Fibrosis Foundation National Patient Registry. Login to comment
107 An earlier article10 reported that 97% of mutations were identified in 90 chromosomes from Ashkenazi Jewish individ- Table 3 CFTR mutation frequencies among Ashkenazi Jewish Caucasian individuals with cystic fibrosis within the recommended minimum testing panel Ordera Mutation Mutation frequency (%) CF Consortiumb Cumulative 5 W1282X 45.92 45.92 1 delF508 31.41 77.33 2 G542X 7.55 84.88 10 3849 ϩ 10kbC Ͼ T 4.77 89.65 6 N1303K 2.78 92.43 12 1717 - 1G Ͼ T 0.67 93.10 7 R553X 0.22 93.32 3 G551D 0.22 93.54 24 3120 ϩ 1G Ͼ T 0.10 93.64 21 1898 ϩ 1G Ͼ A 0.10 93.74 20 2184delA 0.10 93.84 23 I148T 0.10 93.94 11 2789 ϩ 5G Ͼ A 0.10 94.04 14 711 ϩ 1G Ͼ T 0.10 94.14 8 delI507 0.00 94.14 19 R1162X 0.00 94.14 22 R334W 0.00 94.14 4 621 ϩ 1G Ͼ T 0.00 94.14 15 R560T 0.00 94.14 18 G85E 0.00 94.14 9 R117H 0.00 94.14 13 R347P 0.00 94.14 17 3569delC 0.00 94.14 16 A455E 0.00 94.14 25 1078delT 0.00 94.14 Sum 94.14 a The order is based on that found for non-Hispanic Caucasians.3 b Based on between 57 and 503 chromosomes reported by the International Cystic Fibrosis Genetic Analysis Consortium.4 uals with cystic fibrosis, using a panel of 11 mutations. Login to comment
115 In an- Table 4 CFTR mutation frequencies among Asian American individuals with cystic fibrosis within the recommended minimum testing panel Ordera Mutation Mutation frequency (%) Heim et al.1b CF Foundationc Average Cumulative 1 delF508 18.80 59.09 38.95 38.95 10 3849 ϩ 10kbC Ͼ T 0.00 10.61 5.31 44.26 3 G551D 6.30 0.00 3.15 47.41 6 N1303K 0.00 1.52 0.76 48.17 8 delI507 0.00 1.52 0.76 48.93 2 G542X 0.00 0.00 0.00 48.93 4 621 ϩ 1G Ͼ T 0.00 0.00 0.00 48.93 5 W1282X 0.00 0.00 0.00 48.93 7 R553X 0.00 0.00 0.00 48.93 9 R117H 0.00 0.00 0.00 48.93 11 2789 ϩ 5G Ͼ A 0.00 0.00 0.00 48.93 12 1717 - 1G Ͼ T 0.00 0.00 0.00 48.93 13 R347P 0.00 0.00 0.00 48.93 14 711 ϩ 1G Ͼ T 0.00 0.00 0.00 48.93 15 R560T 0.00 0.00 0.00 48.93 16 A455E 0.00 0.00 0.00 48.93 17 3569delC 0.00 0.00 0.00 48.93 18 G85E 0.00 0.00 0.00 48.93 19 R1162X 0.00 0.00 0.00 48.93 20 2184delA 0.00 0.00 0.00 48.93 21 1898 ϩ 1G Ͼ A 0.00 0.00 0.00 48.93 22 R334W 0.00 0.00 0.00 48.93 23 I148T 0.00 0.00 0.00 48.93 24 3120 ϩ 1G Ͼ T 0.00 0.00 0.00 48.93 25 1078delT 0.00 0.00 0.00 48.93 Sum 25.10 72.74 48.93 a The order is based on that found for non-Hispanic Caucasians.3 b Based on 20 chromosomes. Login to comment
173 For exam- Table 7 Estimated number of carriers of the 25 recommended CFTR mutations by racial/ethnic group and weighted average, representing the panethnic population in the United States for 2002 Order CFTR mutation Number of CFTR Mutation Carriers Panethnic frequency, % Non-Hispanic Caucasian Hispanic Caucasian African American Asian American Ashkenazi Jewish Total 1 delF508 64,779 8,207 4,272 886 796 78,940 66.31 2 G542X 2,039 770 141 0 191 3,141 2.64 5 W1282X 1,342 95 23 0 1,164 2,624 2.20 3 G551D 2,013 85 117 72 6 2,293 1.93 4 621 ϩ 1G Ͼ T 1,404 39 108 0 0 1,551 1.30 6 N1303K 1,136 251 34 17 70 1,508 1.27 7 R553X 778 424 225 17 0 1,444 1.21 8 delI507 787 103 181 0 6 1,077 0.90 10 3849 ϩ 10kbC Ͼ T 519 237 16 121 121 1,014 0.85 24 3120 ϩ 1G Ͼ T 72 24 928 0 3 1,027 0.86 9 R117H 626 17 6 0 0 649 0.55 12 1717 - 1G Ͼ T 429 41 36 0 17 523 0.44 11 2789 ϩ 5G Ͼ A 429 24 0 0 3 456 0.38 13 R347P 403 24 6 0 0 433 0.36 14 711 ϩ 1G Ͼ T 385 35 0 0 3 423 0.36 22 R334W 125 269 47 0 0 441 0.37 15 R560T 340 0 16 0 0 356 0.30 19 R1162X 206 88 64 0 0 358 0.30 17 3569delC 304 20 6 0 0 330 0.28 16 A455E 304 8 0 0 0 312 0.26 18 G85E 259 35 12 0 0 306 0.26 20 2184delA 152 24 5 0 3 184 0.15 21 1898 ϩ 1G Ͼ A 143 8 6 0 3 160 0.13 23 I148T 80 14 5 0 3 102 0.09 25 1078delT 18 14 0 0 0 32 0.03 All 79,072 10,856 6,193 1,113 2,389 99,684 84.00 Bolded numbers indicate mutations that are more likely to be found in a racial/ethnic group other than non-Hispanic Caucasians. Login to comment

[hide] Premarital and prenatal screening for cystic fibro... Genet Med. 2004 Sep-Oct;6(5):415-20. Kornreich R, Ekstein J, Edelmann L, Desnick RJ
Premarital and prenatal screening for cystic fibrosis: experience in the Ashkenazi Jewish population.
Genet Med. 2004 Sep-Oct;6(5):415-20., [PMID:15371906]

Abstract [show]
PURPOSE: Since the early 1990s, Dor Yeshorim (DY) and the Mount Sinai School of Medicine (MSSM) have conducted premarital and prenatal carrier screening for cystic fibrosis (CF) in the Ashkenazi Jewish (AJ) population as part of their genetic testing programs, respectively. Together, over 170,000 screenees have been tested. In this study, we report the CF mutation frequencies in over 110,000 screenees who reportedly were of 100% AJ descent from the DY program and MSSM. In addition, the CF mutation frequencies in a group of > 7,000 screenees for AJ diseases who were of < 100% AJ descent are reported. METHODS: Testing for CF mutations was performed by either PCR and restriction digestion or ASO hybridization analyses at MSSM or sent to various academic and commercial laboratories by DY. RESULTS: The overall (and individual) carrier frequency for the five common AJ mutations, W1282X (0.020), DeltaF508 (0.012), G542X (0.0024), 3849+10kb C>T (0.0020), and N1303K (0.0016), among screenees who were 100% AJ was 1 in 26; when D1152H and the rare 1717-1G>A were included, the overall carrier frequency increased to approximately 1 in 23. In four families with D1152H, five compound heterozygotes for D1152H and W1282X (n = 2), DeltaF508 (1) or 3849+10kb C>T (1) were identified. In contrast, the carrier frequency for screenees reporting < 100% AJ descent was approximately 1 in 30 for the seven mutations. CONCLUSIONS: The carrier frequency for five common CF mutations in a large 100% AJ sample increased from 1 in 26 to 1 in 23 when D1152H was included in the panel. Addition of D1152H to mutation panels when screening the AJ population should be considered because compound heterozygosity is associated with a variable disease phenotype. Further studies to delineate the phenotype of CF patients with this mutation are needed.

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14 Although the demand for screening was great, widespread population screening was not actually recommended by the American College of Obstetrics and Gynecology (ACOG) and American College of Medical Genetics (ACMG) until 2001.5 Based on a frequency of at least 0.1% of mutant alleles in a large CF patient database, a panel of 25 mutations was recommended even though the sensitivity was still below 90% in most ethnic groups.6 The results of screening with this mutation panel were reviewed in 20027 and most recently, in this issue.8 The Ashkenazi Jewish (AJ) population represents a unique group for genetic screening as common mutations occur in prevalent recessive diseases due to founder effect and/or selection.9,10 With regard to CF, five CFTR mutations (W1282X, ⌬F508, G542X, 3849ϩ10kb CϾT, N1303K) account for 97% of the mutant alleles in AJ CF patients.11 This population has a very high acceptance rate for genetic screening and has had a positive experience with carrier screening, beginning with Tay-Sachs disease in 1970.12,13 In this study, we report our experiences with CF carrier screening in the AJ population using two different approaches: premarital (Dor Yeshorim, DY) and prenatal screening (Mount Sinai School of Medicine, MSSM). Login to comment
19 DOI: 10.1097/01.GIM.0000139510.00644.F7 September/October 2004 ⅐ Vol. 6 ⅐ No. 5 a r t i c l e Genetics IN Medicine Jewish community, began premarital screening in 1983 for Tay-Sachs.14 CF was added to their premarital testing panel in 1993.15 The Genetic Testing Laboratory at MSSM has been conducting prenatal CF carrier screening since 1992 and has performed over 60,000 CF tests.16 Both programs initially screened for five, reported common AJ mutations (W1282X, ⌬F508, G542X, 3849ϩ10kb CϾT, N1303K).11 In 2000, DY observed the presence of the D1152H CF mutation in the AJ population and, therefore, added this mutation along with 1717-1 GϾA to its routine AJ screening panel. Login to comment
32 For the DY cohort, all individuals were initially tested for five mutations (W1282X, ⌬F508, G542X, 3849ϩ10kb CϾT, N1303K). Login to comment
36 At MSSM, for the period January 1997 through December 2000 for which data and ethnic information are available, five mutations (W1282X, ⌬F508, G542X, 3849ϩ10kb CϾT, N1303K) were analyzed by PCR amplification and restriction enzyme analyses. Login to comment
45 The G542X, 3849ϩ10 kb CϾT, and N1303K were tested in over 117,000 screenees and occurred at similar frequencies in both programs, with overall detection rates of 1 in 413 (0.0024), 1 in 490 (0.0020), and 1 in 633 (0.0016), respectively. Login to comment
47 For the five mutations (W1282X, ⌬F508, G542X, 3849ϩ10kb CϾT, N1303K) for which over 117,000 AJ screenees were tested, a total of 4,498 carriers were identified (1 in 26 or 0.038). Login to comment
68 Of the remaining 31 prenatal diagnoses, 24 fetuses were heterozygotes and Table 1 Carrier frequencies of seven routinely tested AJ CF mutations CF mutation No. individuals tested Frequency Combined frequency Previous studies DY MSSM DY MSSM Ref 3 n ϭ 6076 Ref 15 n ϭ 6850 W1282X 110889 6247 1 in 49.7 (0.0201) 1 in 48.8 (0.0205) 1 in 49.6 (0.0202) 1 in 48.2 (0.0207) 1 in 48.2 (0.0207) ⌬F508 110898 6247 1 in 81.7 (0.0122) 1 in 86.8 (0.0115) 1 in 82.0 (0.0122) 1 in 79.7 (0.0126) 1 in 77.9 (0.0128) D1152H 42208 2322 1 in 189.2 (0.00528) 1 in 193.5 (0.00517) 1 in 189.5 (0.00528) 1 in 113.5a (0.00881) NDb G542X 110893 6247 1 in 410.7 (0.00243) 1 in 446.2 (0.00224) 1 in 412.5 (0.00242) 1 in 338.3 (0.00296) 1 in 506.3 (0.00197) 3849ϩ10kb CϾT 110888 6247 1 in 490.7 (0.00204) 1 in 480.5 (0.00208) 1 in 490.1 (0.00204) 1 in 402.9 (0.00248) 1 in 607.6 (0.00165) N1303K 110894 6247 1 in 637.2 (0.00157) 1 in 567.9 (0.00176) 1 in 633.2 (0.00158) 1 in 913.3 (0.00109) 1 in 552.4 (0.00181) 1717-1 GϾA 57869 2322 1 in 7233.6 (0.000138) 1 in 2322 (0.000431) 1 in 6687.9 (0.000150) NDb NDb Five mutations (Without D1152H and 1717-1 GϾA) 1 in 26.1 (0.0384) 1 in 26.2 (0.0381) 1 in 26.1 (0.0384) 1 in 25.1 (0.0398) 1 in 25.7 (0.0389) Seven mutations 1 in 22.8 (0.0438) 1 in 22.9 (0.0437) 1 in 22.8 (0.0438) a n ϭ 1305. b Not determined. Login to comment
69 Table 2 Distribution of CF mutations in DY and MSSM carriers for five and seven common AJ mutations CF mutation % of AJ carriers (5 mutations) % of AJ carriers (7 mutations) DY MSSM Combined DY MSSM Combined W1282X 52.3 53.8 53.1 45.9 46.9 46.4 ⌬F508 31.9 30.2 31.0 27.9 26.3 27.1 G542X 6.3 5.9 6.1 5.5 5.1 5.3 3849ϩ10kb CϾT 5.3 5.5 5.4 4.7 4.8 4.8 N1303K 4.1 4.6 4.4 3.6 4.0 3.8 D1152H - - - 12.1 11.8 12.0 1717-1GϾA - - - 0.3 1.0 0.6 seven did not carry either parental mutation. Login to comment
83 Previous studies of AJ patients with CF indicated that screening for five mutations (W1282X, ⌬F508, G542X, 3849ϩ10kb CϾT, N1303K) would detect 97% of alleles causing CF in the AJ population.11 Based on the data presented in this study, the largest dataset on AJ carrier screening reported to date, the CF carrier frequency in the 100% AJ population is about 1 in 26 for these five mutations (Table 1). Login to comment
86 For example, W1282X was the most prevalent mutation among Ashkenazi Jews, present in 46.4% of AJ carriers, whereas it was present in only 1.5% of non-Hispanic Caucasian CF patients.8 ⌬F508, the most common mutation in non-Hispanic Caucasian CF pa- Table 3 Frequency of CFTR mutations among screenees reporting 100% AJ or Ͻ 100% AJ descent who requested carrier testing for CF and at least one other AJ recessive disease CF mutation % of mutations in carriers reporting 100% AJ descent (n ϭ 45,530-117,145) % of mutations among carriers reporting Ͻ100% AJ descent (n ϭ 7,393) % of Non-Hispanic Caucasian CF patient chromosomes8 (n ϭ 37,263) W1282X 46.4 (n ϭ 117,136) 32.3 1.5 ⌬F508 27.1 (n ϭ 117,145) 35.7 71.5 D1152H 12.0 (n ϭ 44,530) 8.7 0.03 G542X 5.3 (n ϭ 117,140) 6.1 2.3 3849ϩ10kb CϾT 4.8 (n ϭ 117,135) 4.9 0.7 N1303K 3.8 (n ϭ 117,141) 3.0 1.3 1717-1GϾA 0.6 (n ϭ 60,191) 1.9 0.7 R117H a 2.7 0.8 I148T a 2.3 0.05 R334W - 0.76 0.16 A455E - 0.76 0.19 G551D a 0.38 2.5 R553X - 0.38 1.0 a These mutations were detected when screening Ϸ2,300 100% AJ individuals with the ACMG recommended panel. Login to comment
99 However, previous reports indicate that the D1152H mutation together with a classic CF mutation results in variant CF phenotypes, including an adult (D1152H/ ⌬F508) with mild pulmonary disease.22 The D1152H/⌬F508 genotype also has been reported in four adults with mild pulmonary symptoms, two with Pseudomonas colonization, and another with pancreatic insufficiency.23,24 This mutation has also been observed in males with CBAVD.25,26 However, this mutation was present along with G542X in a fetus with hyperechogenic bowel loops and meconium ileus, suggesting a more severe course would occur in this case.27 In contrast to the limited number of CFTR mutations that were detected in screenees who reported themselves to be 100% AJ, the distribution of CFTR mutations in the over 7,000 MSSM screenees who were of Ͻ 100% AJ descent differed significantly. Login to comment
104 Based on the results of these studies, it is recommended that premarital/prenatal carrier screening for CF be performed by testing the five common AJ CFTR mutations (W1282X, DF508, G542X, 3849ϩ10kb CϾT, N1303K), along with D1152H, for individuals who report that they are 100% AJ. Login to comment

[hide] Genotype-phenotype correlation and frequency of th... Genet Med. 2004 Sep-Oct;6(5):421-5. Monaghan KG, Highsmith WE, Amos J, Pratt VM, Roa B, Friez M, Pike-Buchanan LL, Buyse IM, Redman JB, Strom CM, Young AL, Sun W
Genotype-phenotype correlation and frequency of the 3199del6 cystic fibrosis mutation among I148T carriers: results from a collaborative study.
Genet Med. 2004 Sep-Oct;6(5):421-5., [PMID:15371907]

Abstract [show]
PURPOSE: We expect that the mutation panel currently recommended for preconception/prenatal CF carrier screening will be modified as new information is learned regarding the phenotype associated with specific mutations and allele frequencies in various populations. One such example is the I148T mutation, originally described as a severe CF mutation. After implementation of CF population-based carrier screening, we learned that I148T exists as a complex allele with 3199del6 in patients with clinical CF, whereas asymptomatic compound heterozygotes for I148T and a second severe CF mutation were negative for 3199del6. METHODS: We performed reflex testing for 3199del6 on 663 unrelated specimens, including I148T heterozygotes, compound heterozygotes, and a homozygous individual. RESULTS: Less than 1% of I148T carriers were also positive for 3199del6. Excluding subjects tested because of a suspected or known CF diagnosis or positive family history, 0.6% of I148T-positive individuals were also positive for 3199del6. We identified 1 I148T homozygote and 6 unrelated compound heterozygous individuals with I148T and a second CF variant (2 of whom also carried 3199del6). In addition, one fetus with echogenic bowel and one infertile male were heterozygous for I148T (3199del6 negative). CONCLUSIONS: Reflex testing for 3199del6 should be considered whenever I148T is identified. Reflex testing is of particular importance for any symptomatic patient or whenever one member of a couple carries a deleterious CF mutation and the other member is an I148T heterozygote. Further population data are required to determine if I148T, in the absence of 3199del6, is associated with mild or atypical CF or male infertility.

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97 3199del6 was reported to the CF Consortium in 1998 in a pancreatic insufficient CF patient with I148T and 3199del6onthesamechromosomeand⌬F508ontheotherchro- mosome.1 Another study noted a patient with severe CF and 3199del6 and I148T, though it is not noted specifically whether thesemutationsarein-cisorin-trans.16 3199del6alsooccursinthe absence of I148T, as a patient with severe CF has been reported with 3199del6 (negative for I148T) and G542X.17 Recent data suggests that 3199del6 is the deleterious mutation among I148T/3199del6 complex alleles.9 It is puzzling that early reports of patients with classic CF and I148T/⌬F508 did not identify 3199del6, despite the fact that 2 reports described performing mutation analysis of the entire CFTR coding region and splice site junctions either by a screening method (DDGE) or DNA sequencing.5,7 However, a recent report identified 3199del6 in 24 French-Canadian CF patients originally described as compound heterozygous for I148T and a severe CF mutation.11 Though there is little doubt that 3199del6 is a deleterious CF mutation, the clinical significance of I148T in the absence of 3199del6 is unclear, as we have identified this genotype in 3 males with infertility, two of the obstructive type. Login to comment

[hide] Use of MALDI-TOF mass spectrometry in a 51-mutatio... Genet Med. 2004 Sep-Oct;6(5):426-30. Buyse IM, McCarthy SE, Lurix P, Pace RP, Vo D, Bartlett GA, Schmitt ES, Ward PA, Oermann C, Eng CM, Roa BB
Use of MALDI-TOF mass spectrometry in a 51-mutation test for cystic fibrosis: evidence that 3199del6 is a disease-causing mutation.
Genet Med. 2004 Sep-Oct;6(5):426-30., [PMID:15371908]

Abstract [show]
PURPOSE: We developed a 51-mutation extended cystic fibrosis (CF) panel that incorporates the 25 previously recommended CFTR mutations, plus 26 additional mutations including 3199del6, which was associated with I148T. METHODS: This assay utilizes an integrated matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry system. RESULTS: CF testing was performed on over 5,000 individuals, including a 3-year-old Hispanic-American patient with a compound heterozygous G542X/3199del6 genotype. He is negative for I148T, or other mutations assessed by CFTR gene sequencing. CONCLUSION: These results demonstrate the successful implementation of MALDI-TOF mass spectrometry in CF clinical testing, and establish 3199del6 as a disease-causing CF mutation.

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2 Results: CF testing was performed on over 5,000 individuals, including a 3-year-old Hispanic-American patient with a compound heterozygous G542X/3199del6 genotype. Login to comment
67 Identification of a CF patient with a G542X/3199del6 genotype In the course of clinical testing with the extended CF panel, we studied a 3-year old Hispanic-American male referred by the Baylor Cystic Fibrosis Care Center at Texas Children`s Hospital. Login to comment
77 This assay also demonstrated heterozygosity for the G542X mutation, and reflex testing for the 5T variant at CFTR intron 8 showed a genotype of 7T/9T in this patient (data not Table 3 Description of the 16 multiplex assays designed to analyze 51 CFTR mutations Multiplex Mutations Exon 1 1078delT, G314E, R352Q, G330X 7 2 R347H, R347P, R334W, 1717-1A 7, 11 3 R553X, S549N, R1162X 11, 19 4 A559T, R560T, G551D 11 5 G542X, S549R, 621ϩ1T, Y122X 4, 11 6 W1282X, 3876delA, 3905insT, D1152H 18, 20 7 3849ϩ4G, 3659delC, 1898ϩ1A 12, 19 8 405ϩ1A, 405ϩ3C, 3120A, 3120ϩ1A 3, 16 9 394delTT, E60X, G85E 3 10 A455E, ⌬F508a 9, 10 11 G480C, Q493X, V520F 10 12 711ϩ1T, G178R, 3199del6 5, 17a 13 2143delT, 2184delA, K710X, F316L 7, 13 14 I148T, R117H, R117C 4 15 N1303K, 2789ϩ5A, 3849ϩ10kbT 14b, intron19, 21 16 ⌬I507a 10 17 5Tb intron 8 a F508C and I507V, I506V, I506M variants are tested for concurrently with the ⌬F508 and ⌬I507 assays respectively. Login to comment
80 Compound heterozygosity for the 3199del6 and G542X mutations was confirmed by parental studies, which identified 3199del6 in the patient`s mother and G542X in the father. Login to comment
84 No definitive mutations were identified in addition to G542X and 3199del6. Login to comment
113 The identification of a CF patient with a compound heterozygous 3199del6/G542X genotype represents the first report of 3199del6 that is not associated with I148T on a CF chromosome. Login to comment

[hide] Cystic fibrosis carrier screening: validation of a... Genet Med. 2004 Sep-Oct;6(5):431-8. Edelmann L, Hashmi G, Song Y, Han Y, Kornreich R, Desnick RJ
Cystic fibrosis carrier screening: validation of a novel method using BeadChip technology.
Genet Med. 2004 Sep-Oct;6(5):431-8., [PMID:15371909]

Abstract [show]
PURPOSE: To validate a novel BeadChip assay system for cystic fibrosis (CF) mutation testing using the panel of 25 ACMG recommended mutations and D1152H. METHODS: DNA from 519 individuals originally tested for CF mutation status by allele specific oligonucleotide hybridization (ASOH) were blindly analyzed by the BeadChip assay and the results were compared. The elongation mediated multiplexed analysis of polymorphisms (eMAP) protocol, which combines multiplex amplification of genomic DNA and multiplex detection of mutations on color-coded bead arrays, was used to analyze 26 CF mutations in two separate groups. RESULTS: The system accurately distinguished the 26 CF genotypes and had 100% concordance with the ASOH technique with an assay failure rate of 1.7%. Benign variants of exon 10 codons 506, 507, and 508 did not interfere with mutation identification and reflex testing for the 5/7/9T IVS8 polymorphism was performed on a separate array. CONCLUSIONS: The BeadChip assay system provided accurate and rapid identification of the ACMG recommended CF mutations.

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35 Mutation controls included DNA from previously identified positive patient samples (I148T, D1152H, W1282X, R117H, G85E, A455E, delF508, N1303K) and DNA from NIGMS Human Genetic Cell Repositories (Coriell Cell Repositories) (delF508, delI507, G542X, R560T, 3849ϩ10kbCϾT, N1303K, G85E; G551D, R553X, 621ϩ1GϾT, 1717-1GϾA, A455E, R334W, R347P, R1162X, 3659delC; 711ϩ1GϾT, 2789ϩ5GϾA, 3120ϩ1GϾA). Login to comment
46 Mutant ASOs were end-labeled with ␥-32 P-ATP and pooled into three subgroups (IA-IC) for Group I and four subgroups (IIA-IID) for Group II mutations with the following breakdown of mutations: IA: delF508, delI507, W1282X, R117H; IB: G542X, R560T, 3849ϩ10kbCϾT, N1303K, G85E; IC: G551D, R553X, 621ϩ1GϾT, 1717-1GϾA, I148T; IIA: A455E, R334W, D1152H; IIB: R347P, 1078delT, R1162X, 3659delC; IIC: 711ϩ1GϾT, 1898ϩ1GϾA, 2789ϩ5GϾA, 3120ϩ1GϾA; IID: 2184delA. Login to comment
84 Certain mutations including 711ϩ1GϾA, R117H, G542X, R560T, and W1282X, required a heterozygous allelic ratio with an upper limit set at 2.50. Login to comment
160 I II III IV V VI VII VIII Totals Samples tested 87 57 69 72 66 35 72 61 519 Controls testedk 0h 17h 20 29 22 16 20 21 145 PCR Failuresi 4 4 2 1 1 2 1 3 18 (3.5%) Assay Failuresi 2 0 1 0 2 2 1 1 9 (1.7%) Positives 4a 3b 0 3c 4d 2e 2f 1g 19 (3.7%) a W1282X, delF508, D1152H, W1282X b delF508, delF508, D1152H c delF508, R117H, R117H d G542X, delF508, D1152H, N1303K (does not include proficiency samplesj ) e W1282X, delF508 f I148T, 3849ϩ10kbCϾT g I148T h Runs I and II were amplified with the same master mix and used the same control samples. Login to comment

[hide] CFTR mutations and polymorphisms in male infertili... Int J Androl. 2004 Oct;27(5):251-6. Cuppens H, Cassiman JJ
CFTR mutations and polymorphisms in male infertility.
Int J Androl. 2004 Oct;27(5):251-6., [PMID:15379964]

Abstract [show]
Apart from cystic fibrosis, mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are also involved in congenital bilateral absence of the vas deferens (CBAVD). A mutation is identified in about 80% of the CFTR genes derived from CBAVD patients; the genetic defect in the remainder is yet unknown. In contrast to CF patients, when CFTR is involved, at least one of the mutant CFTR genes of CBAVD patients harbors a mild mutation. A polyvariant mutant CFTR gene is the most frequent CBAVD causing mutant CFTR gene. Here, combinations of particular alleles at several polymorphic loci yield insufficient functional CFTR. The fact that most CBAVD patients, that carry mutations on both CFTR genes, have no lung disease is most probably explained by tissue specific alternative splicing, which is increased in vas deferens compared to bronchial tissue. It has also been reported that CBAVD may be involved in other forms of infertility than CBAVD, however this has not always been confirmed in other studies. Because of techniques such as intracytoplasmic sperm injection, CBAVD patients are now able to father children, however such couples have an increased risk of having a child with cystic fibrosis, and therefore genetic testing and counselling should be provided.

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34 Examples include the G542X, G551D, R553X, W1282X and N1303K mutations. Login to comment

[hide] Toward the pharmacogenomics of cystic fibrosis--an... Pharmacogenomics. 2004 Oct;5(7):861-78. Sangiuolo F, D'Apice MR, Gambardella S, Di Daniele N, Novelli G
Toward the pharmacogenomics of cystic fibrosis--an update.
Pharmacogenomics. 2004 Oct;5(7):861-78., [PMID:15469408]

Abstract [show]
Cystic fibrosis (CF) is the most common autosomal recessive disorder in Caucasians, with a frequency of approximately 1 in 3000 live births. The mutated gene is a defective chloride channel in epithelial cells, named cystic fibrosis transmembrane conductance regulator (CFTR). Several different protocols for the scanning of the entire gene have aided molecular diagnosis and improved our understanding of the disorder's pathophysiology, but also showed the disease's complexity. Therefore, CF phenotype remains difficult to predict from CFTR mutation data alone: several studies have suggested that additional genes could modulate its clinical outcome. Gene replacement therapy is still far from being used in patients with CF, mostly due to the difficulties with targeting the appropriate cells. In this review, we summarize recent advances, both in the pharmacological and gene therapy field, aimed for the treatment of the disease.

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412 31. Hamosh A, Rosenstein BJ, Cutting GR: CFTR nonsense mutations G542X and W1282X associated with severe reduction of CFTR mRNA in nasal epithelial cells. Login to comment

[hide] Pharmacologic therapy for stop mutations: how much... Curr Opin Pulm Med. 2004 Nov;10(6):547-52. Kerem E
Pharmacologic therapy for stop mutations: how much CFTR activity is enough?
Curr Opin Pulm Med. 2004 Nov;10(6):547-52., [PMID:15510065]

Abstract [show]
PURPOSE OF REVIEW: The purpose of this review is to summarize the recent approaches using mutation-specific therapy to correct the genetic defect according to the molecular mechanism by which the mutation causes the defects in cystic fibrosis transmembrane conductance regulator (CFTR). Premature stop mutations (class I mutations) account for 5 to 10% of the total mutant alleles in cystic fibrosis patients, and in certain subpopulations the incidence is much higher. RECENT FINDINGS: The aminoglycoside antibiotics can suppress premature termination codons by permitting translation to continue to the normal termination of the transcript. The susceptibility to suppression by aminoglycosides depends on the stop codon itself and on the sequence context surrounding it. In vitro studies in cell lines expressing stop mutations and in mice have shown that aminoglycosides caused a dose-dependent increase in CFTR expression and restored functional CFTR to the apical membrane. Clinical studies also provided evidence that the aminoglycoside gentamicin can suppress these CFTR premature stop mutations in affected patients. A recent double-blind, placebo-controlled, crossover study has demonstrated restoration of CFTR function by topical application of gentamicin to the nasal epithelium of cystic fibrosis patients carrying stop mutations. In 21% of the patients there was a complete normalization of all the electrophysiologic abnormalities caused by the CFTR defect, and in 68% there was restoration of either chloride or sodium transport. Furthermore, immunohistochemical staining to the C-terminal part of the CFTR was demonstrated via peripheral staining for CFTR in scraped nasal epithelial cells of patients carrying stop mutations. Inconsistent results were reported regarding the required level of corrected CFTR that has to be reached to achieve normal function. Achieving CFTR activity of 10 to 35% might be needed to prevent significant pulmonary morbidity. SUMMARY: It is as yet unknown how much corrected mutant CFTR must reach the apical membrane to induce a clinically relevant beneficial effect. The future goal is to maximize the effect of stop-codon supressors on CFTR while minimizing side effects, but further studies must be performed to find a safer compound that may be administered in small children from the time of diagnosis.

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82 Howard et al. [21] demonstrated in Hela cells transfected with plasmid vector carrying the CFTR nonsense mutations G542X and R553X that aminoglycosides caused a dose-dependent increase in CFTR expression. Login to comment
86 Zsembery et al. [23] isolated cholangiocytes from the liver of a patient carrying the G542X mutation, and incubated them with gentamicin, and they exhibited cAMP activated chloride transport. Login to comment
89 Immunofluorescence staining of intestinal tissues from Cftr-/- mouse carrying a human CFTR-G542X transgene revealed that gentamicin treatment resulted in the appearance of human CFTR protein at the apical surface of the glands of treated mice. Login to comment
92 When taken together, these results indicate that gentamicin, and to a lesser extent tobramycin, can restore the synthesis of functional human CFTR protein by suppressing the human CFTR-G542X premature stop mutation in vivo. Login to comment
133 Quantification studies have shown that after aminoglycoside incubation, the amount of full-length CFTR produced is as much as 25% (in the R553X mutation) to 35% (in the G542X mutation) of that observed in cells transfected with a wild-type CFTR complementary DNA [18,22]. Login to comment

[hide] Nasal airway ion transport is linked to the cystic... Thorax. 2004 Nov;59(11):971-6. Fajac I, Hubert D, Guillemot D, Honore I, Bienvenu T, Volter F, Dall'Ava-Santucci J, Dusser DJ
Nasal airway ion transport is linked to the cystic fibrosis phenotype in adult patients.
Thorax. 2004 Nov;59(11):971-6., [PMID:15516474]

Abstract [show]
BACKGROUND: This study was conducted to determine whether the major nasal airway ion transport abnormalities in cystic fibrosis (that is, defective cAMP regulated chloride secretion and basal sodium hyperabsorption) are related to the clinical expression of cystic fibrosis and/or to the genotype. METHODS: Nasal potential difference was measured in 79 adult patients with cystic fibrosis for whom clinical status, respiratory function, and CFTR genotype were determined. RESULTS: In univariate and multivariate analysis, patients with pancreatic insufficiency were more likely to have low responses to low chloride (odds ratio (OR) 8.6 (95% CI 1.3 to 58.5), p = 0.03) and isoproterenol (OR 11.2 (95% CI 1.3 to 93.9), p = 0.03) solutions. Similarly, in univariate and multivariate analysis, patients with poor respiratory function (forced expiratory volume in 1 second <50% of predicted value) were more likely to have an enhanced response to amiloride solution (OR 3.7 (95% CI 1.3 to 11.0), p = 0.02). However, there was no significant relationship between nasal potential difference and the severity of the genotype. CONCLUSIONS: Nasal epithelial ion transport in cystic fibrosis is linked to the clinical expression of the disease. The pancreatic status appears to be mostly related to the defect in epithelial chloride secretion whereas the respiratory status is mostly related to abnormal sodium transport and the regulatory function of the CFTR protein.

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219 RESULTS Patients Four of the 79 CF patients included in the study had a normal sweat test; three were compound heterozygous for the F508D mutation and the R117H, D1152H and R347H mutations, respectively, and one patient was compound heterozygous for the G542X and 3849+10 kb (C)R (T) mutations. Login to comment

[hide] Rapid detection of CFTR gene rearrangements impact... J Med Genet. 2004 Nov;41(11):e118. Niel F, Martin J, Dastot-Le Moal F, Costes B, Boissier B, Delattre V, Goossens M, Girodon E
Rapid detection of CFTR gene rearrangements impacts on genetic counselling in cystic fibrosis.
J Med Genet. 2004 Nov;41(11):e118., [PMID:15520400]

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207 Gender Current age Phenotype Genotype Origin Age at diagnosis Pancr. status Lung disease Other Sweat test Allele 1 Allele 2 rearrangement involving exon(s) Parental Geographic 1 M 10 years 1 month PI Severe 114 F508del 1 Father North eastern Italy 2 M 16 years Birth PI Severe 130 A561E 2 Father Southern Italy 3 M 10 years 1 year PI Severe + R553X 17b Mother France 4 F 13 years 4 years PI Severe NP + F508del 14b-17b Father Eastern France 5 F 24 years 1 month PI Severe 100 F508del 17a-17b Mother ND 6 F 21 years Childhood PI Moderate + F508del 17a-17b Father Eastern France 7 M 35 years 1 year PI Severe CBAVD, NP 103 F508del 17a-17b Father Eastern France 8* 2 F Deceased at 2 and 6 months Birth PI Severe ND F508del 17a-17b Father Eastern France 9 F Deceased at 15 years 5 years PI Severe 300 1812- 1GRA 3-10,14b-16À Mother Kabylie (Algeria)/ Brittany (France) 10 M 37 years 37 years PS None CBAVD ND R117H(-7T) 1-24 Mother France 11 M Deceased at 31 years 3 months PI Severe DB 90 G542X 4-8 Mother Eastern France CBAVD, congenital bilateral absence of the vas deferens; DB, disseminated bronchiectasis; del, deletion; dup, duplication; F, female; M, male; NP, nasal polyposis; Pancr., pancreatic; PI, pancreatic insufficiency; PS, pancreatic sufficiency. Login to comment
237 **The linked haplotype was hypothesised, considering the most frequent haplotype IVS8(CA)23-IVS17b(TA)33-IVS17b(CA)13 linked to G542X.43 Considering the CFTR deletions already described and those we report here, it clearly appears that some CFTR sequences may be prone to rearrangements. Login to comment

[hide] The role of cystic fibrosis gene mutations in dete... Gastroenterol Clin North Am. 2004 Dec;33(4):817-37, vii. Cohn JA, Mitchell RM, Jowell PS
The role of cystic fibrosis gene mutations in determining susceptibility to chronic pancreatitis.
Gastroenterol Clin North Am. 2004 Dec;33(4):817-37, vii., [PMID:15528020]

Abstract [show]
This article reviews current concepts regarding the pathobiology of cystic fibrosis pancreatic disease. It summarizes recent studies on the relationship between CFTR mutations and pancreatitis, and it reviews several unresolved issues in the field.

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78 The European data Table 1 Abnormal CFTR and PSTI genotypes detected in two studies of idiopathic chronic pancreatitis* CFTR genotype category N Genotypes detected in individual subjects US study (Noone et al [47]) CFsev / CFm-v compound heterozygotes 8 DF508 / R117H-7T**; DF508 / 5T; DF508 / 5T; DF508 / D1152H; DF508 / D1152H; DF508 / P574H; DF508 / 3120G>A; 621þ1G>T/G1069R CFm-v / CFm-v compound heterozygotes 1 5T / 5T** CFsev / - (CF carriers) 1 N1303K / - CFm-v / - 7 R117H-7T / -; 5T / -**; 5T / -; 5T / -; 5T / -; 5T / -; 5T / - Normal (- / -) CFTR genotype 22 1 was homozygous for the N34S PSTI mutation; 5 were N34S carriers European study (Audrezet et al [50]) CFsev / CFm-v compound heterozygotes 4 DF508/R352Q; DF508/P5L; DF508/Q1476X; W1282X/5T*** CFm-v / CFm-v compound heterozygotes 2 V562I/5T; E217G/A1136T CFsev / - (CF carriers)**** 3 DF508 / -; DF508 / -; G542X / - CFm-v / - 9 L967S/-**; IVS18-20T>C/-**; c.4575þ2G>A/-; IVS3-6T>C; 5T/-; 5T/-; 5T/-; 5T/-; 5T/- Normal (- / -) CFTR genotype 17 1 was homozygous for the N34S PSTI mutation; 1 was a N34S carrier * CFTR mutations were classified as causing either severe (CFsev ) or mild-variable loss-of-function (CFm-v ) [18,47]; all detected CFsev mutations are CF-causing mutations according to current consensus criteria [79]. Login to comment

[hide] A large-scale study of the random variability of a... Eur J Hum Genet. 2005 Feb;13(2):184-92. Modiano G, Bombieri C, Ciminelli BM, Belpinati F, Giorgi S, Georges M, Scotet V, Pompei F, Ciccacci C, Guittard C, Audrezet MP, Begnini A, Toepfer M, Macek M, Ferec C, Claustres M, Pignatti PF
A large-scale study of the random variability of a coding sequence: a study on the CFTR gene.
Eur J Hum Genet. 2005 Feb;13(2):184-92., [PMID:15536480]

Abstract [show]
Coding single nucleotide substitutions (cSNSs) have been studied on hundreds of genes using small samples (n(g) approximately 100-150 genes). In the present investigation, a large random European population sample (average n(g) approximately 1500) was studied for a single gene, the CFTR (Cystic Fibrosis Transmembrane conductance Regulator). The nonsynonymous (NS) substitutions exhibited, in accordance with previous reports, a mean probability of being polymorphic (q > 0.005), much lower than that of the synonymous (S) substitutions, but they showed a similar rate of subpolymorphic (q < 0.005) variability. This indicates that, in autosomal genes that may have harmful recessive alleles (nonduplicated genes with important functions), genetic drift overwhelms selection in the subpolymorphic range of variability, making disadvantageous alleles behave as neutral. These results imply that the majority of the subpolymorphic nonsynonymous alleles of these genes are selectively negative or even pathogenic.

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33 In the Tajima`s test,19 the null hypothesis of neutrality is rejected if a statistically significant difference between p Common and rare nonsynonymous and synonymous cSNSs G Modiano et al European Journal of Human Genetics Table 1 List of the 61 cSNSsa encountered in the present survey The random samples of genes (and the technique utilized) cSNS variants found NE Italy (DGGE) Central Italy (DGGE) Southern France (DGGE) Northern France (DHPLC) Spain (SSCA) Czechia (DGGE) Hb Â 104 Exon Exon Length (bp) Ref. no. SNS SASc 1st 100d 2nd 500 1st 100d 2nde 1st 100d 2nd 500 1st 100 2nde 82d 72 Abs. Freq. Total sample size q Â 104 se Â 104 NSf Sf 1g 53 0 0 0 0 0/452 0 924 2 111 1 223C4T R31C 1 1 1/500 1 1 0 0/450 0 5 (11) 1 932 (2 432) 45.23 13.61 90 2 224G4T R31L 0 0 0/500 0 0 0 1/450 0 1 1 932 5.17 5.17 10 3 257C4T S42F 0 0 1/500 0 0 0 0/450 0 1 1 932 5.17 5.17 10 3 109 4 334A4G K68E 1 0 0 0/498 0 0 0 0/452 0 0 1 2 504 3.99 3.99 8 5 352C4T R74W 0 0 0 0/498 0 0 0 1/452 0 0 1 2 504 3.99 3.99 8 6 356G4A R75Q 1 7 1 7/498 2 9 2 9/452 0 2 40 (40) 2 504 (2 544) 157.23 24.66 310 7 386G4A G85E 0 0 1 1/498 0 0 0 0/452 0 0 2 2 504 7.99 5.65 16 4 216 8 482G4A R117H 0 0 0 0/292 0 2 0 1/456 0 0 3 2 302 13.03 7.52 26 9 528T4G I132M 0 0 0 0/292 0 0 0 1/456 0 0 1 2 302 4.34 4.34 8 10 575T4C I148T 1 2 0 1/292 0 0 0 1/456 0 1 6 2 302 26.06 10.63 52 5 90 11 640C4T R170C 0 0 0 0/6 0 0 1/448 0 1 1 436 6.96 6.96 14 12 641G4A R170H 1 1 0 0/6 0 0 2/448 0 4 (4) 1 436 (1 930) 20.73 10.35 41 6a 164 0 0 0/6 0 0 0/432 0 0 992 6b 126 0 0 0/6 0 0 0/454 0 942 7 247 0 0 0/6 0 0 0/796 0 1 284 8 93 13 1281G4A L383 0 0 0 0/6 0 0 1/456 0 0 1 1 516 6.60 6.60 13 9 183 14 1402G4A G424S 0 0 0/6 0 0 1/454 0 1 940 10.64 10.64 21 15 1459G4T D443Y 0 0 0/6 0 0 1/454 0 1 940 10.64 10.64 21 10 192 16 1540A4G M470Vh 42 197 30 37/96 39 199 (i) (i) 27 571(736) 1 484 (1 912) 3849.37 111.28 4 735 17 1598C4A S489X 0 0 0 0/96 0 0 0 1/796 0 1 2 374 4.21 4.21 8 18 1648A4G I506V 1 0 0 0/96 0 0 0 0/796 0 1 2 374 4.21 4.21 8 19 1655T4G F508C 0 1 0 0/96 0 0 0 1/796 0 2 2 038 8.42 5.96 17 20 1716G4A Q528 2 16 1 0/96 0 19 i I 5 43 (58) 1 478 (2 024) 286.56 37.08 557 11 95 21 1756G4T G542X 0 2 0 0/134 0 0 0/796 0 0 2 1 984 10.08 7.12 20 22 1764T4G G544 0 0 0 0/134 0 0 1/796 0 0 1 1 984 5.04 5.04 10 23 1784G4A G551D 0 0 0 0/134 0 0 1/796 0 0 1 1 984 5.04 5.04 10 12 87 24 1816G4A V562I 0 0 0 0 1 0 0/450 0 0 1 (1) 2 004 (2 504) 3.99 3.99 8 25 1816G4C V562L 0 0 0 1 0 0 1/450 0 0 2 (3) 2 004 (2 504) 11.98 6.91 24 26 1859G4C G576A 1 2 0 1 11 0 8/450 0 0 23 (27) 2 004 (2 538) 106.38 20.36 213 13 724j 449 27 1997G4A G622D 0 0 0/80 0/96 1 0 0 0/444 0 1 2 002 5.00 5.00 10 28 2082C4T F650 1 0 0/80 0/20 0 0 0 0/444 0 1 (1) 1 926 (2 412) 4.15 4.15 8 29 2134C4T R668C 1 2 0/80 0/96 1 11 0 12/444 0 27(32) 2 002 (2 558) 125.10 21.98 247 275 30 2377C4T L748 0 0 0/6 0 1 1 388 25.77 25.77 52 14a 129 31 2670G4A W846X 0 0 0/6 0 1 0/452 0/80 0 1 1 010 9.90 9.90 20 32 2694T4G T854 33 23 0/6 33 38 149/452 14/80 11 301 1 010 2980.20 143.92 4 184 33 2695G4A V855I 0 0 0/6 0 0 1/452 0/80 0 1 1 010 9.90 9.90 20 14b 38 0 0 0 0/520 0 0 0 0/446 0 2 448 15 251 34 2816G4C S895T 0 0 0/6 0 0 2/436 0 0 2 996 20.08 14.18 40 35 2831A4C N900T 0 0 0/6 0 0 1/436 0 0 1 996 10.04 10.04 20 36 2988G4C M952I 0 0 0/6 0 0 1/436 0 0 1 996 10.04 10.04 20 37 3030G4A T966 (2)k (1)k 0 6/436 0 6 (25)k 618 (1814)k 137.82 27.37 272 38 3032T4C L967S 0 0 0/6 0 0 1/436 0 0 1 996 10.04 10.04 20 16 80 0 0 0/498 0 0 0/450 0 0 1 502 17a 151 39 3123G4C L997F 0 2 2 1/494 0 7 1 4/454 0 0 17 2 502 67.95 16.42 135 40 3157G4A A1009T 0 2 0 0/494 0 0 0 0/454 0 0 2 2 502 7.99 5.65 16 41 3212T4C I1027T 1 0 0 0/494 0 0 0 0/454 0 0 1 2 502 4.00 4.00 8 17b 228 42 3286T4G F1052V 1 1 0 1/194 0 0 0 0/452 0 0 3 (3) 2 200 (2 240) 13.39 7.73 27 43 3337G4A G1069R 0 1 0 0/194 0 0 0 0/452 0 0 1 2 200 4.55 4.55 9 CommonandrarenonsynonymousandsynonymouscSNSs GModianoetal 186 EuropeanJournalofHumanGenetics 44 3345G4T Q1071H 0 0 0 0/194 0 1 0 0/452 0 0 1 2 200 4.55 4.55 9 45 3417A4T T1995 1 3 0 0/194 1 1 0 0/452 0 0 6 (8) 2 200 (2 506) 31.92 11.27 64 46 3419T4G L1096R 0 0 0 0/194 1 0 0 0/452 0 0 1 2 200 4.55 4.55 9 47 3477C4A T1115 0 0 0 0/194 0 0 0 1/452 0 0 1 2 200 4.55 4.55 9 18 101 48 3523A4G I1131V 0 0 1 0/10 0 0 0/448 0 0 1 (2) 1 512 (1 908) 10.48 7.07 21 49 3586G4C D1152H 0 0 0 0/10 0 0 1/448 0 0 1 1 512 6.61 6.61 13 19 249 50 3617G4T R1162L 0 0 1 1/494 0 0/260 0 0/454 0 0 2 2 262 8.84 6.25 18 51 3690A4G Q1186 0 0 0 0/494 0 0/260 0 0/454 1 0 1 2 262 4.42 4.42 9 52 3813A4G L1227 0 1 0 0/494 0 0/260 0 0/454 0 0 1 2 262 4.42 4.42 9 53 3837T4G S1235R 1 1 0 1/494 0 4/260 0 7/454 0 1 15 (15) 2 262 (2 310) 69.94 16.71 140 20 156 54 4002A4G P1290 2 3 0/6 3 5 18/454 3/80 2 36 1 012 357.73 58.22 690 21 90 55 4009G4A V1293I 0 0 0/6 0 0/300 0 1/456 0 0 1 1 316 7.60 7.60 15 56 4029A4G T1299 1 0 0/6 0 1/300 0 1/456 0 0 3 (8) 1 316 (2 330) 34.33 12.12 69 57 4041C4G N1303K 1 0 0/6 0 0/300 0 0/456 0 0 1 1 316 7.60 7.60 15 58 4085T4C V1318A 0 0 0/6 0 0/300 0 1/456 0 0 1 1 316 7.60 7.60 15 22 173 0 0 0/18 0 0 0/450 0 0 1 022 23 106 0 0 0 0/6 0 0 0/448 0 1 436 24l 198+3 59 4404C4T Y1424 1 0 0/6 1 2 5/420 0 2 11 (32) 980 (2 516) 127.19 22.34 251 60m 4521G4A Q1463 (21) (16) (3/32) (14/80) (30) (94/420) 15/76 (17) 15 (227) 76 (1052) 2142.86 131.07 3 367 61 4563T4C D1477 0 0 0/6 0 1 0/420 0 0 1 980 10.20 10.20 20 Totals 6 525 9 584 16 109 The bracketed figures include also the RFLP analysis data (see Materials and methods); the NE Italy, Central Italy, Southern and Northern France are each subdivided into two samples where the 1st is made up of 100 genes. 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[hide] First study of CF mutations in the CFTR gene of Ir... J Trop Pediatr. 2004 Dec;50(6):359-61. Jalalirad M, Houshmand M, Mirfakhraie R, Goharbari MH, Mirzajani F
First study of CF mutations in the CFTR gene of Iranian patients: detection of DeltaF508, G542X, W1282X, A120T, R117H, and R347H mutations.
J Trop Pediatr. 2004 Dec;50(6):359-61., [PMID:15537723]

Abstract [show]
Thirty-seven unrelated Iranian CF families were screened for the presence of seven common mutations (DeltaF508, G542X, W1282X, G551D, N1303K, 1717-1G-->A, and 621-1G-->T) using ARMS PCR and exons 4 and 7 of the CFTR gene by SSCP method. This study resulted in the identification of 26.8 per cent of all CF alleles: DeltaF508 (16.2 per cent), W1282X (4 per cent), G542X (2.7 per cent), R117H (1.3 per cent), R347H (1.3 per cent), and A120T (1.3 per cent) mutations were detected. To the best of our knowledge, it is the first report of an Asian subject carrying the A120T mutation. Our findings suggest heterogeneity in the Iranian population, stressing the need to draw attention to sequence analysis in order to find population-specific mutations.

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12 The next most common mutations were W1282X (4 per cent) and G542X (2.7 per cent). Login to comment
15 Most of the families in whom ∆F508, W1282X, and G542X mutations BRIEF REPORTS Journal of Tropical Pediatrics Vol. 50, No. Login to comment
16 6 359 First Study of CF Mutations in the CFTR Gene of Iranian Patients: Detection of ∆F508, G542X, W1282X, A120T, R117H, and R347H Mutations by M. Jalalirad,a,b M. Houshmand,a R. Mirfakhraie,a M. H. Goharbari,a and F. Mirzajania a National Research Center for Genetic Engineering and Biotechnology (NRCGEB),Tehran, Iran b Biology Department, Gilan University, Rasht, Iran Summary Thirty-seven unrelated Iranian CF families were screened for the presence of seven common mutations (∆F508, G542X, W1282X, G551D, N1303K, 1717-1G→A, and 621-1G→T) using ARMS PCR and exons 4 and 7 of the CFTR gene by SSCP method. Login to comment
17 This study resulted in the identification of 26.8 per cent of all CF alleles: ∆F508 (16.2 per cent), W1282X (4 per cent), G542X (2.7 per cent), R117H (1.3 per cent), R347H (1.3 per cent), and A120T (1.3 per cent) mutations were detected. Login to comment
32 The next two most common mutations were W1282X (4 per cent) and G542X (2.7 per cent), which have high frequencies in Mediterranean countries.11 R347H, which has the highest incidence in Turkey,8 was detected in a Turkish child residing in north-west Iran with normal sweat chloride values. Login to comment
31 The next two most common mutations were W1282X (4 per cent) and G542X (2.7 per cent), which have high frequencies in Mediterranean countries.11 R347H, which has the highest incidence in Turkey,8 was detected in a Turkish child residing in north-west Iran with normal sweat chloride values. Login to comment

[hide] Late diagnosis defines a unique population of long... Am J Respir Crit Care Med. 2005 Mar 15;171(6):621-6. Epub 2004 Dec 10. Rodman DM, Polis JM, Heltshe SL, Sontag MK, Chacon C, Rodman RV, Brayshaw SJ, Huitt GA, Iseman MD, Saavedra MT, Taussig LM, Wagener JS, Accurso FJ, Nick JA
Late diagnosis defines a unique population of long-term survivors of cystic fibrosis.
Am J Respir Crit Care Med. 2005 Mar 15;171(6):621-6. Epub 2004 Dec 10., 2005-03-15 [PMID:15591474]

Abstract [show]
Although the median survival for patients with cystic fibrosis (CF) is 32.9 years, a small group of patients live much longer. We analyzed the genotype and phenotype of CF patients 40 years and older seen between 1992 and 2004 at the National Jewish Medical and Research Center (n = 55). These patients were divided into two groups according to age at diagnosis: an early diagnosis (ED) group, median age at diagnosis 2.0 years (range 0.1-15 years, n = 28), and a late diagnosis (LD) group, median age of diagnosis 48.8 years (range 24-72.8 years, n = 27). Consistent with the hypothesis that the CFTR genotype affects the age at diagnosis, CFTR DeltaF508 homozygous individuals were more common in the ED group. Although patients in the ED group were predominantly male, the majority of LD patients were female. Patients with CF diagnosed late had a significantly lower prevalence of pancreatic insufficiency and CF-related diabetes, and better lung function. Fewer patients in the LD groups were infected with Pseudomonas aeruginosa, whereas a greater percentage had cultures positive for nontuberculous mycobacteria. This is the largest cohort of older patients with CF described to date, and our findings indicate that patients diagnosed as adults differ distinctly from survivors of long-term CF diagnosed as children.

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117 GENOTYPE DISTRIBUTION Early Diagnosis Late Diagnosis ⌬F508/⌬F508 10 1 ⌬F508/⌬I507 1 ⌬F508/G551D 1 ⌬F508/M1101K 1 ⌬F508/P67L/11027T 1 ⌬F508/3120G-A 1 ⌬F508/2789ϩ5G-A 1 2 ⌬F508/W1282X 1 ⌬F508/621ϩ1G-T 1 ⌬F508/R347P 1 ⌬F508/3849ϩ10kbC-T 1 1 ⌬F508/A455E 2 ⌬F508/R347H 2 ⌬F508/D1152H 1 ⌬508/I148T 1 ⌬F508/R117H 1 ⌬F508/Y109N 1 ⌬F508/IVS8-5T 1 ⌬F508/unknown 3 5 S1251N/D1152H 1 G542X/R117C 1 R117H/G551D 1 W1282X/D1152H 1 Unknown 4 4 Values represent number of individuals in each diagnostic group with each genotype. Login to comment

[hide] The DeltaF508 cystic fibrosis mutation impairs dom... Nat Struct Mol Biol. 2005 Jan;12(1):17-25. Epub 2004 Dec 26. Du K, Sharma M, Lukacs GL
The DeltaF508 cystic fibrosis mutation impairs domain-domain interactions and arrests post-translational folding of CFTR.
Nat Struct Mol Biol. 2005 Jan;12(1):17-25. Epub 2004 Dec 26., [PMID:15619635]

Abstract [show]
Misfolding accounts for the endoplasmic reticulum-associated degradation of mutant cystic fibrosis transmembrane conductance regulators (CFTRs), including deletion of Phe508 (DeltaF508) in the nucleotide-binding domain 1 (NBD1). To study the role of Phe508, the de novo folding and stability of NBD1, NBD2 and CFTR were compared in conjunction with mutagenesis of Phe508. DeltaF508 and amino acid replacements that prevented CFTR folding disrupted the NBD2 fold and its native interaction with NBD1. DeltaF508 caused limited alteration in NBD1 conformation. Whereas nonpolar and some aliphatic residues were permissive, charged residues and glycine compromised the post-translational folding and stability of NBD2 and CFTR. The results suggest that hydrophobic side chain interactions of Phe508 are required for vectorial folding of NBD2 and the domain-domain assembly of CFTR, representing a combined co- and post-translational folding mechanism that may be used by other multidomain membrane proteins.

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362 Du, M. et al. Aminoglycoside suppression of a premature stop mutation in a CFTR-/- mouse carrying a human CFTR-G542X transgene. Login to comment

[hide] Comprehensive cystic fibrosis mutation epidemiolog... Ann Hum Genet. 2005 Jan;69(Pt 1):15-24. Castaldo G, Polizzi A, Tomaiuolo R, Cazeneuve C, Girodon E, Santostasi T, Salvatore D, Raia V, Rigillo N, Goossens M, Salvatore F
Comprehensive cystic fibrosis mutation epidemiology and haplotype characterization in a southern Italian population.
Ann Hum Genet. 2005 Jan;69(Pt 1):15-24., [PMID:15638824]

Abstract [show]
We screened the whole coding region of the cystic fibrosis transmembrane regulator (CFTR) gene in 371 unrelated cystic fibrosis (CF) patients from three regions of southern Italy. Forty-three mutations detected 91.5% of CF mutated chromosomes by denaturing gradient gel electrophoresis analysis, and three intragenic CFTR polymorphisms predicted a myriad of rare mutations in uncharacterized CF chromosomes. Twelve mutations are peculiar to CF chromosomes from southern Italy: R1158X, 4016insT, L1065P and 711 + 1G > T are present in 6.3% of CF chromosomes in Campania; G1244E and 852del22 are present in 9.6% of CF chromosomes in Basilicata and 4382delA, 1259insA, I502T, 852del22, 4016insT, D579G, R1158X, L1077P and G1349D are frequent in Puglia (19.6% of CF alleles). Several mutations frequently found in northern Italy (e.g., R1162X, 711 + 5G > T) and northern Europe (e.g., G551D, I507del and 621 + 1G > T) are absent from the studied population. The I148T-3195del6 complex allele was present in two CF chromosomes, whereas I148T was present in both alleles (as a single mutation) in another CF patient and in five CF carriers; this could result from crossover events. The haplotype analysis of three intragenic polymorphisms (IVS8CA, IVS17bTA and IVS17bCA) compared with data from other studies revealed that several mutations (3849 + 10kbC > T, 1717-1G > A, E585X, 3272-26G > A, L558S, 2184insA and R347P) originated from multiple events, whereas others (R1158X and S549R) could be associated with one or more intragenic recombinant events. Given the large population migration from southern Italy, knowledge of the CF molecular epidemiology in this area is an important contribution to diagnosis, counselling and interlaboratory quality control for molecular laboratories worldwide.

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33 The 13 mutations in this panel are: F508del, N1303K, G542X, W1282X, 2183AA>G, 1717-1G>A, R553X, I148T, R1158X, 711+1G>T, 4016insT, L1065P and G1244E. Login to comment
52 We also identified three homozygotes for G542X, three for 852del22, two for 2183AA>G, and one each for N1303K, 1717-1G>A, 4016insT, R553X, R1158X and L1065P. Login to comment
62 A procedure for the large-scale analysis of several mutations peculiar to southern Italy is also indicated Mutation Analytical CF alleles Campania Basilicata Puglia Total procedure n = 340 n = 52 n = 350 n = 742 DF508 55.6 55.8 46.8 51.5 N1303K 7.3 3.8 7.7 7.3 G542X 5.0 3.8 7.1 5.9 W1282X 3.5 3.8 0.6 2.2 2183 AA>G 2.3 5.8 0.8 1.9 852del22 0 5.8 3.2 1.9 3% agarose 1717-1G>A 2.3 1.9 1.1 1.8 4382delA 0 0 3.7 1.8 RE (Ear I -) 1259insA 0 0 3.1 1.5 4016insT 2.1 0 1.1 1.5 ASO R553X 1.5 0 1.7 1.5 R1158X 1.5 0 1.3 1.2 ASO or RE (Sfa N 1 -) L1077P 0.6 0 1.9 1.2 I502T 0.3 0 2.0 1.1 RE (Mse I -) 3849+10kbC>T 0 1.9 1.6 0.9 D579G 0 0 1.6 0.8 RE (Avr II +) G1244E 0.9 3.8 0.3 0.8 ASO or RE (Mbo II +) G1349D 0 0 1.7 0.8 RE (Sty I -) 2789+5 G>A 0.6 0 0.8 0.7 711+1 G>T 1.5 0 0 0.7 ASO L1065P 1.2 0 0 0.5 ASO or RE (Mnl I +) R347P 0.3 0 0.9 0.5 2522insC 0.9 0 0 0.4 E585X 0.6 0 0 0.3 G85E 0.6 0 0 0.3 G178R 0.6 0 0 0.3 D1152H 0.3 0 0.3 0.3 I148T-3195del6 0.6 0 0 0.3 I148T (alone) 0 0 0.3 0.1 R334W 0 0 0.3 0.1 DI507 0 0 0.3 0.1 I1005R 0 0 0.3 0.1 3272-26A>G 0.3 0 0 0.1 2711delT 0.3 0 0 0.1 L558S 0 1.9 0 0.1 W1063X 0 0 0.3 0.1 D110H 0.3 0 0 0.1 S549R (A>C) 0 1.9 0 0.1 2184insA 0.3 0 0 0.1 3131del22 0.3 0 0 0.1 R709N 0 0 0.3 0.1 A349V 0 0 0.3 0.1 4015insA 0 0 0.3 0.1 Y849X 0 1.9 0 0.1 Cumulative 91.6 92.1 91.7 91.5 Unknown 8.4 7.9 8.3 8.5 Total 100,0 100,0 100,0 100,0 RE: restriction enzyme (-/+: abolition or introduction of a RE site); ASO: allele specific oligonucleotide Figure 2 Multiplex denaturing gradient gel electrophoretic analysis of exons 8, 5 and 18 of the cystic fibrosis transmembrane regulator gene in a cystic fibrosis patient (case n. Login to comment

[hide] Rational approach to genetic testing of cystic fib... Andrologia. 2005 Feb;37(1):1-9. Mennicke K, Klingenberg RD, Bals-Pratsch M, Diedrich K, Schwinger E
Rational approach to genetic testing of cystic fibrosis (CF) in infertile men.
Andrologia. 2005 Feb;37(1):1-9., [PMID:15644056]

Abstract [show]
Male infertility as a result of isolated congenital bilateral absence of the vas deferens (CBAVD) is one primary genital form of cystic fibrosis (CF) and occurs in 1-2% of infertile men. Assisted fertilization in patients with CBAVD increases the risk of transmitting mutations in the CF gene. We developed a rational approach to genetic CF testing in infertile men. A total of 282 infertile male patients were screened for the most common CF mutations (DeltaF508, R117H, IVS8-5T). Clinical data including medical history, examination, semen analysis, sweat tests, karyotypes and hormonal values were analysed. We identified 23 patients carrying mutations in the CF gene (DeltaF508: 10 patients; R117H: six patients; IVS8-5T: 11 patients). Two patients were compound heterozygote for DeltaF508/R117H, two others for DeltaF508/IVS8-5T. Correlating these molecular analyses with the clinical data pertaining to serum follicle-stimulating hormone concentration, semen pH, sperm count and total testicular volume, we were able to develop a score with a high specificity (98.4) for the presence of a cystic fibrosis transmembrane conductance regulator (CFTR) mutation, but only with a low sensitivity (positive post-test likelihood: 62.5%; negative post-test likelihood: 6.3%). With regard to the low sensitivity and the high number of CFTR mutations found in this heterogeneous group of infertile men, we still recommend genetic CF testing before assisted fertilization.

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49 In the presence of CFTR mutations, further genetic screening for the seven most frequently identified CF mutations [G542X, N1303K, 1717-1(GoA), W1282X, G551D, R553X, DI507; The Cystic Fibrosis Analysis Consortium, 1990] was performed. Login to comment

[hide] Systemic inflammatory mediators and cystic fibrosi... Clin Exp Med. 2004 Oct;4(2):99-102. Augarten A, Paret G, Avneri I, Akons H, Aviram M, Bentur L, Blau H, Efrati O, Szeinberg A, Barak A, Kerem E, Yahav J
Systemic inflammatory mediators and cystic fibrosis genotype.
Clin Exp Med. 2004 Oct;4(2):99-102., [PMID:15672947]

Abstract [show]
Morbidity and mortality in cystic fibrosis patients is mainly attributed to pulmonary infection and inflammation. Chemokines play a pivotal role in the inflammatory process. Although genotype-phenotype correlation in cystic fibrosis patients has been defined, a clear relationship between the defect in the cystic fibrosis transmembrane regulator (CFTR) gene and pulmonary inflammation has not been established. The aim of this study was to assess whether serum chemokines levels in cystic fibrosis patients correlate with genotype and pulmonary function tests, as well as with other clinical characteristics. Serum levels of interleukin-8, RANTES, and monocyte chemoattractant protein-1 were measured in 36 cystic fibrosis patients grouped according to their genotype. Group A included 25 patients who carried two mutations associated with a pathological sweat test and pancreatic insufficiency (deltaF508, W1282X, G542X, N1303K, S549R). Group B included 11 compound heterozygote patients who carried one mutation known to cause mild disease with borderline or normal sweat test and pancreatic sufficiency (3849+10kb C to T, 5T). Associations between chemokine levels, genotype, pulmonary function, Pseudomonas aeruginosa colonization, age, sweat chloride level, and pancreatic and nutritional status were examined. Mean interleukin-8 and monocyte chemoattractant protein-1 levels were significantly higher in group A than group B (11.4 +/- 2.1 pg/ml vs. 5 +/- 0.9 pg/ml and 157 +/- 16 pg/ml vs. 88.8 +/- 16.4 pg/ml, respectively) (P < 0.01). No difference in RANTES levels were found between groups. interleukin-8 levels were inversely related to forced expiratory volume in 1 s (r = -0.37, P < 0.02), while there was no association between the latter and RANTES and monocyte chemoattractant protein-1 levels. The Pseudomonas colonization rate was higher among group A patients than group B (88% vs. 40%, P < 0.01). No relationship was found between measured chemokines and age, sweat chloride levels, and pancreatic and nutritional status. Our study demonstrates an association between interleukin-8, forced expiratory volume, and cystic fibrosis genotype. Hence, elevated interleukin-8 serum levels could serve as an indicator of an early inflammatory process and encourage the initiation of anti-inflammatory treatment.

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5 Group A included 25 patients who carried two mutations associated with a pathological sweat test and pancreatic insufficiency (∆F508, W1282X, G542X, N1303K, S549R). Login to comment
28 Group A included 25 patients who carried two mutations associated with a pathological sweat test and pancreatic insufficiency (∆F508, W1282X, G542X, N1303K, S549R) [2]. Login to comment
67 : Systemic inflammatory mediators and cystic fibrosis genotype 101 Table 1 Clinical characteristics of cystic fibrosis (CF) patients Group Aa (n=25) Group Bb (n=11) P Age (years) 16.9±7.2 17.7±9.1 NS Pancreatic sufficiency 0% 0/25 36.3% 4/11 <0.01 Sweat chloride (mmol/l) 105±28 92±18.6 NS Weight percentiles 19±19.8 57.2±25.2 <0.01 Sputum Pseudomonas 88% 22/24 40% 4/10 <0.01 a Group A CF patients carrying two mutations associated with severe disease presentation (∆F508, W1282X, G542X, N1303K, S549R) b Group B CF patients carrying mutations associated with mild disease presentation (3849+10kb CǞT, 5T) Table 2 Comparison of serum chemokine levels between groups (IL-8 interleukin-8, MCPI monocyte chemoattractant protein-1) Chemokine Group Aa Group Bb P IL-8 (pg/ml) 11.4±2.1 5.0±0.9 0.01 MCP1(pg/ml) 157±16 88.8+16.4 0.01 RANTES (pg/ml) 323±48 287.5±93 NS a Group A CF patients carrying two mutations associated with severe disease presentation (∆F508, W1282X, G542X, N1303K, S549R) b Group B CF patients carrying mutations associated with mild disease presentation (3849+10kb CÞT, 5T) Fig. 1 Relationship between interleukin-8 (IL-8) levels and forced expiratory volume in 1 s (FEV1) values defective protein production; class II, defective protein processing; class III, defective chloride channel regulation; and class IV, defective chloride channel conduction. Login to comment

[hide] Diagnosis of cystic fibrosis after newborn screeni... Pediatr Pulmonol. 2005 May;39(5):440-6. Massie J, Clements B
Diagnosis of cystic fibrosis after newborn screening: the Australasian experience--twenty years and five million babies later: a consensus statement from the Australasian Paediatric Respiratory Group.
Pediatr Pulmonol. 2005 May;39(5):440-6., [PMID:15704202]

Abstract [show]
Newborn screening for cystic fibrosis has been used in Australia and New Zealand for over 20 years. In that time, considerable experience has been developed regarding the early diagnosis of cystic fibrosis after newborn screening. To date, there has not been a consensus on the process of screening and clinical evaluation leading to the diagnosis of cystic fibrosis in infants, many of whom are not symptomatic at time of notification of the screening result. The aim of this paper is to provide some consensus on the important issues of a cystic fibrosis diagnosis arising from newborn screening, based on the experience gained in Australia and New Zealand over the last 20 years.

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47 Management of infants with a borderline sweat test after NBS ABBREVIATIONS CF Cystic fibrosis CFTR Cystic fibrosis transmembrane conductance regulator Cl Chloride DNA Deoxyribonucleic acid IRT Immunoreactive trypsinogen MI Meconium ileus Na Sodium NBS Newborn screening NPD Nasal potential difference TABLE 1- Newborn Screening for CF in Australia and New Zealand1 State/country Year screening started Babies screened (to end of 2003) New South Wales 1981 1,940,000 Victoria 1989 913,181 Queensland 1983 878,905 South Australia (including Tasmania and Northern Territory) 1990 407,625 Western Australia 2001 63,000 New Zealand 1981 1,098,329 Total 5,301,040 1 Mutations screened: New South Wales, DF508; Victoria DF508; South Australia (including Tasmania and Northern Territory), DF508, DI507, G551D, G542X, and R553X; Western Australia, DF508, G551D, G542X, and 621 þ 1G ! Login to comment
48 T; Queensland, DF508, DI507, G551D, G542X, 621 þ 1G ! Login to comment
49 T, R553X, N1303K, and R117H; New Zealand, DF508, G551D, and G542X. Login to comment

[hide] Molecular pathology of the CFTR locus in male infe... Reprod Biomed Online. 2005 Jan;10(1):14-41. Claustres M
Molecular pathology of the CFTR locus in male infertility.
Reprod Biomed Online. 2005 Jan;10(1):14-41., [PMID:15705292]

Abstract [show]
Congenital bilateral absence of the vas deferens (CBAVD) is a form of infertility with an autosomal recessive genetic background in otherwise healthy males. CBAVD is caused by cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations on both alleles in approximately 80% of cases. Striking CFTR genotypic differences are observed in cystic fibrosis (CF) and in CBAVD. The 5T allele is a CBAVD mutation with incomplete penetrance. Recent evidence confirmed that a second polymorphic locus exists and is a major CFTR modifier. The development of minigene models have led to results suggesting that CFTR exon 9 is skipped in humans because of unusual suboptimal 5' splice sites. An extremely rare T3 allele has been reported and it has recently been confirmed that the T3 allele dramatically increases exon 9 skipping and should be considered as a 'CF' mutation. Routine testing for the most prevalent mutations in the CF Caucasian population will miss most CFTR gene alterations, which can be detected only through exhaustive scanning of CFTR sequences. Finally, a higher than expected frequency of CFTR mutations and/or polymorphisms is now found in a growing number of monosymptomatic disorders, which creates a dilemma for setting nosologic boundaries between CF and diseases related to CFTR.

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159 Examples include nonsense (G542X), frameshifl (3659delC) or severe splicing (1717-lG-^A) mutations. Login to comment

[hide] Platelet activation in cystic fibrosis. Blood. 2005 Jun 15;105(12):4635-41. Epub 2005 Feb 10. O'Sullivan BP, Linden MD, Frelinger AL 3rd, Barnard MR, Spencer-Manzon M, Morris JE, Salem RO, Laposata M, Michelson AD
Platelet activation in cystic fibrosis.
Blood. 2005 Jun 15;105(12):4635-41. Epub 2005 Feb 10., 2005-06-15 [PMID:15705796]

Abstract [show]
Cystic fibrosis (CF) is caused by a mutation of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). We examined platelet function in CF patients because lung inflammation is part of this disease and platelets contribute to inflammation. CF patients had increased circulating leukocyte-platelet aggregates and increased platelet responsiveness to agonists compared with healthy controls. CF plasma caused activation of normal and CF platelets; however, activation was greater in CF platelets. Furthermore, washed CF platelets also showed increased reactivity to agonists. CF platelet hyperreactivity was incompletely inhibited by prostaglandin E(1) (PGE(1)). As demonstrated by Western blotting and reverse-transcriptase-polymerase chain reaction (RT-PCR), there was neither CFTR nor CFTR-specific mRNA in normal platelets. There were abnormalities in the fatty acid composition of membrane fractions of CF platelets. In summary, CF patients have an increase in circulating activated platelets and platelet reactivity, as determined by monocyte-platelet aggregation, neutrophil-platelet aggregation, and platelet surface P-selectin. This increased platelet activation in CF is the result of both a plasma factor(s) and an intrinsic platelet mechanism via cyclic adenosine monophosphate (cAMP)/adenylate cyclase, but not via platelet CFTR. Our findings may account, at least in part, for the beneficial effects of ibuprofen in CF.

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44 Clinical characteristics of cystic fibrosis patients Patient Age, y Vitamin E, mg/L* FEV1, % predicted† Inpatient or outpatient‡ Genotype Platelet studies§ 1 20 6.6 25 In ␦F508/unk A 2 20 3.6 70 In ␦F508/G542X A 3 11 16.8 92 Out ␦F508/dF508 A 4 16 5.4 101 Out ␦F508/G542X A 5 9 3.9 124 Out ␦F508/dF508 A,F 6 6 5.1 118 Out ␦F508/dF508 A,F 7 13 8.1 119 Out ␦F508/dF508 A,F 8 10 9.7 104 Out ␦F508/dF508 A,F 9 22 9.0 58 In ␦F508/dF508 A 10 19 8.0 57 Out ␦F508/N1303K A 11 17 7.0 24 Out ␦F508/dF508 A,D,E 12 20 3.2 55 Out ␦F508/dF508 A,D 13 15 5.8 41 In ␦F508/dF508 A,D,E 14 26 12.7 88 Out ␦F508/dF508 A,D 15 11 16.3 72 Out ␦F508/W1282X A,D 16 18 10.0 58 In ␦F508/dF508 A,D 17 22 10.5 50 Out ␦F508/dF508 A,D 18 35 8.6 87 Out ␦F508/C276X A,E 19 17 16.2 62 In ␦F508/dF508 B,E 20 14 4.1 85 In ␦F508/dF508 B 21 22 2.3 62 In ␦F508/G542X B 22 21 7.7 54 In ␦F508/N1303K B 23 19 2.4 69 In ␦F508/Y1092X B 24 19 4.6 87 In ␦F508/dF508 B, C, E 25 21 8.2 58 In R334W/unk C 26 22 5.8 85 In ␦F508/dF508 C,E 27 22 2.9 67 In unk/unk C 28 20 6.7 77 In ␦F508/dF508 E 29 18 13.3 92 In ␦F508/dF508 E 30 22 8.8 71 In ␦F508/394delTT E 31 15 13.0 68 In ␦F508/dF508 E 32 14 unk 97 Out ␦F508/dF508 E unk indicates unknown. Login to comment

[hide] Chloride transport in nasal ciliated cells of cyst... Am J Respir Crit Care Med. 2005 May 1;171(9):1026-31. Epub 2005 Feb 11. Sermet-Gaudelus I, Dechaux M, Vallee B, Fajac A, Girodon E, Nguyen-Khoa T, Marianovski R, Hurbain I, Bresson JL, Lenoir G, Edelman A
Chloride transport in nasal ciliated cells of cystic fibrosis heterozygotes.
Am J Respir Crit Care Med. 2005 May 1;171(9):1026-31. Epub 2005 Feb 11., 2005-05-01 [PMID:15709055]

Abstract [show]
Studying subjects heterozygous for mutations of the cystic fibrosis (CF) gene may help clarify the impact on disease onset of CF transmembrane conductance regulator protein (CFTR-)-dependent chloride secretion. CFTR-mediated chloride transport was evaluated in 52 heterozygous subjects, 32 healthy control subjects, and 77 patients with CF with class I or II mutations. We measured the change in nasal potential difference in response to chloride-free isoproterenol solution for each subject and used a video-imaging fluorescent dye assay to assess the percentage of nasal ciliated cells with cAMP-dependent anion conductance. Our findings did not confirm the standard assumption that heterozygosity implies 50% of normal CFTR function. Half the heterozygous subjects had CFTR-mediated chloride transport levels below 50% of the normal range, and one-third had levels similar to those of the patients with CF. This reduced CFTR function was not associated with an elevated prevalence of CF-like symptoms in heterozygous subjects but was highly related to respiratory status in the patients with CF. These data suggest that CFTR-dependent chloride conductance does not directly modulate disease severity but may be part of a more global defect in patients with CF involving other CFTR functions or currently unknown modulatory factors.

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57 Ten had class I mutations: 3659delC, 1078delT, 3791delC, 1717-1GϾA, 2183AAϾG, S466X, W1282X, R553X, or G542X (n ϭ 2). Login to comment

[hide] Spectrum of cystic fibrosis mutations in Serbia an... Genet Test. 2004 Fall;8(3):276-80. Radivojevic D, Djurisic M, Lalic T, Guc-Scekic M, Savic J, Minic P, Antoniadi T, Tzetis M, Kanavakis E
Spectrum of cystic fibrosis mutations in Serbia and Montenegro and strategy for prenatal diagnosis.
Genet Test. 2004 Fall;8(3):276-80., [PMID:15727251]

Abstract [show]
We have screened 175 patients for molecular defects in the cystic fibrosis transmembrane conductance regulator (CFTR) gene using nondenaturing polyacrylamide gel electrophoresis (PAGE), denaturing gradient gel electrophoresis (DGGE), and sequencing. Six different mutations (F508del, G542X, 621+1G --> T, 2789+5G --> A, R1070Q, and S466X) accounted for 79.71% of CF alleles, with the F508del mutation showing a frequency of 72.28%. Another 12 mutations (R334W, 2184insA, I507del, 1525-1G --> A, E585X, R75X, M1I, 457TAT --> G, 574delA, 2723delTT, A120T, and 2907delTT) covered an additional 3.36%. A novel mutation (2723delTT) was found in one CF patient (F508del/2723delTT). Thus, a total of 18 mutations cover 82.57% of CF alleles. During our study, 72% of families at risk for having a CF child were found to be fully informative for prenatal diagnosis. Prenatal diagnosis was performed on 56 families; 76 analyses resulting in 16 affected, 38 carriers, and 22 healthy fetuses. These results imply that the molecular basis of CF in Serbia and Montenegro is highly heterogeneous, as is observed in other eastern and southern European populations. Because we detected more then 80% of CFTR alleles, results could be used for planning future screening and appropriate genetic counseling programs in our country.

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2 Six different mutations (F508del, G542X, 621؉1G Ǟ T, 2789؉5G Ǟ A, R1070Q, and S466X) accounted for 79.71% of CF alleles, with the F508del mutation showing a frequency of 72.28%. Login to comment
40 Six different mutations (F508del, 621ϩ1G Ǟ T, G542X, S466X, R1070Q, and 2789ϩ5G Ǟ A) accounted for 79.71% of the CF alleles in Yugoslavian population, of which the F508del mutation had a frequency of 72.28% (253/350). Login to comment
44 CFTR MUTATIONS IDENTIFIED IN 175 YUGOSLAVIAN CF PATIENTS Location Number of positive Frequency Mutation (exon/intron) CF alleles (percentage) F508del Exon 10 253 72.28 621 ϩ 1G → T Intron 4 10 2.86 G542X Exon 11 9 2.57 S466X Exon 10 3 0.86 2789 ϩ 5 G → A Intron 14b 2 0.57 R1070Q Exon 17b 2 0.57 MI1 Exon 1 1 0.28 R75X Exon 3 1 0.28 457TAT → G Exon 4 1 0.28 574delA Exon 4 1 0.28 A120T Exon 4 1 0.28 R334W Exon 7 1 0.28 1525-1 G → A Intron 9 1 0.28 I507del Exon 10 1 0.28 E585X Exon 12 1 0.28 2184insA Exon 13 1 0.28 2723delTTa Exon 14a 1 0.28 2907delTT Exon 15 1 0.28 Unknown - 61 17.43 aNew frameshift mutation. Login to comment
55 Nine different mutations were detected: F508del, 2907delTT, S466X, 457TAT Ǟ G, R75X, 2184insA, G542X, 621ϩ1G Ǟ T, and R1070Q in a total of 76 prenatal analyses (Table 2). Login to comment
63 A few studies on CF patients from the former Yugoslavian Republic have been published to date, but in only one, based on a relatively small number of CF patients, two mutations were found in affected children from Serbia and Montenegro (F508del, 70%; G542X, 4%) (Dabovic et al., 1992). Login to comment
67 Six of the molecular defects identified in Yugoslavian patients belong to the 24 most common mutations worldwide (F508del, G542X, 621ϩ1G Ǟ T, I507del, 2789ϩ5G Ǟ A, and R334W) (CFGCA, 1994) (Table 1). Login to comment
70 The third most common mutation in our population, G542X, is one of the most frequent in European populations (2.6%), is detected in 6.1% CF alleles in Mediterranean countries, and is found TABLE 2. Login to comment
71 RESULTS OF PRENATAL DIAGNOSIS OF CF IN SERBIA AND MONTENEGRO Number of prenatal Genotype Material diagnoses Outcome F508del/F508del CVS, AF, CBa 51 11 Affected, 25 carriers, 15 normal, F508del/2907delTT CVS 2 1 Affected, 1 carrier F508del/S466X CVS, AF 2 2 Carriers F508del/457TATϾG CVS 1 1 Carrier F508del/2184insA CVS 1 1 Affected F508del/621ϩ1GϾT CVS 1 1 Normal F508del/R1070Q CVS 1 1 Normal G542X/621ϩ1GϾT CVS 4 1 Affected, 2 carriers, 1 normal G542X/R7X CVS 3 2 Carriers, 1 normal F508del/? Login to comment

[hide] The CFTR 3849+10kbC->T and 2789+5G->A alleles are ... Eur Respir J. 2005 Mar;25(3):468-73. Dugueperoux I, De Braekeleer M
The CFTR 3849+10kbC->T and 2789+5G->A alleles are associated with a mild CF phenotype.
Eur Respir J. 2005 Mar;25(3):468-73., [PMID:15738290]

Abstract [show]
Most cystic fibrosis (CF) transmembrane receptor mutations are rare. The French CF Registry offers an opportunity to study the genotype-phenotype relationship of these rare alleles. Since 1992, 39 CF patients carrying one copy of the 3849+10kbC->T mutation and 88 the 2789+5G->A allele have been seen at least once in a CF care centre. Among them, 16 carrying the 3849+10kbC->T/Delta F508 genotype and 34 with the 2789+5G->A/Delta F508 genotype were seen in 2000. Their age at diagnosis, sweat chloride concentration, anthropometric and lung function results, and clinical aspects were compared with those homozygous for the Delta F508 mutation matched for sex, age and CF care centre. Major differences, most of them statistically significant, in the age at diagnosis, prevalence of pancreatic insufficiency, and other clinical signs, anthropometric and lung function measures were observed between both compound heterozygote groups and their matched Delta F508/Delta F508 groups. The mean sweat chloride concentration was also lower (close to normal values) among 3849+10kbC->T/Delta F508 patients, but not among 2789+5G->A/Delta F508 patients. In conclusion, both mutations studied here are associated with a milder course of cystic fibrosis disease. The 3849+10kbC->T and 2789+5G->A alleles are splice site mutations, leading to abnormal mRNA; however, a small amount of normally spliced transcripts can also be detected. The presence of these small amounts of normal cystic fibrosis transmembrane receptor protein in these cystic fibrosis patients is likely to be responsible for the milder severity of disease and a better life expectancy.

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51 Two genotypes accounted for almost 80% of the patients, 3849+10kbC-.T/DF508 (n527, 69.2%) and 3849+10kbC-.T/ G542X (n54, 10.3%), and two siblings shared the G1244E allele (5.2%). Login to comment
63 Although only borderline significant, lung function was definitely better in the 3849+10kbC-.T/DF508 group (FEV1 83.0% and FVC 91.6% pred) than in the DF508 homozygote group (FEV1 59.9% TABLE 1 Genotypes identified among cystic fibrosis patients sharing the 3849+10kbC-.T or the 2789+5G-.A mutation Genotypes 3849+10kbC-.T 2789+5G-.A DI507 2 DF508 27 61 1525-1G-.A 1 1717-1G.A 1 2183AA.G 3 3129del4 1 3659delC 1 G542X 4 6 G551D 1 G970R 2 G1244E 2 L558S 1 M1V 1 N1303K 1 R347P 1 R553X 1 1 R1066C 1 S1251N 1 Unknown 1 6 Total 39 88 I. DUGUE´PE´ROUX AND M. DE BRAEKELEER MILD PHENOTYPE ASSOCIATED WITH TWO CFTR MUTATIONS c and FVC 76.9% pred). Login to comment
71 Three genotypes accounted for almost 80% of the patients: 2789+5G-.A/DF508 (n561, 69.3%), 2789+5G-.A/G542X (n56, 6.8%) and 2789+5G- .A/2183AA-.G (n53, 3.4%). Login to comment

[hide] Frequency of large CFTR gene rearrangements in Ita... Eur J Hum Genet. 2005 May;13(5):687-9. Bombieri C, Bonizzato A, Castellani C, Assael BM, Pignatti PF
Frequency of large CFTR gene rearrangements in Italian CF patients.
Eur J Hum Genet. 2005 May;13(5):687-9., [PMID:15741992]

Abstract [show]
In most populations, an appreciable fraction of cystic fibrosis transmembrane regulator (CFTR) gene mutations in patients affected by cystic fibrosis (CF) cannot be identified, and large gene rearrangements might be missed by standard analyses. We have searched large gene rearrangements in a sample of 25 North East Italian CF patients who, after an extensive gene analysis of 188 patients, still bear one or two unidentified CF mutations. A systematic gene screening by quantitative multiplex PCR of short fluorescent fragments was performed. Overall, 5/26 (19.2%) rearranged alleles were detected, bearing mutation 3120+1Kbdel8.6Kb (three patients), and c.4_IVS1+69del119bpins299bp (two patients). These mutations were observed in compound heterozygotes with F508del or termination mutations, and a pancreatic insufficient form of CF. These findings confirm the frequency of CFTR gene rearrangements recently observed in French CF patients.

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36 In all patients, the rearrangements are present in compound heterozygosity with a common CF mutation (F508del, G542X, or Q552X). Login to comment

[hide] The impact of cystic fibrosis and PSTI/SPINK1 gene... Clin Lab Med. 2005 Mar;25(1):79-100. Cohn JA, Mitchell RM, Jowell PS
The impact of cystic fibrosis and PSTI/SPINK1 gene mutations on susceptibility to chronic pancreatitis.
Clin Lab Med. 2005 Mar;25(1):79-100., [PMID:15749233]

Abstract [show]
This article reviews current concepts regarding the pathobiology of cystic fibrosis pancreatic disease. It summarizes recent studies on the relationship between CFTR mutations and pancreatitis, and it reviews several unresolved issues in the field.

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90 Table 1 Abnormal CFTR and PSTI genotypes detected in two studies of idiopathic chronic pancreatitis* CFTR genotype category N Genotypes detected in individual subjects US study (Noone et al [47]) CFsev / CFm-v compound heterozygotes 8 DF508 / R117H-7T**; DF508 / 5T; DF508 / 5T; DF508 / D1152H; DF508 / D1152H; DF508 / P574H; DF508 / 3120G>A; 621þ1G>T/G1069R CFm-v / CFm-v compound heterozygotes 1 5T / 5T** CFsev / - (CF carriers) 1 N1303K / - CFm-v / - 7 R117H-7T / -; 5T / -**; 5T / -; 5T / -; 5T / -; 5T / -; 5T / - Normal (- / -) CFTR genotype 22 1 was homozygous for the N34S PSTI mutation; 5 were N34S carriers European study (Audrezet et al [50]) CFsev / CFm-v compound heterozygotes 4 DF508/R352Q; DF508/P5L; DF508/Q1476X; W1282X/5T*** CFm-v / CFm-v compound heterozygotes 2 V562I/5T; E217G/A1136T CFsev / - (CF carriers)**** 3 DF508 / -; DF508 / -; G542X / - CFm-v / - 9 L967S/-**; IVS18-20T>C/-**; c.4575þ2G>A/-; IVS3-6T>C; 5T/-; 5T/-; 5T/-; 5T/-; 5T/- Normal (- / -) CFTR genotype 17 1 was homozygous for the N34S PSTI mutation; 1 was a N34S carrier * CFTR mutations were classified as causing either severe (CFsev ) or mild-variable loss-of-function (CFm-v ) [18,47]; all detected CFsev mutations are CF-causing mutations according to current consensus criteria [79]. Login to comment

[hide] Reduced CFTR function and the pathobiology of idio... J Clin Gastroenterol. 2005 Apr;39(4 Suppl 2):S70-7. Cohn JA
Reduced CFTR function and the pathobiology of idiopathic pancreatitis.
J Clin Gastroenterol. 2005 Apr;39(4 Suppl 2):S70-7., [PMID:15758663]

Abstract [show]
Idiopathic chronic pancreatitis (ICP) is the leading cause of chronic pancreatitis in children and nonalcoholic adults. The risk of developing ICP is increased in individuals who have mutations of the cystic fibrosis gene (CFTR) and of a trypsin inhibitor gene (PSTI). In studies from the United States and France, the risk of ICP is increased about 40-fold by having two abnormal copies of the CFTR gene, about 14-fold by having the N34S PSTI mutation, and about 500-fold by having both. When ICP patients have two abnormal copies of the CFTR gene, there is also evidence of reduced residual CFTR protein function in extrapancreatic tissues based on clinical findings and nasal ion transport responses. Thus, pancreatitis risk is highest in individuals who have abnormalities in both the pancreatic ducts (CFTR) and acini (PSTI). These findings indicate that PSTI is a modifier gene for CFTR-related ICP and have implications for the diagnosis and pathogenesis of pancreatitis.

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69 Abnormal CFTR and PSTI Genotypes Detected in Two Studies of ICP CFTR Genotype Category* N Genotypes Detected in Individual Subjects U.S. study (Noone et al47 ) CFsev / CFm-v compound heterozygotes 8 DF508 / R117H-7T †; DF508 / 5T; DF508 / 5T; DF508 / D1152H; DF508 / D1152H; DF508 / P574H; DF508 / 3120G.A; 621 + 1G.T/G1069R CFm-v / CFm-v compound heterozygotes 1 5T / 5T † CFsev / 2 (CF carriers) 1 N1303K / 2 CFm-v / 2 7 R117H-7T / 2; 5T / 2 †; 5T / 2; 5T / 2; 5T / 2; 5T / 2; 5T / 2 Normal (2 / 2) CFTR genotype 22 1 was homozygous for the N34S PSTI mutation; 5 were N34S carriers French study (Audrezet et al50 ) CFsev / CFm-v compound heterozygotes 4 DF508/R352Q; DF508/P5L; DF508/Q1476X; W1282X/5T‡ CFm-v / CFm-v compound heterozygotes 2 V562I/5T; E217G/A1136T CFsev / 2 (CF carriers)§ 3 DF508 / 2; DF508 / 2; G542X / 2 CFm-v / 2 9 L967S/2 †; IVS18-20T.C/ 2†; c.4575+2G.A/2; IVS3-6T.C; 5T/2; 5 /2; 5T/ 2; 5T/2; 5T/ 2 Normal (2 / 2) CFTR genotype 17 1 was homozygous for the N34S PSTI mutation; 1 was a N34S carriers *Mutations of the cystic fibrosis (CF) gene (CFTR) were classified as causing either severe (CFsev ) or mild-variable loss-of-function (CFm-v )18,47 ; all detected CFsev mutations are CF-causing mutations according to current consensus criteria.68 In the U.S. study, most patients were tested for rare mutations by DNA sequencing47 ; in the French study, most patients were tested by dHPL.50 †These patients were also carriers for the N34S mutation of a trypsin inhibitor gene (PSTI). Login to comment

[hide] Misprocessing of the CFTR protein leads to mild cy... Hum Mutat. 2005 Apr;25(4):360-71. Clain J, Lehmann-Che J, Dugueperoux I, Arous N, Girodon E, Legendre M, Goossens M, Edelman A, de Braekeleer M, Teulon J, Fanen P
Misprocessing of the CFTR protein leads to mild cystic fibrosis phenotype.
Hum Mutat. 2005 Apr;25(4):360-71., [PMID:15776432]

Abstract [show]
Cystic fibrosis (CF) is mainly caused by mutations that interfere with the biosynthetic folding of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The aim of this study was to determine the mechanism of dysfunction of a disease-causing mutation associated with variable phenotypes. In order to attain these objectives, we studied the effect of the p.L206W mutation on CFTR protein production and function, and we examined the genotype-phenotype correlation of [p.L206W]+[p.F508del] patients. We showed that p.L206W is a processing (class II) mutation since the CFTR biosynthetic pathway was severely impaired, whereas single-channel measurements indicated ion conductance similar to the wild-type protein. These data raise the larger question of the phenotypic variability of class II mutants, including p.F508del. Since multiple potential partners could modify the processing of the CFTR protein during its course to the cell surface, environmental and other genetic factors might contribute to this variability.

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112 For most patients (30/36), p.L206W was combined with a severe mutation (p.F508del, p.I507del, p.G542X, p.W216X, p.R851X, and p.E60X) on the other CFTR allele. Login to comment
143 ]c 179 [p.L206W]+[p.F508del] [1540A]+[1540A] [(TG)9(T)9]+[(TG)10(T)9] [16;7;17]+[23;31;13] 422 [p.L206W]+[p.G542X] [1540A]+[1540A] [(TG)9(T)9]+[(TG)10(T)9] [16;7;17]+[23;33;13] 1720 [p.L206W]+[p.F508del] [1540A]+[1540A] [(TG)9(T)9]+[(TG)10(T)9] [16;7;17]+[17;32;13]c 1878 [p.L206W]+[p.F508del] [1540A]+[1540A] [(TG)9(T)9]+[(TG)10(T)9] [16;7;17]+[17;31;13]c 626 [p.L206W]+[p.E60X] [1540A]+[1540G] [(TG)9(T)9]+[(TG)11(T)7] [16;7;17]+[16;31;13] 1455 [p.L206W]+[1342-6(T)5]] [1540A]+[1540G] [(TG)9(T)9]+[(TG)12(T)5] [16;7;17]+[16;31;14]c 2104 [p.L206W]+[p.F508del] [1540A]+[1540A] [(TG)9(T)9]+[(TG)10(T)9] [16;7;17]+[23;31;13]c 652 [p.L206W]+[p.E216X] [1540A]+[1540A] [(TG)9(T)9]+[(TG)10(T)9] [16;7;17]+[23;32;13] 2345 [p.L206W]+[p.F508del] [1540A]+[1540A] [(TG)9(T)9]+[(TG)10(T)9] [16;7;17]+[17;32;13] a The DNA and mutation numbering follows the CFTR mutation database, (www.genet.sickkids.on.ca/cftr), the A of the ATG translation start codon being numbered +133 (GeneBank NM_000492.2).We followed the approved nomenclature format in mutation names at the protein level and in genotype writing. Login to comment
148 [1995] [p.L206W]+[p.G542X]II/I2237MCBAVDPS^c 65711(3)Desgeorgesetal. Login to comment
165 [1999] [p.L206W]+[p.W216X]II/I0.116F^PSNo75(1)Thisstudy [p.L206W]+[p.F508del]II/II0.2d 2F^PSe BronchialhyperreactivityPositiveThisstudy [p.L206W]+[p.F508del]II/II216F^PSNo54714(6)Thisstudy [p.L206W]+[p.F508del]II/II24M^PSe Bronchitis65(1)Thisstudy [p.L206W]+[p.F508del]II/II23M^PSe Bronchitis9672(2)Thisstudy [p.L206W]+[p.F508del]II/II47F^PIBronchitis5478(2)Thisstudy [p.L206W]+[p.F508del]II/I56F^PSAsthma7576(2)Thisstudy [p.L206W]+[1342-6(T)5]II/-2833MCBAVDPSBronchitis^Thisstudy [p.L206W]+[p.G542X]II/I3243MCBAVDPSNo^Thisstudy [p.L206W]+[p.F508del]II/II3740MCBAVD^^^Thisstudy [p.L206W]+[p.E60X]II/I2938MCBAVDPSNo64(1)Thisstudy [p.L206W]+[p.F508del]II/II3536MCBAVDPINo93(1)Thisstudy a Theclassi'cationofmissensemutationswasbasedonfunctionalstudies[Lietal.,1993;Chengetal.,1990;Champignyetal.,1995]. Login to comment

[hide] Increased prevalence of chronic rhinosinusitis in ... Arch Otolaryngol Head Neck Surg. 2005 Mar;131(3):237-40. Wang X, Kim J, McWilliams R, Cutting GR
Increased prevalence of chronic rhinosinusitis in carriers of a cystic fibrosis mutation.
Arch Otolaryngol Head Neck Surg. 2005 Mar;131(3):237-40., [PMID:15781764]

Abstract [show]
OBJECTIVE: To explore whether there is an increased prevalence of chronic rhinosinusitis (CRS) in known cystic fibrosis (CF) carriers. Self-reported CRS affects 13% to 14% of the US population and clusters in families, which suggests that genetic factors may play an etiologic role. Cystic fibrosis is an inherited recessive disorder that invariably affects the sinuses. The frequency of CF mutations has been reported to be higher in patients with CRS than in unaffected controls. PATIENTS: Obligate CF carriers (parents of patients with CF) were recruited from the Johns Hopkins CF clinic. The presence of signs and symptoms of CRS was assessed by a sinus disease questionnaire. A subgroup of participants was evaluated by a physician experienced in the diagnosis of CRS. RESULTS: Fifty-three (36%) of 147 obligate CF carriers who returned a completed questionnaire had self-reported CRS. Twenty-three CF carriers (14 with and 9 without CRS based on self-reporting in the questionnaire) were clinically evaluated. Seven were diagnosed as having CRS (all 7 with self-reported CRS), while another 6 had allergic rhinitis or recurrent acute rhinosinusitis (all 6 with self-reported CRS), and 10 had no evidence of active sinus disease (1 with self-reported CRS). The sensitivity (100%) and specificity (56%) of the questionnaire for physician-diagnosed CRS was similar to that of other survey instruments used to estimate the prevalence of self-reported CRS in the general population. CONCLUSION: Carriers of a single CF mutation have a higher prevalence of CRS than the general population.

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94 Distribution of CFTR Alleles in CRS and Non-CRS Obligate CF Carriers Using a Screen for 16 CF Alleles CF Allele CRS (n = 26) Non-CRS (n = 27) Allele Frequency, % (n = 53) ⌬F508 18 20 71.7 R117H 0 1 1.9 G542X 0 1 1.9 G551D 0 2 3.8 W1282X 0 2 3.8 N1303K 1 0 1.9 3849 + 10 kb C→T 1 0 1.9 Not detected 6 1 12.8 Abbreviations: CF, cystic fibrosis; CRS, chronic rhinosinusitis; kb, kilobase. Login to comment

[hide] Lack of association of common cystic fibrosis tran... Am J Gastroenterol. 2005 Apr;100(4):874-8. Gallegos-Orozco JF, E Yurk C, Wang N, Rakela J, Charlton MR, Cutting GR, Balan V
Lack of association of common cystic fibrosis transmembrane conductance regulator gene mutations with primary sclerosing cholangitis.
Am J Gastroenterol. 2005 Apr;100(4):874-8., [PMID:15784035]

Abstract [show]
BACKGROUND: Primary sclerosing cholangitis (PSC) is a chronic progressive cholestatic liver disease of uncertain etiology. However, the histologic features of PSC liver disease can resemble those in cystic fibrosis (CF), an inherited disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We sought to determine if PSC patients have a higher frequency of common CF alleles than disease controls. METHODS: DNA was extracted from peripheral lymphocytes of patients with end-stage liver disease. Samples were obtained before liver transplantation from 59 PSC patients and from three groups of control patients (20 each with primary biliary cirrhosis, autoimmune hepatitis, or hepatitis C). DNA samples were genotyped for 32 common CF mutations, the intron 8 T tract variants, and the M470V variant. RESULTS: One of 59 PSC patients (1.7%) had the common CF mutation (DeltaF508) in one CFTR gene. Two controls (3.3%) carried a single CF mutation (DeltaF508 in one primary biliary cirrhosis patient; W1282X in one hepatitis C patient). These rates do not differ from expected in the general population. The frequency of CFTR variants (5T and M470V) was also similar between PSC patients and controls. CONCLUSIONS: Despite anatomical similarities between CF liver disease and PSC, we could not confirm that PSC patients carried common CF mutations or common CFTR variants in higher than expected frequencies. These data suggest that CFTR dysfunction does not influence the pathogenesis of PSC.

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55 CFTR Mutations and Associated Phenotype Classic Nonclassic Cystic Fibrosis Cystic Fibrosis Variant Normal 621 + 1G→T R117H G85E* 7T 711 + 1G→T R334W 5T† 9T 1078delT R347P M470V‡ F508C I507 A455E I507V F508 2789 + 5G → A I506V 1717 - 1G→A 3849 + 10kbC→T G542X G551D R553X R560T R1162X 3659delC W1282X N1303K * Classic cystic fibrosis and nonclassic cystic fibrosis. Login to comment

[hide] Cystic fibrosis prenatal screening in genetic coun... J Genet Couns. 2005 Feb;14(1):1-15. Langfelder-Schwind E, Kloza E, Sugarman E, Pettersen B, Brown T, Jensen K, Marcus S, Redman J
Cystic fibrosis prenatal screening in genetic counseling practice: recommendations of the National Society of Genetic Counselors.
J Genet Couns. 2005 Feb;14(1):1-15., [PMID:15789152]

Abstract [show]
For over a decade, prenatal screening for cystic fibrosis (CF) has been considered a model for the integration of genetic testing into routine medical practice. Data from pilot studies and public policy discourse have led to recommendations by some professional organizations that CF screening should be offered or made available to pregnant women and their partners, and to couples planning a pregnancy. It is crucial that genetic counselors gain thorough understanding of the complexities of CF and the implications of positive test results, so that they may serve as a reliable, educated referral base and resource for health care providers and their patients. While not all pregnant women will be referred for genetic counseling prior to CF carrier testing, genetic counselors often will be asked to counsel clients after they have a positive test result, or who are found to be at increased risk. Genetic counselors can play an important role in providing accurate and current information as well as support for patients' informed decisions. These recommendations were created by a multicenter working group of genetic counselors with expertise in CF and are based on personal clinical experience, review of pertinent English language medical articles, and reports of expert committees. The recommendations should not be construed as dictating an exclusive course of management, nor does the use of such recommendations guarantee a particular outcome. These recommendations do not displace a health care provider's professional judgment based on the clinical circumstances of a particular client.

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155 Mutations described as "severe,"forexample, F508, I507,G542X,G551D, W1282X, N1303K, R553X, 621 + 1G>T, and 1717-1G>A, are usually categorized as Class I, II, or III, and the expected pancreatic insufficient phenotype occurs when one of these mutations is inherited in trans with a second mutation, of Class I, II, or III. Login to comment

[hide] Cystic fibrosis carriers have higher neonatal immu... Am J Med Genet A. 2005 Jun 1;135(2):142-4. Castellani C, Picci L, Scarpa M, Dechecchi MC, Zanolla L, Assael BM, Zacchello F
Cystic fibrosis carriers have higher neonatal immunoreactive trypsinogen values than non-carriers.
Am J Med Genet A. 2005 Jun 1;135(2):142-4., 2005-06-01 [PMID:15832355]

Abstract [show]
Following cystic fibrosis (CF) neonatal screening implementation, a high frequency of heterozygotes has been reported among neonates with elevated immunoreactive trypsinogen (IRT) and normal sweat chloride levels. We studied the relationship between normal IRT values and CF heterozygosity: 10,000 neonates were screened for CF by IRT measurement and tested for 40 CF mutations; the 294 carriers detected were coupled with newborns negative to the same genetic testing, and the two groups' IRT levels compared. Heterozygotes had higher IRT levels than their controls (mean 35.32 vs. 27.58 microg/L, P<0.001). Even within normal trypsinogen range, the probability of being a CF carrier increases with neonatal IRT concentration.

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40 Distribution and Classification of the Tested Mutations in the Normal IRT Heterozygote Population Under Study Mutations Type of mutation Class of mutation Number of cases F508del Severe II 161 N1303K Severe II 19 G542X Severe I 19 711 þ 5G > A - V 15 R117H Mild IV 13 R1162X Severe I 13 R553X Severe I 11 G85E - IV 8 2183AA > G Severe I 8 1717-1G > A Severe I 8 R334Q Mild - 4 Q552X Severe I 4 W1282X Severe I 3 2789 þ 5G > A Mild V 2 1898 þ 3A > G Mild V 2 T338I Mild IV 1 R709X Severe I 1 R347H Mild IV 1 3849 þ 10KbC > T Mild V 1 Total 294 Other tested mutations: 1078delTn1609delCAn1717-8g/an394delTTn457TAT> Gn541delCn621 þ 1g/tn711 þ 1g/tnA559TnDI507nG551DnR1158XnR334Wn R347PnR352QnS549InS549NnS549Ra/cn2790-2G > An1811 þ 1.2KbA > G; 711þ5G > A and G85E not categorized in type of mutation; R334Q not categorized in class of mutation. Login to comment

[hide] Time-motion analysis of 6 cystic fibrosis mutation... Clin Chem. 2005 Jul;51(7):1116-22. Epub 2005 Apr 28. Krafft AE, Lichy JH
Time-motion analysis of 6 cystic fibrosis mutation detection systems.
Clin Chem. 2005 Jul;51(7):1116-22. Epub 2005 Apr 28., [PMID:15860566]

Abstract [show]
BACKGROUND: A dramatic increase in requests for routine cystic fibrosis (CF) carrier screening prompted us to conduct a time-motion analysis comparing commercially available CF testing platforms. Questions addressed in the study included: (a) How much time is required to perform each step involved in carrying out the assay procedure? (b) Which system requires the minimum number of manual manipulations to complete a typical run? (c) What workflow benefits can be achieved by automation? METHODS: We used a 96-sample run for comparisons and analyzed each of the 6 methods to determine the number of pipetting steps and manual manipulations, the labor and instrument time, and the total time required to perform the assay. The survey participants included a staff of 4 technologists who perform complex molecular assays regularly. Time required for each procedure was determined by direct observation and from work logs completed by the technologists. RESULTS: The total number of pipetting motions varied from 78 to 344. Labor time ranged from 2.6 to 8.4 h, and total assay time from 7.6 to 13.7 h. CONCLUSION: Time-motion analysis allowed identification of a method that minimized pipetting motions and thus reduced the risk of repetitive stress injury.

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43 These included 58 patient DNA samples initially characterized by CF Gold 1.0, of which 28 were wild type and 30 contained 1 of the following 16 mutant alleles: F508del, R553X, 2184delA, 3120 ϩ 1GϾA, I507del, G542X, G551D, W1282X, N1303K, 621 ϩ 1GϾT, R117H, 1717-1GϾA, R560T, R334W, R347P, and I148T. Login to comment

[hide] Cystic fibrosis lung disease: genetic influences, ... Pediatr Radiol. 2005 Aug;35(8):739-57. Epub 2005 May 3. Moskowitz SM, Gibson RL, Effmann EL
Cystic fibrosis lung disease: genetic influences, microbial interactions, and radiological assessment.
Pediatr Radiol. 2005 Aug;35(8):739-57. Epub 2005 May 3., [PMID:15868140]

Abstract [show]
Cystic fibrosis (CF) is a multiorgan disease caused by mutation of the CF transmembrane conductance regulator (CFTR) gene. Obstructive lung disease is the predominant cause of morbidity and mortality; thus, most efforts to improve outcomes are directed toward slowing or halting lung-disease progression. Current therapies, such as mucolytics, airway clearance techniques, bronchodilators, and antibiotics, aim to suppress airway inflammation and the processes that stimulate it, namely, retention and infection of mucus plaques at the airway surface. New approaches to therapy that aim to ameliorate specific CFTR mutations or mutational classes by restoring normal expression or function are being investigated. Because of its sensitivity in detecting changes associated with early airway obstruction and regional lung disease, high-resolution CT (HRCT) complements pulmonary function testing in defining disease natural history and measuring response to both conventional and experimental therapies. In this review, perspectives on the genetics and microbiology of CF provide a context for understanding the increasing importance of HRCT and other imaging techniques in assessing CF therapies.

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48 Class 1 alleles (second panel), such as one with a nonsense mutation in codon 542 that normally encodes glycine (G542X allele), contain premature stop codons that result in truncated RNA messages. Login to comment
71 Modifier genes as modulators of CF lung phenotype Some DF508 homozygous individuals develop severe obstructive lung disease during the first and second decades of life despite maximal medical therapy, while others with the same genotype have little or no obstructive lung disease despite minimal therapy during Table 2 Examples of disease-associated CFTR alleles CFTR allele Allele class Usual clinical status (when compounded with a severe CFTR allelea ) Allele frequency in Caucasiansb General populationc CF CAVD G542X (9T) 1 Pancreatic-insufficient CF 0.001 0.023 0.003 DF508 (9T) 2 Pancreatic-insufficient CF 0.012-0.016 0.694 0.20 G551D (7T) 3 Pancreatic-insufficient CF 0.001 0.022 0.01 R117H (5T) 4 Pancreatic-sufficient CF 0.0001 0.004 ND R117H (7T) 4 CAVD or carrier 0.002-0.003 0.003 0.04 A455E (9T) 5 Pancreatic-sufficient CF ND 0.001 ND 3849+10kbC fi T 5 Pancreatic-sufficient CF ND 0.007 ND WT (5T) 5 CAVD or carrier 0.042 d 0.19 Other allelese 1-5 Variable 0.002-0.006 0.247 0.55 WT (7T or 9T) Wild-type Carrier 0.935 e e a Severe CFTR allele is defined as a class 1, 2, or 3 allele. Login to comment
89 This suppression of translational termination might be of therapeutic benefit for genetic disease caused by nonsense mutations such as the CFTR Class 1 allele G542X [36-38]. Login to comment

[hide] Screening of mutations in the CFTR gene in 1195 co... Eur J Hum Genet. 2005 Aug;13(8):959-64. Stuppia L, Antonucci I, Binni F, Brandi A, Grifone N, Colosimo A, De Santo M, Gatta V, Gelli G, Guida V, Majore S, Calabrese G, Palka C, Ravani A, Rinaldi R, Tiboni GM, Ballone E, Venturoli A, Ferlini A, Torrente I, Grammatico P, Calzolari E, Dallapiccola B
Screening of mutations in the CFTR gene in 1195 couples entering assisted reproduction technique programs.
Eur J Hum Genet. 2005 Aug;13(8):959-64., [PMID:15870824]

Abstract [show]
Genetic testing of the cystic fibrosis transmembrane conductance (CFTR) gene is currently performed in couples undergoing assisted reproduction techniques (ART), because of the high prevalence of healthy carriers in the population and the pathogenic relationship with congenital bilateral absence of vas deferens (CBAVD). However, discordant data have been reported concerning the usefulness of this genetic test in couples with no family history of cystic fibrosis (CF). In this study, we report the results of CFTR molecular screening in 1195 couples entering ART. Genetic testing was initially carried out in a single partner of each couple. CFTR mutations were detected in 55 subjects (4.6%), a percentage that overlaps with the one reported in the general population. However, significantly higher frequencies of were found in CBAVD individuals (37.5%) and in males with nonobstructive azoospermia (6.6%). The 5T allele was found in 78 patients (6.5%). This figure was again significantly different in males with nonobstructive-azoospermia (9.9%) and in those with CBAVD (100%). All together, 139 subjects (11.6%) had either a CFTR mutation or the 5T allele. Subsequent molecular analysis of their partners disclosed a CFTR mutation or 5T allele in nine cases (6.5%). However, none of these couples had CFTR alterations in both members, a CFTR mutation being invariably present in one partner and the 5T allele in the other. In order to improve genetic counselling of these couples, the TG-M470V-5T association was analyzed, and a statistically significant relationship between 12TG-V470 and CBAVD was detected.

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64 of detected carriers Prevalence among detected CFTR mutations DF508 40 (3.34%) 65.58% DI507 0 0 G542X 6 (0.50%) 9.84% 1717-1G-A 1 (0.08%) 1.64% G551D 0 0 R553X 0 0 R560T 0 0 Q552X 0 0 W1282X 7 (0.58 %) 11.48% S1251N 0 0 N1303K 3 (0.20%) 4.91% 394delTT 0 0 G85E 3 (0.25%) 4.91% E60X 0 0 621+1G-T 0 0 R117H 0 0 1078delT 0 0 R347P 0 0 R334W 0 0 2143delT 0 0 2183AA-G 0 0 2184delA 0 0 711+5G-A 0 0 2789+5G-A 1 (0.08%) 1.64% R1162X 0 0 3659del5 0 0 3849+10kbC-T 0 0 A455E 0 0 5T 78 (6.52%) Table 2 Distribution of CFTR mutations and 5T allele according to phenotype for the 1195 individuals Phenotype CF/WT 5T/WT CF/5T WT/WT Infertile males (non-CBAVD), N ¼ 304 20 (6.58%) 30 (9.87%) 0 254 (83.55%) Infertile males (CBAVD), N ¼ 16 0 10 (62.50%) 6 (37.50 %) 0 Infertile females, N ¼ 93 5 (5.37%) 7 (7.53%) 0 81 (87.10%) Unexplained infertility, N ¼ 782 30 (3.84%) 31 (3.96%) 0 721 (92.20%) Total ¼ 1195 55 (4.60%) 78 (5.50%) 6 (0.50%) 1056 (88.40%) CFTR alteration was detected, including a mutation in three cases and the 5T polymorphism in the remaining six. Login to comment
85 Altogether, we detected a CFTR mutation or the 5T allele in 139 (11.6%) single partners of our couples, in agreement with the figure Table 3 Couples with both partners carriers of a CFTR mutation or a 5T allele First partner Second partner W1282X/5T 5T/wt 1717-1G4A/5T 5T/wt G542X/5T 5T/wt DF508/wt 5T/wt DF508/wt 5T/wt DF508/wt 5T/wt 5T/wt G542X/wt 5T/wt 1717-1G4A/wt 5T/wt 5T/wt Table 4 Distribution of the different TG-M470V-5T associations in relation to the phenotype for the 67 investigated males Phenotype TG12-V470 TG12-M470 TG11-V470 TG11-M470 Total Fertile men 7 0 9 8 24 CBAVD 11 0 0 2 13 Azoospermia 4 0 0 2 6 Oligozoospermia 8 2 7 7 24 Total 30 2 16 19 67 expected in the general Caucasian population. Login to comment

[hide] Pharmacological induction of CFTR function in pati... Pediatr Pulmonol. 2005 Sep;40(3):183-96. Kerem E
Pharmacological induction of CFTR function in patients with cystic fibrosis: mutation-specific therapy.
Pediatr Pulmonol. 2005 Sep;40(3):183-96., [PMID:15880796]

Abstract [show]
CFTR mutations cause defects of CFTR protein production and function by different molecular mechanisms. Mutations can be classified according to the mechanisms by which they disrupt CFTR function. This understanding of the different molecular mechanisms of CFTR dysfunction provides the scientific basis for the development of targeted drugs for mutation-specific therapy of cystic fibrosis (CF). Class I mutations are nonsense mutations that result in the presence of a premature stop codon that leads to the production of unstable mRNA, or the release from the ribosome of a short, truncated protein that is not functional. Aminoglycoside antibiotics can suppress premature termination codons by disrupting translational fidelity and allowing the incorporation of an amino acid, thus permitting translation to continue to the normal termination of the transcript. Class II mutations cause impairment of CFTR processing and folding in the Golgi. As a result, the mutant CFTR is retained in the endoplasmic reticulum (ER) and eventually targeted for degradation by the quality control mechanisms. Chemical and molecular chaperones such as sodium-4-phenylbutyrate can stabilize protein structure, and allow it to escape from degradation in the ER and be transported to the cell membrane. Class III mutations disrupt the function of the regulatory domain. CFTR is resistant to phosphorylation or adenosine tri-phosphate (ATP) binding. CFTR activators such as alkylxanthines (CPX) and the flavonoid genistein can overcome affected ATP binding through direct binding to a nucleotide binding fold. In patients carrying class IV mutations, phosphorylation of CFTR results in reduced chloride transport. Increases in the overall cell surface content of these mutants might overcome the relative reduction in conductance. Alternatively, restoring native chloride pore characteristics pharmacologically might be effective. Activators of CFTR at the plasma membrane may function by promoting CFTR phosphorylation, by blocking CFTR dephosphorylation, by interacting directly with CFTR, and/or by modulation of CFTR protein-protein interactions. Class V mutations affect the splicing machinery and generate both aberrantly and correctly spliced transcripts, the levels of which vary among different patients and among different organs of the same patient. Splicing factors that promote exon inclusion or factors that promote exon skipping can promote increases of correctly spliced transcripts, depending on the molecular defect. Inconsistent results were reported regarding the required level of corrected or mutated CFTR that had to be reached in order to achieve normal function.

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53 This class may include promoter mutations that reduce transcription TABLE 1- Classes of CFTR Mutations1 Class Mutations I Stop codons: W1282X, G542X, R1162X, R553X, E822X Splicing mutations that completely abolish protein synthesis: 1717 À 1G ! Login to comment
74 Howard et al. demonstrated in HeLa cells transfected with plasmid vector carrying the CFTR nonsense mutations G542X and R553X that aminoglycosides caused a dose-dependent increase in CFTR expression.14 Subsequently, the same group showed that functional CFTR was restored to the apical membrane, and the relative level of mRNA transcript increased in bronchial cell line IB3-1 expressing the W1282X mutation. Login to comment
75 Following incubation with aminoglycosides, cAMP-activated chloride conductance and the expression of functional CFTR were restored to the apical membrane.15 Zsembery et al. isolated cholangiocytes from the liver of a patient carrying the G542X mutation and incubated them with gentamicin. Login to comment
77 Du et al. daily administered the aminoglycoside antibiotics gentamicin or tobramycin.17 Immunofluorescence staining of intestinal tissues from CftrÀ/À hCFTR-G542X mice revealed that gentamicin treatment resulted in the appearance of CFTR protein at the apical surface of the glands of treated mice. Login to comment
80 When taken together, these results indicate that gentamicin, and to a lesser extent tobramycin, can restore the synthesis of functional CFTR protein by suppressing the G542X premature stop mutation in vivo. Login to comment
243 Quantification studies showed that after aminoglycoside incubation, the amount of full-length CFTR produced is as much as 25% (in the R553X mutation) to 35% (in the G542X mutation) of that observed in cells transfected with a wild-type CFTR complementary DNA (cDNA).14,15 This increase in functional CFTR might be above the threshold required for normal respiratory epithelial cell function. Login to comment

[hide] Cystic fibrosis. N Engl J Med. 2005 May 12;352(19):1992-2001. Rowe SM, Miller S, Sorscher EJ
Cystic fibrosis.
N Engl J Med. 2005 May 12;352(19):1992-2001., 2005-05-12 [PMID:15888700]

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120 About 5 to 10 percent ofCFTR mutations are due toprematuretruncationornonsensealleles(designated by "X," such as G542X, a class I mutation). Login to comment

[hide] Novel, mechanism-based therapies for cystic fibros... Curr Opin Pediatr. 2005 Jun;17(3):385-92. Rubenstein RC
Novel, mechanism-based therapies for cystic fibrosis.
Curr Opin Pediatr. 2005 Jun;17(3):385-92., [PMID:15891431]

Abstract [show]
PURPOSE OF REVIEW: Cystic fibrosis results from disruption of the biosynthesis or function of the cystic fibrosis transmembrane conductance regulator. Cystic fibrosis transmembrane conductance regulator plays a critical role in the regulation of epithelial ion transport. Restoration of cystic fibrosis transmembrane conductance regulator function should improve the cystic fibrosis phenotype. RECENT FINDINGS: Recent investigations affording a better understanding of the mechanism of dysfunction of mutant cystic fibrosis transmembrane conductance regulators, as well as the roles of cystic fibrosis transmembrane conductance regulator in regulating epithelial ion transport, have led to development of therapeutic strategies based on repair or bypass of mutant cystic fibrosis transmembrane conductance regulator dysfunction. The former strategy, coined 'protein repair therapy,' is aimed at improving or restoring the function of mutant cystic fibrosis transmembrane conductance regulators, whereas the latter approach aims to augment epithelial ion transport to compensate for the absent function mutant cystic fibrosis transmembrane conductance regulator. SUMMARY: Strategies to improve mutant cystic fibrosis transmembrane conductance regulator function or to bypass mutant cystic fibrosis transmembrane conductance regulator function hold great promise for development of novel therapies aimed at correcting the underlying pathophysiology of cystic fibrosis.

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23 Such mutations are relatively infrequent in the general CF population (G542X, 2.4%; R553X, 0.9%; W1282X, 1.4% of mutant alleles in the 2003 Cystic Fibrosis Foundation Patient Registry). Login to comment
25 Treatment of cells expressing these 'X` mutations with aminoglycoside antibiotics such as gentamicin or G418 (Geneticin, Life Technologies, Inc., Gaithersburg, MD, USA) causes expression of a full-length, functional CFTR protein from G542X [8], R553X [8], R1162X [9], and W1282X [9] alleles. Login to comment
27 Similar effects were noted in a murine cftr(ÿ/ÿ) knockout model where the G542X allele was expressed under control of an intestine-specific promoter. Login to comment

[hide] Multiple mutation analysis of the cystic fibrosis ... Mol Hum Reprod. 2005 Jun;11(6):463-8. Epub 2005 May 20. Sanchez-Garcia JF, Benet J, Gutierrez-Mateo C, Luis Seculi J, Monros E, Navarro J
Multiple mutation analysis of the cystic fibrosis gene in single cells.
Mol Hum Reprod. 2005 Jun;11(6):463-8. Epub 2005 May 20., [PMID:15908456]

Abstract [show]
PGD is becoming an alternative to prenatal diagnosis. The combination of IVF techniques with the PCR technology allows for the detection of genetic abnormalities in first polar bodies from oocytes and blastomeres from cleavage-stage embryos. Dealing with a genetic disease with a heterogeneous spectrum of mutations like cystic fibrosis, one of the objectives of centres offering PGD is the application of simple and efficient protocols that allow for the detection of a wide range of mutations with a single procedure. In the present work, 29 normal loci and the 31 most frequent cystic fibrosis transmembrane conductance regulator (CFTR) mutations in Southern Europe could be detected at the same time in single cells applying a modified and improved primer extension preamplification-PCR. Two different Taq polymerases were tested in isolated buccal cells heterozygous for several mutations. The protocol that gave statistically significant better results was also successful in oocytes and their first polar bodies.

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38 Cell samples Isolated buccal cells were collected by mouthwashes from three normal individuals, three patients affected by mutations DF508, DF508/1078delT and DF508/3849þ10kbC.T and five heterozygous carriers for CFTR mutations DF508, N1303K, G542X, R347P and 2183AA.G. Login to comment
61 The mutations assayed are: DF508, DI507, Q493X, V520F, 1717-1G.A, G542X, G551D, R560T, S459R, S459N and R553X labelled with FAM (blue), 3849þ10kbC.T, 3849 þ 4A . Login to comment
88 A similar result was seen in heterozygous cells for G542X and 3849þ10kbC.T. Login to comment
121 250 S549 20 25 60 15 75 30 13.3 46.7 40 86.7 R553 20 15 65 20 85 30 6.7 46.7 46.7 93.3 G551 20 15 70 15 85 30 10 46.7 43.3 90 V520 20 5 40 55 95 30 0 13.3 86.7 100 I507 20 5 45 50 95 30 0 26.7 73.3 100 F508 20 10 80 10 90 30 10 50 40 90 dF508 (*) 12 25 66.7 8.3 75 19 10.5 52.6 36.8 89.5 Q493 20 10 75 15 90 30 10 56.7 33.3 90 1717-G1 20 25 60 15 75 30 6.7 46.7 46.7 93.3 G542 20 20 65 15 80 30 10 50 40 90 G542X (*) 2 50 50 0 50 7 14.3 85.7 0 85.7 R560 20 20 50 30 80 30 6.7 43.3 50 93.3 R347 20 5 70 25 95 30 0 16.7 83.3 100 R347P (*) - - - - - 3 0 33.3 66.7 100 3849 þ 4A 20 15 55 30 85 30 3.3 50 46.7 96.7 W1282 20 20 40 40 80 30 13.3 30 56.7 86.7 R334 20 10 75 15 90 30 0 13.3 86.7 100 1078 20 25 60 15 75 30 0 13.3 86.7 100 1078 del T (*) 2 0 100 0 100 - - - - - 3849 þ 10kbC 20 20 65 15 80 30 3.3 56.7 40 96.7 3849 þ 10kbC . Login to comment

[hide] Risk calculations for cystic fibrosis in neonatal ... Genet Med. 2005 May-Jun;7(5):317-27. Ogino S, Flodman P, Wilson RB, Gold B, Grody WW
Risk calculations for cystic fibrosis in neonatal screening by immunoreactive trypsinogen and CFTR mutation tests.
Genet Med. 2005 May-Jun;7(5):317-27., [PMID:15915083]

Abstract [show]
PURPOSE: Although neonatal screening (or newborn screening) for cystic fibrosis (CF) is commonly practiced, systematic methods for accurate risk calculations are currently lacking. METHODS AND RESULTS: We evaluated characteristics of the immunoreactive trypsinogen (IRT) test using the published data. The probability that a neonate has a positive IRT test, if the neonate is affected, a carrier, or a noncarrier, is approximately 1, 0.041, or 0.011, respectively. We provide methods to calculate genetic risks for a variety of commonly encountered scenarios in which neonates are positive by the IRT test. CONCLUSION: Our Bayesian methods permit CF disease probabilities to be calculated accurately, taking into account all relevant information.

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32 Table 1 Summary of CF carrier frequencies, overall mutation detection rates by the ACMG panel, and frequencies of major mutations for each major ethnic group (adapted from Watson et al.10 and Richards et al.1) Ethnic group CF carrier frequency Overall mutation detection rate by the ACMG CFTR 23-mutation panel10 Fraction of F508del among all disease alleles Other major mutations (fraction)a Non-Hispanic Caucasian 1/25 88.29% 72.42% G542X (2.28%) G551D (2.25%) 621ϩ1GϾT (1.57%) W1282X (1.50%) N1303K (1.27%) Ashkenazi Jewish 1/25 94.04% 31.41% W1282X (45.92%) G542X (7.55%) 3849ϩ10kbCϾT (4.77%) N1303K (2.78%) African American 1/65 64.46% 44.07% 3120ϩ1GϾA (9.57%) R553X (2.32%) I507del (1.87%) G542X (1.45%) G551D (1.21%) 621ϩ1GϾT (1.11%) Hispanic Caucasian 1/46 71.72% 54.38% G542X (5.10%) R553X (2.81%) R334W (1.78%) N1303K (1.66%) 3849ϩ10kbCϾT (1.57%) Asian American 1/90 48.93% 38.95% 3849ϩ10kbCϾT (5.31%) G551D (3.15%) Bayesian analysis to calculate CF risks for neonates with a positive IRT test A fraction of each major CFTR disease allele among all CFTR disease alleles and a mutation detection rate are summarized for each of five major ethnic groups (Table 1). Login to comment

[hide] Mutation spectrum in Jewish cystic fibrosis patien... Am J Med Genet A. 2005 Jul 30;136(3):246-8. Quint A, Lerer I, Sagi M, Abeliovich D
Mutation spectrum in Jewish cystic fibrosis patients in Israel: implication to carrier screening.
Am J Med Genet A. 2005 Jul 30;136(3):246-8., 2005-07-30 [PMID:15948195]

Abstract [show]
We have tested 144 unrelated Jewish patients suffering from the classical form of cystic fibrosis. The patients were screened for a panel of 12 mutations including the six Ashkenazi founder mutations (DeltaF508, W1282X, N1303K, G542X, 3849 + 10 kb C-->T, 1717-1G > A) and six mutations that were found in non-Ashkenazi Jewish patients (S549R (T-->G), G85E, 405 + 1G-->A, W1089X, Y1092, and D1152H). Patients of Georgian origin were tested also for the Q359K/T360K mutation. In addition, all the patients were tested for the IVS-8 variant (9T/7T/5T). Of all the cystic fibrosis (CF)-bearing chromosomes, 94% (264/281) were accounted for by one of the known mutations, and none of the patients had the 5T allele of the IVS-8 variant. Single strand conformation polymorphism (SSCP) analysis of the coding sequence of the CFTR gene followed by sequencing showed eight mutations on ten CF chromosomes, leaving seven chromosomes (2.5%) with unknown mutations. We identified three mutations in two or more CF chromosomes, 2571 + 1insT in Jews from Iraq, 3121-1G > A in patients from Kurdistan and I1234V in Yemenite Jewish patients. The other five mutations appeared on a single allele and are considered "private mutations." In this study we have identified 99% of CF alleles in Ashkenazi Jewish patients, 91% in Jews of North African origin and 75% in Jewish patients from Iraq. The significance of these findings to the population screening in Israel is discussed.

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25 MUTATION ANALYSIS The following mutations are routinely tested in Jewish patients: the Ashkenazi founder mutations, DF508, W1282X, N1303K, G542X, 3849 þ 10 kb C!T, 1717-1G > A [Abeliovich et al., 1992], mutations commonly found in non-Ashkenazi patients, S549R (T!G), G85E, 405 þ 1G!A, W1089X, Y1092X, D1152H. Login to comment
36 The G542X, N1303K, 3849 þ 10 kb C!T, and 1717-1G!A mutations were found on 21.6% of the CF chromosomes. Login to comment
37 In this group we revealed two additional mutations L165S and A455E, each was identified on a single chromosome and one mutation remained unidentified. The relative frequencies of the Ashkenazi founder mutations in Ashkenazi patients were W1282X (43%), DF508 (33%), G542X (10%), 3849 þ 10 kb C!T (5%), N1303K (5%), and 1717 (1%). Login to comment
44 Patients from the Balkan countries, Greece and Turkey (21 alleles), had some of the Ashkenazi founder mutations (W1282X, DF508, G542X, and 3849 þ 10 kb C!T), in addition to two other mutations, G85E and W1089X that were not found in Jewish patients from other origins. Login to comment
58 Mutations in the CF Bearing Alleles in the Jewish Patients According to the Ethnic Origin Country of origin Ashkenazi Morocco Tunisia Balkan Iraq Iran/ Kurdistan Georgia Yemen Total Number of alleles (%) 193 (69.0) 34 (12.1) 12 (4.3) 21 (7.5) 8 (2.8) 3 (0.7) 8 (2.8) 2 (0.7) 281 W1282X (%) 83 (42.8) 1 (8.3) 4 (19.0) 88 (31.3) DF508 (%) 65 (33.5) 24 (70.6) 3 (25.0) 7 (33.3) 1 100 (35.6) N1303K (%) 10 (5.2) 10 (3.6) G542X (%) 19 (10.3) 4 (19.0) 24 (8.5) 3849-10 kbC!T (%) 10 (5.1) 1 (2.9) 2 (9.5) 13 (4.6) 1717-1G!A (%) 2 (1.0) 2 (0.7) D1152H (%) 1 (0.5) 1 (0.4) S549R (T!G) (%) 4 (11.8) 4 (1.4) G85E (%) 2 (9.5) 2 (0.7) 405 þ 1G!A (%) 8 (66.7) 8 (2.8) Y1092X (%) 3 (37.5) 3 (1.1) W1089X (%) 2 (9.5) 2 (0.7) Q359K/T360K (%) 8 (100) 8 (2.8) I1234V (%) 2 (100) 2 (0.7) 2751 þ 1insT (%) 2 (25.0) 2 (0.7) 3121-1G > A (%) 1 1 (0.4) M952I (%) 1 (12.5) 1 (0.4) L165S (%) 1 (0.5) 1 (0.4) A455E (%) 1 (0.5) 1 (0.4) L997F (%) 1 (2.9) 1 (0.4) G1244E (%) 1 (2.9) 1 (0.4) Unkown (%) 1 (0.5) 3 (8.8) 2 (25.0) 1 7 (2.5) Mutation Spectrum in Jewish CF Patients [Wahab, 2003]. Login to comment
69 We suggest that 15 mutations that were found on two or more CF chromosomes from unrelated patients (DF508, W1282X, N1303K, G542X, 3849 þ 10 kb C!T, 1717-1 G!A, S549R (T!G), G85E, 405 þ 1G!A, W1089X, Y1092X, 2751 þ 1insT, 3121-1G!A, Q359K/T360K, I1234V) be tested in the CF screening of all Jewish individuals regardless of their origin. Login to comment

[hide] A CFTR mutation (D1152H) in a family with mild lun... Clin Genet. 2005 Jul;68(1):88-90. Highsmith WE Jr, Friedman KJ, Burch LH, Spock A, Silverman LM, Boucher RC, Knowles MR
A CFTR mutation (D1152H) in a family with mild lung disease and normal sweat chlorides.
Clin Genet. 2005 Jul;68(1):88-90., [PMID:15952991]

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15 The three siblings with the clinical syndrome of CF were compound heterozygotes, D1152H/G542X. Login to comment
29 The lung disease attributable to the D1152H/G542X genotype in this family, even with the additional insult of prolonged smoking, is associated with survival into the seventh and eighth decade of life. Login to comment

[hide] The prevalence and clinical characteristics of cys... Arch Dis Child. 2005 Jul;90(7):675-9. Mei-Zahav M, Durie P, Zielenski J, Solomon M, Tullis E, Tsui LC, Corey M
The prevalence and clinical characteristics of cystic fibrosis in South Asian Canadian immigrants.
Arch Dis Child. 2005 Jul;90(7):675-9., [PMID:15970608]

Abstract [show]
BACKGROUND: Cystic fibrosis (CF) is considered to be rare among individuals from the Indian subcontinent. Furthermore, affected individuals are reported to experience a more severe clinical course. AIMS: It was hypothesised that CF is under diagnosed in people of South Asian origin and therefore the prevalence may be higher than previously estimated. METHODS: The prevalence of CF in the South Asian and in the general population living in the same geographic region (Metropolitan Toronto) were compared between 1996 and 2001. Population data were obtained from the Canadian census survey. CF phenotype and genotype data were obtained from the Toronto CF database. RESULTS: Among 381 patients with CF, 15 were of South Asian descent. The age related prevalence of CF among the South Asian and general populations was: 0-14 years, 1:9200 versus 1:6600; 15-24 years, 1:13,200 versus 1:7600; older than 25 years, 1:56,600 versus 1:12,400. Age at diagnosis, duration and severity of symptoms at diagnosis, current nutritional status, and FEV(1) were similar in the two groups. While not significant, FEV1 tended to be lower (48% versus 57% predicted) among adult South Asians, compared to the general CF population. Also, the percentage with pancreatic sufficiency was higher (27% versus 16%) and the frequency of DeltaF508 allele was lower (50% versus 65.1%). CONCLUSIONS: These data suggest that the prevalence and natural history of CF in South Asians is similar to that among individuals of European origin. The relatively lower prevalence among older South Asians may reflect an improving recognition of CF in this ethnic subgroup.

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303 Table 3 CFTR gene mutations among CF patients of South Asian origin and all patients living in the same geographic region in the CF population Mutation South Asian CF population Mutation General CF population (number, % of total alleles) (number, % of total alleles) No. identified % of alleles No. identified % of alleles DF508 13 50 DF508 375 65.1 L218X 2 7.7 W1282X 16 2.8 1525-1GRA 1 3.8 G551D 15 2.6 S549N 1 3.8 G542X 10 1.7 3849+10kbCRT 1 3.8 621+1GRT 10 1.7 V392G 1 3.8 R117H 7 1.2 N1303K 7 1.2 49 others (,1%) 89 16.4 Unidentified 7 26.9 Unidentified 47 8.2 What is already known on this topic N CF is rare in populations not of European Caucasian origin N More severe disease has been reported in South Asian CF patients N DF508, the most common mutation in Caucasians, is less prevalent in South Asians What this study adds N Prevalence and clinical course of CF in children of South Asian origin is similar to that in the general Toronto population N Previous reports reflect inadequate awareness of CF in this ethnic group N The prevalence of DF508 is confirmed to be lower in South Asians than other Caucasian groups Mei-Zahav, Durie, Zielenski, et al www.archdischild.com Authors` affiliations . Login to comment

[hide] Genotype-phenotype correlation for pulmonary funct... Thorax. 2005 Jul;60(7):558-63. de Gracia J, Mata F, Alvarez A, Casals T, Gatner S, Vendrell M, de la Rosa D, Guarner L, Hermosilla E
Genotype-phenotype correlation for pulmonary function in cystic fibrosis.
Thorax. 2005 Jul;60(7):558-63., [PMID:15994263]

Abstract [show]
BACKGROUND: Since the CFTR gene was cloned, more than 1000 mutations have been identified. To date, a clear relationship has not been established between genotype and the progression of lung damage. A study was undertaken of the relationship between genotype, progression of lung disease, and survival in adult patients with cystic fibrosis (CF). METHODS: A prospective cohort of adult patients with CF and two CFTR mutations followed up in an adult cystic fibrosis unit was analysed. Patients were classified according to functional effects of classes of CFTR mutations and were grouped based on the CFTR molecular position on the epithelial cell surface (I-II/I-II, I-II/III-V). Spirometric values, progression of lung disease, probability of survival, and clinical characteristics were analysed between groups. RESULTS: Seventy four patients were included in the study. Patients with genotype I-II/I-II had significantly lower current spirometric values (p < 0.001), greater loss of pulmonary function (p < 0.04), a higher proportion of end-stage lung disease (p < 0.001), a higher risk of suffering from moderate to severe lung disease (odds ratio 7.12 (95% CI 1.3 to 40.5)) and a lower probability of survival than patients with genotype I-II/III, I-II/IV and I-II/V (p < 0.001). CONCLUSIONS: The presence of class I or II mutations on both chromosomes is associated with worse respiratory disease and a lower probability of survival.

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209 To study the decline in pulmonary function between groups the ANOVA method (repeated measures) was used with baseline and current spirometric values as dependent variables, genotype groups as the independent variable, and age and evolution time as Table 1 CFTR mutation according to functional classification Class Molecular dysfunction Mutation I Defective protein production G542X, 711+1GRT, 1609delCA, R1162X, 1717-8GRA, W1282X, 1782delA, Q890X, 1898+3ARG, CFTRdele19, 936delTA II Defective protein processing F508del, N1303K, I507del, R1066C III Defective protein regulation D1270N, G551D IV Defective protein conductance L206W, R334W, R117H, R347H, D836Y, P205S V Partially defective production or processing 2789+5GRA, 1811+1.6kbARG, 3849+10kbCRT, 3272+26GRA Table 2 Groups based on genotype in CF adult patients Functional classes Genotype No of subjects I-I G542X/W1282X 1 R1162X/1898+3ARG 1 R1162X/CFTRdele19 1 I-II F508del/G542X 5 F508del/711+1GRT 2 F508del/1717-8GRA 1 F508del/936delTA 1 F508del/R1162X 1 N1303K/1609delCA 1 I-III G542X/D1270N+R74W 1 711+1G-T/G551D 1 I-IV G542X/P205S 1 Q890X/R334W 1 1609delCA/R347H 1 I-V G542X/2789+5GRT 2 G542X/1811+1.6kbARG 1 1782delA/2789+5GRA 1 1609delCA/1811+1.6kbARG 1 II-II F508del/F508del 21 F508del/N1303K 1 F508del/R1066C 1 II-III F508del/D1270N+R74W 1 I507del/D1270N+R74W 1 II-IV F508del/L206W 4 F508del/R334W 3 F508del/R117H 3 F08del/R347H 2 F508del/D836Y 1 II-V F508del/2789+5GRA 5 F508del/3849+10kbCRT 2 F508del/1811+1.6kbARG 2 F508del/3272+26GRA 1 N1303K/1811+1.6kbARG 1 N1303K/2789+5GRA 1 adjusted variables. Login to comment

[hide] Analysis of most common CFTR mutations in patients... Eur Arch Otorhinolaryngol. 2005 Dec;262(12):982-6. Epub 2005 Jun 17. Kostuch M, Klatka J, Semczuk A, Wojcierowski J, Kulczycki L, Oleszczuk J
Analysis of most common CFTR mutations in patients affected by nasal polyps.
Eur Arch Otorhinolaryngol. 2005 Dec;262(12):982-6. Epub 2005 Jun 17., [PMID:16075239]

Abstract [show]
Nasal polyps, a chronic inflammatory disease occurring in the nose and para-nasal sinuses, result from several different causes, including cystic fibrosis (CF). Forty-four patients affected by nasal polyps were admitted to the Department of Otolaryngology, Lublin University School of Medicine, Lublin, Poland, and screened for the most-commonly identified CFTR mutations [DeltaF508, G542X, N1303 K, 1717-1 (G to A), W1282X, G551D, R553X and DeltaI507] by applying the INNO-LIPA CF2 test strips. None of the patients had symptoms that allowed for the diagnosis of CF, including the negative sweat test. We detected 5 of 44 (11.4%) carriers of the CFTR mutations. All patients positive for this test were heterozygous carriers of DeltaF508. In the control group, only 1 of 70 (1.4%) cases showed DeltaF508 heterozygosity. The frequency of DeltaF508 mutation herein reported was significantly higher than in the control group (P = 0.0312) and in the general Polish population as well (P = 0.0059). Our data suggest that a heterozygous manifestation of the DeltaF508 may exist in a selected group of patients affected by nasal polyps, who have no other clinical features of CF.

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1 Forty-four patients affected by nasal polyps were admitted to the Department of Otolaryngology, Lublin University School of Medicine, Lublin, Poland, and screened for the most-commonly identified CFTR mutations [DF508, G542X, N1303 K, 1717-1 (G to A), W1282X, G551D, R553X and DI507] by applying the INNO-LIPA CF2 test strips. Login to comment
48 Using the INNO-LIPA CF2 test strips, it is possible to detect eight mutations simultaneously within the CFTR gene: DF508, G542X, N1303 K, 1717-1 (G to A), W1282X, G551D, R553X and DI507 [14]. Login to comment
83 Positive bands are seen for wild-types [DF508, G542X, N1303 K, 1717-1 (G to A), W1282X, G551D and R553X], but the only positive band for mutant types is DF508 this mutation reported in the control subjects (see Results). Login to comment

[hide] Genetics of cystic fibrosis. Semin Respir Crit Care Med. 2003 Dec;24(6):629-38. Gallati S
Genetics of cystic fibrosis.
Semin Respir Crit Care Med. 2003 Dec;24(6):629-38., [PMID:16088579]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes a protein expressed in the apical membrane of exocrine epithelial cells. CFTR functions principally as a cyclic adenosine monophosphate (cAMP)-induced chloride channel and appears capable of regulating other ion channels. Mutations affect CFTR through a variety of molecular mechanisms, which can produce little or no functional gene product at the apical membrane. More than 1000 different disease-causing mutations within the CFTR gene have been described. The potential of a mutation to contribute to the phenotype depends on its type, localization in the gene, and the molecular mechanism as well as on interactions with secondary modifying factors. Genetic testing can confirm a clinical diagnosis of CF and can be used for infants with meconium ileus, for carrier detection in individuals with positive family history and partners of proven CF carriers, and for prenatal diagnostic testing if both parents are carriers. Studies of clinical phenotype in correlation with CFTR genotype have revealed a very complex relationship demonstrating that some phenotypic features are closely determined by the underlying mutations, whereas others are modulated by modifier genes, epigenetic mechanisms, and environment.

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43 Mutations (missense, nonsense, frameshift, splice, small and large in-frame deletions or insertions) con- Table 1 Distribution of theWorldwide 24 Most Common Cystic Fibrosis Mutationsa Exon/ Northern Southern North South Austral- Relative Mutation Intron Europe Europe America America asia Africa Asia Frequency G85E E 03 30 14 16 n.a. n.a. 0 7 0.15 R117H E 04 62 3 61 n.a. 7 0 0 0.30 621+1G→T I 04 97 37 154 n.a. 27 0 0 0.72 711+1G→T I 05 15 13 21 n.a. n.a. n.a. 0 0.11 1078delT E 07 53 2 1 n.a. 1 n.a. 0 0.13 R334W E 07 18 21 12 n.a. 2 0 0 0.12 R347P E 07 55 24 26 n.a. 1 0 0 0.24 A455E E 09 35 0 27 n.a. n.a. n.a. 0 0.14 ⌬I507 E 10 57 5 20 2 9 0 0 0.21 ⌬F508 E 10 14,866 4007 6901 342 2309 351 173 66.02 1717-1G→A I 10 160 65 44 n.a. 12 0 3 0.65 G542X E 11 439 259 234 38 56 9 27 2.42 S549N E 11 18 2 5 1 3 1 0 0.07 G551D E 11 356 37 206 1 117 0 0 1.64 R553X E 11 165 44 96 5 11 1 0 0.73 R560T E 11 40 0 24 0 3 0 0 0.15 1898+1G→A I 12 41 10 2 n.a. n.a. n.a. 0 0.12 2184delA E 13 14 7 8 n.a. n.a. n.a. 0 0.07 2789+5G→A I 14b 27 10 17 n.a. n.a. n.a. 0 0.12 R1162X E 19 36 68 19 0 2 0 0 0.28 3659delC E 19 39 1 14 n.a. n.a. n.a. 0 0.12 3849+10kbC→T I 19 23 8 57 n.a. n.a. n.a. 16 0.24 W1282X E 20 120 43 245 n.a. 6 2 120 1.22 N1303K E 21 209 179 130 11 23 8 29 1.34 Chromosomes 21,154 7281 10438 758 3095 515 608 screened Detection rate 80.2 66.7 79.9 52.8 83.7 72.2 61.7 aAccording to the Cystic Fibrosis Genetic Analysis Consortium, http://www.genet.sickkids.on.ca/cftr/. Login to comment
67 SSCP analysis is one of the most popular methods for the detection of sequence variants in polymerase chain reaction (PCR) amplified DNA fragments.29 The princi- Table 3 Cystic Fibrosis Mutations Detected by Commercial Kits INNO-LiPA Mutations CF2 ⌬F508, ⌬I507, G542X, 1717-1G→A, G551D, R553X, W1282X, N1303K CFTR12 ⌬F508, ⌬I507, G542X, 1717-1G→A, G551D, R553X, W1282X, N1303K, S1251N, R560T, 3905insT, Q552X CFTR17+Tn 394delTT, G85E, 621+1G→T, R117H, 1078delT, R347P, R334W, E60X, 2183AA→G, 2184delA, 711+5G→A, 2789+5G→A, R1162X, 3659delC, 3849+10kbC→T, 2143delT, A455E, (5T/7T/9T) Elucigene CF4 ⌬F508, G542X, G551D, 621+1G→T CF12 ⌬F508, G542X, G551D, N1303K, W1282X, 1717-1G→A, R553X, 621+1G→T, R117H, R1162X, 3849+10kbC→T, R334W CF20 1717-1G→A, G542X, W1282X, N1303K, ⌬F508, 3849+10kbC→T, 621+1G→T, R553X, G551D, R117H, R1162X, R334W, A455E, 2183AA→G, 3659delC, 1078delT, ⌬I507, R345P, S1251N, E60X CF Poly-T 5T/7T/9T OLA CF OLA assay ⌬F508, F508C, ⌬I507, Q493X, V520F, 1717-1G→A, G542X, G551D, R553X, R560T, S549R, S549N, 3849+10kbC→T, 3849+4A→G, R1162X, 3659delC, W1282X, 3905insT, N1303K, G85E, 621+1G→T, R117H, Y122X, 711+1G→T, 1078delT, R347P, R347H, R334W, A455E, 1898+1G→A, 2183AA→G, 2789+5G→A b Figure 2 Mutation screening of exon 19 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene using polymerase chain reaction (PCR) followed by single-strand conformation polymorphism/heteroduplex (SSCP/HD) analysis on a silver-stained polyacrylamide gel. Login to comment

[hide] Intracellular chloride channels: critical mediator... Curr Pharm Des. 2005;11(21):2753-64. Suh KS, Yuspa SH
Intracellular chloride channels: critical mediators of cell viability and potential targets for cancer therapy.
Curr Pharm Des. 2005;11(21):2753-64., [PMID:16101453]

Abstract [show]
The passage of ions to form and maintain electrochemical gradients is a key element for regulating cellular activities and is dependent on specific channel proteins or complexes. Certain ion channels have been the targets of pharmaceuticals that have had impact on a variety of cardiovascular and neurological diseases. Chloride channels regulate the movement of a major cellular anion, and in so doing they in part determine cell membrane potential, modify transepithelial transport, and maintain intracellular pH and cell volume. There are multiple families of chloride channel proteins, and respiratory, neuromuscular, and renal dysfunction may result from mutations in specific family members. Interest in chloride channels related to cancer first arose when the multidrug resistance protein (MDR/P-glycoprotein) was linked to volume-activated chloride channel activity in cancer cells from patients undergoing chemotherapy. More recently, CLC, CLIC, and CLCA intracellular chloride channels have been recognized for their contributions in modifying cell cycle, apoptosis, cell adhesion, and cell motility. Moreover, advances in structural biology and high-throughput screening provide a platform to identify chemical compounds that modulate the activities of intracellular chloride channels thereby influencing chloride ion transport and altering cell behavior. This review will focus on several chloride channel families that may contribute to the cancer phenotype and suggest how they may serve as novel targets for primary cancer therapy.

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86 A variety of other mutations have been detected in CF patients [39] leading to ablation of protein synthesis (nonsense G542X, frameshift 394delTT, or splice junction 1717 G/A), blocked protein processing (missense N1303K or AA deletion in F508), blocked protein regulation (missense at G551D), altered conductance (missense R117H or R347P), and reduced protein synthesis (missense A455E, alternative splicing 3849 + 10kbC/T) [40]. Login to comment

[hide] Modifier genetics: cystic fibrosis. Annu Rev Genomics Hum Genet. 2005;6:237-60. Cutting GR
Modifier genetics: cystic fibrosis.
Annu Rev Genomics Hum Genet. 2005;6:237-60., [PMID:16124861]

Abstract [show]
Cystic fibrosis (CF) is the most common lethal autosomal recessive disorder in the Caucasian population, affecting about 30,000 individuals in the United States. The gene responsible for CF, the CF transmembrane conductance regulator (CFTR), was identified 15 years ago. Substantial variation in the many aspects of the CF phenotype among individuals with the same CFTR genotype demonstrates that factors independent of CFTR exert considerable influence on outcome in CF. To date, the majority of published studies investigating the cause of disease variability in CF report associations between candidate genes and some aspect of the CF phenotype. However, a definitive modifier gene for CF remains to be identified. Despite the challenges posed by searches for modifier effects, studies of affected twins and siblings indicate that genetic factors play a substantial role in intestinal manifestations. Identifying the factors contributing to variation in pulmonary disease, the primary cause of mortality, remains a challenge for CF research.

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133 As expected, mutations highly associated with pancreatic insufficiency, such as F508 and G542X, exist at higher frequency in patients with meconium ileus than those without meconium ileus (44, 79). Login to comment
544 Two cystic fibrosis patients with the genotype G542X/G551D. Login to comment

[hide] Gender-sensitive association of CFTR gene mutation... Mol Hum Reprod. 2005 Aug;11(8):607-14. Epub 2005 Aug 26. Morea A, Cameran M, Rebuffi AG, Marzenta D, Marangon O, Picci L, Zacchello F, Scarpa M
Gender-sensitive association of CFTR gene mutations and 5T allele emerging from a large survey on infertility.
Mol Hum Reprod. 2005 Aug;11(8):607-14. Epub 2005 Aug 26., [PMID:16126774]

Abstract [show]
Human infertility in relation to mutations affecting the cystic fibrosis transmembrane regulator (CFTR) gene has been investigated by different authors. The role of additional variants, such as the possible forms of the thymidine allele (5T, 7T and 9T) of the acceptor splice site of intron 8, has in some instances been considered. However, a large-scale analysis of the CFTR gene and number of thymidine residues, alone and in combination, in the two sexes had not yet been addressed. This was the aim of this study. Two groups were compared, a control group of 20,532 subjects being screened for perspective reproduction, and the patient group represented by 1854 idiopathically infertile cases. Analyses involved PCR-based CFTR mutations assessment, reverse dot-blot IVS8-T polymorphism analyses, denaturing gradient gel electrophoresis (DGGE) and DNA sequencing. The expected 5T increase in infertile men was predominantly owing to the 5/9 genotypic class. The intrinsic rate of 5T fluctuated only slightly among groups, but some gender-related differences arose when comparing their association. Infertile men showed a significantly enriched 5T + CFTR mutation co-presence, distributed in the 5/9 and 5/7 classes. In contrast, females, from both the control and the infertile groups, showed a trend towards a pronounced reduction of such association. The statistical significance of the difference between expected and observed double occurrence of 5T + CFTR traits in women suggests, in line with other reports in the literature, a possible survival-hampering effect. Moreover, regardless of the 5T status, CFTR mutations appear not to be involved in female infertility. These results underline the importance of (i) assessing large sample populations and (ii) considering separately the two genders, whose genotypically opposite correlations with these phenomena may otherwise tend to mask each other.

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47 CFTR gene alterations were first scored by PCR and reverse dot blot (Chehab and Wall, 1992), targeted to the detection of the following mutations: ∆F508, G85E, 541∆C, D110H, R117H, 621+1G→T, 711+5G→A, R334W, R334Q, T338I, 1078∆T, R347H, R352Q, ∆I507, 1609∆CA, E527G, 1717-1G→A, 1717-8G→A, G542X, R347P, S549N, S549R A→C, Q552X, R553X, A559T, D579G, Y577F, E585X, 1898+3A→G, 2183AA→G, R709X, 2789+5G→A, 3132∆TG, 3272-26A→G, L1077P, L1065P, R1070Q, R1066H, M1101K, D1152H, R1158X, R1162X, 3849+10KbC→T, G1244E, W1282R, W1282X, N1303K and 4016∇T. Login to comment
77 All these rare mutations, having been sought only in one partner, and only in the appropriate cases, are not included in the data discussed in Tables I, II and IV. Finally, as regards the mutations found in women of the control group, who bore 5T and a CFTR mutation, these 15 subjects presented eight cases of ∆F508 and single instances of the following: R117H, G542X, W1282X, R1162X, N1303K, 2183 aa/g and D1152H. Login to comment
101 Mutations Women (987) Men (867) N IVS8-T genotype N IVS8-T genotype ∆F508 16 15(7/9); 1(9/9) 26 15(7/9)*; 11(5/9) N1303K 4 4(7/9) 1 7/7 3849+10KbC→T 1 5/7 1 5/7 G542X 2 7/9 1 7/9† 2183AA→G 2 7/7 4 7/7 R553X 2 7/7 0 - R1162X 2 7/7 6 5(7/7)‡; 1(7/9) D1152H 0 - 3 2(7/7); 7/9† 711+5G→A 0 - 3 7/7 1717-8G→A 0 - 1 5/7 1717-1G→A 1 7/7 0 - Y577F 0 - 1 7/7 R117H 1 7/7 1 7/9* 621+3A→G 1 7/9 0 - W1282X 1 7/7 0 - deltaI1507 1 7/7 0 - T3381 1 7/7 1 7/9 R1066H 0 - 1 7/7§ R334Q 0 - 1 7/9 2789+5G→A 1 7/7 2 7/7‡§ Total 36¶ 53¶ records, all these mutations are normally found in trans with respect of 5T. Login to comment

[hide] Combining immunoreactive trypsinogen and pancreati... J Pediatr. 2005 Sep;147(3):302-5. Sarles J, Berthezene P, Le Louarn C, Somma C, Perini JM, Catheline M, Mirallie S, Luzet K, Roussey M, Farriaux JP, Berthelot J, Dagorn JC
Combining immunoreactive trypsinogen and pancreatitis-associated protein assays, a method of newborn screening for cystic fibrosis that avoids DNA analysis.
J Pediatr. 2005 Sep;147(3):302-5., [PMID:16182665]

Abstract [show]
OBJECTIVES: To evaluate the performance of a strategy in which, after immunoreactive trypsinogen (IRT) determination, genetic analysis is replaced by a biological test, the pancreatitis-associated protein (PAP) enzyme-linked immunosorbent assay (ELISA). STUDY DESIGN: The French newborn screening program includes cystic fibrosis (CF) screening by the IRT/CFTR mutation strategy. PAP was assayed on screening cards, in parallel with IRT, in all newborns from 5 French regions (n = 204,749). Analysis of PAP values in CF and non-CF newborns with elevated IRT allowed direct comparison between the current strategy and the proposed IRT/PAP strategy. RESULTS: A protocol in which newborns with IRT >50 ng/mL and PAP >1.8 ng/mL and those with IRT >100 ng/mL and PAP >1.0 ng/mL are directly recalled for sweat testing would have the same performance as the IRT/CFTR mutation strategy. CONCLUSIONS: The IRT/PAP strategy is an alternative for CF newborn screening, which avoids the drawbacks of genetic analysis and is cheaper and easier to implement than the current IRT/CFTR mutation strategy.

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44 A closer look at the results revealed that among the newborns with CF with moderately elevated IRT (50 to <100 ng/mL), 10 had genuine forms of the disease, their genotypes being DF508/DF508 (n = 6), DF508/P574H (n = 1), DF508/G542X (n = 1), DF508/G149R (n = 1), or DF508/? Login to comment

[hide] A comparison of high-resolution melting analysis w... Am J Clin Pathol. 2005 Sep;124(3):330-8. Chou LS, Lyon E, Wittwer CT
A comparison of high-resolution melting analysis with denaturing high-performance liquid chromatography for mutation scanning: cystic fibrosis transmembrane conductance regulator gene as a model.
Am J Clin Pathol. 2005 Sep;124(3):330-8., [PMID:16191501]

Abstract [show]
High-resolution melting analysis (HRMA) was compared with denaturing high-performance liquid chromatography (dHPLC) for mutation scanning of common mutations in the cystic fibrosis transmembrane conductance regulator gene. We amplified (polymerase chain reaction under conditions optimized for melting analysis or dHPLC) 26 previously genotyped samples with mutations in exons 3, 4, 7, 9, 10, 11, 13, 17b, and 21, including 20 different genotypes. Heterozygous mutations were detected by a change in shape of the melting curve or dHPLC tracing. All 20 samples with heterozygous mutations studied by both techniques were identified correctly by melting (100% sensitivity), and 19 were identified by dHPLC (95% sensitivity). The specificity of both methods also was good, although the dHPLC traces of exon 7 consistently revealed 2 peaks for wild-type samples, risking false-positive interpretation. Homozygous mutations could not be detected using curve shape by either method. However, when the absolute temperatures of HRMA were considered, G542X but not F508del homozygotes could be distinguished from wild type. HRMA easily detected heterozygotes in all single nucleotide polymorphism (SNP) classes (including A/T SNPs) and 1- or 2-base-pair deletions. HRMA had better sensitivity and specificity than dHPLC with the added advantage that some homozygous sequence alterations could be identified. HRMA has great potential for rapid, closed-tube mutation scanning.

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6 However, when the absolute temperatures of HRMA were considered, G542X but not F508del homozygotes could be distinguished from wild type. Login to comment
18 Materials and Methods Sample Source and Study Design Eleven commercially genotyped samples were obtained from Coriell Cell Repositories, Coriell Institute for Medical Research, Camden, NJ (Y122X, R334W, R347P, A455E, I507del, F508del, F508C, G542X/G542X, R553X, R560T, and M1101K). Login to comment
31 ❚Table 1❚ Mutations Analyzed in the Study Position From 5' Exon (or Intron) Genotype* No. of Samples Nucleotide Change SNP Class† End/Amplicon Size (bp) 3 394delTT 1 Del‡ - 132/234 4 R117H 1 G→A 1 83/270 Y122X 1 T→A 4 99/270 I148T 2 T→C 1 176/270 Intron 4 621+1 2 G→T 2 233/270 7 R334W 1 C→T 1 208/345 R347P 1 G→C 3 248/345 9 A455E 2 C→A 2 155/263 10 I507del 1 Del‡ - 171/292 F508del 3 Del‡ - 174/292 F508del/F508del 1 Del - 174/292 F508C 1 T→G 2 175/292 11 G542X 1 G→T 2 90/175 G542X/G542X 1 G→T 2 90/175 G551D 1 G→A 1 118/175 R553X 2 C→T 1 123/175 R560T 1 G→C 3 145/175 13 2184delA 1 Del‡ - 356/458 17b M1101K 1 T→A 4 196/292 21 N1303K 1 C→G 3 175/250 bp, base pairs; SNP, single nucleotide polymorphism. Login to comment
32 * All genotypes were heterozygous except homozygous F508del and G542X. Login to comment
49 Of 20 heterozygous samples, all were detected by melting and 19 by dHPLC for sensitivities of 100% and 95%, respectively. Homozygous mutations F508del and G542X could not be identified by melting analysis or dHPLC when only curve shapes were compared. Login to comment
50 However, when melting curve position and shape were considered, G542X homozygotes could be identified by high-resolution melting. Login to comment
75 Additional mutations in exons 9, 10, 11, and 21 included 7 heterozygous SNPs (A455E, F508C, G542X, G551D, R553X, R560T, and N1303K) and 2 heterozygous 3-base deletions (I507del and F508del). Login to comment
78 The dHPLC traces of homozygous G542X and F508del mutations were the same as wild-type control samples (Figure 3). Login to comment
80 G542X but not F508del homozygotes could be discerned from their wild types if curve position (absolute temperature) instead of curve shape was used for comparison. Login to comment
81 Nearest-neighbor calculations predict that the ∆Tm between wild type and G542X homozygotes is Time (min) Absorbance(mV) 0 1 2 3 4 5 20 - 19 - 18 - 17 - 16 - 15 - 14 - 13 - 12 - 11 - 10 - 9 - 8 - 7 - 6 - 5 - 4 - 3 - 2 - 1 - 0 - R347P het R334W het WT Temperature (°C) Fluorescence 82 83 84 85 86 87 100 - 90 - 80 - 70 - 60 - 50 - 40 - 30 - 20 - 10 - 0 - R334W het C::A T::G R347P het C::C G::G WT G::C Temperature (°C) Fluorescence 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 100 - 90 - 80 - 70 - 60 - 50 - 40 - 30 - 20 - 10 - 0 - WT 0 50 100 150 200 250 300 350 100 90 80 70 60 50 40 30 20 10 Temperature(°C) Base Pairs Temperature (°C) Fluorescence 75 76 77 78 79 80 100 - 90 - 80 - 70 - 60 - 50 - 40 - 30 - 20 - 10 - 0 - A B C D ❚Figure 2❚ High-resolution melting and denaturing high-performance liquid chromatography (dHPLC) analysis of exon 7 of the cystic fibrosis transmembrane conductance regulator gene. Login to comment
86 Analysis of 5 different wild types and the single available G542X homozygote revealed a shifted curve position (Figure 4), suggesting that absolute temperature differences may be used to identify homozygous sequence alterations. Login to comment
98 In comparison, Ravnik-Glavac et al28 reported correct detection of heterozygous Time (min) Absorbance(mV) 0 1 2 3 20 - 19 - 18 - 17 - 16 - 15 - 14 - 13 - 12 - 11 - 10 - 9 - 8 - 7 - 6 - 5 - 4 - 3 - 2 - 1 - 0 - G542X het G542X hom WT Time (min) Absorbance(mV) 0 1 2 3 20 - 19 - 18 - 17 - 16 - 15 - 14 - 13 - 12 - 11 - 10 - 9 - 8 - 7 - 6 - 5 - 4 - 3 - 2 - 1 - 0 - F508del het F508del hom WT A B ❚Figure 3❚ Denaturing high-performance liquid chromatography analysis of mutations G542X (exon 11) and F508del (exon 10) of the cystic fibrosis transmembrane conductance regulator gene. Login to comment
99 A, Elution patterns of the wild-type (WT), heterozygous (het) G542X, and homozygous (hom) G542X samples. Login to comment
101 Temperature (°C) Fluorescence 77 78 79 80 81 82 83 84 85 86 100 - 90 - 80 - 70 - 60 - 50 - 40 - 30 - 20 - 10 - 0 - G542X het WT G542X hom ❚Figure 4❚ High-resolution melting analysis of exon 11. Login to comment
102 Melting curves shown are 5 wild-type (WT) samples, 1 G542X heterozygote (het), and 1 G542X homozygote (hom). Login to comment
131 Homozygous SNP detection has been reported in PCR products up to 544 bp.19 In the present study, dHPLC missed the G542X homozygote, but melting analysis was able to identify the homozygous change using Tm differences. Login to comment

[hide] Newborn screening for cystic fibrosis in Wisconsin... J Pediatr. 2005 Sep;147(3 Suppl):S73-7. Rock MJ, Hoffman G, Laessig RH, Kopish GJ, Litsheim TJ, Farrell PM
Newborn screening for cystic fibrosis in Wisconsin: nine-year experience with routine trypsinogen/DNA testing.
J Pediatr. 2005 Sep;147(3 Suppl):S73-7., [PMID:16202788]

Abstract [show]
OBJECTIVE: To describe the development and follow-up confirmatory results of the routine cystic fibrosis (CF) newborn screening (NBS) program in Wisconsin. METHODS: CF NBS has been performed on a routine clinical basis in Wisconsin since July 1994. The 2-tiered immunoreactive trypsinogen (IRT)/DNA technique was used on dried blood on filter paper spots. From July 1994 to February 2002, mutation analysis was for the DeltaF508 allele. Beginning in March 2002, multimutation analysis of 25 CF mutations was performed. Infants with a positive result on NBS were seen in certified CF centers for sweat testing by means of quantitative pilocarpine iontophoresis, and families received genetic counseling. RESULTS: From July 1994 to February 2002, there were 120 cases of CF detected by means of NBS (509,794 infants screened), with 53 DeltaF508 homozygotes and 67 compound heterozygotes. There were 8 clinically diagnosed cases of CF (no DeltaF508 allele). The CF incidence was 1:3983 (95%CI, 1:3373-1:4774). From March 2002 to June 2003, multimutation analysis identified 21 cases of classic CF (90,142 infants screened). Sweat tests were successfully performed in infants younger than 1 month. CONCLUSIONS: Early diagnosis of CF through NBS was successfully performed, with an estimated sensitivity rate of 99% using the IRT/25 CFTR multimutation assay.

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30 Mutations included in this assay are 2184delA, A455E, DI507, DF508, G542X, G551D, R553X, R560T, 1717-1G>A, R1162X, 3659delC, N1303K, W1282X, R334W, R347P, 1078delT, R117H, I148T, 62111G>T, 278915G>A, 3849110kbC>T, G85E, 109811G>A, 71111G>T and 312011G>A. Login to comment
64 Two alleles, with 1 being an R117H: 5 cases of DF508/R117H(7T/9T), 1 case of G542X/ R117H(7T/9T), 1 case of R117H/R117H(5T/7T), and 1 case of I148T/R117H(7T/9T). Login to comment

[hide] Hyperacidity of secreted fluid from submucosal gla... Am J Physiol Cell Physiol. 2006 Mar;290(3):C741-9. Epub 2005 Oct 5. Song Y, Salinas D, Nielson DW, Verkman AS
Hyperacidity of secreted fluid from submucosal glands in early cystic fibrosis.
Am J Physiol Cell Physiol. 2006 Mar;290(3):C741-9. Epub 2005 Oct 5., [PMID:16207791]

Abstract [show]
Prior studies have shown that fluid secretions from airway submucosal glands in cystic fibrosis (CF) are reduced and hyperviscous, possibly contributing to the pathogenesis of CF airway disease. Because the CF transmembrane conductance regulator (CFTR) protein can transport both chloride and bicarbonate, we investigated whether gland fluid pH is abnormal in early CF, using nasal biopsies from pediatric subjects having minimal CF lung disease. Gland fluid pH, measured in freshly secreted droplets under oil stained with BCECF-dextran, was 6.57 +/- 0.09 (mean +/- SE) in biopsies from six CF subjects, significantly lower than 7.18 +/- 0.06 in eight non-CF biopsies (P < 0.01). To rule out the possibility that the apparent gland fluid hyperacidity in CF results from modification of fluid pH by the airway surface, a microcannulation method was used to measure pH in fluid exiting gland orifices. In pig trachea and human bronchi, gland fluid pH was reduced by up to 0.45 units by CFTR inhibitors, but was not affected by amiloride. Acid base transport in the surface epithelium of pig trachea was studied from pH changes in 300-nl fluid droplets deposited onto the oil-covered airway surface. The droplets had specified ionic composition/pH and/or contained transporter activators/inhibitors. We found evidence for CFTR-dependent bicarbonate transport by the tracheal surface epithelium as well as ATP/histamine-stimulated proton secretion, but not for sodium/proton or chloride/bicarbonate exchange. These results provide evidence for intrinsic hyperacidity in CF gland fluid secretions, which may contribute to CF airway pathology.

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90 Clinical characteristics of study subjects CF Subjects Age, Sex Genotype FEV1 Shwachman Brasfield CF1 13, F ⌬F508 - E60X 117% 85 22 CF2 22, M G542X - unknown 58% 70 15 CF3 10, M ⌬F508 - unknown 99% 80 23 CF4 20, M ⌬F508 - ⌬F508 71% 90 23 CF5 16, F ⌬F508 - unknown 79% 65 15 CF6 16, M ⌬F508 - ⌬F508 115% 75 19 Non-CF Subjects Age, Sex Diagnosis Procedure N1 3, F OSA T & A N2 2, M OSA T & A N3 10, F Recurrent tonsillitis T & A N4 10, M Recurrent tonsillitis Tonsillectomy N5 13, M Chronic cough T & A and BAL N6 4, M OSA T & A N7 6, F Recurrent tonsillitis T & A N8 3, M OSA T & A CF, cystic fibrosis; N, non-CF; OSA, obstructive sleep apnea; T & A, tonsillectomy and adenoidectomy; BAL, bronchoalveolar lavage; FEV1, forced expiratory volume in the first second. Login to comment
124 Genotype analysis showed that all subjects carried at least one copy of the ⌬F508 gene, except for one subject, whose genotype was G542X/ unknown. Login to comment

[hide] Cystic fibrosis transmembrane regulator gene carri... Gut. 2005 Nov;54(11):1661-2. McWilliams R, Highsmith WE, Rabe KG, de Andrade M, Tordsen LA, Holtegaard LM, Petersen GM
Cystic fibrosis transmembrane regulator gene carrier status is a risk factor for young onset pancreatic adenocarcinoma.
Gut. 2005 Nov;54(11):1661-2., [PMID:16227367]

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277 R McWilliams Department of Oncology and Department of Medicine, Mayo Clinic, Rochester, Minnesota, USA W E Highsmith Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA K G Rabe, M de Andrade, L A Tordsen Department of Health Sciences Research, Mayo Clinic, Rochester, Minnesota, USA Conflict of interest: None declared. Table 1 Comparison of CFTR mutation frequencies detected in the young onset pancreatic cancer cohort versus the clinical database Young onset pancreatic cancer cases (,60 y old at diagnosis, n = 166) Mayo Clinic clinical database reference group (n = 5349) No % No % CFTR mutation non-carriers 152 91.6 5132 95.9 CFTR mutation carriers 14 8.4 217 4.1 Mutation distribution DF508 12 85.7 155 71.4 R177H 1 7.1 28 12.9 G551D 6 2.8 2789+5G.A 6 2.8 G542X 4 1.8 N1303K 1 7.1 3 1.4 1717-1G.T 2 0.9 3849+10kbC.T 2 0.9 A455E 2 0.9 R1162X 2 0.9 R347H 1 0.5 R553X 1 0.5 3905insT 1 0.5 621+1G.T 1 0.5 W1282X 1 0.5 1898+1G.A 1 0.5 R560T 1 0.5 Young onset pancreatic cancer cases were more frequent carriers of the CFTR mutations compared with patients in the control database (odds ratio 2.18 (95% confidence interval 1.24-3.29); p = 0.006). Login to comment

[hide] Markedly elevated neonatal immunoreactive trypsino... Arch Dis Child. 2006 Mar;91(3):222-5. Epub 2005 Oct 21. Massie J, Curnow L, Tzanakos N, Francis I, Robertson CF
Markedly elevated neonatal immunoreactive trypsinogen levels in the absence of cystic fibrosis gene mutations is not an indication for further testing.
Arch Dis Child. 2006 Mar;91(3):222-5. Epub 2005 Oct 21., [PMID:16243854]

Abstract [show]
AIMS: To investigate the immunoreactive trypsinogen (IRT) values above the usual 99th centile laboratory cut-off and determine the value of offering further testing to those infants with a markedly elevated IRT but no cystic fibrosis transmembrane regulator (CFTR) gene mutation identified by the screening programme. METHODS: All babies born in Victoria, Australia, between 1991 and 2003, were screened by IRT followed by CF gene mutation analysis. RESULTS: Of the 806,520 babies born, 9268 with the highest IRT levels had CFTR mutation analysis. There were 123 DeltaF508 homozygotes and 703 heterozygotes (86 with CF, 617 carriers). A total of 8442 babies had no CFTR gene mutation, of whom 18 (0.21%) had CF. The total number of CF babies with IRT greater than the laboratory cut-off was 227 (2.4%). The IRT results of the CF patients were distributed normally, with the majority above the laboratory cut-off of newborn IRT results. There was no evidence of an excess of babies with CF in the very highest levels of IRT above the 99th centile. CONCLUSIONS: Only a small proportion of babies with a neonatal IRT >99th centile have CF. Additional CF testing for infants with an elevated IRT but no CFTR gene mutation has an extremely low yield, no matter how high the IRT result.

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220 Table 2 shows the relatively poor PPV of Table 1 Details of cystic fibrosis patients with IRT .99th centile but no DF508 mutation, Victoria, Australia, 1991-2003 Patient IRT (MoM) Genotype* Presentation Patient 1 2.77 R117H/2 Sibling with CF Patient 2 4.57 N1303K/2 Meconium ileus/sibling with CF Patient 3 3.28 2/2 Failure to thrive Patient 4 18.16 N1303K/N1303K Failure to thrive/recurrent cough Patient 5 2.98 V520F/2 Meconium ileus Patient 6 3.79 2/2 Meconium ileus Patient 7 6.65 G551D/3849 Failure to thrive/recurrent cough Patient 8 8.32 2/2 Failure to thrive Patient 9 6.45 2/2 Failure to thrive Patient 10 3.69 2/2 Clinical details not available Patient 11 13.81 2/2 Failure to thrive Patient 12 6.64 G542X/2 Recurrent chest infection Patient 13 5.51 2/2 Affected sibling Patient 14 3.95 G542X/2 Meconium ileus Patient 15 6.92 2/2 Recurrent chest infection Patient 16 6.82 2/2 Failure to thrive Patient 17 7.31 2/2 Failure to thrive Patient 18 7.66 2/2 Sibling with CF IRT, immunoreactive trypsinogen; MoM, multiple of median. Login to comment
222 *All patients underwent an extended CFTR mutation analysis for the following mutations in addition to DF508: G551D, R553X, G542X, R117H, N1303K, 621+1G-T, A455E, V520F, 1717-1G-A, W1282X, R1162X, 3849+10kbC-T, R347P, R334W, R560T, S549N. Login to comment

[hide] Haplotype block structure study of the CFTR gene. ... Eur J Hum Genet. 2006 Jan;14(1):85-93. Pompei F, Ciminelli BM, Bombieri C, Ciccacci C, Koudova M, Giorgi S, Belpinati F, Begnini A, Cerny M, Des Georges M, Claustres M, Ferec C, Macek M Jr, Modiano G, Pignatti PF
Haplotype block structure study of the CFTR gene. Most variants are associated with the M470 allele in several European populations.
Eur J Hum Genet. 2006 Jan;14(1):85-93., [PMID:16251901]

Abstract [show]
An average of about 1700 CFTR (cystic fibrosis transmembrane conductance regulator) alleles from normal individuals from different European populations were extensively screened for DNA sequence variation. A total of 80 variants were observed: 61 coding SNSs (results already published), 13 noncoding SNSs, three STRs, two short deletions, and one nucleotide insertion. Eight DNA variants were classified as non-CF causing due to their high frequency of occurrence. Through this survey the CFTR has become the most exhaustively studied gene for its coding sequence variability and, though to a lesser extent, for its noncoding sequence variability as well. Interestingly, most variation was associated with the M470 allele, while the V470 allele showed an 'extended haplotype homozygosity' (EHH). These findings make us suggest a role for selection acting either on the M470V itself or through an hitchhiking mechanism involving a second site. The possible ancient origin of the V allele in an 'out of Africa' time frame is discussed.

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30 The T2A rate was much lower than 1 Frequencies of the CFTR variants within the M or the V alleles exon or intron VARIANT SITES in the M genes (MM subjects) in the V genes (VV subjects) A 5' UTR 125 g/c 8/144 (0.056) 3/356 (0.008) -80 1 2 R31C 5/226 (0.004) 1/576 (0.002) -56 in M genes in V genes 6 2 R75Q 1/226 (0.004) 15/576 (0.026) -51 M V (ttga)n 0.461 0.017 7 3 G85E 0/226 (0) 1/576 (0.002) -51 2.214 0.362 (tg)n 0.616 0.114 B i 3 406-6 t/c 0/226 (0) 6/576 (0.010) -29 (t)n 0.499 0.036 8 4 R117H 2/226 (0.009) 0/576 (0) -29 10 4 I148T 3/224 (0.013) 0/576 (0) -29 C i 4 621+3 a/g 1/224 (0.004) 0/576 (0) -29 12 5 R170H 1/158 (0.006) 0/402 (0) -26 D i 6a 875+40 a/g 6/36 (0.167)c 0/118 (0)c -25 i 6b (ttga)6 13/36 (0.361) 1/118 (0.008) -23 E i 6b 1001+11 c/t 5/60 (0.083) 0/166 (0) -23 F i 8 1341+28 c/t 1/152 (0.007) 0/464 (0) -18 i 8 (tg)10 39/76 (0.513) 5/218 (0.023) -11 i 8 (tg)11 21/76 (0.276) 205/218 (0.940) -11 i 8 (tg)12 16/76 (0.211) 8/218 (0.037) -11 i 8 t5 4/76 (0.053) 2/218 (0.009) -11 i 8 t7 48/76 (0.632) 214/218 (0.982) -11 i 8 t9 24/76 (0.316) 2/218 (0.009) -11 16 10 M470V H ex 10 F508del 3/226 (0.013) 0/572 (0) 0 19 10 F508C 0/226 (0) 1/572 (0.002) 0 20 10 1716g/a 15/226 (0.066) 0/572 (0) 0 21 11 G542X 1/158 (0.006) 0/400 (0) +28 24 12 V562I 1/226 (0.004) 0/576 (0) +30 25 12 V562L 1/226 (0.004) 0/576 (0) +30 26 12 G576A 3/226 (0.013) 0/576 (0) +30 28 13 2082c/t 1/104 (0.010) 0/226 (0) +32 29 13 R668C 3/224 (0.013) 0/562 (0) +32 32 14a 2694t/g 45/70 (0.643) 9/208 (0.043) +35 I i 14a 2752-15 c/g 0/226 (0) 5/576 (0.009) +44 37 15 3030g/a 1/158 (0.006) 7/402 (0.017) +44 O i 15 3041-71 g/c 5/226 (0.022) 0/576 (0) +47 39 17a L997F 1/226 (0.004) 4/576 (0.007) +51 40 17a A1009T 0/226 (0) 1/572 (0.002) +51 42 17b F1052V 1/226 (0.004) 0/572 (0) +52 43 17b G1069R 1/226 (0.004) 0/572 (0) +52 44 17b Q1071H 1/226 (0.004) 0/572 (0) +52 45 17b 3417a/t 0/226 (0) 4/572 (0.007) +52 46 17b L1096R 1/226 (0.004) 0/572 (0) +52 52 19 3813a/g 0/118 (0) 1/484 (0.002) +68 53 19 S1235R 3/100 (0.030) 0/294 (0) +68 54 20 4002a/g 5/56 (0.089) 1/168 (0.006) +83 q in the M alleles q in the V alleles 56 21 4029a/g 0/194 (0) 3/506 (0.006) +93 57 21 N1303K 1/92 (0.011) 0/272 (0) +93 59 24 4404c/t 3/226 (0.013) 14/576 (0.024) +107 60 24 4521g/a 21/56 (0.375) 2/172 (0.012) +107 "slow evolution" markers "fast evolution" markers (i.e. STRs) H is the sum of the degrees of heterozygosity of all the markers Ref.No.a ABSOLUTE AND RELATIVE FREQUENCIES distance from the M470V siteb (Kb) H associated with the…. Login to comment

[hide] Mutations of the CFTR gene in idiopathic pancreati... Pancreas. 2005 Nov;31(4):350-2. Gullo L, Mantovani V, Manca M, Migliori M, Bastagli L, Pezzilli R
Mutations of the CFTR gene in idiopathic pancreatic hyperenzymemia.
Pancreas. 2005 Nov;31(4):350-2., [PMID:16258369]

Abstract [show]
OBJECTIVES: Idiopathic pancreatic hyperenzymemia is a new syndrome that is characterized by a chronic increase of serum pancreatic enzymes in the absence of pancreatic disease. The aim of this study was to assess whether mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene may have a role in the etiology of this hyperenzymemia. METHODS: Seventy subjects with idiopathic pancreatic hyperenzymemia, 44 men and 26 women (mean age, 48 years; range, 8-74 years), were studied. Thirteen of these 70 subjects had the familial form of the syndrome. The mutation analysis of the CFTR gene was carried out using diagnostic commercial kits for the simultaneous detection of 29 mutations and Tn polymorphism. RESULTS: Among the 70 subjects studied, 7 (10.0%) had CFTR gene mutations. None of these 7 subjects had the familial form of pancreatic hyperenzymemia. These mutations were DeltaF 508 in 1 subject, 2789 + 5 G > A in another subject, and T5 allele in the remaining 5. All these mutations were heterozygous, with the exception of 1 T5 allele that was homozygous in 1 subject. CONCLUSIONS: The frequencies of the mutations of the CFTR gene found in these subjects are similar to the carrier frequencies in the general Italian population. This finding does not support a role for CFTR gene mutations in the etiology of idiopathic pancreatic hyperenzymemia.

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52 A (i) 5 1078delT, R347P, R334W 7 A455E, Tn (i) 8, 9 DF508, DI507 10 G542X, 1717-1 G . Login to comment

[hide] Adherence of airway neutrophils and inflammatory r... Am J Physiol Lung Cell Mol Physiol. 2006 Mar;290(3):L588-96. Epub 2005 Nov 4. Tabary O, Corvol H, Boncoeur E, Chadelat K, Fitting C, Cavaillon JM, Clement A, Jacquot J
Adherence of airway neutrophils and inflammatory response are increased in CF airway epithelial cell-neutrophil interactions.
Am J Physiol Lung Cell Mol Physiol. 2006 Mar;290(3):L588-96. Epub 2005 Nov 4., [PMID:16272177]

Abstract [show]
Persistent presence of PMN in airways is the hallmark of CF. Our aim was to assess PMN adherence, percentage of apoptotic airway PMN (aPMN), and IL-6 and IL-8 production when aPMN are in contact with airway epithelial cells. Before coculture, freshly isolated CF aPMN have greater spontaneous and TNF-alpha-induced apoptosis compared with blood PMN from the same CF patients and from aPMN of non-CF patients. We then examined cocultures of PMN isolated from CF and non-CF airways with bronchial epithelial cells bearing mutated cftr compared with cftr-corrected bronchial epithelial cells. After 18-h coculture, the number of CF aPMN adhered on cftr-deficient bronchial epithelial cells was 2.3-fold higher compared with the coculture of non-CF aPMN adhered on cftr-corrected bronchial epithelial cells. The percentage of CF apoptotic aPMN (9.5 +/- 0.2%) adhered on cftr-deficient bronchial epithelial cells was similar to the percentage of non-CF apoptotic aPMN adhered on cftr-corrected bronchial epithelial cells (10.3 +/- 0.7%). IL-6 and IL-8 levels were enhanced 6.5- and 2.9-fold, respectively, in coculture of CF aPMN adhered on cftr-deficient bronchial epithelial cells compared with coculture of non-CF aPMN adhered on cftr-corrected bronchial epithelial cells. Moreover, blocking surface adhesion molecules ICAM-1, VCAM-1, and E-selectin on cftr-deficient bronchial epithelial cells with specific MAbs inhibited the adherence of CF aPMN by 64, 51, and 50%, respectively. Our data suggest that in CF patients a high number of nonapoptotic PMN adhered on airway epithelium associated with elevated IL-6 and IL-8 levels may contribute to sustained and exaggerated inflammatory response in CF airways.

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66 Sex Age, yr CF Genotype Clinical Score FVC, % FEV1, % Pseudomonas aeruginosa Colonization 1 M 15 ⌬F508/⌬F508 30 32 21 yes 2 M 16 ⌬F508/⌬F508 60 70 43 yes 3 M 17 ⌬F508/⌬F508 30 41 23 yes 4 F 4 ⌬F508/⌬F508 80 NA NA no 5 F 16 ⌬F508/⌬F508 50 53 41 yes 6 M 15 ⌬F508/⌬F508 30 33 20 yes 7 M 13 ⌬F508/⌬F508 50 61 37 yes 8 M 15 ⌬F508/⌬F508 70 70 50 no 9 F 21 R1066C/NI 50 60 77 yes 10 F 14 2183A/2183A 50 48 39 yes 11 M 14 G542X/2176insC 30 34 25 yes 12 M 17 ⌬F508/R560C 30 47 27 yes 13 M 17 IVS8 (5T)/IVS8 (5T) NA NA NA yes 14 F 18 3906insT/1609⌬CA 40 47 30 yes 15 F 13 G551D/S1235Rϩ5T 70 84 86 no 16 F 15 N1303K/347 del70 70 44 35 yes Clinical score, Schwachman-Kulczycki score; FVC, forced vital capacity; FEV1, forced expiratory volume in 1 s; M, male; F, female; NA, not applicable. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Tohoku J Exp Med. 2005 Dec;207(4):279-85. Uzun S, Gokce S, Wagner K
Cystic fibrosis transmembrane conductance regulator gene mutations in infertile males with congenital bilateral absence of the vas deferens.
Tohoku J Exp Med. 2005 Dec;207(4):279-85., [PMID:16272798]

Abstract [show]
Congenital bilateral absence of the vas deferens (CBAVD) is characterized by azoospermia and male infertility. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are associated with cystic fibrosis (CF), the most common autosomal recessive disorder in Caucasians. Recent publications on CBAVD raised the question whether CFTR gene mutations are responsible for CBAVD occurrence or not. This study was conducted to explore the role of CFTR gene mutations in the occurrence of CBAVD-dependent male infertility. Forty-four chromosomes of 22 CBAVD patients from Austrian ancestry were studied. For detection of the most common mutation DeltaF508, a deletion of phenylalanine at the 508th position of mature CFTR chloride channel protein, the 10th exon of the gene was screened by heteroduplex analysis. In order to identify non-DeltaF508 mutations, we also analyzed the entire coding regions, exon/intron boundaries of 27 exons and the 5'- and 3'-untranslated regions of the gene by denaturing gradient gel electrophoresis (DGGE) after polymerase chain reaction. All exons showing different banding patterns on the DGGE gels were sequenced to define existing DNA sequence variations. Among the analyzed 44 chromosomes of 22 patients, disease producing mutations were found in 31.8% (14/44). The most common mutation was DeltaF508 with a frequency of 43% (6/14), followed by R117H with 29% (4/14). Our results indicate that CFTR gene mutations are common but not the only reason for the occurrence of CBAVD-dependent male infertility. We recommend screening of the CFTR gene in these patients.

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28 Other CF mutations, G542X, G551D, D1152H, M470W, R334W, R74W, M952I, W1282X, N1303K, and G85E, are known to be involved in CBAVD etiology (Wang et al. 2002; Danziger et al. 2004). Login to comment
34 ΔF508 and G542X are known as severe alleles and R117H as a mild allele. Login to comment
63 The remaining two patients were heterozygous for the nonsense mutation G542X and missense mutation D1152H (Table 1). Login to comment
89 D1152H / -- 1725 ΔF508 mut. het ΔF508 / -- 1827 G542X Nonsense mutation, het. Login to comment
90 G542X / -- 1879 ΔF508 mut. het ΔF508 / -- 2097 ΔF508 mut. het ΔF508 / -- 2162 ΔF508 mut. het M952I Missense mutation, het. Login to comment
92 G542X Mutation. A G to T transversion at nucleotide position 1756 in exon 11 generates a nonsense mutation. Login to comment

[hide] Up-regulation of AMP-activated kinase by dysfuncti... J Biol Chem. 2006 Feb 17;281(7):4231-41. Epub 2005 Dec 18. Hallows KR, Fitch AC, Richardson CA, Reynolds PR, Clancy JP, Dagher PC, Witters LA, Kolls JK, Pilewski JM
Up-regulation of AMP-activated kinase by dysfunctional cystic fibrosis transmembrane conductance regulator in cystic fibrosis airway epithelial cells mitigates excessive inflammation.
J Biol Chem. 2006 Feb 17;281(7):4231-41. Epub 2005 Dec 18., 2006-02-17 [PMID:16361706]

Abstract [show]
AMP-activated kinase (AMPK) is a ubiquitous metabolic sensor that inhibits the cystic fibrosis (CF) transmembrane conductance regulator (CFTR). To determine whether CFTR reciprocally regulates AMPK function in airway epithelia and whether such regulation is involved in lung inflammation, AMPK localization, expression, and activity and cellular metabolic profiles were compared as a function of CFTR status in CF and non-CF primary human bronchial epithelial (HBE) cells. As compared with non-CF HBE cells, CF cells had greater and more diffuse AMPK staining and had greater AMPK activity than their morphologically matched non-CF counterparts. The cellular [AMP]/[ATP] ratio was higher in undifferentiated than in differentiated non-CF cells, which correlated with AMPK activity under these conditions. However, this nucleotide ratio did not predict AMPK activity in differentiating CF cells. Inhibiting channel activity in non-CF cells did not affect AMPK activity or metabolic status, but expressing functional CFTR in CF cells reduced AMPK activity without affecting cellular [AMP]/[ATP]. Therefore, lack of functional CFTR expression and not loss of channel activity in CF cells appears to up-regulate AMPK activity in CF HBE cells, presumably through non-metabolic effects on upstream regulatory pathways. Compared with wild-type CFTR-expressing immortalized CF bronchial epithelial (CFBE) cells, DeltaF508-CFTR-expressing CFBE cells had greater AMPK activity and greater secretion of tumor necrosis factor-alpha and the interleukins IL-6 and IL-8. Further pharmacologic AMPK activation inhibited inflammatory mediator secretion in both wild type- and DeltaF508-expressing cells, suggesting that AMPK activation in CF airway cells is an adaptive response that reduces inflammation. We propose that therapies to activate AMPK in the CF airway may be beneficial in reducing excessive airway inflammation, a major cause of CF morbidity.

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57 Six of the 10 CF HBE cell lines were from patients who were homozygous for the ⌬F508 CF mutation, and the remaining lines were from compound heterozygotes who had one ⌬F508 allele along with a Class I or III mutation in the other allele, including G542X, W1282X, and G551D (one each); one mutation was unknown. Login to comment

[hide] Novel and recurrent rearrangements in the CFTR gen... Hum Genet. 2006 Mar;119(1-2):126-36. Epub 2005 Dec 17. Hantash FM, Redman JB, Starn K, Anderson B, Buller A, McGinniss MJ, Quan F, Peng M, Sun W, Strom CM
Novel and recurrent rearrangements in the CFTR gene: clinical and laboratory implications for cystic fibrosis screening.
Hum Genet. 2006 Mar;119(1-2):126-36. Epub 2005 Dec 17., [PMID:16362824]

Abstract [show]
Because standard techniques used to detect mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene do not detect single or multiple exonic rearrangements, the importance of such rearrangements may be underestimated. Using an in-house developed, single-tube, semi-quantitative fluorescent PCR (SQF PCR) assay, we analyzed 36 DNA samples submitted for extensive CFTR sequencing and identified ten samples with rearrangements. Of 36 patients with classic CF, 10 (28%) harbored various deletions in the CFTR gene, accounting for 14% of CF chromosomes. A deletion encompassing the CFTR promoter and exons 1 and 2 was detected in a sample from one proband, and in the maternal DNA as well. In another family, a deletion of the promoter and exon 1 was detected in three siblings. In both of these cases, the families were African American and the 3120+1G > A splice site mutation was also identified. These promoter deletions have not been previously described. In a third case, a deletion of exons 17a, 17b, and 18 was identified in a Caucasian female and the same mutation was detected in the paternal DNA. In the other seven cases, we identified the following deletions: exons 2 and 3 (n = 2); exons 4, 5, and 6a; exons 17a and 17b; exons 22 and 23; and exons 22, 23, and 24 (n = 2). In our series, the frequency of CFTR rearrangements in classic CF patients, when only one mutation was identified by extensive DNA sequencing, was >60% (10/16). Screening for exon deletions and duplications in the CFTR gene would be beneficial in classic CF cases, especially when only one mutation is identified by standard methodologies.

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122 DNA from patient 5, a 4-month-old Ashkenazi Jewish infant with severe CF, harbored two point mutations: G542X (in Fig. 2 a Detection of a deletion of exons 17a-18 in a patient (b) and her father (c). Login to comment
127 The early onset of severe CF phenotype in the 4-month-old patient is likely the result of the G542X and the deletion of exons 4-6. Login to comment

[hide] Indirect CFTR mutation identification by PCR/OLA a... Genet Test. 2005 Winter;9(4):285-91. Stanziale P, Savino M, De Bonis P, Granatiero M, Zelante L, Bisceglia L
Indirect CFTR mutation identification by PCR/OLA anomalous electropherograms.
Genet Test. 2005 Winter;9(4):285-91., [PMID:16379540]

Abstract [show]
Mutations of CFTR gene are responsible for cystic fibrosis (CF) and other clinical conditions such as congenital absence of the vas deferens (CAVD), chronic pancreatitis (IP), and idiopathic disseminated bronchiectasis (DBE) classified as CFTR-related disorders. The PCR/OLA assay is designed to detect 31 known mutations including the 24 most common CF mutations worldwide, as identified by the CF Consortium. In order to define the CFTR genotype a series of 1812 individuals from central-southern Italy with and without CF manifestations were screened by using the PCR/OLA assay. Here we report the description of five cases of anomalous electropherograms obtained after PCR/OLA analysis, that led to the identification, in the homozygous state, of two point mutations (D110H and S589N) not included in the assay test panel, a large gene deletion (CFTRdel14b_17b), and an exonic polymorphism (c.4002A > G). Haplotype and real time PCR analysis were also performed in the subject carrying the large CFTR deletion. The study demonstrates that the PCR/OLA assay, besides being an efficient and user-friendly method to screen known mutations in the CFTR gene, may also function as a mutation/polymorphism-scanning assay, at least for certain nucleotide changes located in some critical regions of the gene.

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50 FREQUENCY DISTRIBUTION OF CFTR MUTATIONS IDENTIFIED IN 116 PATIENTS WITH CYSTIC FIBROSIS ORIGINATING FROM CENTRAL-SOUTHERN ITALY Mutations Allele frequency (%) F508del 47.41 G542X 9.48 N1303K 5.60 G85E 5.17 2789ϩ5GϾA 1.29 621ϩ1G-ϾT 1.29 R347P 1.29 R553X 1.29 S589N 1.29 W1282X 1.29 CFTRdele14b-17b 0.86 1717-1G-ϾA 0.43 2183 AA-ϾG 0.43 R1162X 0.43 R334W 0.43 711ϩ5G-ϾA 0.43 3849ϩ1OKbC-ϾT 0.43 Unidentified 21.12 A B C D GTTG-3Ј), 14bF (5Ј-GGGAGGAATAGGTGAAGAT-3Ј) and 14bR (5Ј-AATCCACTATGTTTGTATGTA-3Ј), 17bF (5Ј-AA- TGACATTTGTGATATGAT-3Ј) and 17bR (5Ј-ACTTTAG- CTAAGCATTTAAG-3Ј), respectively. Login to comment

[hide] Cystic fibrosis: terminology and diagnostic algori... Thorax. 2006 Jul;61(7):627-35. Epub 2005 Dec 29. De Boeck K, Wilschanski M, Castellani C, Taylor C, Cuppens H, Dodge J, Sinaasappel M
Cystic fibrosis: terminology and diagnostic algorithms.
Thorax. 2006 Jul;61(7):627-35. Epub 2005 Dec 29., [PMID:16384879]

Abstract [show]
There is great heterogeneity in the clinical manifestations of cystic fibrosis (CF). Some patients may have all the classical manifestations of CF from infancy and have a relatively poor prognosis, while others have much milder or even atypical disease manifestations and still carry mutations on each of the CFTR genes. It is important to distinguish between these categories of patients. The European Diagnostic Working Group proposes the following terminology. Patients are diagnosed with classic or typical CF if they have one or more phenotypic characteristics and a sweat chloride concentration of >60 mmol/l. The vast majority of CF patients fall into this category. Usually one established mutation causing CF can be identified on each CFTR gene. Patients with classic CF can have exocrine pancreatic insufficiency or pancreatic sufficiency. The disease can have a severe course with rapid progression of symptoms or a milder course with very little deterioration over time. Patients with non-classic or atypical CF have a CF phenotype in at least one organ system and a normal (<30 mmol/l) or borderline (30-60 mmol/l) sweat chloride level. In these patients confirmation of the diagnosis of CF requires detection of one disease causing mutation on each CFTR gene or direct quantification of CFTR dysfunction by nasal potential difference measurement. Non-classic CF includes patients with multiorgan or single organ involvement. Most of these patients have exocrine pancreatic sufficiency and milder lung disease. Algorithms for a structured diagnostic process are proposed.

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361 Examples include the G542X, G551D, R553X, W1282X and N1303K mutations. Login to comment

[hide] [New concepts of pathophysiology and therapy in cy... Pneumologie. 2005 Nov;59(11):811-8. Hirche TO, Loitsch S, Smaczny C, Wagner TO
[New concepts of pathophysiology and therapy in cystic fibrosis].
Pneumologie. 2005 Nov;59(11):811-8., [PMID:16385442]

Abstract [show]
Today, the majority of cystic fibrosis (CF) patients treated in Germany have reached adulthood. However, with increasing age the morbidity and frequency of severe pulmonary complications continues to rise. Further optimization of conventional therapy alone will be insufficient to compensate for this development. In recent years, there has been impressive progress in our understanding of the molecular basis of the CF gene and its product, the cystic fibrosis transmembrane conductance regulator (CFTR). This knowledge can now be applied to develop new therapeutic strategies. However, important questions remain to be solved, i. e., little is known about the pathways that link the malfunctioning of the CFTR protein with the observed clinical phenotype. This review briefly touches on CF genetics as it applies to lung disease and will focus on the current hypotheses of CFTR (dys)function and its impact on pulmonary fluid homeostasis. New treatment options that target the molecular basis of the disease will be discussed.

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61 1 Verteilung und Klassifikation der 10 häufigsten CFTR Mutationen in Deutschland 2003 (modifiziert nach [2]) CFTR Mutation identifizierte Mutationen häufigste Mutationen CFTR Mutationsklassea n (%) (%) I II III IV V ˜F508 6593 65,8 88,0 X R553X 172 1,7 2,3 X G542X 160 1,6 2,1 X N1303K 154 1,5 2,0 X G551D 141 1,4 1,9 X R347P 100 1,0 1,3 X 1717 ±1G fi A 61 0,6 0,8 X 3849 + 10 Kb C fi T 49 0,5 0,7 X W1282X 35 0,4 0,5 X R117H 25 0,3 0,4 X andere 524 5,1 gesamt n = 8014 79,9% 100% 7,6%b 88,0% 1,9% 1,7% 0,8% a Zur Einteilung der CFTR Mutationsklassen vergleiche Abb. 3. b Anteil der CFTR Mutationsklasse an den 10 häufigsten Mutationen [%] teinsynthese proportional zu der Schwere der pulmonalen Erkrankung war. Login to comment

[hide] Association of common haplotypes of surfactant pro... Pediatr Pulmonol. 2006 Mar;41(3):255-62. Choi EH, Ehrmantraut M, Foster CB, Moss J, Chanock SJ
Association of common haplotypes of surfactant protein A1 and A2 (SFTPA1 and SFTPA2) genes with severity of lung disease in cystic fibrosis.
Pediatr Pulmonol. 2006 Mar;41(3):255-62., [PMID:16429424]

Abstract [show]
Most individual cystic fibrosis transmembrane conductance regulator (CFTR) mutations appear not to correlate directly with severity of lung damage in cystic fibrosis (CF). Components of innate immunity, namely, mannose-binding lectin (MBL2), and surfactant protein A1 and A2 genes (SFTPA1 and SFTPA2), were shown to be critical in pulmonary host defenses. A pilot association study was conducted to identify genetic modifiers of lung disease in adult patients with CF. The structural and promoter (-221x/y) variants of MBL2, variants at codons 19, 50, 62, and 219 of SFTPA1, and at codons 9, 91, and 223 for SFTPA2, were studied in 135 adults with CF and compared to their forced expired volume in 1 sec (FEV1), diffusion of CO (DLCO), and other pulmonary scores. Predicted FEV1 was significantly lower in adults with the SFTPA1 6A3 allele and SFTPA2 1A1) allele (P = 0.01 and 0.009, respectively). The extended haplotype 6A3/1A1, which includes SFTPA1 and SFTPA2, was associated with lower pulmonary function, using FEV1 (P = 0.005) and poor pulmonary scores which were determined by American Medical Association, American Thoracic Society, and modified Shwachman-Kulczycki scores. Lower FEV1 and DLCO values were associated with MBL2 coding variants in those who had the DeltaF508 CFTR mutation (P = 0.03 and 0.004, respectively). These results support the current hypothesis that variants in pulmonary host defense molecules are potentially genetic modifiers of pulmonary disease in CF. Further work in larger populations is required to provide important new insights into the pathogenesis of CF.

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33 Complementary mutations were identified in 51 CF subjects: R117H (4), R347H (1), R347P (1), G542X (7), G551D (4), 1717-1G-A (2), 2789 þ 5G > A(3), 3120 þ 1G > A (2), 3659delC (3), 3849 þ 10kbC>T (6), 394delTT (1), 621 þ 1G>T (4), 711 þ 1G > T (1), G85E (1), I507 (1), N1303K (2), R352Q (1), R553X (2), R560T (1), and W1282X (4). Login to comment
35 Eleven subjects had rare mutations such as G551D/G551D, G551D/3659delC, G551D/I507, G551D/ Neg (2), E60X/Q493X, R1162X/G542X, W1282X/ W1282X (3), and 1717 À G > A/Neg. Login to comment

[hide] Detection of F508del mutation in cystic fibrosis t... Singapore Med J. 2006 Feb;47(2):129-33. Zilfalil BA, Sarina S, Liza-Sharmini AT, Oldfield NJ, Stenhouse SA
Detection of F508del mutation in cystic fibrosis transmembrane conductance regulator gene mutation among Malays.
Singapore Med J. 2006 Feb;47(2):129-33., [PMID:16435054]

Abstract [show]
INTRODUCTION: Cystic fibrosis (CF) is one of the common genetic disorders in the western world. It has been reported to be very rare in Asian populations. According to the Cystic Fibrosis Genetic Analysis Consortium, more than 1,000 mutations of the CF gene have been identified. The CF gene, named the cystic fibrosis transmembrane conductance regulator (CFTR), is located on chromosome 7 and composed of 27 exons. This study aims to detect possible CFTR gene mutations in Malays. METHODS: We analysed 50 blood samples from healthy Malays with no symptoms of CF. DNA was extracted from blood using commercially available extraction kits (Eppendorf, Germany). Identification of CFTR gene mutation was performed using the CF OLA (Oligonucleotide Ligation Assay) kit (Applied Biosystems, USA). The PCR-ligation products were electrophoresed on eight percent sequagel using an ABI PRISM 377 genetic analyser (Applied Biosystems, USA). Electrophoresis data was analysed using the Genotyper software and a report of the CF genotype for all loci tested was created using the CF Genotyper Template software. Out of 50, one sample (two percent) was detected to have the F508del mutation (3bp deletion at exon 10), which is one of the most common CFTR gene mutations in Caucasians. RESULTS: The F508del mutation allele was detected in one subject. This indicates that she was a CF carrier. CONCLUSION: We report the finding of a carrier of the F508del mutation of the CFTR gene in the Malay population. Our finding revealed that CF could also affect the Malay population. Larger studies are necessary to determine the exact gene frequency of this population.

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55 MUTATIONS R553X G551D 1507 del F508 del 1717-1 G>A G542X R560T R347P W1282X R334W 1078 Del T 3849 + 10KB C>T R1162X N1303K 3659 Del C A455E R117H 2183 AA>G 2789+5 G>A 1898 +1 G>A 621+1 G>T 711+1 G>T G85E S549N S549R V520F Q493X R347H 3849 +4 A>G 3905 INS T Y122X 4 software before running the gel electrophoresis in 1X TBE using ABI PRISM® 377 Genetic Analyzer (Applied Biosystems, USA) for 45 minutes. Login to comment

[hide] On the discovery and development of CFTR chloride ... Curr Pharm Des. 2006;12(4):471-84. Becq F
On the discovery and development of CFTR chloride channel activators.
Curr Pharm Des. 2006;12(4):471-84., [PMID:16472140]

Abstract [show]
Chloride channels play important roles in vital cellular signalling processes contributing to homeostasis in both excitable and non-excitable cells. Since 1987, more than ten ion channel genes have been identified as causing human hereditary diseases among them the genes for the voltage-dependent chloride channel ClC-1 (myotonia) and the cystic fibrosis transmembrane conductance regulator (CFTR) protein (cystic fibrosis). The CFTR gene was cloned in 1989 and its protein product identified as an ATP-gated and phosphorylation-regulated chloride channel during the following two years. Since then, searching for potent and specific small molecules able to modulate normal and mutated CFTR has become a crucial endpoint in the field for both our understanding of the physiological role that CFTR plays in epithelial cells and more importantly for the development of therapeutic agents to cure cystic fibrosis (CF). It is predicted that a pharmacological approach would help not only to restore the defective transport activity of mutant CFTR but also to correct the regulatory function of CFTR. This review describes the evolution of CFTR pharmacology and how during the last five years, high throughput screening assays have been developed to identify novel molecules, some of them probably constituting a reservoir of future therapeutic agents for CF.

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38 Corresponding proteins are degraded rapidly or alternatively no protein is produced (e.g. stop mutations: W1282X, G542X). Login to comment

[hide] Analyzing DNA from buccal cells is a reliable meth... Genet Med. 2006 Mar;8(3):175-7. de Vries TW, Ajubi N, Slomp J, Storm H
Analyzing DNA from buccal cells is a reliable method for the exclusion of cystic fibrosis. Results of a pilot study.
Genet Med. 2006 Mar;8(3):175-7., [PMID:16540752]

Abstract [show]
PURPOSE: In children there is frequently a reason to exclude cystic fibrosis. Sweat testing is used for this. Because sweat testing has some disadvantages we investigated whether analyzing DNA for the local most common CFTR mutations, harvested from buccal cells, is reliable as a method to exclude cystic fibrosis. METHODS: In patients in whom a sweat test had been ordered during the period January 1, 2002 to December 31, 2004, we harvested buccal cell DNA for analysis. When blood was available, DNA from leukocytes was also analyzed. RESULTS: A total of 73 sweat tests were ordered during the two-year study period, mostly because of recurrent pulmonary infections (36; 49%), failure to thrive (20; 27%) and chronic diarrhea (10, 14%). In 70, children the results of the sweat test were normal, in three patients the results were borderline. Sixty buccal smears were analyzed and no patient was homozygous for cystic fibrosis, two were heterozygous for cystic fibrosis. In none of the children the diagnosis of cystic fibrosis was established. CONCLUSION: Analyzing DNA in cells, harvested from the buccal cells, is a reliable alternative to exclude cystic fibrosis. It is safe, simple, and child-friendly.

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36 3 b r i e f r e p o r t Genetics IN Medicine Polymerase Chain Reaction (PCR) PCR followed by restriction-fragment length polymorphism (RFLP) was performed to analyze delta F508, G542X, G551D, R553X, N1303K, and A455E according to previously described methods on a Perkin Elmer PE 2400 thermocycler.5 As an alternative, the delta F508 mutation was also analyzed by means of amplification refraction mutation system (ARMS) using the following primer combination: common reverse primer 5=GGGTAGTGTGAAGGGTTCATATGCATAATC3=, Wildtype Forward primer 5=GCCTGGCACCATTAAAGAA- AATATCATCTT3=, and Mutant Forward primer 5=GCCTG- GCACCATTAAAGAAAATATCATTGG3=.6 After PCR was performed, amplicons were digested (in the case of RFLP) using appropriate restriction enzymes as previously described. Login to comment

[hide] Clinical doses of amikacin provide more effective ... J Mol Med (Berl). 2006 Jul;84(7):573-82. Epub 2006 Mar 16. Du M, Keeling KM, Fan L, Liu X, Kovacs T, Sorscher E, Bedwell DM
Clinical doses of amikacin provide more effective suppression of the human CFTR-G542X stop mutation than gentamicin in a transgenic CF mouse model.
J Mol Med (Berl). 2006 Jul;84(7):573-82. Epub 2006 Mar 16., [PMID:16541275]

Abstract [show]
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the disease cystic fibrosis. We previously reported that gentamicin administration suppressed a CFTR premature stop mutation in a Cftr-/- mouse model carrying a human CFTR-G542X (hCFTR-G542X) transgene, resulting in the appearance of hCFTR protein and function. However, the high doses used in that study resulted in peak serum levels well beyond the levels typically administered to humans. To address this problem, we identified doses of both gentamicin and amikacin that resulted in peak serum levels within their accepted clinical ranges. We then asked whether these doses could suppress the hCFTR-G542X mutation in the Cftr-/- hCFTR-G542X mouse model. Our results indicate that low doses of each compound restored some hCFTR protein expression and function, as shown by immunofluorescence and short-circuit current measurements. However, we found that amikacin suppressed the hCFTR-G542X premature stop mutation more effectively than gentamicin when administered at these clinically relevant doses. Because amikacin is also less toxic than gentamicin, it may represent a superior choice for suppression therapy in patients that carry a premature stop mutation in the CFTR gene.

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6 David M. Bedwell Clinical doses of amikacin provide more effective suppression of the human CFTR-G542X stop mutation than gentamicin in a transgenic CF mouse model Received: 31 October 2005 / Accepted: 9 January 2006 / Published online: 16 March 2006 # Springer-Verlag 2006 Abstract Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the disease cystic fibrosis. Login to comment
7 We previously reported that gentamicin administration suppressed a CFTR premature stop mutation in a Cftr-/- mouse model carrying a human CFTR-G542X (hCFTR-G542X) transgene, resulting in the appearance of hCFTR protein and function. Login to comment
9 We then asked whether these doses could suppress the hCFTR-G542X mutation in the Cftr-/- hCFTR-G542X mouse model. Login to comment
11 However, we found that amikacin suppressed the hCFTR-G542X premature stop mutation more effectively than gentamicin when administered at these clinically relevant doses. Login to comment
42 We previously reported that the aminoglycoside gentamicin is capable of suppressing CFTR premature stop mutations in a CF transgenic mouse model, in which an hCFTR cDNA containing the G542X premature stop mutation was expressed under the control of the rat intestinal fatty acid binding protein (FABP) promoter in a Cftr-/- mouse. Login to comment
46 This hCFTR-G542X Cftr-/- mouse was used to show that once daily administration of 34 mg/kg of gentamicin or tobramycin via subcutaneous injections could suppress the G542X nonsense mutation and partially restore expression of functional hCFTR protein [20]. Login to comment
50 In a previous study, we showed that gentamicin suppressed the hCFTR-G542X nonsense mutation in vivo, although peak serum concentrations used were well above the levels allowed during the recommended clinical use of these compounds [20]. Login to comment
52 In the current study, we examined whether gentamicin and amikacin could suppress the G542X mutation when administered at doses within the recommended therapeutic range of serum peak and trough levels. Login to comment
53 We obtained evidence that both compounds could suppress the hCFTR-G542X mutation in the Cftr-/- mouse model and partially restore functional CFTR expression when administered at doses that produced serum levels in the recommended range. Login to comment
54 However, our results indicate that amikacin suppressed the hCFTR-G542X mutation in vivo much more effectively than gentamicin under these conditions, suggesting that it may represent a better choice for the suppression of the hCFTR-G542X stop mutation (and possibly other premature stop mutations that cause CF). Login to comment
55 Materials and methods Plasmid construction and generation of transgenic mice Expression of the hCFTR cDNA transgene that contained the G542X (UGA) premature stop mutation was driven by the rat FABP promoter. Login to comment
56 The plasmid construction of the FABP-hCFTR-G542X transgene and generation of the Cftr-/- hCFTR-G542X were described previously [20]. Login to comment
57 In vitro translation system A modified form of a readthrough reporter system, previously used to monitor the suppression of stop mutations by aminoglycosides [4, 5], was used to determine whether amikacin (Calbiochem) could suppress the hCFTR-G542X mutation in an in vitro rabbit reticulocyte lysate (RRL) translation system (Promega TNT system). Login to comment
60 In the present study, the readthrough cassette contained the hCFTR-G542X premature stop codon and six upstream and downstream codons from the hCFTR gene. Login to comment
64 Efficient translation termination at the G542X stop mutation produced a 25-kDa protein, while suppression of the stop codon resulted in the synthesis of a 35-kDa protein. Login to comment
66 Suppression of the G542X mutation was expressed as percent readthrough, which was calculated as the 35 kda protein= 25 kda þ 35 kda proteinsð Þ½ Â 100: Aminoglycoside treatment Aminoglycoside treatment of homozygous Cftr-/- mice carrying the FABP-hCFTR-G542X transgene was initiated 16 days after birth (1 week before weaning). Login to comment
91 Results Amikacin suppresses the hCFTR-G542X premature stop mutation in a mammalian in vitro translation system The ability of an aminoglycoside to suppress a premature stop mutation depends on several factors, including the specific features of its chemical structure, the identity of the stop codon to be suppressed, and the sequence context surrounding the stop codon in the mRNA [4, 5]. Login to comment
93 Although amikacin has been shown to suppress premature stop mutations in several mammalian mRNAs in vitro [5, 17], it has not yet been shown to suppress the CFTR-G542X mutation in vitro. Login to comment
94 We previously reported that gentamicin and tobramycin suppressed the hCFTR-G542X mutation in a mammalian RRL translation system [20]. Login to comment
96 Accordingly, we first asked whether amikacin, an aminoglycoside commonly used to treat bacterial infections, could also suppress the hCFTR-G542X mutation using the RRL translation system. Login to comment
97 Initially, we cloned the hCFTR-G542X premature stop codon and six flanking codons on either side into a readthrough reporter plasmid (Fig. 1) [20]. Login to comment
98 In this reporter system, a 25-kDa protein is produced if termination occurs at the G542X premature stop mutation, while a 35-kDa protein is produced if the G542X stop codon is suppressed. Login to comment
99 This construct was expressed in a RRL-based coupled transcription/translation system in the presence of increasing concentrations of gentamicin or amikacin, and the concentration of each aminoglycoside that induced the maximum level of readthrough of the G542X mutation (without inhibiting total protein synthesis) was determined. Login to comment
100 While both compounds were found to suppress termination at the G542X mutation, gentamicin induced readthrough at much lower concentrations than amikacin. Login to comment
101 The maximum level of G542X readthrough induced by amikacin (11%) was similar to that induced by gentamicin (14%). Login to comment
106 These results demonstrate that amikacin can effectively suppress the hCFTR-G542X mutation without inhibiting total protein synthesis as observed with gentamicin. Login to comment
112 In a previous study, we reported that the subcutaneous injection of 34 mg/kg gentamicin once daily suppressed the G542X mutation in Cftr-/- hCFTR-G542X mice and restored a significant level of functional hCFTR protein [20]. Login to comment
114 In the current study, we sought to determine whether aminoglycosides could suppress the hCFTR-G542X mutation in our mouse model when administered at concentrations that produced serum levels well below the maximum recommended for humans. To determine an acceptable dose of each aminoglycoside, mice were injected subcutaneously with various concentrations of each compound. Login to comment
118 The hCFTR-G542X UGA mutation (boxed) and surrounding context are indicated by the expanded sequence. Login to comment
119 Termination at the G542X mutation results in a 25-kDa protein, while suppression of that mutation allows continued elongation and the production of a 35-kDa protein. Login to comment
120 b Data showing suppression of the G542X mutation in an RRL in vitro translation system. Login to comment
121 c Quantitation of G542X readthrough from data shown in b samples were then collected at specified times after administration and serum concentrations were determined using FPIA (Fig. 2). Login to comment
129 A clinical dose of gentamicin suppresses the hCFTR-G542X mutation but restores only a barely detectable amount of hCFTR protein We initially used an immunofluorescence assay to ask whether a once daily, low dose of 5 mg/kg gentamicin can suppress the hCFTR-G542X mutation. Login to comment
131 A similar immunofluorescence assay was performed using intestinal tissues from untreated Cftr-/- hCFTR-G542X mice or from mice treated with either 34 mg/kg or 5 mg/kg gentamicin (Fig. 3a). Login to comment
133 In the samples treated with the hCFTR-specific antiserum, no hCFTR protein was detected in the intestinal tissue from untreated Cftr-/- hCFTR-G542X mice, while a strong hCFTR protein signal was detected at the apical surface of epithelial cells in the submucosal glands of the duodenum from mice treated with 34 mg/kg gentamicin. Login to comment
135 These results suggest that this lower, clinically relevant dose of gentamicin can suppress the G542X mutation and restore hCFTR protein in Cftr-/- hCFTR-G542X mice, although the level of hCFTR protein detected was substantially less than was observed in animals treated with the higher dose. Login to comment
136 A clinical dose of amikacin suppresses the hCFTR-G542X mutation and restores a significant amount of hCFTR protein expression We next asked whether amikacin could suppress the hCFTR-G542X mutation in the Cftr-/- hCFTR-G542X mouse. Login to comment
138 Because 34 mg/kg gentamicin was well-tolerated by Cftr-/- hCFTR-G542X mice in our previous study [20] and the doses of amikacin routinely recommended for human use are several folds higher than gentamicin [28], we initially chose to compare a high dose of 170 mg/kg amikacin and a low dose of 15 mg/kg amikacin. Login to comment
140 To determine the ability of amikacin to suppress the hCFTR-G542X mutation, the same administration protocol was used and intestinal tissues from mice were assayed by immunofluorescence using either preimmune serum or an hCFTR-specific polyclonal antiserum. Login to comment
141 No hCFTR protein was detected in samples from untreated Cftr-/- hCFTR-G542X mice using hCFTR-specific antiserum or in samples from treated mice using preimmune serum. Login to comment
144 These results indicate that amikacin can restore a significant amount of hCFTR protein in the Cftr-/- hCFTR-G542X mice when administered at either a very high dose or at a lower dose that produces a peak serum level well within the recommended clinical range. Login to comment
147 The aminoglycoside concentration in each serum sample was determined by fluorescence polarization immunoassay Analysis of hCFTR activity in Cftr-/- hCFTR-G542X mice after treatment with clinical doses of gentamicin or amikacin The hCFTR protein is a cyclic adenosine monophosphate (cAMP)-activated chloride channel that facilitates transepithelial chloride conductance upon activation by cAMP agonists such as forskolin. Login to comment
148 We previously demonstrated that cAMP-dependent transepithelial chloride conductance could be detected in intestinal tissues of Cftr-/- hCFTR-G542X mice that had been treated with 34 mg/kg gentamicin [20]. Login to comment
150 Figure 4 shows representative short-circuit current tracings obtained from Cftr-/- hCFTR-G542X mouse intestinal tissues that were harvested from untreated Cftr-/- hCFTR-G542X mice, or from Cftr-/- hCFTR-G542X mice treated once daily with either 5 mg/kg gentamicin or 15 mg/kg amikacin for 2 to 3 weeks. Login to comment
152 Table 1 provides a summary of data collected from short-circuit current tracings of intestinal tissues harvested from untreated Cftr+/+ hCFTR-G542X mice, +/+ hCFTR-G542X mice treated with low doses of gentamicin or amikacin, untreated Cftr-/- hCFTR-G542X mice, and Cftr-/- hCFTR-G542X mice treated with high or low doses of gentamicin or amikacin. Login to comment
158 a Samples from the duodenum of homozygous Cftr -/-hCFTR-G542X mice (untreated or treated with 5 or 34 mg/kg gentamicin). Login to comment
159 b Samples from the duodenum of homozygous Cftr-/-hCFTR-G542X mice (untreated or treated with 15 or 170 mg/kg amikacin). Login to comment
163 A P<0.05 value was considered as significant In untreated Cftr-/- hCFTR-G542X mice used as negative controls, we observed a cAMP-stimulated short-circuit current in only 8% of the samples (1/12), resulting in an average current of only 0.2 μA/cm2 . Login to comment
164 In our experimental samples, we found that 63% of samples (5/8) from Cftr-/- hCFTR-G542X mice treated with 34 mg/kg gentamicin yielded cAMP-activated short-circuit currents after forskolin addition, resulting in an average current of 1.67 μA/cm2 . Login to comment
165 In contrast, 35% of samples (7/20) from Cftr-/- hCFTR-G542X mice treated with 5 mg/kg of gentamicin yielded cAMP-stimulated short-circuit currents, resulting in an average current of 0.82 μA/cm2 . Login to comment
167 They also demonstrate that the administration of a clinically relevant dose of gentamicin to Cftr-/- hCFTR-G542X mice can restore a statistically significant increase (P value<0.05) in cAMP-stimulated chloride currents relative to untreated controls, consistent with a partial restoration of CFTR protein and activity. Login to comment
169 We found that treatment of Cftr-/- hCFTR-G542X mice with 170 mg/kg of amikacin once daily for 23 weeks yielded a short-circuit response in 75% of intestinal tissues assayed (9/12), resulting in an average current of 1.77 μA/cm2 . Login to comment
173 Discussion The results of this study show that amikacin, like gentamicin, can effectively suppress the hCFTR-G542X stop mutation in a transgenic CF mouse model. Login to comment
174 In fact, we found that the low dose of amikacin tested in this study appears to suppress the hCFTR-G542X mutation more effectively than the low dose of gentamicin. Login to comment
182 a Tracing from the ileum of a homozygous Cftr-/- hCFTR-G542X mouse without any prior aminoglycoside treatment. Login to comment
183 b Tracing from the ileum of a homozygous Cftr-/- hCFTR-G542X mouse after 5 mg/kg gentamicin treatment once daily for 2-3 weeks. Login to comment
184 c Tracing from the ileum of a homozygous Cftr-/- hCFTR-G542X mouse after 15 mg/kg amikacin treatment once daily for 2-3 weeks. Login to comment
210 The expression of the human CFTR-G542X cDNA in this animal will undoubtedly be different than the expression of endogenous mouse Cftr that contains a premature stop mutation. Login to comment
212 In our study, we found that the administration of amikacin at doses that produced serum levels within the clinically accepted range could suppress the hCFTR-G542X mutation and restore functional hCFTR protein. Login to comment
213 While gentamicin was also found to suppress the hCFTR-G542X mutation when administered in doses that produced serum levels in the accepted clinical range, the levels of suppression appeared to be significantly less than those observed with amikacin. Login to comment

[hide] Association of improved pulmonary phenotype in Iri... Pediatr Pulmonol. 2006 Jun;41(6):584-91. Courtney JM, Plant BJ, Morgan K, Rendall J, Gallagher C, Ennis M, Kalsheker N, Elborn S, O'Connor CM
Association of improved pulmonary phenotype in Irish cystic fibrosis patients with a 3' enhancer polymorphism in alpha-1-antitrypsin.
Pediatr Pulmonol. 2006 Jun;41(6):584-91., [PMID:16617455]

Abstract [show]
Modifier genes other than CFTR are thought to influence lung disease phenotype in cystic fibrosis (CF). In this study, we investigated the relationship between a polymorphism (1237 G --> A) in the 3' enhancer region of the alpha-1-antitrypsin (AAT) gene and pulmonary disease severity in 320 CF patients recruited from two independent adult referral centers in Ireland, and evaluated the in vivo effect of the polymorphism on AAT levels during acute infection. When corrected for confounding variables, the polymorphism was found to make a small but significant contribution to variance in percent predicted forced expired volume in 1 sec (FEV1) (1.1%, P = 0.05), with possession of the A allele being associated with better pulmonary function (AA/AG genotype: percent predicted FEV1, 70.8 +/- 3.9; GG genotype: percent predicted FEV1, 62.0 +/- 1.4). As would be expected of a modifier effect, the influence of the polymorphism was more marked in patient groups traditionally associated with more severe lung disease, contributing 3.2% (P = 0.033) to the variance in percent predicted FEV1 in patients homozygous for DF508, 3.3% (P = 0.007) to those infected with Pseudomonas aeruginosa, and 3% (P = 0.024) in female patients. In each instance, a positive association between possession of the A variant and higher percent predicted FEV1 was observed. We did not, however, find any evidence that possession of the A allele effected upregulation of AAT during acute infection in vivo. This lack of a demonstrable functional effect in vivo suggests that the polymorphism is a marker for a modifying effect on pulmonary phenotype in the Irish CF population by a mechanism that is yet to be explained.

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80 T 1.2% 1.7% 1.4% Nonmild R560T 1.5% 1.3% 1.4% Nonmild G542X 0.7% 1.7% 1.1% Nonmild E60X 0.5% 0.9% 0.6% Mild R553X 0.0% 1.3% 0.5% Nonmild N103K 0.7% 0.0% 0.5% Nonmild 9DELTT 0.0% 0.9% 0.3% Mild 3849 þ 10 kb C > T 0.0% 0.9% 0.3% Mild R75Q 0.0% 0.9% 0.3% Mild 1717 þ 1 G > A/À 0.5% 0.0% 0.3% Mild D1507 0.5% 0.0% 0.3% Mild Minor alleles 9.5% 27.4% 15.9% Mild 1 Alleles listed individually occur at a frequency of !0.5% in either population. Login to comment

[hide] The relevance of sweat testing for the diagnosis o... Clin Biochem Rev. 2005 Nov;26(4):135-53. Mishra A, Greaves R, Massie J
The relevance of sweat testing for the diagnosis of cystic fibrosis in the genomic era.
Clin Biochem Rev. 2005 Nov;26(4):135-53., [PMID:16648884]

Abstract [show]
Cystic fibrosis (CF) is the most common inherited disorder of childhood. The diagnosis of CF has traditionally been based on clinical features with confirmatory evidence by sweat electrolyte analysis. Since 1989 it has been possible to also use gene mutation analysis to aid the diagnosis. Cloning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has advanced our understanding of CF, in particular the molecular basis of an expanded CF phenotype. However, because there are over 1000 mutations and 200 polymorphisms, many without recognised effects on CFTR, the molecular diagnosis can be troublesome. This has necessitated measurement of CFTR function with renewed interest in the sweat test. This review provides an overview of the clinical features of CF, the diagnosis and complex genetics. We provide a detailed discussion of the structure and function of CFTR and the classification of CFTR mutations. Sweat electrolyte analysis is discussed, from the physiology of sweating to the rigours of a properly performed sweat test and its interpretation. With this information it is possible to understand the relevance of the sweat test in the genomic era.

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94 These mechanisms of CFTR dysfunction are intended to provide a framework for understanding the molecular basis of epithelial cell abnormalities in CF.71 Class 1: Defective Protein Synthesis Mutations in this class include the most severe CF phenotypes resulting in no protein being synthesised.2,59,67 The most common Class I mutation is G542X, which either prevents the synthesis of a stable protein or results in the production of a truncated protein due to the creation of a premature termination codon.67 The aminoglycoside antibiotics can suppress premature termination codons by permitting translation to continue to the normal termination of the transcript.72 This has shown to be promising in in vitro and in clinical trials but further studies need to be performed to make it a safer compound that may be administered to children with this class of mutation from the time of diagnosis.72 Class II: Defective Protein Processing Mutations in this class result in a CFTR protein that fails to traffic to the correct cellular localisation due to mis-folding of the protein. Login to comment
131 However there is a diminishing chance of detecting a mutation after ∆F508 which has an allele frequency in CF of 70%, when one considers that the next most common mutations (in Australia) are G551D (5%), G542X (2.4%), N1303K (1.3%) andmostothermutationshaveanallelefrequencysignificantly less than 0.5%. Login to comment

[hide] Variants in the glutamate-cysteine-ligase gene are... Am J Respir Crit Care Med. 2006 Aug 15;174(4):415-9. Epub 2006 May 11. McKone EF, Shao J, Frangolias DD, Keener CL, Shephard CA, Farin FM, Tonelli MR, Pare PD, Sandford AJ, Aitken ML, Kavanagh TJ
Variants in the glutamate-cysteine-ligase gene are associated with cystic fibrosis lung disease.
Am J Respir Crit Care Med. 2006 Aug 15;174(4):415-9. Epub 2006 May 11., 2006-08-15 [PMID:16690975]

Abstract [show]
BACKGROUND: Chronic progressive lung disease is the most serious complication of cystic fibrosis (CF). Glutathione plays an important role in the protection of the CF lung against oxidant-induced lung injury. OBJECTIVES: We hypothesized that a polymorphism in a novel candidate gene that regulates glutathione synthesis might influence CF lung disease. METHODS: In a cross-sectional study, subjects were recruited from CF clinics in Seattle and multiple centers in Canada. We tested for an association between CF lung disease and a functional polymorphism in the glutamate-cysteine ligase catalytic subunit (GCLC) gene. Multiple linear regression was used to test for association between polymorphisms of GCLC and severity of CF lung disease while adjusting for age, Pseudomonas aeruginosa infection, and cystic fibrosis transmembrane conductance regulator (CFTR) genotype. Analysis was repeated for patients with CF stratified by CFTR genotype. MEASUREMENTS AND MAIN RESULTS: A total of 440 subjects with CF participated in the study (51% male; mean [+/- SD] age, 26 +/- 11 yr; mean FEV(1), 62 +/- 28% predicted). In the total population, there was a trend toward an association between GCLC genotypes and CF lung disease (linear regression coefficient [SEM], 1.68 [1.0]; p = 0.097). In the stratified analysis, there was a highly significant association between GCLC genotype and CF lung function in subjects with a milder CFTR genotype (linear regression coefficient [SEM], 5.5 (1.7); p = 0.001). CONCLUSIONS: In patients with CF with a milder CFTR genotype, there is a strong association between functional polymorphisms of the GCLC gene and CF lung disease severity.

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62 * Severe cystic fibrosis transmembrane conductance regulator (CFTR) mutations (Class I-III) ϭ G542X, R553X, W1282X, R1162X, 621-1G→T, 1717-1G→A, 1078⌬T, 3659⌬C, ⌬F508, ⌬I507, N1303K, S549N, G551D, R560T. Login to comment

[hide] Diagnosing CF: sweat, blood and years. Thorax. 2006 Jul;61(7):556-7. Elborn JS, Bradley JM
Diagnosing CF: sweat, blood and years.
Thorax. 2006 Jul;61(7):556-7., [PMID:16807389]

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193 Over 1000 mutations of the CFTR gene have now been described, but only a proportion are associated with disease.4 Mutations of the CFTR gene which cause disease can be classified as follows: class 1, defective protein synthesis (e.g. G542X); class 2, defective protein processing (e.g. DF508); class 3, defective protein regulation (e.g. G551D). Login to comment

[hide] Polymorphic markers suggest a gene flow of CFTR ge... J Hered. 2006 Jul-Aug;97(4):313-7. Epub 2006 Jul 12. Cabello GM, Cabello PH, Llerena JC Jr, Fernandes O
Polymorphic markers suggest a gene flow of CFTR gene from Sub-Saharan/Arabian and Mediterranean to Brazilian Population.
J Hered. 2006 Jul-Aug;97(4):313-7. Epub 2006 Jul 12., [PMID:16837565]

Abstract [show]
The analysis of 2 diallelic loci (M470V and T854T) and a microsatellite IVS8(T)n of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has shown different haplotype distribution in Brazilian cystic fibrosis (CF) chromosomes carrying different CF mutations. The DeltaF508 mutation was in absolute linkage disequilibrium with 1-1 haplotype (M470V-T854T). Most of DeltaF508 chromosomes (84%) were found to carry the IVS8-9T. The most frequent haplotypes IVS8-7T and 2-1 (M470V-T854T) were found associated with Non-DeltaF508 mutations. Although there is a remarkable linkage disequilibrium between these markers with CFTR locus, the mutations R334W (7T-1-2 and 7T-2-1) and the 3120 + 1G --> A (7T-1-2 and 9T-1-2) are associated with two different haplotypes probably introduced in the Brazilian population by migration. These findings suggest that recombination events from the original haplotype and gene flow among different ethnic groups (sub-Saharan and Mediterranean) might have resulted in CF mutations associated with different haplotypes by independent introductions.

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23 A previous screening of the whole coding region and flanking intronic sequences from the 23 exons of the CFTR gene in 190 chromosomes allowed us to identify 11 different mutations: DF508 (28.4%), G85E (4.7%), 3120 þ 1G / A (3.7%), R334W (2.6%), G542X (2.1%), P205S (1.0%), G551D (0.5%), R1162X (0.5%), Y1092X (0.5%), S549R (0.5%), and S4X (0.5%) (Cabello GMK, Cabello PH, Otsuki, and others 2005). Login to comment
68 The most common 9T-1-1 haplotype among Caucasians was also the most frequent haplotype linked to DF508 and G542X mutations in our patients. Login to comment
88 The 9T-1-1 haplotype was also found to be associated with G542X mutation. Login to comment

[hide] Analysis of CFTR, SPINK1, PRSS1 and AAT mutations ... J Pediatr Gastroenterol Nutr. 2006 Sep;43(3):299-306. Sobczynska-Tomaszewska A, Bak D, Oralewska B, Oracz G, Norek A, Czerska K, Mazurczak T, Teisseyre M, Socha J, Zagulski M, Bal J
Analysis of CFTR, SPINK1, PRSS1 and AAT mutations in children with acute or chronic pancreatitis.
J Pediatr Gastroenterol Nutr. 2006 Sep;43(3):299-306., [PMID:16954950]

Abstract [show]
OBJECTIVES: Defects of PRSS1, SPINK1, CFTR and AAT are considered causative or predisposing to pancreatitis. The aim of this study was to evaluate the impact of these defects into molecular pathology of chronic pancreatitis (CP) and acute recurrent pancreatitis (ARP). METHODS: Ninety-two children with CP or ARP, 55 family members and 50 controls were investigated. The subjects were screened for PRSS1 mutations: R122H, R122C, A16V, N29I; SPINK1 N34S variant; panel of 14 CFTR defects: INNOLiPA CFTR12, CFTRdele2,3 and IVS8-T variant or panel of 3 CFTR defects-F508del, CFTRdele2,3 and IVS8-T; AAT mutations: E264V, E342K. RESULTS: We identified 1 mutated allele in at least 1 of 4 genes in 31 of 92 patients and 12 of 50 controls (P = 0.157). Mutations in SPINK1 and PRSS1 were most frequent. PRSS1 mutations were identified mainly in CP patients (9.6% of CP vs 2.5% of ARP alleles, P = 0.094), whereas N34S SPINK1 mutation was present with comparable frequency in CP and ARP patients (7.7% vs 10.0%, P = 0.768). The frequency of mutations in CFTR alleles was similar to controls (4.9% vs 5%, P = 0.587). Overall frequency of AAT mutations was lower than in the controls. Family studies showed that defects in the examined genes did not always segregate with disease. CONCLUSIONS: PRSS1 defects seem to be causative for pancreatitis, whereas defects in SPINK1 are suggested to be associated with the disease. No association between CFTR mutations and pancreatitis was observed. The importance of AAT variants remains speculative.

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64 For the first 50 patients enrolled in this study, the CFTR mutations F508del, G542X, G551D, R553X, N1303K, W1282X, 1717-1G/A, I507del, S1251N, R560T, 3905insT, Q552X (INNO-LiPA CFTR12, Innogenetics, Gent, Belgium), CFTRdele2,3 (16) and polyT variant in intron 8 (IVS8-T) (17) were analyzed. Login to comment

[hide] Effect of allergic bronchopulmonary aspergillosis ... Am J Respir Crit Care Med. 2006 Dec 1;174(11):1211-20. Epub 2006 Sep 7. Kraemer R, Delosea N, Ballinari P, Gallati S, Crameri R
Effect of allergic bronchopulmonary aspergillosis on lung function in children with cystic fibrosis.
Am J Respir Crit Care Med. 2006 Dec 1;174(11):1211-20. Epub 2006 Sep 7., 2006-12-01 [PMID:16959918]

Abstract [show]
RATIONALE: The relationship between sensitization to Aspergillus fumigatus and progression of pulmonary function is not yet established in cystic fibrosis (CF). OBJECTIVES: We aimed to evaluate onset of A. fumigatus sensitization and development of allergic bronchopulmonary aspergillosis (ABPA), as well as to determine the physiologic factors of lung function influencing these mechanisms in CF. METHODS: Serial annual lung function tests performed in 122 children with CF (62 males; 60 females; age: 6-18 yr) provided data pertaining to FRC measured by plethysmography, lung clearance index, volume of trapped gas, effective specific airway resistance, and forced expiratory indices (FEV1, FEF at 50% VC). Specific IgE to recombinant A. fumigatus allergens, rAspf1 and rAspf3, served as marker for sensitization, and to rAspf4 and rAspf6 as indications for a serologic ABPA, were clinically diagnosed (Nelson criteria). By linear mixed-effect model analysis, five patient groups, (1) not sensitized and free from Pseudomonas aeruginosa, (2) intermittently P. aeruginosa colonized, (3) chronically P. aeruginosa infected, (4) sensitized, and (5) with ABPA, were retrospectively evaluated. MEASUREMENTS AND MAIN RESULTS: A. fumigatus sensitization was best reflected by increased rAspf1+3-specific IgE levels, whereas, in most patients, sensitization was preceded by P. aeruginosa infection. Patients with ABPA demonstrated the most severe progression in all lung function parameters, and FEF at 50% VC, volume of trapped gas, and effective specific airway resistance were the best predictors (p < 0.0001). However, regarding distinction between sensitization to A. fumigatus and development of ABPA in the course of CF, chronic P. aeruginosa infection has to be taken into account. CONCLUSIONS: Airway narrowing, gas trapping, and small airway disease are the major targets for functional derangement in ABPA.

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74 * Miscellaneous-numbers in brackets: ⌬F508 and 1717-1GϾA(3), W1282X(2), 2347delG (1), 621ϩ1GϾT(1), Q525X(2), N1303K(1), 2176insC (1), 394delTT (1), 4005ϩ1G-A(1), 420DEL9 (1), E585X(1), G126D(1), G85E(1), R347P(1); 3905insT and 1717-1GϾA(1), K710X(1), M1101K(1), Q39X(1); R553X and R553X(1), 3905insT (1); G542X and T5(3), G542X(1); Q542X and K1200E(2); N1303K and 2347delG (1), 2789ϩ5GϾA(1); 1199delG and R560S(1). Login to comment

[hide] Comprehensive genetic analysis of the cystic fibro... Genet Med. 2006 Sep;8(9):557-62. Kammesheidt A, Kharrazi M, Graham S, Young S, Pearl M, Dunlop C, Keiles S
Comprehensive genetic analysis of the cystic fibrosis transmembrane conductance regulator from dried blood specimens--implications for newborn screening.
Genet Med. 2006 Sep;8(9):557-62., [PMID:16980811]

Abstract [show]
PURPOSE: In the United States, approximately 1/3,700 babies is born with cystic fibrosis each year. The >1,300 documented sequence variants pose a challenge for detection of cystic fibrosis through genetic screening. To investigate whether comprehensive characterization of the cystic fibrosis gene is feasible using dried newborn blood specimens, we modified the whole blood Ambry Test: CF and determined its sensitivity by testing DNA from individuals with cystic fibrosis who still had unknown mutations after commercial mutation panel testing. METHODS: DNA from 42 archived newborn dried blood specimens of affected Hispanic, African-American and Caucasian individuals in California was analyzed by temporal temperature gradient electrophoresis screening and targeted sequencing, and by gross deletion analysis. RESULTS: Excluding two specimens that could not be analyzed due to poor DNA quality, we report a 100% sensitivity and clinical detection rate in the remaining 40 patients. Eighty-three mutations representing 40 different variants were detected, including 8 novel mutations. CONCLUSIONS: This study demonstrates the feasibility of temporal temperature gradient electrophoresis-based full sequence analysis and targeted sequencing from DNA in newborn blood specimens. The Ambry Test: CF, as an additional step in cystic fibrosis newborn screening models, can be used to dramatically reduce the number of cystic fibrosis carrier sweat test referrals.

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73 Apart from delta F508, nine other mutations were present more than once in this data set, with several mutations occurring 4-5 times (1288insTA, 2055del9insA, 406-1GϾA, G542X and H199Y). Login to comment
98 In states with single specimenmodels,originalspecimensaretestedforthepresenceof themostcommonmutation,deltaF508,and/orotherdeleterious Table 1 Genotype data from panel testing and comprehensive Ambry TestTM : CF analysis Case Ethnicity ABI-31 Mutation 1 ABI-31 Mutation 2 Genzyme-87 Mutation 1 Genzyme-87 Mutation 2 Ambry Mutation 1 Ambry Mutation 2 Ambry Mutation 3 1 Hispanic delF508a 4382delAa 2 Hispanic delF508 N/I delF508 N/I delF508a 1248ϩ1GϾAa 3 African-American N/I N/I N/I N/I M150K CFTRdele17A,17Bb 4 Hispanic G542X N/I G542X N/I G542Xa 1288insTAa 5 African-American N/I N/I 3120ϩ1GϾA N/I 3120ϩ1GϾAa Q98Xa 3849؉72G>A 6 Hispanic delF508 N/I delF508 N/I delF508a 2289del10ins5a 7c Hispanic N/I N/I N/I N/I H199Ya 406-1GϾAa 8 Hispanic delF508 N/I delF508 N/I delF508a CFTRdele2,3(21kbb 9 Hispanic delF508 N/I delF508 N/I delF508a 2105-2117del13insAGAAAa 10 Hispanic G542X N/I G542X N/I G542X M952I Y914X 11 Hispanic N/I N/I N/I N/I 663delT L558S 12 Hispanic N/I N/I delF311 N/I delF311a 406-1GϾAa 13 Hispanic N/I N/I 2055del9insAa 2055del9insAa 14 Hispanic delF508 N/I delF508 N/I delF508 2055del9insA 15 Hispanic delF508 N/I delF508 N/I delF508 E257X 16 Hispanic N/I N/I N/I N/I V232D V232D 17 Hispanic delF508 N/I delF508 N/I delF508 H199Y 18 Hispanic delF508 N/I delF508 4160insGGGG 19 Caucasian delF508 N/I delF508 297-1GϾA 20 Hispanic 2183delAAϾG N/I 2183delAAϾG N/I 2183de1AAϾG 3500-2AϾG 21 Hispanic delF508 N/I delF508 S492F 22 Hispanic delF508 N/I delF508 N/I delF508 935delA 23 Caucasian R1162X N/I R1162X N/I R1162X 3940delG 24 Hispanic 711ϩ1GϾT N/I 711ϩ1GϾT T465N 25 Hispanic delF508 N/I delF508 N/I delF508 406-1GϾA 26 Hispanic delF508 N/I delF508 2055del9insA 27 Hispanic delF508 N/I delF508 N/I delF508 V232D 28 Hispanic delF508 N/I delF508 N/I delF508 S1235R 29 Hispanic G542X N/I G542X N/I G542X 297-1GϾA 30 Hispanic delF508 N/I delF508 N/I delF508 Q1100P 31 Hispanic delF508 N/I delF508 W216X 32 Hispanic N/I N/I N/I N/I 406-1GϾA H199Y 33 Hispanic N/I N/I N/I N/I 3272-26AϾG R75X 34 Hispanic N/I N/I Q890X N/I Q890X 2055del9insA 35 Hispanic delF508 N/I delF508 N/I delF508 W216X 36 Hispanic delF508 N/I delF508 N/I delF508 H199Y 37 Hispanic delF508 N/I delF508 N/I delF508 1288insTA I1027T 38 Hispanic G542X N/I G542X N/I G542X 663delT 39 Hispanic delF508 N/I delF508 N/I delF508 1288insTA 40 Hispanic delF508 N/I delF508 1288insTA mutations using mutation panels. Login to comment
111 del) AA F 572.9 No 2 mo 99 9 y 134 27 5 Q98X 3120ϩ1GϾA 3849؉72G>A (c.3717؉72G>A) AA F 253.1 No 6 mo 143 7 y 116 20 6 delF508 2289del10ins5a (c.2158_2167delACAA ATGAATinsGTAAG; p.L719fs) H M 70.8 No 1 y 104 14 y N/A N/A 8 delF508 CFTRdele2,3 (21 kb)b H F 214.2 No 3 y 103 5 y 108 18.6 10 G542X M952I Y914X (c.2742T>A;p.Y914X) H M 250.5 No 3 mo 95 6 y 112 22.2 15 delF508 E257X (c.769G>T; p.E257X) H M 301.3 No 0 mo 89 5 y N/A N/A 23 R1162X 3940delG (c.3808delG; p.D1270fs) C F N/A No 4 mo 86 8 mo 65 5.2 24 711ϩ1GϾT T465N (c.1394C>A; p.T465N) H F N/A N/A N/A N/A Deceased N/A N/A AA, African-American; H, Hispanic; C, Caucasian; MI, meconium ileus; IRT, immunoreactive trypsinogen; N/A, not available. Login to comment

[hide] Identification of CFTR, PRSS1, and SPINK1 mutation... Pancreas. 2006 Oct;33(3):221-7. Keiles S, Kammesheidt A
Identification of CFTR, PRSS1, and SPINK1 mutations in 381 patients with pancreatitis.
Pancreas. 2006 Oct;33(3):221-7., [PMID:17003641]

Abstract [show]
OBJECTIVES: Chronic pancreatitis is a progressive inflammatory disorder leading to irreversible exocrine and/or endocrine impairment. It is well documented that mutations in the cationic trypsinogen (PRSS1) gene can cause hereditary pancreatitis. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) and the serine protease inhibitor Kazal type 1 (SPINK1) genes are also associated with pancreatitis. METHODS: We analyzed 381 patients with a primary diagnosis of chronic or recurrent pancreatitis using the Ambry Test: Pancreatitis to obtain comprehensive genetic information for the CFTR, SPINK1, and PRSS1 genes. RESULTS: The results identified 32% (122/381) of patients with 166 mutant CFTR alleles, including 12 novel CFTR variants: 4375-20 A>G, F575Y, K598E, L1260P, G194R, F834L, S573C, 2789 + 17 C>T, 621+83 A>G, T164S, 621+25 A>G, and 3500-19 G>A. Of 122 patients with CFTR mutations, 5.5% (21/381) also carried a SPINK1 mutation, and 1.8% (7/381) carried a PRSS1 mutation. In addition, 8.9% (34/381) of all patients had 1 of 11 different SPINK1 mutations. Another 6.3% (24/381) of the patients had 1 of 8 different PRSS1 mutations. Moreover, 1.3% of the patients (5/381) had 1 PRSS1 and 1 SPINK1 mutation. A total 49% (185/381) of the patients carried one or more mutations. CONCLUSIONS: Comprehensive testing of the CFTR, PRSS1, and SPINK1 genes identified genetic variants in nearly half of all subjects considered by their physicians as candidates for genetic testing. Comprehensive test identified numerous novel variants that would not be identified by standard clinical screening panels.

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54 Patients With More Than 1 CFTR Mutation CFTR Mutation 1 CFTR Mutation 2 CFTR Mutation 3 No. of Patients deltaF508 5T 3 deltaF508 D1152H 1 deltaF508 deltaF508 1 deltaF508 F575Y 1 deltaF508 K598E 1 deltaF508 T164S 1 deltaF508 R74W D1270N 1 deltaF508 Q1476X 1 deltaF508 L997F 1 R553X D1152H 1 R553X G1069R 1 2789+5 G9A 2183 AA9G 1 3849+10kb C9T L1260P 1 711+3 A to G I1139V 1 1341+1 G9A G194R 5T 1 621+25 A9G 3500-19 C9T 1 R74W V855I 1 G542X R117H 1 G551D F311L 1 G576A R668C 2 K710X L997F 1 L997F L320V 1 G1069R 5T 1 1818+18 G9A 5T 1 F1074L 5T 1 F834L 5T 1 R74Q R297Q 1 R74Q R297Q 5T 1 R785Q 5T 1 R117H 5T 3 deltaF508 I1027T 1 Total patients 36 MutationsinboldfacewouldnothavebeendetectedbytheAmericanCollegeofObstetrics and Gynecology (ACOG)/American College of Medical Genetics (ACMG) mutation panel. Login to comment
71 Patients With 1 CFTR Mutation CFTR Mutation 1 No. of Patients 1717-1 G9A 1 2789+5 G9A 1 3849+10kb C9T 2 3849+45 G9A 1 621+3 A9G 2 A1364V 1 A349V 1 A455E 1 D1152H 1 D1445N 1 deltaF508 16 E217G 1 F1286C 1 F316L 1 G542X 1 G551D 1 I148T 1 I807M 1 L206W 1 L967S 2 L997F 2 P55S 1 Q179K 1 Q220X 1 R117H 3 R1453W 1 R297Q 1 R31C 1 R668C 2 S1235R 1 S573C 1 S945L 1 V562A 1 V754M 2 Y1092X 1 Total patients 58 MutationsinboldfacewouldnothavebeendetectedbytheACOG/ACMGmutationpanel. Login to comment

[hide] Prospective analysis of cystic fibrosis transmembr... Chest. 2006 Oct;130(4):995-1002. Ziedalski TM, Kao PN, Henig NR, Jacobs SS, Ruoss SJ
Prospective analysis of cystic fibrosis transmembrane regulator mutations in adults with bronchiectasis or pulmonary nontuberculous mycobacterial infection.
Chest. 2006 Oct;130(4):995-1002., [PMID:17035430]

Abstract [show]
BACKGROUND: Bronchiectasis and pulmonary infection with nontuberculous mycobacteria (NTM) may be associated with disease-causing mutations in the cystic fibrosis transmembrane regulator (CFTR). METHODS: Fifty adult patients at Stanford University Medical Center with a diagnosis of bronchiectasis and/or pulmonary NTM infection were prospectively characterized by sweat chloride measurement, comprehensive mutational analysis of CFTR, and sputum culture results. RESULTS: A de novo diagnosis of cystic fibrosis (CF) was established in 10 patients (20%). Patients with CF were more likely than those without CF to have mucus plugging seen on chest high-resolution CT, and women with a CF diagnosis were thinner, with a significantly lower mean body mass index than the non-CF subjects. Thirty CFTR mutations were identified in 24 patients (50% prevalence). Sweat chloride concentration was elevated > 60 mEq/dL (diagnostic of CF) in seven patients (14%), and from 40 to 60 mEq/dL in eight patients (16%). The frequency of CFTR mutations was elevated above that expected in the general population: heterozygous DeltaF508 (12% vs 3%), R75Q (14% vs 1%), and intron 8 5T (17% vs 5 to 10%). Other known CFTR mutations identified were V456A, G542X, R668C, I1027T, D1152, R1162L, W1282X, and L183I. Three novel CFTR mutations were identified: A394V, F650L, and C1344S. CONCLUSIONS: Mutations in CFTR that alter RNA splicing and/or functional chloride conductance are common in this population, and are likely to contribute to the susceptibility and pathogenesis of adult bronchiectasis and pulmonary NTM infection. Careful clinical evaluation for disease cause should be undertaken in this clinical context.

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11 Other known CFTR mutations identified were V456A, G542X, R668C, I1027T, D1152, R1162L, W1282X, and L183I. Login to comment
79 Other identified mutations included R75Q, G542X, V4566A, D1152H, F650L, I1027T, W1282X, and the intron 8 polymorphism IVS 8 5T. Login to comment
94 Comparison of pulmonary function test data did not demonstrate significant differences between the Table 1-Subjects With Diagnosis of CF* Patient No. Age, yr Sex Bronch NTM† Other Infection‡ CFTR Mutations M470V Alleles IVS8 PolyT§ Sweat Chloride Level, mEq/dL 1 63 F Y MAC, Mch ⌬F508, I1027T 1 7T/9T 41 2 58 F Y MAC 2 7T/7T 65 3 66 F Y MAC, Mka PA 1 7T/7T 70 4 62 F Y MAC R75Q 2 5T/7T 67 5 53 F Y MAC G542X 0 5T/7T 60 6 74 F Y MAC SA ⌬F508, D1152H 1 9T/9T 46 7 33 F Y N PA ⌬F508, V456A 1 9T/9T 74 8 49 M Y N 1 7T/7T 77 9 73 F Y N S malto W1282X 1 7T/7T 63 10 52 F N Msi 2 2 7T/7T 68 *Bronch ϭ bronchiectasis (Bronch); F ϭ female; M ϭ male; Y ϭ yes; N ϭ no. Login to comment

[hide] CFTR genotype as a predictor of prognosis in cysti... Chest. 2006 Nov;130(5):1441-7. McKone EF, Goss CH, Aitken ML
CFTR genotype as a predictor of prognosis in cystic fibrosis.
Chest. 2006 Nov;130(5):1441-7., [PMID:17099022]

Abstract [show]
STUDY RATIONALE: Certain CFTR genotypes are associated with reduced mortality. The accuracy of using CFTR genotype as a predictor of survival and the mechanisms through which CFTR genotype influences survival are unknown. PARTICIPANTS: All patients with cystic fibrosis (CF) enrolled in the US Cystic Fibrosis Foundation national registry between 1993 and 2002. DESIGN: We examined the prognostic value of CFTR genotype, grouped into "high-risk" and "low-risk" categories based on the effect of their CFTR genotype on phenotype and protein production. MEASUREMENTS AND RESULTS: Clinical and genetic data were available from 15,651 patients with CF. Patients with a high-risk CFTR genotype had a greater than twofold increased risk of death compared to patients with a low-risk CFTR genotype (relative risk, 2.25; 95% confidence interval [CI], 1.77 to 2.84; p < 0.001). This association was partly explained by lung function, nutritional status, pancreatic insufficiency, and Pseudomonas aeruginosa colonization. Of the 1,672 patients who died, median age at death for the high-risk CFTR genotype was 24.2 years (interquartile range, 18.4 to 32.0 years) and for the low-risk CFTR genotype was 37.6 years (interquartile range, 28.8 to 47.9 years; p < 0.001). The positive predictive value of this classification method as a test to identify patients who died before or after their 30th birthday was 69% (95% CI, 67 to 72%) with a negative predictive value of 71% (95% CI, 60 to 80%). CONCLUSIONS: Grouping patients into high-risk and low-risk CFTR genotype categories is associated with significant differences in survival and median age at death. These differences are not fully explained by lung function, nutritional measures, pancreatic insufficiency, or P aeruginosa colonization. Modest reassurance about the likelihood of a milder than average course can be provided for CF patients with a low-risk CFTR genotype, although it should be acknowledged that substantial phenotypic variability exists.

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46 Alleles High-risk CFTR genotype Class I 2,131 G542X, R553X, W1282X, R1162X, 621-1G3T, 1717-1G3A, 1078⌬T, 3659⌬C Class II 11,231 ⌬F508, ⌬I507, N1303K, S549N, G85E Class III 783 G551D, R560T Low-risk CFTR genotype Class IV 391 R117H, R334W, R347P Class V 421 3849 ϩ 10KbC3T, 2789 ϩ 5G3A, A455E *Patients with both CFTR alleles in either class I, class II, or class III were grouped together as a high-risk genotype, while patients with at least one mutant allele in class IV and V were considered to have low-risk genotypes; 380 patients had both mutations in either class I, II, or III, while 314 patients had both mutations in either class IV or V (total, n ϭ 15,651). Login to comment

[hide] Progression of pulmonary hyperinflation and trappe... Respir Res. 2006 Nov 30;7:138. Kraemer R, Baldwin DN, Ammann RA, Frey U, Gallati S
Progression of pulmonary hyperinflation and trapped gas associated with genetic and environmental factors in children with cystic fibrosis.
Respir Res. 2006 Nov 30;7:138., [PMID:17137500]

Abstract [show]
BACKGROUND: Functional deterioration in cystic fibrosis (CF) may be reflected by increasing bronchial obstruction and, as recently shown, by ventilation inhomogeneities. This study investigated which physiological factors (airway obstruction, ventilation inhomogeneities, pulmonary hyperinflation, development of trapped gas) best express the decline in lung function, and what role specific CFTR genotypes and different types of bronchial infection may have upon this process. METHODS: Serial annual lung function tests, performed in 152 children (77 males; 75 females) with CF (age range: 6-18 y) provided data pertaining to functional residual capacity (FRCpleth, FRCMBNW), volume of trapped gas (VTG), effective specific airway resistance (sReff), lung clearance index (LCI), and forced expiratory indices (FVC, FEV1, FEF50). RESULTS: All lung function parameters showed progression with age. Pulmonary hyperinflation (FRCpleth > 2SDS) was already present in 39% of patients at age 6-8 yrs, increasing to 67% at age 18 yrs. The proportion of patients with VTG > 2SDS increased from 15% to 54% during this period. Children with severe pulmonary hyperinflation and trapped gas at age 6-8 yrs showed the most pronounced disease progression over time. Age related tracking of lung function parameters commences early in life, and is significantly influenced by specific CFTR genotypes. The group with chronic P. aeruginosa infection demonstrated most rapid progression in all lung function parameters, whilst those with chronic S. aureus infection had the slowest rate of progression. LCI, measured as an index of ventilation inhomogeneities was the most sensitive discriminator between the 3 types of infection examined (p < 0.0001). CONCLUSION: The relationships between lung function indices, CFTR genotypes and infective organisms observed in this study suggest that measurement of other lung function parameters, in addition to spirometry alone, may provide important information about disease progression in CF.

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99 420del9(1), E585X(1), G126D(1), G85E(1), R347P(1), 1078delT(1); Miscellaneous 43 28.3 3905insT and1717-1G>A(1),K710X(1), M1101K(1), Q39X(1), P5L(1), R553X(1); R553X andR553X(1); G542X and T5(3), G542X(1); Q542X and3732delA(2); N1303K and2347delG(1), 2789+5G>A(1); 1199delG andR560S(1). Login to comment

[hide] Prediction of cellular immune responses against CF... Am J Respir Cell Mol Biol. 2007 May;36(5):529-33. Epub 2007 Jan 11. Figueredo J, Limberis MP, Wilson JM
Prediction of cellular immune responses against CFTR in patients with cystic fibrosis after gene therapy.
Am J Respir Cell Mol Biol. 2007 May;36(5):529-33. Epub 2007 Jan 11., [PMID:17218617]

Abstract [show]
Different classes of mutations (class I-VI) of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene are responsible for lung/pancreatic disease. The most common mutation, DeltaF508, is characterized by expression of precursor forms of CFTR but no functional CFTR. Since only 5-10% of normal CFTR function is required to correct the electrophysiologic defect across the airway epithelium, gene therapy holds promise for treatment of patients with CF lung disease. However, efficient delivery and transgene expression are not the only parameters that may influence the success of gene therapy. Host-specific immune responses generated against the therapeutic CFTR protein may pose a problem, especially when the coding sequence between the normal CFTR and mutated CFTR differ. This phenomenon is more pertinent to class I mutations in which large fragments of the protein are not expressed. However, T cells directed against epitopes that span sequences containing class II-V mutations are also possible. We used MHC-binding prediction programs to predict the probability of cellular immune responses that may be generated against CFTR in DeltaF508 homozygote patients. Results obtained from running the prediction algorithms yielded a few high-scoring MHC-Class I binders within the specific sequences, suggesting that there is a possibility of the host to mount a cellular immune response against CFTR, even when the difference between therapeutic and host CFTR is a single amino acid (F) at position 508.

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25 Most of the other CFTR mutations are rare, with only four mutations (G542X, N1303K, G551D, and W1282X) having overall frequencies above 1%. Login to comment

[hide] Cystic fibrosis is associated with a defect in api... J Am Soc Nephrol. 2007 Mar;18(3):707-18. Epub 2007 Feb 7. Jouret F, Bernard A, Hermans C, Dom G, Terryn S, Leal T, Lebecque P, Cassiman JJ, Scholte BJ, de Jonge HR, Courtoy PJ, Devuyst O
Cystic fibrosis is associated with a defect in apical receptor-mediated endocytosis in mouse and human kidney.
J Am Soc Nephrol. 2007 Mar;18(3):707-18. Epub 2007 Feb 7., [PMID:17287432]

Abstract [show]
Inactivation of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) causes cystic fibrosis (CF). Although CFTR is expressed in the kidney, no overwhelming renal phenotype has been documented in patients with CF. This study investigated the expression, subcellular distribution, and processing of CFTR in the kidney; used various mouse models to assess the role of CFTR in proximal tubule (PT) endocytosis; and tested the relevance of these findings in patients with CF. The level of CFTR mRNA in mouse kidney approached that found in lung. CFTR was located in the apical area of PT cells, with a maximal intensity in the straight part (S3) of the PT. Fractionation showed that CFTR co-distributed with the chloride/proton exchanger ClC-5 in PT endosomes. Cftr(-/-) mice showed impaired (125)I-beta(2)-microglobulin uptake, together with a decreased amount of the multiligand receptor cubilin in the S3 segment and a significant loss of cubilin and its low molecular weight (LMW) ligands into the urine. Defective receptor-mediated endocytosis was found less consistently in Cftr(DeltaF/DeltaF) mice, characterized by a large phenotypic heterogeneity and moderate versus mice that lacked ClC-5. A significant LMW proteinuria (and particularly transferrinuria) also was documented in a cohort of patients with CF but not in patients with asthma and chronic lung inflammation. In conclusion, CFTR inactivation leads to a moderate defect in receptor-mediated PT endocytosis, associated with a cubilin defect and a significant LMW proteinuria in mouse and human. The magnitude of the endocytosis defect that is caused by CFTR versus ClC-5 loss likely reflects functional heterogeneity along the PT.

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96 Genotyping identified the ⌬F508 mutation in all patients (homozygous in 25 of 30; heterozygous with N1303K, G542X, or 3849 10 kb C⌸T in three of 30; second mutation not identified in two of 30). Login to comment

[hide] Nonsense-mediated mRNA decay affects nonsense tran... J Clin Invest. 2007 Mar;117(3):683-92. Epub 2007 Feb 8. Linde L, Boelz S, Nissim-Rafinia M, Oren YS, Wilschanski M, Yaacov Y, Virgilis D, Neu-Yilik G, Kulozik AE, Kerem E, Kerem B
Nonsense-mediated mRNA decay affects nonsense transcript levels and governs response of cystic fibrosis patients to gentamicin.
J Clin Invest. 2007 Mar;117(3):683-92. Epub 2007 Feb 8., [PMID:17290305]

Abstract [show]
Aminoglycosides can readthrough premature termination codons (PTCs), permitting translation of full-length proteins. Previously we have found variable efficiency of readthrough in response to the aminoglycoside gentamicin among cystic fibrosis (CF) patients, all carrying the W1282X nonsense mutation. Here we demonstrate that there are patients in whom the level of CF transmembrane conductance regulator (CFTR) nonsense transcripts is markedly reduced, while in others it is significantly higher. Response to gentamicin was found only in patients with the higher level. We further investigated the possibility that the nonsense-mediated mRNA decay (NMD) might vary among cells and hence governs the level of nonsense transcripts available for readthrough. Our results demonstrate differences in NMD efficiency of CFTR transcripts carrying the W1282X mutation among different epithelial cell lines derived from the same tissue. Variability was also found for 5 physiologic NMD substrates, RPL3, SC35 1.6 kb, SC35 1.7 kb, ASNS, and CARS. Importantly, our results demonstrate the existence of cells in which NMD of all transcripts was efficient and others in which the NMD was less efficient. Downregulation of NMD in cells carrying the W1282X mutation increased the level of CFTR nonsense transcripts and enhanced the CFTR chloride channel activity in response to gentamicin. Together our results suggest that the efficiency of NMD might vary and hence have an important role in governing the response to treatments aiming to promote readthrough of PTCs in many genetic diseases.

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83 It is important to note that in all previous clinical trials in CF patients, aminoglycosides (22, 23, 29) or other molecules (41-44) affected only the chloride transport Table 1 Study patients and response to gentamicin treatment Patient Genotype Basal NPD Chloride transport Response number Before treatment After gentamicin Before treatment After gentamicin 1 W1282X/ΔF508 -38 -18 -1 -6 + 2 W1282X/ΔF508 -41 -41 -3 -5 + 3 W1282X/ΔF508 -43 -30 -1 -4 + 4 W1282X/ΔF508 -40 -30 4 -4 + 5 W1282X/ΔF508 -40 -30 -2 -2 +/-A 6 W1282X/W1282X -47 -32 0 -9 + 7 W1282X/G542X -43 -45 -2 -11 + 8 W1282X/W1282X -40 -35 11 -9 + 9 W1282X/W1282X -32 -33 2 4 - 10 W1282X/3849+10 kb C→T -65 -56 3 -3 - All values are expressed as mV. Login to comment
218 Methods Patients. Five of the patients were heterozygous for the W1282X and the ΔF508 mutations (patients 1-5); 3 were homozygous for the W1282X mutation (patients 6, 8, and 9); 1 was heterozygous for the W1282X and G542X mutations (patient 7); and 1 was heterozygous for W1282X and 3849+10 kb C→T mutations (patient 10), which can lead to inclusion of an 84-bp cryptic exon harboring a PTC. Login to comment
328 Du, M., et al. 2002. Aminoglycoside suppression of a premature stop mutation in a Cftr-/- mouse carrying a human CFTR-G542X transgene. Login to comment
432 CFTR nonsense mutations G542X and W1282X associated with severe reduction of CFTR mRNA in nasal epithelial cells. Login to comment

[hide] No detectable improvements in cystic fibrosis tran... Am J Respir Cell Mol Biol. 2007 Jul;37(1):57-66. Epub 2007 Mar 8. Clancy JP, Rowe SM, Bebok Z, Aitken ML, Gibson R, Zeitlin P, Berclaz P, Moss R, Knowles MR, Oster RA, Mayer-Hamblett N, Ramsey B
No detectable improvements in cystic fibrosis transmembrane conductance regulator by nasal aminoglycosides in patients with cystic fibrosis with stop mutations.
Am J Respir Cell Mol Biol. 2007 Jul;37(1):57-66. Epub 2007 Mar 8., [PMID:17347447]

Abstract [show]
Cystic fibrosis (CF) is an autosomal recessive disorder caused by many types of genetic defects, including premature stop codons. Gentamicin can suppress stop mutations in CF transmembrane conductance regulator (CFTR) in vitro and in vivo, leading to improvements in CFTR-dependent ion transport and protein localization to the apical surface of respiratory epithelial cells. The primary objective of this study was to test whether nasally administered gentamicin or tobramycin could suppress premature stop mutations in CFTR, resulting in full-length, functional protein. A secondary objective was to obtain data to aid in the design of multicenter trials using the nasal potential difference as a study endpoint. A multicenter study was conducted in two cohorts of patients with CF, those heterozygous for stop mutations in the CFTR gene and those without nonsense mutations, to investigate the effects of both gentamicin and tobramycin administered over a 28-d period on sequential nasal potential difference and airway cell immunofluorescence endpoints. Eleven patients with CF with stop mutations were enrolled in a randomized, double-blinded, crossover fashion to receive each drug, while 18 subjects with CF without stop mutations were randomized 1:1 in a parallel fashion to receive one drug. After demonstration of drug delivery, neither aminoglycoside produced detectable changes in nasal ion transport or CFTR localization in brushed cells from either study group. These results with first-generation suppressive agents suggest the need for improved drug delivery methods and/or more potent suppressors of nonsense mutations to confer CFTR correction in subjects with CF heterozygous for nonsense mutations. The study provides valuable information on parameters of the nasal potential difference measurements for use in future multicenter clinical trials.

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50 GENOTYPE AND DEMOGRAPHIC INFORMATION OF STUDY SUBJECTS Age (yr) Sex Genotype Premature stop mutation subjects 16 Male 621ϩ1G-T/E60X 16 Male ⌬F508/G542X 22 Male ⌬F508/G542X 12 Female ⌬F508/G542X 22 Female ⌬F508/G542X 11 Male ⌬F508/R553X 15 Female 621ϩ1G-T/R553X 27 Female ⌬F508/R553X 32 Female ⌬F508/Y1092X 28 Male ⌬F508/R1162X 11 Female ⌬F508/W1282X Mean yr (SD) 20.2 (8.9) M:F 5:6 (six separate stop alleles represented) Control subjects 8 Male ⌬F508/⌬F508 14 Male ⌬F508/⌬F508 16 Female ⌬F508/⌬F508 16 Female ⌬F508/⌬F508 16 Male ⌬F508/⌬F508 18 Female ⌬F508/⌬F508 18 Male ⌬F508/⌬F508 20 Male ⌬F508/⌬F508 20 Female ⌬F508/⌬F508 20 Male ⌬F508/⌬F508 24 Female ⌬F508/⌬F508 32 Female ⌬F508/⌬F508 35 Male ⌬F508/⌬F508 42 Female ⌬F508/⌬F508 29 Male ⌬F508/G551D 59 Female ⌬F508/2789ϩ5G-T 16 Male ⌬F508/3905InsT 15 Female ⌬F508/N1303K Mean yr (SD) 23.2 (12.3) M:F 9:9 ⌬F508/⌬F508: 14:18 were provided (with 25% overfill) at Days 0, 7, 42, and 49 for the premature stop group, and at Days 0 and 7 for the control group. Login to comment
165 (B) Low-power fields of nasal cells obtained from a normal subject, a patient with CF homozygous for the ⌬F508 CFTR mutation, and a subject with CF possessing a premature stop mutation (G542X/⌬F508). Login to comment
168 The final panel demonstrates minimal CFTR staining (G542X/⌬F508 subject). Login to comment
176 Figure 4 provides examples of cytoplasmic and surface CFTR staining of nasal cells brushed from normal control subjects, perinuclear CFTR staining from a ⌬F508/⌬F508 subject with CF, and sparse staining from a subject with CF heterozygous for a premature stop mutation (G542X/⌬F508; all staining shown was from cells isolated from subjects off of treatment). Login to comment
230 The four most common stop mutations (G542X, R553X, R1162X, W1282X CFTR) all contain a UGA codon, and all have been shown to be suppressed by aminoglycoside treatment in vitro using heterologous expression systems. Login to comment

[hide] Heteropolymeric triplex-based genomic assay to det... PLoS One. 2007 Mar 21;2(3):e305. Daksis JI, Erikson GH
Heteropolymeric triplex-based genomic assay to detect pathogens or single-nucleotide polymorphisms in human genomic samples.
PLoS One. 2007 Mar 21;2(3):e305., [PMID:17375191]

Abstract [show]
Human genomic samples are complex and are considered difficult to assay directly without denaturation or PCR amplification. We report the use of a base-specific heteropolymeric triplex, formed by native duplex genomic target and an oligonucleotide third strand probe, to assay for low copy pathogen genomes present in a sample also containing human genomic duplex DNA, or to assay human genomic duplex DNA for Single Nucleotide Polymorphisms (SNP), without PCR amplification. Wild-type and mutant probes are used to identify triplexes containing FVL G1691A, MTHFR C677T and CFTR mutations. The specific triplex structure forms rapidly at room temperature in solution and may be detected without a separation step. YOYO-1, a fluorescent bis-intercalator, promotes and signals the formation of the specific triplex. Genomic duplexes may be assayed homogeneously with single base pair resolution. The specific triple-stranded structures of the assay may approximate homologous recombination intermediates, which various models suggest may form in either the major or minor groove of the duplex. The bases of the stable duplex target are rendered specifically reactive to the bases of the probe because of the activity of intercalated YOYO-1, which is known to decondense duplex locally 1.3 fold. This may approximate the local decondensation effected by recombination proteins such as RecA in vivo. Our assay, while involving triplex formation, is sui generis, as it is not homopurine sequence-dependent, as are "canonical triplexes". Rather, the base pair-specific heteropolymeric triplex of the assay is conformation-dependent. The highly sensitive diagnostic assay we present allows for the direct detection of base sequence in genomic duplex samples, including those containing human genomic duplex DNA, thereby bypassing the inherent problems and cost associated with conventional PCR based diagnostic assays.

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125 Sequence bglIR-WT25C 1 59-TATTTTGATTATAGGACATGAAGAT-39 DR01-WT15 2 59-GAGCCGAAGGGGCAG-39 CFTR delta F508-WT25C 3 59-TAGGAAACACCAAAGATGATATTTT-39 CFTR delta F508-MUT25C 4 59-ATAGGAAACACCA---ATGATATTTTCT-39 CFTR delta I507-WT25C 5 59-TAGGAAACACCAAAGATGATATTTT-39 CFTR delta I507-MUT25C 6 59-ATAGGAAACACCAAAGA---TATTTTCT-39 CFTR 3659delC-WT25C 7 59-TGGTTTGGTTGACTTGGTAGGTTTA-39 CFTR 3659delC-MUT25C 8 59-ATGGTTTGGTTGACTTG-TAGGTTTA-39 CFTR 3849+10kbCRT-WT25C 9 59-GTGTCTTACTCGCCATTTTAATACT-39 CFTR 3849+10kbCRT-MUT25C 10 59-GTGTCTTACTCACCATTTTAATACT-39 CFTR 2789+5GRA-WT25C11 59-AATAGGACATGGAATACTCACTTTC-39 CFTR 2789+5GRA-MUT25C 12 59-AATAGGACATGGAATATTCACTTTC-39 CFTR G551D-WT25C 13 59-ATTCTTGCTCGTTGACCTCCACTCA-39 CFTR G551D-MUT25C 14 59-ATTCTTGCTCGTTGATCTCCACTCA-39 CFTR 621+1GRT-WT25C 15 59-AAGTATTACCTTCTTATAAATCAAA-39 CFTR 621+1GRT-MUT25C16 59-AAGTATTAACTTCTTATAAATCAAA-39 CFTR R1162X-WT25C 17 59-AACTTAAAGACTCGGCTCACAGATC-39 CFTR R1162X-MUT25C 18 59-AACTTAAAGACTCAGCTCACAGATC-39 CFTR 1717-1GRA-WT25C 19 59-TGGAGATGTCCTATTACCAAAAATA-39 CFTR 1717-1GRA- MUT25C 20 59-TGGAGATGTCTTATTACCAAAAATA-39 CFTR A455E-WT25C 21 59-CCAGCAACCGCCAACAACTGTCCTC-39 CFTR A455E-MUT25C 22 59-CCAGCAACCTCCAACAACTGTCCTC-39 CFTR G542X-WT25C 23 59-ATTCCACCTTCTCCAAGAACTATAT-39 CFTR G542X-MUT25C 24 59-ATTCCACCTTCTCAAAGAACTATAT-39 CFTR N1303K-WT25C 25 59-TAGGGATCCAAGTTTTTTCTAAATG-39 CFTR N1303K-MUT25C 26 59-TAGGGATCCAACTTTTTTCTAAATG-39 CFTR R560T-WT25C 27 59-AGTTATTCACCTTGCTAAAGAAATT-39 CFTR R560T-MUT25C 28 59-AGTTATTCACGTTGCTAAAGAAATT-39 CFTR W1282X-WT25C 29 59-TTTCCTCCACTGTTGCAAAGTTATT-39 CFTR W1282X-MUT25C 30 59-TTTCCTTCACTGTTGCAAAGTTATT-39 MTHFR C677T-WT25C 31 59-TGATGATGAAATCGGCTCCCGCAGA-39 MTHFR C677T-MUT25C 32 59-TGATGATGAAATCGACTCCCGCAGA-39 FVL G1691A-WT25C 33 59-CCCTCTGTATTCCTCGCCTGTCCAG-39 FVL G1691A-MUT25C 34 59-CCCTCTGTATTCCTTGCCTGTCCAG-39 All 25-mer probes listed were antisense. Login to comment
704 Mutation Source Number of tests Percentage GC in probe sequence Percentage difference of mismatched TAF relative to perfect match TAF CFTR delta F508 blood 102 28% 2100% to 281% CFTR delta I507 blood 6 28% 2100% to 285% CFTR 3659delC blood 11 40% 2100% to 255% CFTR 3849+10kbCRT blood 9 36% 2100% to 282% CFTR 2789+5GRA blood 16 36% 2100% to 275% CFTR 2789+5GRA saliva 13 36% 2100% to 266% CFTR G551D blood 11 48% 2100% to 261% CFTR 621+1GRT blood 5 20% 2100% to 257% CFTR R1162X blood 6 44% 267% to 236% CFTR 1717-1GRA blood 12 32% 2100% to 258% CFTR A455E blood 9 60% 2100% to 289% CFTR G542X blood 6 36% 2100% to 260% CFTR N1303K blood 8 32% 2100% to 283% CFTR R560T blood 6 28% 2100% to 254% CFTR W1282X blood 14 36% 2100% to 274% MTHFR C677T blood 55 52% 2100% to 272% FVL G1691A blood 34 60% 2100% to 281% TAF indicates Triplex-Associated Fluorescence. doi:10.1371/journal.pone.0000305.t004 ..................................................................................... brighter when intercalated into complexes of identical short oligonucleotides, such as the probes used in our assay, than when a like number of YOYO-1 molecules were in the presence of genomic duplex DNA. Login to comment

[hide] Testing and reporting ACMG cystic fibrosis mutatio... Genet Test. 2007 Spring;11(1):11-31. Lebo RV, Grody WW
Testing and reporting ACMG cystic fibrosis mutation panel results.
Genet Test. 2007 Spring;11(1):11-31., [PMID:17394390]

Abstract [show]
Testing strategies and summary reports for pregnant patients and symptomatic patients being tested for cystic fibrosis (CF; MIM 219700) were developed based upon calculated after (posterior) test risk tables incorporating patient and family histories, ethnicities, and prior testing status. This manuscript defines the proportion of all mutations detected by the American College of Medical Genetics (ACMG)-recommended 23-mutation cystic fibrosis transmembrane conductance regulator (CFTR) gene core panel when testing all patient categories with severe symptoms, including pregnant couples with no family history as well as CF patients, their partners, and other family members. Reference tables incorporate prior and posterior test risks sufficient to complete >99% of all tested cases and to report the results according to HIPAA guidelines. These tables were calculated based on the assumption that all patient samples have been collected, labeled, analyzed, and reported correctly, including the patient's reported relationship to a known affected or carrier relative, even though the template letter states that the likelihood is about 99% that each reported result is accurate. Pedigrees and tables with the prior (before; a priori) test risks of patients offered CF screening with a family history of a CF patient and/or a known carrier patient are provided for ready reference with each risk frequency, dependent upon the assumption that the patient's pedigree reflects familial relationships correctly. Comparison of tables emphasizes the value of asking the tested partner to ask a relative known to have CF or who tested positive for a CF mutation to donate a sample as a DNA test control and/or to obtain a copy of a prior molecular test result and/or extracted DNA sample. These tables posterior test risks also indicate that when one partner with no family history tests negative for the 23 mutation panel, no further prenatal testing is indicated.

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141 Alternatively, when the genotype of a more closely related carrier has been reported, this information becomes the most informative (i.e., uncle is a ⌬F508 carrier rather than affected second cousin is a compound heterozygote for the ⌬F508 and the G542X mutations). Login to comment

[hide] In vitro prediction of stop-codon suppression by i... BMC Med. 2007 Mar 29;5:5. Sermet-Gaudelus I, Renouil M, Fajac A, Bidou L, Parbaille B, Pierrot S, Davy N, Bismuth E, Reinert P, Lenoir G, Lesure JF, Rousset JP, Edelman A
In vitro prediction of stop-codon suppression by intravenous gentamicin in patients with cystic fibrosis: a pilot study.
BMC Med. 2007 Mar 29;5:5., [PMID:17394637]

Abstract [show]
BACKGROUND: Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which acts as a chloride channel activated by cyclic AMP (cAMP). The most frequent mutation found in 70% of CF patients is F508del, while premature stop mutations are found in about 10% of patients. In vitro aminoglycoside antibiotics (e.g. gentamicin) suppress nonsense mutations located in CFTR permitting translation to continue to the natural termination codon. Pharmacologic suppression of stop mutations within the CFTR may be of benefit to a significant number of patients. Our pilot study was conducted to determine whether intravenous gentamicin suppresses stop codons in CF patients and whether it has clinical benefits. METHODS: A dual gene reporter system was used to determine the gentamicin-induced readthrough level of the most frequent stop mutations within the CFTR in the French population. We investigated readthrough efficiency in response to 10 mg/kg once-daily intravenous gentamicin perfusions in patients with and without stop mutations. Respiratory function, sweat chloride concentration, nasal potential difference (NPD) and CFTR expression in nasal epithelial cells were measured at baseline and after 15 days of treatment. RESULTS: After in vitro gentamicin incubation, the readthrough efficiency for the Y122X mutation was at least five times higher than that for G542X, R1162X, and W1282X. In six of the nine patients with the Y122X mutation, CFTR immunodetection showed protein at the membrane of the nasal epithelial cells and the CFTR-dependent Cl- secretion in NPD measurements increased significantly. Respiratory status also improved in these patients, irrespective of the gentamicin sensitivity of the bacteria present in the sputum. Mean sweat chloride concentration decreased significantly and normalised in two patients. Clinical status, NPD and sweat Cl- values did not change in the Y122X patients with no protein expression, in patients with the other stop mutations investigated in vitro and those without stop mutations. CONCLUSION: Suppression of stop mutations in the CFTR gene with parenteral gentamicin can be predicted in vitro and is associated with clinical benefit and significant modification of the CFTR-mediated Cl- transport in nasal and sweat gland epithelium.

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8 Results: After in vitro gentamicin incubation, the readthrough efficiency for the Y122X mutation was at least five times higher than that for G542X, R1162X, and W1282X. Login to comment
27 Methods Readthrough quantification in cell culture A dual gene reporter system was used to quantify the readthrough efficiency directed by the most frequent stop mutations in the French population (Y122X, G542X, R1162X and W1282X) [10], in the presence or absence of gentamicin. Login to comment
65 Table 1: Oligonucleotide sequences used in the dual reporter gene assay, corresponding to the Y122X, G542X, R1162X and W1292X mutations and the TQ in frame control. Login to comment
67 Readthrough level (%)* Mutation Oligonucleotides** 0 600 μg/ml gentamicin Y122X w 5' CGCTCTATCGCGTAACTAGGCATAGGC 3'; c 5' GCCTATGCCTAGTTACGCGATAGAGCG 3' 0.52 1.6 W1282X w 5` AATATAGTTCTTTGAGAAGGTGGAATC 3` c 5` GATTCCACCTTCTCAAAGAACTATATT 3` 0.115 0.35 R1162X w 5' CGATCTGTGAGCTGAGTCTTTAAGTTC 3'; c 5' GAACTTAAAGACTCAGCTCACAGATCG 3' 0.023 0.22 G542X w 5' ACTTTGCAACAGTGAAGGAAAGCCTTT 3'; c 5' AAAGGCTTCCTTCACTGTTGCAAAGT 3' 0.017 0.26 TQ: in frame control w 5' GCAGGAACACAACAGCAATTACAG 3' c 5' CTGTAATTGCTGTTGTGTTCCTGC 3' 100 100 *At least five independent experiments were performed with each construct and showed less than 20% variation. Login to comment
78 The R1162X and the G542X mutations yielded basal readthrough of less than 0.03% and increased by a factor of 10 and 15 respectively after gentamicin. Login to comment
79 The basal readthrough of W1282X was ~10 times higher than those of R1162X and G542X and tripled after gentamicin. Login to comment
80 Y122X had a basal readthrough level five times higher than that for the W1282X mutation, 22 times that for R1162X and 30 times that for G542X. Login to comment
81 After gentamicin incubation, Y122X readthrough efficiency remained at least 4.5 times higher than that for W1282X, six times that for G542X and 7.3 that for R1162X. Login to comment
85 Four had another stop mutation: one was homozygous for G542X, one for R1162X, and two were compound heterozygous for W1282X/F508del and R553X/CFTRdele17b (Group B). Login to comment
123 Genotype Sputum colonisation Age (year) Δscore FEV1var FVCvar FEF25-75var Sweat Cl- at D0 Sweat Cl- at D15 ΔCl-free-iso at D0 ΔCl-free-iso at D15 ICC Y122X+/+ SA 11 -4 24 23 31 126 91 0 0 - Y122X+/+ PA* 16 -2 -12 -6 -15 79 37 NP 0 - Y122X+/+ PA*,SA 18 -4 2 -2 -8 109 115 0 NP + Y122X+/+ PP* 15 -5 25 19 86 90 91 -0.5 0 + Y122X+/+ PP* 13 -15 18 8 96 103 46 -1.6 -3.8 + Y122X+/+ SA 22 -13 3 0 7 108 100 -3.7 -17.6 + Y122X+/+ BC* 21 -22 18 24 150 136 135 0 -4 + Y122X+/+ PA* 12 -12 3 -9 NP 119 86 0 -8.2 NP Y122X+/F508del SA* 10.5 -3 21 21 45 114 65 -1 -3.3 + R1162X +/+ SA 14 -2 0.4 0 4 116 131 0 0 - F508del/W1282X PA 13 -2 15 14 27 103 100 0 -1.3 NP G542X +/+ SA 11 -4 21 17 20 113 105 0 0 NP R553X/CFTRdele17b PA* 10 0 NP NP NP 115 NP -4 NP NP PA: Pseudomonas aeruginosa; SA: Staphylococcus aureus; PP: Pseudomonas putida; BC: Burkholderia cepacia; * bacteria resistant to gentamicin. Login to comment
172 In contrast, patients with mutations producing lower levels of translational readthrough in the cell culture assay (G542X, R1162X and W1282X) did not show significant changes in clinical status, chloride secretion in either the nasal or sweat gland epithelia after gentamicin treatment. Login to comment
192 The +4C nucleotide could account for the better response to gentamicin, because it is associated with greater readthrough efficiency, whereas the other mutations tested in our study, R1162X and G542X, imply a +4G nucleotide, which is associated with a poor readthrough [11]. Login to comment

[hide] Molecular characterization of the cystic fibrosis ... Genet Med. 2007 Mar;9(3):163-72. Grangeia A, Sa R, Carvalho F, Martin J, Girodon E, Silva J, Ferraz L, Barros A, Sousa M
Molecular characterization of the cystic fibrosis transmembrane conductance regulator gene in congenital absence of the vas deferens.
Genet Med. 2007 Mar;9(3):163-72., [PMID:17413420]

Abstract [show]
PURPOSE: Approximately 20% of patients with congenital absence of the vas deferens remain without two mutations identified. We applied a strategy of serial screening steps to 45 patients with congenital absence of the vas deferens and characterized cystic fibrosis transmembrane conductance regulator gene mutations in all cases. METHODS: DNA samples of 45 patients with congenital absence of the vas deferens were screened by successive different molecular genetics approaches. RESULTS: Initial screening for the 31 most frequent cystic fibrosis mutations, IVS8 poly(TG)m, poly(T)n, and M470V polymorphisms, identified 8 different mutations in 40 patients (88.9%). Extensive cystic fibrosis transmembrane conductance regulator gene analysis by denaturing gradient gel electrophoresis, denaturing high-performance liquid chromatography, and DNA sequencing detected 17 further mutations, of which three were novel. Cystic fibrosis transmembrane conductance regulator gene rearrangements were searched by semiquantitative fluorescent multiplex polymerase chain reaction, which detected a CFTRdele2,3 (21 kb) large deletion and confirmed two homozygous mutations. Overall, 42 patients (93.3%) had two mutations and 3 patients (6.7%) had one mutation detected. CONCLUSIONS: The present screening strategy allowed a higher mutation detection rate than previous studies, with at least one cystic fibrosis transmembrane conductance regulator gene mutation found in all patients with congenital absence of the vas deferens.

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93 DeltaF508 was the second most common mutation, representing 21 (23.3%) of total alleles, followed by R334W (6, Table 1 CFTR gene mutations and polymorphisms in patients with congenital absence of the vas deferens Mutation Location Nucleotide alteration Effect Method 1 CFTRdele2,3 Exons 2-3 Deletion of exons 2 and 3 Frameshift QFM-PCR 2 R117H Exon 4 G¡A at 482 AA substitution 31 mutation panel 3 P205S Exon 6a C¡T at 745 AA substitution DGGE/dHPLC 4 L206W Exon 6a T¡G at 749 AA substitution DGGE/dHPLC 5 R258G Exon 6b A¡G at 904 AA substitution DGGE/dHPLC 6 R334W Exon 7 C¡T at 1132 AA substitution 31 mutation panel 7 T5 allele Intron 8 Deletion of 2T at 1342-12 to -6 Aberrant splicing DGGE/DNA sequencing 8 P439S Exon 9 C¡T at 1447 AA substitution DGGE/dHPLC 9 D443Ya Exon 9 G¡T at 1459 AA substitution DGGE/dHPLC 10 I507del Exon 10 Deletion of 3 bp at 1648-1653 AA deletion 31 mutation panel 11 DeltaF508 Exon 10 Deletion of 3 bp at 1652-1655 AA deletion 31 mutation panel 12 G542X Exon 11 G¡T at 1756 Truncation 31 mutation panel 13 V562I Exon 12 G¡A at 1816 AA substitution DGGE/dHPLC 14 G576Aa Exon 12 G¡C at 1859 Aberrant splicing DGGE/dHPLC 15 D614G Exon 13 A¡G at 1973 AA substitution DGGE/dHPLC 16 R688Ca Exon 13 C¡T at 2134 AA substitution DGGE/dHPLC 17 V754M Exon 13 G¡A at 2392 AA substitution DGGE/dHPLC 18 E831X Exon 14a G¡T at 2623 Truncation DGGE/dHPLC 19 3272-26AϾG Intron 17a A¡G at 3272-26 Aberrant splicing DGGE/dHPLC 20 2789ϩ5G¡A Intron 14b G¡A at 2789ϩ5 Aberrant splicing 31 mutation panel 21 V1108L Exon 17b G¡C at 3454 AA substitution DGGE/dHPLC 22 L1227S Exon 19 T¡C at 3812 AA substitution DGGE/dHPLC 23 S1235R Exon 19 T¡G at 3837 AA substitution DGGE/dHPLC 24 P1290S Exon 20 C¡T at 4000 AA substitution DGGE/dHPLC 25 N1303K Exon 21 C¡G at 4041 AA substitution 31 mutation panel 26 E1401K Exon 23 G¡A at 4333 AA substitution DGGE/dHPLC Polymorphisms 1 TG repeats Intron 8 9-13 copies at 1342-12 to -35 Sequence variation DGGE/DNA sequencing 2 M470V Exon 10 A or G at 1540 Sequence variation DNA sequencing 3 125G/C Exon 1 G¡C at 125 Sequence variation DGGE/dHPLC 4 1001ϩ11T/C Intron 6b C¡4T at 1001ϩ11 Sequence variation DGGE/dHPLC 5 1716G/A Exon 10 G¡A at 1716 Sequence variation DGGE/dHPLC 6 1899-136T/G Intron 12 T¡G at 1899-136 Sequence variation DGGE/dHPLC 7 T854T Exon 14a T¡G at 2694 Sequence variation DGGE/dHPLC 8 3601-65C/A Intron 18 C¡A at 3601-65 Sequence variation DGGE/dHPLC 9 4521G/A Exon 24 G¡A at 4521 Sequence variation DGGE/dHPLC QFM-PCR, semiquantitative fluorescent multiplex polymerase chain reaction; bp, base pair; DGGE, denaturing gradient gel electrophoresis; dHPLC, denaturing high-performance liquid chromatography. Login to comment
97 The allelic frequency of the other mutations was 4.4% for R117H, G576A, and R668C, 3.3% for S1235R and 3272-26A¡G, and 2.2% for P205S, L206W, D443Y, G542X, D614G, and N1301K, whereas the remaining 12 mutations were present in single patients (Table 3). Login to comment
101 The missense M470V polymorphism was evaluated in all 45 pa- tientswithCAVD(Table2).TheallelicfrequencyoftheM470variant Table 2 CFTR genotypes identified in patients with congenital absence of the vas deferens CFTR mutation genotypes [(TG)mTn] genotype M470V Patients N % DeltaF508 (TG)10T9 (TG)12T5 M V 11 24.4 DeltaF508 (TG)10T9 (TG)11T5 M M 1 2.2 DeltaF508 R117H (TG)10T9 (TG)10T7 M M 2 4.4 G542X (TG)10T9 (TG)12T5 M V 2a 4.4 DeltaF508 R334W (TG)10T9 (TG)11T7 M V 1 2.2 DeltaF508 D443Y-G576A-R668C (TG)10T9 (TG)10T7 M M 1 2.2 DeltaF508 D614G (TG)10T9 (TG)11T7 M V 1 2.2 DeltaF508 E831X (TG)10T9 (TG)11T7 M V 1 2.2 DeltaF508 L1227S (TG)10T9 (TG)11T7 M M 1 2.2 DeltaF508 E1401K (TG)10T9 (TG)11T7 M V 1 2.2 I507del D614G (TG)11T7 (TG)10T7 M V 1 2.2 N1303K L206W (TG)10T9 (TG)9T9 M M 1 2.2 R117H P205S (TG)11T7 (TG)10T7 M V 1 2.2 R117H R334W (TG)10T7 (TG)11T7 M V 1 2.2 R334W P439S (TG)11T7 (TG)11T7 M V 1 2.2 R334W R334Wb (TG)11T7 (TG)11T7 V V 1 2.2 R334W V562I (TG)11T7 (TG)11T5 V M 1 2.2 D443Y-G576A-R668C 3272-26A¡G (TG)10T7 (TG)10T7 M M 1 2.2 G576A-R668C V754Mb (TG)10T7 (TG)11T7 M M 1 2.2 S1235R S1235Rb (TG)13T5 (TG)13T5 M M 1 2.2 2789ϩ5G¡A S1235Rb (TG)10T7 (TG)13T5 M M 1 2.2 3272-26A¡G P1290S (TG)11T7 (TG)10T7 M V 1 2.2 P205S (TG)11T7 (TG)12T5 V V 1 2.2 G576A-R668C b (TG)10T7 (TG)11T5 M M 1 2.2 V1108L b (TG)11T7 (TG)11T5 V M 1 2.2 N1303K (TG)10T9 (TG)12T5 M V 1 2.2 3272-26A¡G b (TG)10T7 (TG)12T5 M V 1 2.2 CFTRdele2,3 b (TG)11T7 (TG)13T5 V M 1 2.2 b (TG)11T5 (TG)12T5 M V 1 2.2 b (TG)13T5 (TG)12T5 M V 1 2.2 DeltaF508 - (TG)10T9 (TG)11T7 M V 1a 2.2 L206W -b (TG)9T9 (TG)11T7 M V 1 2.2 R258G -b (TG)11T7 (TG)11T7 V V 1 2.2 a CUAVD. Login to comment
110 Large Table 3 Allelic frequencies of CFTR mutations in patients with congenital absence of the vas deferens CBAVD CUAVD Total Patients 42 3 45 Alleles 84 6 90 Mutations N % N % N % 1 T5 allele 26a 31 2 33.3 28 31.1 2 DeltaF508 20 23.8 1 16.7 21 23.3 3 R334W 6a 7.1 0 0 6 6.7 4 R117H 4 4.8 0 0 4 4.4 5 G576A 4b 4.8 0 0 4 4.4 6 R688C 4b 4.8 0 0 4 4.4 7 S1235R 3a 3.6 0 0 3 3.3 8 3272-26A¡G 3 3.6 0 0 3 3.3 9 P205S 2 2.4 0 0 2 2.2 10 L206W 2 2.4 0 0 2 2.2 11 D443Y 2b 2.4 0 0 2 2.2 13 D614G 2 2.4 0 0 2 2.2 14 N1303K 2 2.4 0 0 2 2.2 12 G542X 0 0 2 33.3 2 2.2 15 R258G 1 1.2 0 0 1 1.1 16 P439S 1 1.2 0 0 1 1.1 17 I507del 1 1.2 0 0 1 1.1 18 V562I 1 1.2 0 0 1 1.1 19 V754M 1 1.2 0 0 1 1.1 20 E831X 1 1.2 0 0 1 1.1 21 2789ϩ5G¡A 1 1.2 0 0 1 1.1 22 V1108L 1 1.2 0 0 1 1.1 23 L1227S 1 1.2 0 0 1 1.1 24 P1290S 1 1.2 0 0 1 1.1 25 E1401K 1 1.2 0 0 1 1.1 26 CFTRdele2,3 1 1.2 0 0 1 1.1 CBAVD, congenital bilateral absence of the vas deferens; CUAVD, congenital unilateral absence of the vas deferens. Login to comment

[hide] Validation of cystic fibrosis mutation analysis us... Diagn Mol Pathol. 2007 Mar;16(1):57-9. Huang CK, Pan Q
Validation of cystic fibrosis mutation analysis using ABI 3130XL genetic analyzer.
Diagn Mol Pathol. 2007 Mar;16(1):57-9., [PMID:17471160]

Abstract [show]
Cystic fibrosis (CF) is one of the most common autosomal recessive diseases in the white population, with a prevalence estimate of 1 in 2500 to 3300 live births. CF is characterized by viscous mucus in the lungs with involvement of digestive and reproductive systems as well as sweat glands (excess salt loss). Treatment for CF patients is palliative. Over 1300 mutations have been identified in the CFTR gene. However, most of the mutations are at frequencies of <0.1% or represent private mutations. Although other methodologies are available for CF testing, the oligonucleotide ligation assay is a unique approach to mutation detection of point mutations, small deletions, and small insertions, and consists of 2 phases. Applied Biosystems 3130 Series Genetic Analyzers are the next-generation platform for low to medium throughput laboratories and deliver improved performance. One disadvantage of the Genetic Analyzers is that there is no template of instrument settings for POP-6 polymer using 36-cm array. The Abbott CF oligonucleotide ligation assay ASRs can be run only using POP-6 polymer. We are the first to have optimized the instrument settings for POP-6 polymer based on the template of Rapidseq36-POP6 for Abbott Diagnostics CF V3 ASRs. Several conditions were tried, and the conditions of sample injection voltage at 10,000 v and sample injection time at 5 seconds gave better results, which were with clearer peaks and lower background signals. Twenty cell line DNA samples from Coriell were analyzed, and the results were matched. In addition, Synthetic Controls from AcroMetrix were analyzed, and the results were same as expected. Also, about 1500 clinical samples were analyzed, and high-quality reportable results were obtained. In conclusion, our modified protocol is robust and reliable on this ABI 3130XL instrument.

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58 Mutation controls: to specifically assess the detection of CF mutations, 20 cell line DNA samples with mutations of R553X, 3659delC/delF508, delF508/Q493X, 711+ 1G>T/621+1G>T, 621+1G>T/delF508, G85E/ 621+1G>T, R560T/delF508, A455E/621+1G>T, N1303K, W1282X, G551D/R553X, 2789+5G>A/ 2789+5G>A, 3849+10C>T/3849+10C>T, 1717-1G>A, delF508/delF508, R347P/G551D, R334W, V520F, R117H/delF508/5T/9T, or G542X/G542X, respectively, from the Coriell Cell Repositories were analyzed. Login to comment

[hide] Clinical evaluation of isolated nonvisualized feta... Prenat Diagn. 2007 Aug;27(8):699-703. Ochshorn Y, Rosner G, Barel D, Bronshtein M, Muller F, Yaron Y
Clinical evaluation of isolated nonvisualized fetal gallbladder.
Prenat Diagn. 2007 Aug;27(8):699-703., [PMID:17510919]

Abstract [show]
OBJECTIVE: Isolated nonvisualized fetal gallbladder (INVFGB) is relatively rare. In most cases, the gallbladder will eventually be detected. In some cases however, INVFGB may be associated with serious abnormalities, cystic fibrosis (CF), aneuploidy, and agenesis of the gall bladder. We describe a clinical evaluation of prenatally diagnosed INVFGB. METHODS: Cases of nonvisualized gallbladder were first evaluated by serial scans. Cases with no additional malformations were designated as INVFGB, and were further evaluated by mutation analysis for CF, and amniocentesis for karyotype and microvillar membrane enzymes (MME). RESULTS: A total of 22 cases of nonvisualized gallbladder were detected. Of these, 2 had additional malformations, and 3 were excluded because of incomplete evaluation. Of the remaining 17 cases, 3 (17.6%) had adverse outcomes: 1 case of CF, 1 case of 47,XXX, and 1 case of multiple congenital anomalies detected only postnatally. Abnormal levels of MMEs were detected in 3 cases, 1 of which was diagnosed with CF. In 2 cases, the gallbladder was not detected even after birth, but development is normal. CONCLUSION: Evaluation of INVFGB should include genetic counselling, amniocentesis for karyotype and MME analysis, CFTR mutation analysis and repeated ultrasound scans.

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32 Mutation analysis for cystic fibrosis: The mutation panel included cystic fibrosis transmembrane conductance regulator (CFTR) mutations that are common in the Israeli population, including F508del, W1282X, N1303K, G542X, and D1152H. Login to comment

[hide] Cystic fibrosis and formes frustes of CFTR-related... Respiration. 2007;74(3):241-51. Southern KW
Cystic fibrosis and formes frustes of CFTR-related disease.
Respiration. 2007;74(3):241-51., [PMID:17534127]

Abstract [show]
Cystic fibrosis (CF) is the commonest genetic cause of bronchiectasis in the Caucasian population. Since identification of the putative gene in 1989, the molecular basis of the condition has become clearer with characterisation of the unique pathophysiology. The small airways are the primary site of lung disease, with an intense but localised inflammatory picture, dominated by neutrophils. The clinical heterogeneity is explained to some degree by the distinct molecular consequences of the many mutations that have been recognised to affect the CF transmembrane conductance regulator (CFTR) gene; however other genes appear to modify the phenotype as well as environmental exposure. It has become increasingly apparent that certain conditions may result from CFTR dysfunction without fulfilling diagnostic criteria for CF. In some cases this may result in single organ disease for which the term CF (or CFTR)-related disease has been advocated. Congenital bilateral absence of the vas deferens is the most clearly characterised of these. In other cases where a mild CF phenotype is apparent, atypical CF is probably a better term. It remains unclear whether carrier status predisposes to certain conditions such as chronic rhinosinusitis or pancreatitis.

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34 Interestingly, the most prevalent CFTR mutations aside from phe508del are thought to originate from ancient populations (G542X, Phoenicians [16]; G551D, Celtic [17], and 394delTT, Nordic [17]), though none approach the prevalence of phe508del. Login to comment
60 Classes of CFTR mutations, with molecular and phenotypic consequences Class Molecular consequence Example Phenotypic consequence I nonsense or frameshift mutations that result in no significant protein product G542X typical CF phenotype II protein product does not negotiate intracellular trafficking pathways phe508del R1066C A561E typical CF phenotype III protein product transported to the cell membrane but no significant ion transport function G551D typical CF phenotype IV protein product transported to cell membrane and functions at a low level R117H R334W associated with pancreatic sufficiency V reduced mRNA expression, protein product normal 5T variant of intron poly T region. Login to comment

[hide] Restoration of W1282X CFTR activity by enhanced ex... Am J Respir Cell Mol Biol. 2007 Sep;37(3):347-56. Epub 2007 May 31. Rowe SM, Varga K, Rab A, Bebok Z, Byram K, Li Y, Sorscher EJ, Clancy JP
Restoration of W1282X CFTR activity by enhanced expression.
Am J Respir Cell Mol Biol. 2007 Sep;37(3):347-56. Epub 2007 May 31., [PMID:17541014]

Abstract [show]
Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Premature termination codons represent a common minority of CFTR mutations, and are caused by base pair substitutions that produce abnormal stop codons in the coding sequence. Select aminoglycosides induce "translational readthrough" of premature stop codons and have been shown to restore full-length functional protein in a number of preclinical and clinical settings. We studied two well-described premature termination codons found in the distal open reading frame of CFTR, W1282X and R1162X, expressed in polarizing and nonpolarizing cells. Our findings indicate that W1282X CFTR-expressing cells demonstrate significantly greater CFTR activity when overexpressed compared with R1162X CFTR cells, even when truncated protein is the predominant form. In addition, our results show that the combination of stimulated expression and stop codon suppression produces additive effects on CFTR-mediated ion transport. These findings provide evidence that W1282X CFTR exhibits membrane localization and retained chloride channel function after enhanced expression, and suggest that patients harboring this mutation may be more susceptible to CFTR rescue.

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85 RESULTS Preferential Enhancement of CFTR Activity in W1282X CFTR Expressing Cells by Sodium Butyrate Previous studies have examined the function of several CFTR molecules containing clinically relevant premature stop codons in transient, high-level expression systems using nonpolarizing cell types (including G542X, R553X, R1162X, and W1282X CFTR), with variable levels of constitutive and regulated CFTR activity described (7, 8, 33). Login to comment
221 In previously published studies, our laboratory has reported that high-level expression of the two most common CFTR premature stop mutations G542X and R553X CFTR (using transient vaccinia-based expression in non-polarizing cells) led to constitutive halide transport function lacking cAMP-dependent regulation (8, 27). Login to comment

[hide] Correlation of chest radiograph pattern with genot... Chest. 2007 Aug;132(2):569-74. Epub 2007 Jun 15. Kaza V, Katz MF, Cumming S, Frost AE, Safdar Z
Correlation of chest radiograph pattern with genotype, age, and gender in adult cystic fibrosis: a single-center study.
Chest. 2007 Aug;132(2):569-74. Epub 2007 Jun 15., [PMID:17573513]

Abstract [show]
INTRODUCTION: Cystic fibrosis (CF) is a common lethal genetic disorder. The aim of this study was to determine the common chest radiograph (CXR) patterns in adult CF, and correlate disease distribution on CXRs with genotype, age, and gender. METHODS: One hundred nine CF patients treated at Baylor Adult Cystic Fibrosis Center were identified. The intake CXR was reviewed and characterized as diffuse bilateral (DB), unilateral, upper lobe (UL), and lower lobe (LL) disease, or relatively normal. Lack of intake CXR, and/or genotype excluded 41 patients from analysis. RESULTS: Of 68 patients, 38 were homozygous for DeltaF508 and 30 were heterozygous. Mean age of the population was 30 +/- 8 years (+/- SD) [range, 18 to 48 years]. The most common CXR pattern was DB; 62% had DB, 28% had UL, and 7% had LL predominance. This is in contrast to the UL-predominant CXR pattern commonly described in the pediatric population. In 18 DB patients, archived pediatric films were available, and the average patient age was 15.7 years. DB pattern was present in 16 of 18 CXRs that antedated adult intake CXRs by an average of 12.7 years. Homozygous DeltaF508 genotype was identified in 56% of patients and did not distinguish radiologic phenotypes. There was no association between radiograph pattern and identified infecting/colonizing organisms and percentage of predicted FEV(1). CONCLUSIONS: CF has commonly been reported as an UL disease. However, in this study of adult patients, the common pattern observed was DB. A small subgroup analysis suggests that DB disease was not a pattern of disease evolution but may be present from disease onset.

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62 Homozygous ⌬F508 38 F508/no ID 10 F508/G542X 4 F508/n3849 ϩ 10KBT 2 F508/N1303K 2 F508/G85E 2 F508/G551D 2 F508/R1179H 1 F508/3849 ϩ 10 KBC.T 1 F508/621ϩIG-T 1 F508/3659deltaC 1 F508/P67L 1 F508/2789 ϩ 5E 1 G551/LL48T 1 G551D/no ID 1 Total 68 570 our adult CF population was UL predominance; 26% of those homozygous for ⌬F508 (group I) and 30% of the other genotypes (group II) had the radiologic appearance of UL disease (Fig 2). Login to comment

[hide] Activation of CFTR-specific T Cells in cystic fibr... Mol Ther. 2007 Sep;15(9):1694-700. Epub 2007 Jun 19. Limberis MP, Figueredo J, Calcedo R, Wilson JM
Activation of CFTR-specific T Cells in cystic fibrosis mice following gene transfer.
Mol Ther. 2007 Sep;15(9):1694-700. Epub 2007 Jun 19., [PMID:17579582]

Abstract [show]
Gene therapy for cystic fibrosis (CF) airway disease has emerged as a potentially successful therapy, because expression of the CF gene would be expected to restore the electrophysiological function of the airway epithelium to normalcy. Although, cellular and humoral immune responses to viral gene transfer vectors have been studied extensively, there has been no evaluation of T cell-mediated responses to the therapeutic human CF gene product. Using an adenovirus vector we demonstrated that T cells against human CF gene protein are elicited in CF gene knockout (KO), heterozygote (Het), and wild-type (wt) mice. A dominant CD8 T cell epitope found in CF gene KO, Het, and wt mice was mapped to NTYLRYITV. In CF gene KO mice we also identified (to a conserved region of the CF gene CSQFSWIMPGTIKEN), a minor T cell epitope that did not show any activity in the Het or wt mice.

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16 The first set of studies attempted to model the consequences of in vivo gene transfer in recipients void of CFTR -expression such as in those with two Class I CFTR gene -mutations, with CFTR being either deleted or substantially truncated (i.e., G542X).16 This clinical situation was modeled by delivering an adenovirus vector expressing hCFTR into mice homozygous for the germ line interruption of CFTR. Login to comment
133 The most common Class I CFTR mutation, which is also the next-to-most frequent mutation in Caucasian populations, is G542X.25 The premature stop codon in this CFTR mutation leads to the production of a truncated non-functional CFTR protein25 resulting in a significant repertoire of CFTR-specific T cells that escape central tolerance and are at risk of activation in the setting of CFTR gene therapy. Login to comment
286 CFTR nonsense mutations G542X and W1282X associated with severe reduction of CFTR mRNA in nasal epithelial cells. Login to comment

[hide] Does cystic fibrosis neonatal screening detect aty... Clin Genet. 2007 Jul;72(1):39-46. Narzi L, Ferraguti G, Stamato A, Narzi F, Valentini SB, Lelli A, Delaroche I, Lucarelli M, Strom R, Quattrucci S
Does cystic fibrosis neonatal screening detect atypical CF forms? Extended genetic characterization and 4-year clinical follow-up.
Clin Genet. 2007 Jul;72(1):39-46., [PMID:17594398]

Abstract [show]
The neonatal screening protocol for cystic fibrosis (CF) is based on a first determination of blood immunoreactive trypsin (IRT1), followed by a first level genetic test that includes the 31 worldwide most common mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene (DNA31), and a second determination of blood immunoreactive trypsin (IRT2). This approach identifies, in addition to affected subjects, a high proportion of newborns with hypertrypsinaemia at birth, in whom only one mutation is identified and who have a negative or borderline sweat test and pancreatic sufficiency. Although it has been suggested that hypertrypsinaemia may be caused by a single CFTR mutation, whether such neonates should be merely considered as healthy carriers remains a matter of debate as hypertrypsinaemia at birth may be a biochemical marker of a CFTR malfunction because of a second mild mutation. We analyzed, by means of an extended sequencing protocol, 32 newborns who tested positive at an IRT1/DNA31/IRT2 screening protocol and in whom only one CFTR mutation was found. The results obtained demonstrate that 62.5% of these newborns were also carrying a second mild CFTR mutation. The high proportion of compound heterozygous subjects, combined with the results of a 4-year follow-up in nine of these subjects all of whom displaying initial CF clinical symptoms, suggest that it may be possible to use the IRT1/DNA31/IRT2 protocol of neonatal screening to identify newborns with atypical forms of CF. In view of these findings, an extended genetic search for subjects with compound heterozygosity and a periodic clinical assessment should be considered.

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48 CFTR genotypes, IRT2 and sweat test values of the 32 newborns analyzed Newborn CFTR genotype IRT2 Sweat test (mmol/l [Cl2 ]) at enrolment True heterozygous subjects 1 N1303K/1 Negative 18 2 2183AAtoG/1 Negative 11 3 G85E/1 Positive 19 4 F508del/1 Negative 21 5 F508del/1 Negative 20 6 R117H/1 Negative 6 7 1717-1GtoA/1 Positive 7 8 W1282X/1 Negative 14 9 278915GtoA/1 Negative 23 10 N1303K/1 Negative 19 11 F508del/1 Negative 14 12 G542X/1 Negative 39 % of positivity ¼ 16.7% Average Æ SD ¼ 18 Æ 9 Compound heterozygous subjects 13 F508del/D806G Positive 24 14 F508del/D836Y Negative 12 15 R347P/R1162L Negative 18 16 F508del/P5L (TG)11T5 Negative 16 17 F508del/L997F Positive 32 18 R347P/D1152H Positive 42 19 F508del/P5L Negative 42 20 278915GtoA/71113AtoG Positive 33 21 F508del/P5L Positive 39 22 F508del (TG)12T7/(TG)12T5 Negative 23 23 N1303K/S1235R (TG)12T7 Negative 30 24 F508del/L997F Positive 34 25 F508del/(TG)12T5 Negative 34 26 R117H/(TG)12T7 Positive 22 27 F508del/P1013L Positive 8 28 F508del/L997F Negative 28 29 N1303K/(TG)12T5 Positive 13 30 F508del/L997F Positive 50 31 R1162X/P5L Negative 31 32 L997F/S549R(AtoC) Positive 38 % of positivity ¼ 55.0% Average Æ SD ¼ 29 Æ 12 CFTR, cystic fibrosis transmembrane conductance regulator. Login to comment
75 Discussion The majority of the mutations found (F508del, R347P, D1152H, 2789 1 5G-.A, 711 1 3A-.G, N1303K, R117H, R1162X, S549R(A-.C), 2183AA-.G, G85E, 1717-1G-.A, G542X, and W1282X) have an established pathogenic role (26-44). Login to comment

[hide] Analysis of cystic fibrosis gene mutations and ass... Genet Test. 2007 Summer;11(2):133-8. Knezevic J, Tanackovic G, Matijevic T, Barisic I, Pavelic J
Analysis of cystic fibrosis gene mutations and associated haplotypes in the Croatian population.
Genet Test. 2007 Summer;11(2):133-8., [PMID:17627383]

Abstract [show]
The aim of this study was to reveal the CFTR gene mutation status in the Croatian population as well as to establish the haplotypes associated with cystic fibrosis (CF) and those associated with specific gene mutations. A total of 48 unrelated CF patients from Croatia were examined. Among 96 tested alleles, we found nine different mutations: DeltaF508, 58.33%; G542X, 3.12%; N1303K, 2.08%; R1162X; 621 + 1G --> T; G85E; Y569C; E585X; and S466X, 1.04%. Analysis of three polymorphic loci revealed 15 different haplotypes. Two of them (21-23-13 and 21-17-13) occurred with a higher frequency (40% and 24%). Both of these haplotypes also carried a CFTR gene mutation (DeltaF508 or G542X) on 27 out of 32 chromosomes. Among 12 (of all together 29) CF alleles on which no mutations were found, we detected 10 different haplotypes. Because there are still no published data on the distribution of polymorphic loci in Croatia, nor haplotypes associated with mutations in the CFTR gene, our results greatly contribute to knowledge regarding the genetic background of CF in this region.

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2 Among 96 tested alleles, we found nine different mutations: ⌬F508, 58.33%; G542X, 3.12%; N1303K, 2.08%; R1162X; 621 ؉ 1G→T; G85E; Y569C; E585X; and S466X, 1.04%. Login to comment
5 Both of these haplotypes also carried a CFTR gene mutation (⌬F508 or G542X) on 27 out of 32 chromosomes. Among 12 (of all together 29) CF alleles on which no mutations were found, we detected 10 different haplotypes. Login to comment
11 Only four other mutations, G542X, G551D, N1303K, and W1282X, are relatively frequent in the European population (1-2.5%) (Morral et al., 1996). Login to comment
39 INNOGENETICS INNO-LIPA CFTR 12 and INNO-LIPA CFTR 7 ϩ Tn diagnostic kits were used to assess the presence of the 29 mutations in CF patients; ⌬F508, ⌬I507, G542X, N1303K, 1717-1G Ǟ A, W1282X, G551D, R553X, S1251N, R560T, 3905insT, Q552X, 394delTT, G85E, E60X, 621 ϩ 1G Ǟ T, R117H, 1078delT, R347P, R334W, 2143delT, 2183AA Ǟ G, 2184delA, 711 ϩ 5G Ǟ A, 2789 ϩ 5G Ǟ A, R1162X, 3659delC, 3849 ϩ 10kbC Ǟ T, and A455E. Login to comment
50 Nine different mutations were found: ⌬F508 (58.33%), G542X (3.12%), N1303K (2.08%), R1162X, 621 ϩ 1G Ǟ T, G85E, Y569C, E585X, and S466X (1.04%). Login to comment
76 Of three CFTR alleles with the G542X mutation, two had the 21-23-13 haplotype. Login to comment
81 MUTATIONS AND CORRESPONDING GENOTYPES OBSERVED IN A CROATION COHORT OF CF PATIENTS Number of affected Number of detected Mutation alleles (%) Genotype genotypes (%) ⌬F508 56 (58.33) ⌬F508/⌬F508 19 (39.58) G542X 3 (3.12)0 ⌬F508/Na 7 (14.58) N1303K 2 (2.08)0 ⌬F508/G542X 3 (6.25)0 R1162X 1 (1.04)0 ⌬F508/N1303K 2 (4.17)0 621ϩ1G→T 1 (1.04)0 ⌬F508/R1162X 1 (2.08)0 G85E 1 (1.04)0 ⌬F508/621ϩ1G→T 1 (2.08)0 Y569C 1 (1.04)0 ⌬F508/G85E 1 (2.08)0 E585X 1 (1.04)0 ⌬F508/Y569C 1 (2.08)0 S466X 1 (1.04)0 ⌬F508/E585X 1 (2.08)0 Na 29 (30.21) ⌬F508/S466X 1 (2.08) Na/Na 11 (22.92) Total 96b Total 48c aAlleles without mutation. Login to comment
89 The most frequent was ⌬F508 (65%), followed by G542X (5%), N1303K, and 1717-1G Ǟ A (3.3%). Login to comment
100 The two most often seen haplotypes (21-23-13 and 21-17-13) carried either ⌬F508 or G542X in 75% of tested chromosomes (27 out of 36). Login to comment
112 ASSOCIATION BETWEEN MICROSATELLITE HAPLOTYPES AND CFTR GENE MUTATIONS IN A CROATIAN COHORT OF CF PATIENTS Haplotype Number of haplotypesa (%) Mutationsb No mutationc 21-23-13 20 (40) ⌬F508 (15) 3 G542X (2) 21-17-13 12 (24) ⌬F508 (10) 2 21-25-13 3 (6)0 ⌬F508 (3) / 21-21-13 2 (4)0 ⌬F508 (1) / 621ϩ1G→T (1) / 20-23-13 2 (4)0 ⌬F508 (2) / 22-17-13 1 (2)0 ⌬F508 (1) / 22-16-13 1 (2)0 G85E (1) / 25-17-13 1 (2)0 1 25-16-14 1 (2)0 1 26-16-13 1 (2)0 1 21-16-13 2 (4)0 2 22-23-13 1 (2)0 1 23-23-13 1 (2)0 1 19-23-13 1 (2)0 1 22-16-17 1 (2)0 1 aNumber of alleles with the corresponding haplotype. Login to comment

[hide] Too much salt, too little soda: cystic fibrosis. Sheng Li Xue Bao. 2007 Aug 25;59(4):397-415. Quinton PM
Too much salt, too little soda: cystic fibrosis.
Sheng Li Xue Bao. 2007 Aug 25;59(4):397-415., 2007-08-25 [PMID:17700961]

Abstract [show]
Cystic fibrosis (CF) of the pancreas is the most widely accepted name of the most common fatal inherited single gene defect disease among Caucasians. Its incidence among other races is thought to be significantly less, but mutations in the gene have been reported in most, if not all, major populations. This review is intended to give general concepts of the molecular as well as physiological basis of the pathology that develops in the disease. First, an overview of the organ pathology and genetics is presented, followed by the molecular structure of the gene product (cystic fibrosis transmembrane conductance regulator, CFTR), its properties, functions, and controls as currently understood. Second, since mutations appear to be expressed primarily as a defect in electrolyte transport, effects and mechanisms of pathology are presented for two characteristically affected organs where the etiology is best described: the sweat gland, which excretes far too much NaCl ("salt") and the pancreas, which excretes far too little HCO3(- )("soda"). Unfortunately, morbidity and mortality in CF develop principally from refractory airway infections, the basis of which remains controversial. Consequently, we conclude by considering possible mechanisms by which defects in anion transport might predispose the CF lung to chronic infections.

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30 Of the other mutations only a few occur with a frequency greater than 1%, including G542X (2.4%), G551D (1.6%), N1303K (1.3%), and W1282X (1.2%). Login to comment

[hide] Cystic fibrosis in a southern Brazilian population... Clin Genet. 2007 Sep;72(3):218-23. Faucz FR, Gimenez J, Ramos MD, Pereira-Ferrari L, Estivill X, Raskin S, Casals T, Culpi L
Cystic fibrosis in a southern Brazilian population: characteristics of 90% of the alleles.
Clin Genet. 2007 Sep;72(3):218-23., [PMID:17718859]

Abstract [show]
Cystic fibrosis (CF) is a genetic disease that frequently leads to death in infancy among Europeans and their descendants. The goals of the present study were to analyze the molecular aspects of CFTR gene characterizing mutations, their frequencies, and the haplotypes formed by four CFTR gene intragenic markers, IVS8-6(T)n, IVS8CA, IVS17bTA and IVS17bCA, in a southern Brazilian population of Caucasian origin. DNA samples from 56 non-related CF patients were analyzed using scanning techniques (single strand conformation polymorphism and denaturing gradient gel electrophoresis), restriction fragment length polymorphism and direct DNA sequencing to identify the mutations. Our results revealed a total of 25 different CF mutations representing nearly 90% of CF alleles, two being novel mutations. Microsatellite haplotypes were defined for CF and normal alleles. The mutational spectrum and the associated haplotypes described for the first time in this study should prove relevant for genetic counselling and CF population screening in Brazil. Moreover, our results suggest the presence of a major Mediterranean component in the contemporary Brazilian CF patient pool.

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20 We previously reported the mutation heterogeneity in Brazilian CF patients by direct analysis of F508del and four other common mutations (G542X, N1303K, G551D and R553X). Login to comment
55 Nine mutations showed a frequency higher than 1%, F508del (45.5%), G542X (6.3%), N1303K (4.5%), G85E, R334W and R1162X (3.6%), 2183AA.G and W1282X Table1.FrequenciesoftheCFTRmutations,theirmicrosatellitehaplotypesandIVS8-6(T)nallelesintheBrazilianCFpatientsa MutationExon/intron ChromosomesParana State/SantaCatarina State(total)%HaplotypesIVS8CA,IVS17bTA,IVS17bCA(n)(T)nlocus(n) DF508Exon1027/24(51)45.5416-7-17(1)/16-29-14(1)/16-31-13(1)/17-30-13 (1)/17-31-13(20)/17-32-13(7)23-31-13(15)/23-32-14 (1)/23-46-13(1)/25-30-13(1)/26-31-13(1)/unknown(1) 9T(44)/7T(3)unknown(4) G542XExon115/2(7)6.2523-32-13(1)/23-33-13(5)/23-34-13(1)9T(7) N1303KExon212/3(5)4.4616-30-13(1)/23-30-13(1)/23-31-13(3)9T(4)/7T(1) G85EExon32/2(4)3.5716-24-13(4)7T(4) R334WExon71/3(4)3.5716-34-13(1)/(16-48-13)(1)/17-33-13(1)/17-41-13(1)7T(3)/unknown(1) R1162XExon191/3(4)3.5717-31-13(4)7T(4) 2183AA.GExon131/2(3)2.6816-31-13(2)/16-31-14(1)7T(2)/unknown(1) W1282XExon201/2(3)2.6817-7-17(3)7T(2)/9T(1) R553XExon112/0(2)1.7817-44-11(1)/17-47-11(1)7T(1)/unknown(1) S4XExon11/0(1)0.89(16-__-13)(1)Unknown(1) 232del18Exon20/1(1)0.8921-36-13(1)Unknown(1) 62111G.TIntron41/0(1)0.89__-34-13(1)Unknown(1) 71111G.TIntron51/0(1)0.8916-25-13(1)7T(1) 71115G.AIntron51/0(1)0.89__-7-17(1)Unknown(1) R347PExon70/1(1)0.8916-32-13(1)7T(1) 1717-1G.AIntron101/0(1)0.8916-7-17(1)7T(1) 1717-8G.AIntron101/0(1)0.8916-33-13(1)9T(1) 1812-1G.AIntron111/0(1)0.8916-31-14(1)9T(1) A561EExon121/0(1)0.8916-44-13(1)7T(1) E585XExon121/0(1)0.89Unknown(1)7T(1) 189811G.AIntron120/1(1)0.8916-45-13(1)7T(1) G1069RExon17b1/0(1)0.8917-30-13(1)Unknown(1) Y1092XExon17b1/0(1)0.8916-30-137T(1) 3849110kbC.TIntron191/0(1)0.8916-7-17(1)7T(1) W1282GExon201/0(1)0.8916-32-14(1)7T(1) Unknown13/0(13)11.6016-7-17(1)/16-29-13(2)/16-30-13(1)/16-31-13 (1)/16-32-13(3)/16-33-13(1)16-34-13(1)/16-38-16 (1)/18-35-13(2) Unknown(13) Total112100 Ôn`,thetotalnumberofchromosomesbearingeachhaplotypeor(T)nlocus;Ôunknown`,usedwhenthehaplotype/(T)nlocuscannotbecharacterized;Ô_`,usedwhenaspecific alleleofthehaplotypecannotbecharacterized. Login to comment
90 Previously, such heterogeneity was indeed identified in Brazilian CF patients of European origin by the screening of five common mutations (F508del, G542X, N1303K, G551D and R553X) (13). Login to comment
96 Our mutational spectrum showed a certain similarity with that reported in the Italian population (F508del, 48.9%; G542X, 5.9%; N1303K, 5.9%; and 2183AA.G, 2.6%) (28). Login to comment
98 For example, common mutations in the Portuguese population show quite different frequencies (G542X, 1.3%; N1303K, 0.7%, and 2183AA.G, 0%) (10). Login to comment

[hide] Negative genetic neonatal screening for cystic fib... Clin Genet. 2007 Oct;72(4):374-7. Girardet A, Guittard C, Altieri JP, Templin C, Stremler N, Beroud C, des Georges M, Claustres M
Negative genetic neonatal screening for cystic fibrosis caused by compound heterozygosity for two large CFTR rearrangements.
Clin Genet. 2007 Oct;72(4):374-7., [PMID:17850636]

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34 If IRT at day 3 is positive (.65 ng/ml), the card is subjected to an ARMS Elucigen kit (Tepnel) testing for 30 common CF mutations (F508del, Y1092X, 1717-1G.A, G542X, W1282X, N1303K, 3849110kbC.T, 394delTT, 62111G.T, S1251N, G551D, R117H, R1162X, R334W, A455E, 2183AA.G, 3659delC, 1078delT, I507del, R347P, R553X, E60X, 1 8 1 1 11 . Login to comment

[hide] Scanning the cystic fibrosis transmembrane conduct... Clin Chem. 2007 Nov;53(11):1891-8. Epub 2007 Sep 21. Montgomery J, Wittwer CT, Kent JO, Zhou L
Scanning the cystic fibrosis transmembrane conductance regulator gene using high-resolution DNA melting analysis.
Clin Chem. 2007 Nov;53(11):1891-8. Epub 2007 Sep 21., [PMID:17890437]

Abstract [show]
BACKGROUND: Complete gene analysis of the cystic fibrosis transmembrane conductance regulator gene (CFTR) by scanning and/or sequencing is seldom performed because of the cost, time, and labor involved. High-resolution DNA melting analysis is a rapid, closed-tube alternative for gene scanning and genotyping. METHODS: The 27 exons of CFTR were amplified in 37 PCR products under identical conditions. Common variants in 96 blood donors were identified in each exon by high-resolution melting on a LightScanner(R). We then performed a subsequent blinded study on 30 samples enriched for disease-causing variants, including all 23 variants recommended by the American College of Medical Genetics and 8 additional, well-characterized variants. RESULTS: We identified 22 different sequence variants in 96 blood donors, including 4 novel variants and the disease-causing p.F508del. In the blinded study, all 40 disease-causing heterozygotes (29 unique) were detected, including 1 new probable disease-causing variant (c.3500-2A>T). The number of false-positive amplicons was decreased 96% by considering the 6 most common heterozygotes. The melting patterns of most heterozygotes were unique (37 of 40 pairs within the same amplicon), the exceptions being p.F508del vs p.I507del, p.G551D vs p.R553X, and p.W1282X vs c.4002A>G. The homozygotes p.G542X, c.2789 + 5G>A, and c.3849 + 10kbC>T were directly identified, but homozygous p.F508del was not. Specific genotyping of these exceptions, as well as genotyping of the 5T allele of intron 8, was achieved by unlabeled-probe and small-amplicon melting assays. CONCLUSIONS: High-resolution DNA melting methods provide a rapid and accurate alternative for complete CFTR analysis. False positives can be decreased by considering the melting profiles of common variants.

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202 With the exception of p.G551D and p.R553X, the melting profile of each genotype is distinct, including the G542X homozygote. Login to comment
9 The homozygotes p.G542X, c.2789 ؉ 5G>A, and c.3849 ؉ 10kbC>T were directly identified, but homozygous p.F508del was not. Login to comment
132 Furthermore, 3 homozygous disease-causing variants were identified in the blinded study: p.G542X, c.2789 ϩ 5GϾA, and c.3849 ϩ 10kbCϾT. Login to comment

[hide] CFTR mutations and reproductive outcomes in a popu... Hum Genet. 2008 Jan;122(6):583-8. Epub 2007 Sep 28. Gallego Romero I, Ober C
CFTR mutations and reproductive outcomes in a population isolate.
Hum Genet. 2008 Jan;122(6):583-8. Epub 2007 Sep 28., [PMID:17901983]

Abstract [show]
Multiple hypotheses have been proposed to explain the high incidence of cystic fibrosis in Caucasian populations. Most rely on a fitness advantage to carriers of CF mutations, either through increased resistance to infectious disease, such as cholera, or through increased fertility. In this study we tested the latter hypothesis in the Hutterites of South Dakota, a genetic isolate with a relatively high CF carrier frequency. Following a population-wide screen for the only two mutations present in the Hutterites (M1101K, DeltaF508), we tested for associations between carrier status and measures of fertility. There was no evidence of nonrandom transmission of mutations (P = 0.409) or skewed sex ratios (P = 0.847) in children of carrier parents. Moreover, carrier status was not associated with overall fertility (P = 0.597 for carrier fathers and 0.694 for carrier mothers). Although carrier males' sibship sizes were larger than carrier females' sibship sizes (P = 0.049), this was not significant after accounting for multiple testing. Overall, our results suggest that if there is a fertility advantage among CF carriers, it is too small to be detected in our sample (85 carriers out of approximately 950 screened), or the effects are confined to DeltaF508 carriers, for which there are too few in our sample to test this specific hypothesis.

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15 F508 accounts for up to 83% of CF mutations in northern European populations, and 66% of mutations worldwide, well above the 2.4% worldwide frequency of the second most common mutation, G542X, in CF patients (Zielenski and Tsui 1995). Login to comment

[hide] Chimeric constructs endow the human CFTR Cl- chann... Proc Natl Acad Sci U S A. 2007 Oct 9;104(41):16365-70. Epub 2007 Oct 3. Scott-Ward TS, Cai Z, Dawson ES, Doherty A, Da Paula AC, Davidson H, Porteous DJ, Wainwright BJ, Amaral MD, Sheppard DN, Boyd AC
Chimeric constructs endow the human CFTR Cl- channel with the gating behavior of murine CFTR.
Proc Natl Acad Sci U S A. 2007 Oct 9;104(41):16365-70. Epub 2007 Oct 3., 2007-10-09 [PMID:17913891]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-) channel gated by ATP-driven nucleotide-binding domain (NBD) dimerization. Here we exploit species differences between human and murine CFTR to investigate CFTR channel gating. Using homologous recombination, we constructed human-murine CFTR (hmCFTR) chimeras with sequences from NBD1, NBD2, or the regulatory domain (RD) of human CFTR replaced by the equivalent regions of murine CFTR. The gating behavior of hmRD and human CFTR were indistinguishable, whereas hmNBD1 and hmNBD2 had subtle effects on channel gating, prolonging both burst duration and interburst interval. By contrast, hmNBD1+2, containing both NBDs of murine CFTR, reproduced the gating behavior of the subconductance state of murine CFTR, which has dramatically prolonged channel openings. The CFTR potentiator pyrophosphate (PP(i)) enhanced human, hmRD, and hmNBD1 CFTR Cl(-) currents, but not those of hmNBD2, hmNBD1+2, and murine CFTR. By analyzing the rate-equilibrium free-energy relationships of chimeric channels, we obtained snapshots of the conformation of the NBDs during ATP-driven dimerization. Our data demonstrate that the conformation of NBD1 changes before that of NBD2 during channel opening. This finding suggests that NBD dimerization does not proceed by a symmetric tweezer-like motion, but instead in an asymmetric fashion led by NBD1. We conclude that the NBDs of murine CFTR determine the unique gating behavior of its subconductance state, whereas NBD2 controls channel potentiation by PP(i).

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241 For example, gentamicin improves the survival of G542X CF mice (27) and rescues CFTR function in CF patients bearing stop codon mutations (28). Login to comment

[hide] Serum zinc concentrations in cystic fibrosis patie... Biol Trace Elem Res. 2007 Oct;119(1):19-26. Van Biervliet S, Van Biervliet JP, Vande Velde S, Robberecht E
Serum zinc concentrations in cystic fibrosis patients aged above 4 years: a cross-sectional evaluation.
Biol Trace Elem Res. 2007 Oct;119(1):19-26., [PMID:17914215]

Abstract [show]
AIM: Assess the risk of zinc (Zn) deficiency in the older cystic fibrosis (CF) population. METHOD: Cross-sectional investigation of all CF patients above the age of 4 followed at the Ghent University center between 2002 and 2003. Data on age, weight, height z-score, pancreatic and pulmonary functions, chronic Pseudomonas infection, and CF transmembrane conductance regulator (CFTR) mutations were collected. Serum Zn, vitamins (vit) A and E, retinol-binding protein (RBP), albumin, sedimentation rate, total IgG, and cholesterol were determined. Serum Zn was compared with a local healthy control group (Van Biervliet et al., Biol Trace Elem Res 94:33-40, 2003) and with literature data (Hotz C, et al. Am J Clin Nutr 78:756-764, 2003). RESULTS: 101 patients (median age 16 years) were included. There was no difference in serum Zn concentration between CF patients and controls. In CF patients no difference in serum Zn concentration between pancreatic-sufficient or pancreatic-insufficient patients was seen. Serum Zn was not associated to nutritional status or height z-score. A significant association serum Zn to serum albumin (p < 0.0005) and to vit A (p < 0.01) was seen. No associations of serum Zn to serum vit E, RBP, cholesterol, or CFTR were present, but there is a significant association serum Zn to forced vital capacity (p < 0.01). Serum Zn was not associated to inflammatory parameters or chronic Pseudomonas infection. CONCLUSION: Comparison of CF patients with local controls revealed no significant differences. However, because persisting steatorrhea increases Zn loss (Easley et al., J Pediatr Gastroenterol Nutr 26:136-139, 1998) and 12.6% of our population has a serum Zn below the p value of 2.5 of the NHANES II study (Hotz C, et al. Am J Clin Nutr 78:756-764, 2003), there could remain an increased risk of Zn deficiency in some CF patients. Furthermore, the association with pulmonary function needs more investigation.

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73 Table 1 Genotype of the 101 CF Patients: Details of the CF Mutations and Classification into Two Groups Genotype Groups Genotype No of Patients A ΔF508/ΔF508 47 ΔF508/E60X 1 ΔF508/G542X 7 ΔF508/N1303K 3 ΔF508/Q493X 1 ΔF508/1717-1G→A 1 ΔF508/Y1092X 1 ΔF508/394delTT 1 ΔF508/R785X 1 ΔF508/R553X 1 ΔF508/ΔI507 1 394delTT/394delTT 1 N1303K/N1303K 2 B ΔF508/3849+10kbC-T 1 ΔF508/306ΔTAGA 1 ΔF508/S1251N 8 ΔF508/L927P 1 G458V/1717-1G→A 1 ΔF508/I336K 2 G542X/622-2 A→C 1 ΔF508/G970R 3 ΔF508/3272-26A→G 2 ΔF508/R117H 2 ΔF508/2789+5G→A 2 1717-1G->A/S1251N 1 G542X/G970R 1 394delTT/Y913C 1 N1303K/deletion exon 19 1 Unidentified/unidentified 2 3600+2insTA/2005 del T 1 ΔF508/1898+1G→A 1 Deletion exon 2/del exon 2 1 There was no difference according to gender or age. Login to comment

[hide] One multiplex control for 29 cystic fibrosis mutat... Genet Test. 2007 Fall;11(3):256-68. Lebo RV, Bixler M, Galehouse D
One multiplex control for 29 cystic fibrosis mutations.
Genet Test. 2007 Fall;11(3):256-68., [PMID:17949287]

Abstract [show]
A simple approach is described to synthesize and clone an inexhaustible supply of any homozygous and/or heterozygous controls diluted with yeast genomic DNA to mimic human genome equivalents for use throughout the entire multiplex mutation assay. As a proof of principle, the 25 cystic fibrosis mutation panel selected by the American College of Medical Genetics and four additional mutant sequences were prepared as a single control mixture. The 29 CFTR mutations were incorporated into 17 gene fragments by PCR amplification of targeted sequences using mutagenic primers on normal human genomic DNA template. Flanking primers selected to bind beyond all published PCR primer sites amplified controls for most assay platforms. The 17 synthesized 433-933-bp CFTR fragments each with one to four homozygous mutant sequences were cloned into nine plasmid vectors at the multiple cloning site and bidirectionally sequenced. Miniplasmid preps from these nine clones were mixed and diluted with genomic yeast DNA to mimic the final nucleotide molar ratio of two CFTR genes in 6 x 10(9) bp total human genomic DNA. This mixture was added to control PCR reactions prior to amplification as the only positive control sample. In this fashion >200 multiplex clinical PCR analyses of >4,000 clinical patient samples have been controlled simultaneously for PCR amplification and substrate specificity for 29 tested mutations without cross contamination. This clinically validated multiplex cystic fibrosis control can be modified readily for different test formats and provides a robust means to control for all mutations instead of rotating human genomic controls each with a fraction of the mutations. This approach allows scores of additional mutation controls from any gene loci to be added to the same mixture annually.

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103 For example, the Intron 10/Exon 11 fragment spans 5 common mutation sites: 1717-1G Ǟ A, G542X, G551D, R553X, and R560T, while the ⌬I507 and ⌬F508 mutations in Exon 10 overlap by one basepair and each delete three basepairs. Login to comment
105 Because the G551D and R553X mutations are within four basepairs, these mutations were also synthesized on independently cloned Intron 10/Exon 11 fragments, both of which carried three other mutations: 1717-1G Ǟ A, G542X, and R560T (Fig. 2, fragments 1 and 3). Login to comment
165 As part of this validation, two different Intron10/Exon11 fragments were sequenced and tested: both contain the 1717-1G Ǟ A, G542X, and R560T mutations, and the first also contains the G551D mutation (Fig. 2, clone 1; Fig. 3, f1), while the second also contains the R553X mutation (Fig. 2, clone 3; Fig. 3, f3). Login to comment
166 When tested individually, clone 1 hybridized uniquely to the G551D mutant allelic site as well as to the other three mutations (1717-1G Ǟ A, G542X, and R560T), but not to the wild-type (normal) R553 allelic site because the G551D mutation sequence interferes with the binding to the wild type R553 probe on the strip (Fig. 3, f1, left strip). Login to comment

[hide] PTC124 is an orally bioavailable compound that pro... Proc Natl Acad Sci U S A. 2008 Feb 12;105(6):2064-9. Epub 2008 Feb 6. Du M, Liu X, Welch EM, Hirawat S, Peltz SW, Bedwell DM
PTC124 is an orally bioavailable compound that promotes suppression of the human CFTR-G542X nonsense allele in a CF mouse model.
Proc Natl Acad Sci U S A. 2008 Feb 12;105(6):2064-9. Epub 2008 Feb 6., 2008-02-12 [PMID:18272502]

Abstract [show]
Nonsense mutations inactivate gene function and are the underlying cause of a large percentage of the individual cases of many genetic disorders. PTC124 is an orally bioavailable compound that promotes readthrough of premature translation termination codons, suggesting that it may have the potential to treat genetic diseases caused by nonsense mutations. Using a mouse model for cystic fibrosis (CF), we show that s.c. injection or oral administration of PTC124 to Cftr-/- mice expressing a human CFTR-G542X transgene suppressed the G542X nonsense mutation and restored a significant amount of human (h)CFTR protein and function. Translational readthrough of the premature stop codon was demonstrated in this mouse model in two ways. First, immunofluorescence staining showed that PTC124 treatment resulted in the appearance of hCFTR protein at the apical surface of intestinal glands in Cftr-/- hCFTR-G542X mice. In addition, functional assays demonstrated that PTC124 treatment restored 24-29% of the average cAMP-stimulated transepithelial chloride currents observed in wild-type mice. These results indicate that PTC124 can effectively suppress the hCFTR-G542X nonsense mutation in vivo. In light of its oral bioavailability, safety toxicology profile in animal studies, and efficacy with other nonsense alleles, PTC124 has the potential to be an important therapeutic agent for the treatment of inherited diseases caused by nonsense mutations.

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2 Using a mouse model for cystic fibrosis (CF), we show that s.c. injection or oral administration of PTC124 to Cftr-/- mice expressing a human CFTR-G542X transgene suppressed the G542X nonsense mutation and restored a significant amount of human (h)CFTR protein and function. Login to comment
4 First, immunofluorescence staining showed that PTC124 treatment resulted in the appearance of hCFTR protein at the apical surface of intestinal glands in Cftr-/- hCFTR-G542X mice. Login to comment
6 These results indicate that PTC124 can effectively suppress the hCFTR-G542X nonsense mutation in vivo. Login to comment
12 Of these mutations, CFTR-G542X is the most common. Login to comment
24 Accordingly, in the present work we show that subcutaneous (s.c.) or oral administration of PTC124 suppressed the G542X mutation in a CF mouse model that expressed a human CFTR-G542X transgene in a Cftr-/- background, leading to a significant restoration of CFTR expression and function. Login to comment
26 Results Subcutaneous Injection of PTC124 Suppresses Nonsense Mutations and Induces hCFTR Expression in Cftr-/- hCFTR-G542X Mice. Login to comment
27 We constructed a CF transgenic mouse model that expressed a human CFTR (hCFTR) cDNA containing the G542X premature stop mutation in a mouse line that carried a knockout of the endogenous Cftr locus (referred to hereafter as the Cftr-/- hCFTR-G542X mouse line) (8). Login to comment
38 Because of these differences, we used the compact, intestine-specific rat fatty acid-binding protein (FABP) promoter to drive expression of the CFTR-G542X transgene. Login to comment
39 This Cftr-/- hCFTR-G542X mouse model was used to show that both gentamicin and amikacin could suppress the hCFTR-G542X mutation and partially restore CFTR protein expression and function (7, 8). Login to comment
40 To examine the ability of PTC124 to suppress the hCFTR-G542X mutation in vivo, once daily s.c. injections of PTC124 were administered to Cftr-/- hCFTR-G542X mice at dosages of 60, 30, or 15 mg/kg body weight for 14-21 days. Login to comment
45 Similarly, no hCFTR protein was detected in intestinal tissues from untreated Cftr-/- hCFTR-G542X mice with hCFTR-specific antiserum. Login to comment
46 However, strong hCFTR staining was observed at the apical surface of epithelial cells in submucosal glands from Cftr-/- hCFTR-G542X mice treated with 60 mg/kg PTC124 and, as observed, in tissues from mice treated with 34 mg/kg gentamicin. Login to comment
48 These results indicate that PTC124 can suppress the G542X mutation and partially restore hCFTR protein expression in Cftr-/- hCFTR-G542X mice. Login to comment
49 Subcutaneous Injection of PTC124 Partially Restores cAMP-Stimulated Chloride Channel Activity in Cftr-/- hCFTR-G542X Mice. Login to comment
51 We reported that cAMP-dependent transepithelial chloride conductance appeared in intestinal tissues of Cftr-/- hCFTR-G542X mice after treatment with 34 mg/kg gentamicin (7, 8). Login to comment
53 Supporting information (SI) Fig. 7 shows representative short-circuit current tracings obtained from Cftrϩ/ϩ mice, untreated Cftr-/- hCFTR-G542X mice, and Cftr-/- hCFTR-G542X mice treated with once daily s.c. injections of either 60 mg/kg PTC124 or 34 mg/kg gentamicin. Login to comment
54 The intestinal tissues harvested from untreated Cftr-/- hCFTR-G542X mice showed no change in short-circuit currents after the addition of forskolin, a cAMP agonist. Login to comment
55 In contrast, forskolin addition frequently induced an increase in short-circuit current in intestinal tissues from Cftr-/- hCFTR-G542X mice injected with 60 mg/kg PTC124 or 34 mg/kg gentamicin once a day for 14-21 days. Login to comment
56 Fig. 2 summarizes the data collected from short-circuit current measurements from intestinal tissues harvested from untreated Cftr-/- hCFTR-G542X mice, Cftr-/- hCFTR-G542X mice treated with three different dosages of PTC124 for 14-21 days, or Cftr-/- hCFTR-G542X mice treated with 34 mg/kg gentamicin for 14-21 days. Login to comment
58 In untreated Cftr-/- hCFTR-G542X mice, we detected cAMP-stimulated short-circuit currents in only 8% of samples (1 of 12), resulting in an average current of 0.20 ␮A/cm2 . Login to comment
59 In the Cftr-/- hCFTR-G542X mice treated with 60 mg/kg PTC124, 47% of samples (8 of 17) showed a positive reaction after the addition of forskolin, resulting in an average current of 1.66 ␮A/cm2 . Login to comment
60 Similarly, Cftr-/- hCFTR-G542X mice treated with 34 mg/kg gentamicin manifested cAMP-stimulated short-circuit currents in 63% of samples (5 of 8), resulting in an average current of 1.67 ␮A/cm2 . Login to comment
62 Samples were prepared from the duodenum of untreated Cftr-/- hCFTR-G542X mice and Cftr-/- hCFTR-G542X mice treated with 15, 30, or 60 mg/kg PTC124. Login to comment
63 Intestinal tissues from Cftr-/- hCFTR-G542X mice treated with 34 mg/kg gentamicin by the same administration protocol were also examined as a positive control. Login to comment
67 PTC124 to Cftr-/- hCFTR-G542X mice once daily by s.c. injection produced a statistically significant increase (P Ͻ 0.05) in cAMP-stimulated transepithelial chloride currents in intestinal tissues relative to untreated controls, resulting in 29% of the mean cAMP-stimulated short currents measured in wild-type mice. Login to comment
69 The s.c. administration of PTC124 at 30 or 15 mg/kg once daily did not show a significant increase in cAMP-stimulated short currents after forskolin addition relative to untreated Cftr-/- hCFTR-G542X mice, indicating that 60 mg/kg PTC124 is the minimal effective dose with this administration protocol. Login to comment
73 Oral Administration of PTC124 Partially Restores hCFTR Protein Expression and cAMP-Stimulated Chloride Channel Activity in Cftr-/- hCFTR-G542X Mice. Login to comment
74 The results above indicated that once daily s.c. injection of 60 mg/kg PTC124 suppressed the G542X mutation in Cftr-/- hCFTR-G542X mice and restored the expression of functional hCFTR protein. Login to comment
75 To determine serum levels resulting from this administration protocol, age-matched Cftrϩ/- hCFTR-G542X mice were injected with 60 mg/kg PTC124, and orbital blood was collected at various times after injection (Fig. 3A). Login to comment
78 Because of the potential for intestinal blockage in Cftr-/- hCFTR-G542X mice, these animals are routinely fed a liquid complete diet (Peptamen Complete Elemental Diet; Nestle´) upon weaning to promote survival. Login to comment
79 To identify an oral dose of PTC124 that might provide a therapeutic benefit, the compound was dissolved in the liquid diet at two concentrations (0.3 and 0.9 mg/ml) and given to Cftrϩ/- hCFTR-G542X mice as the sole source of food and water for 2-6 days. Login to comment
83 Because the average serum levels obtained by oral administration with these doses of PTC124 effectively bracketed the peak serum levels obtained immediately after s.c. injection and the optimal dosing established in the mdx studies, Cftr-/- hCFTR-G542X mice were fed the Peptamen liquid diet containing 0.3 or 0.9 mg/ml PTC124 for 14-21 days to determine whether this dosing protocol could lead to effective suppression of the G542X mutation. Login to comment
86 In mice maintained on Cftr-/- hCFTR-G542X Cftr+/+ UnTreated Gent 34mg/kg PTC124 60mg/kg PTC124 30mg/kg PTC124 15mg/kg UnTreated PTC124 60mg/kg Positive/Total Reactions 1/12 5/8 8/17 2/16 2/13 12/12 10/10 100% 5.78 0.48 101% Positive Reactions (%) 8% 63% 47% 13% 15% 100% Mean Current (µA/cm2 ) 0.20 1.67 1.66 0.29 0.12 5.72 P value - 0.023 0.034 0.36 0.36 - % WT Current 3.5% 29% 29% 5.1% 2.1% 100% ShortCircuitCurrent(µA/cm2 ) 0 2 4 6 8 10 12 14 Fig. 2. Login to comment
87 Effect of PTC124 s.c. injection on cAMP-stimulated transepithelial chloride currents in intestinal tissues from Cftr-/- hCFTR-G542X and Cftrϩ/ϩ mice. Login to comment
93 No hCFTR protein was detected in the intestines of treated Cftr-/- hCFTR-G542X mice with preimmune serum. Login to comment
95 These results indicated that oral administration of PTC124 could partially restore hCFTR protein expression in Cftr-/- hCFTR-G542X mice. Login to comment
97 We found that intestinal tissues harvested from Cftr-/- hCFTR-G542X mice fed the liquid diet with 0.3 mg/ml PTC124 yielded cAMP-stimulated short-circuit currents in 29% of intestinal tissues assayed (7 of 24), resulting in an average currents of 0.91 ␮A/cm2 . Login to comment
100 The mean cAMP-stimulated short-circuit currents observed in Cftr-/- hCFTR-G542X mice fed the liquid diet with 0.9 mg/ml PTC124 was 24% of the average cAMP-stimulated currents measured in wild-type mice. Login to comment
104 Finally, cAMP-stimulated short-circuit currents were not observed in Cftr-/- knockout mice that lacked the hCFTR-G542X transgene, regardless of whether they were untreated or treated with 0.9 mg/ml PTC124. Login to comment
105 This result confirmed that the CFTR function observed after PTC124 treatment depends on the presence of the hCFTR-G542X transgene, consistent with a role for PTC124 in inducing readthrough of premature translation termination codons. Login to comment
108 Hence, although PTC124 is thought to have a direct effect on nonsense codon readthrough (14), it is formally possible that the ability of the drug to suppress the hCFTR-G542X mutation is attributable to an effect on mRNA stability. Login to comment
109 However, RT-PCR analysis demonstrated that there were comparable levels of mRNA from the hCFTR-G542X transgene in untreated mice and in mice treated with 0.9 mg/ml PTC124 in the liquid diet (Fig. 6). Login to comment
118 (A) Blood was taken from different age-matched Cftrϩ/- hCFTR-G542X mice at the indicated times after s.c. injection of 60 mg/kg PTC124. Login to comment
119 (B) Blood samples were taken after feeding Cftrϩ/- hCFTR-G542X mice a liquid diet containing 0.3 or 0.9 mg/ml PTC124 for 2-6 days or Cftr-/- hCFTR-G542X mice a liquid diet containing 0.3 or 0.9 mg/ml PTC124 for 14-21 days. Login to comment
123 Samples were from the duodenum of Cftr-/- hCFTR-G542X mice treated with 0.3 or 0.9 mg/ml PTC124 in the Peptamen liquid diet. Login to comment
126 to Cftr-/- hCFTR-G542X mice by s.c. injection restored 29% of the normal intestinal transepithelial cAMP-stimulated short-circuit currents observed in Cftrϩ/ϩ mice. Login to comment
129 Our data indicate that the human CFTR protein produced by suppression of the nonsense mutation in Cftr-/- hCFTR-G542X mice is located primarily at the epithelial surface of the submucosal glands in the duodenum. Login to comment
131 To determine whether the suppressed level of CFTR expression can prevent the frequent intestinal blockage associated with the lack of functional CFTR protein, we are currently making a knockin Cftr-G542X mouse that will have a normal distribution of CFTR expression. Login to comment
132 We observed an occasional weak cAMP-stimulated current in intestinal tissues from Cftr-/- hCFTR-G542X mice in the absence of any treatment but not in Cftr-/- knockout mice that lacked the transgene (see Fig. 5). Login to comment
133 These data suggest that there may be a low level of endogenous readthrough of the premature stop codon in Cftr-/- hCFTR-G542X mice that we occasionally detect because such residual activity would not be observed in Cftr-/- knockout mice that lack the hCFTR-G542X transgene. Login to comment
134 Previous studies have shown that some nonsense codons (particularly UGA codons like that encoded by the G542X mutation) have a higher basal level of readthrough and are more susceptible to readthrough induced by aminoglycosides than other stop codons (19-21). Login to comment
140 In conclusion, our results demonstrate that PTC124 induces readthrough of the premature translation termination codon encoded by the hCFTR-G542X nonsense mutation, resulting in the partial restoration of CFTR protein and cAMP-activated chloride currents in the intestines of Cftr-/- hCFTR-G542X transgenic mice. Login to comment
142 Cftr-/- hCFTR-G542X Cftr+/+ Cftr-/- UnTreated PTC124 0.3mg/ml PTC124 0.9mg/ml UnTreated PTC124 0.9mg/ml UnTreated PTC124 0.9mg/ml 0/8 0/8 0% 0.00 - 0% 0% 0.00 - Positive/Total Reactions 2/16 7/24 5/11 12/12 0% 13/13 100% 6.15 0.35 108% Positive Reactions (%) 13% 29% 45% 100% Mean Current (µA/cm2) 0.20 0.91 1.35 5.72 P value - 0.021 0.027 - % WT Current 3.5% 16% 24% 100% ShortCircuitCurrent(µA/cm2 ) 0 4 8 12 16 ++++++++ 2 6 10 14 Fig. 5. Login to comment
143 Effect of PTC124 oral administration on cAMP-stimulated transepithelial chloride currents in intestinal tissues from Cftr-/- hCFTR-G542X, Cftrϩ/ϩ, and Cftr-/- mice. Login to comment
147 RT-PCR detection of hCFTR mRNA in untreated Cftr-/- hCFTR-G542X mice and Cftr-/- hCFTR-G542X mice treated with an oral dose of 0.9 mg/ml PTC124. Login to comment
149 The rat FABP promoter was used to drive expression of the hCFTR transgene that contained the G542X (UGA) premature stop mutation. Login to comment
150 The plasmid construction of the FABP-hCFTR-G542X transgene and generation of the Cftr-/- hCFTR-G542X mouse were described in refs. 7 and 8. Login to comment
153 Subcutaneous injections with the indicated doses of PTC124 or gentamicin were made in the hind limb of age-matched Cftr-/- hCFTR-G542X mice. Login to comment
156 Because of the potential for intestinal blockage in Cftr-/- hCFTR-G542X mice, they were maintained on a liquid diet (Peptamen) after weaning, and other food and water were withheld. Login to comment
190 To monitor expression of the hCFTR-G542X transgene, mRNA was isolated from intestinal tissues of untreated Cftr-/- hCFTR-G542X mice and Cftr-/- hCFTR-G542X mice treated with 0.9 mg/ml PTC124 in the liquid diet for 17 days. Login to comment
191 RT-PCR analysis of hCFTR-G542X mRNA was done by using a SuperScript III one-step RT-PCR system with platinum Taq polymerase (Invitrogen). Login to comment
193 The primers used to amplify a 1,557-bp fragment from the hCFTR-G542X mRNA were DB985 (5Ј-CAAGATAGAA AGAGGACAGT TGTT-3Ј) and DB986 (5Ј-TTGAGGGTTG ACATAGGTGC TTGAA-3Ј). Login to comment
217 Du M, et al. (2006) Clinical doses of amikacin provide more effective suppression of the human CFTR-G542X stop mutation than gentamicin in a transgenic CF mouse model. Login to comment
220 Du M, et al. (2002) Aminoglycoside suppression of a premature stop mutation in a Cftr-/- mouse carrying a human CFTR-G542X transgene. Login to comment

[hide] The changing face of the exocrine pancreas in cyst... Eur J Gastroenterol Hepatol. 2008 Mar;20(3):164-8. Augarten A, Ben Tov A, Madgar I, Barak A, Akons H, Laufer J, Efrati O, Aviram M, Bentur L, Blau H, Paret G, Wilschanski M, Kerem BS, Yahav Y
The changing face of the exocrine pancreas in cystic fibrosis: the correlation between pancreatic status, pancreatitis and cystic fibrosis genotype.
Eur J Gastroenterol Hepatol. 2008 Mar;20(3):164-8., [PMID:18301294]

Abstract [show]
OBJECTIVES: The aims of this study were to determine the current pancreatic status of the entire cystic fibrosis (CF) population of Israel, to analyze the clinical characteristics of the pancreatic sufficient (PS) patients, and to characterize the correlation between pancreatic status, pancreatitis, and CF genotype. METHODS: The Israeli CF database includes 505 patients. These patients were defined as being PS or insufficient according to their fecal pancreatic elastase level or by coefficient fat absorption findings. Mutations were categorized as severe (DeltaF508, W1282X, G542X, S549R, N1303K, Q359K/T360K, 405+1G, and 1717) or mild/variable (3849+10 kb, D1152H, G85E, I1234V, R334W, and 5T) based on disease severity in patients carrying these mutations. Age at diagnosis, presenting symptoms, sweat-chloride concentrations, occurrence of pancreatitis, presence of diabetes, and liver disease were recorded. RESULTS: One hundred and thirty-nine (27.5%) of the CF patients were PS. None carried two mutations associated with severe disease. Over one third (34%) had normal or borderline sweat tests; 20 of these 139 patients had pancreatitis (14.3%) but none of the 366 pancreatic insufficient patients had it. Four initially PS patients became pancreatic insufficient: conversion followed several events of pancreatitis in three of them. Nasal potential differences were all pathological in 35 tested PS patients. None had either diabetes or liver disease. CONCLUSIONS: A substantial number of CF patients are PS. All of them carry at least one mild mutation enabling production of a sufficient amount of normal mRNA to maintain exocrine pancreatic function. Pancreatitis occurs only in CF patients who are PS. These patients are at risk of progressing to pancreatic insufficiency.

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23 The mutations DF508, W1282X, G542X, S549R, Q359K/T360K, 405 + 1G, 1717, and N1303K were defined as severe and the mutations 3849 + 10 kb, D1152H, G85E, I1234V, R334W, and 5T were defined as mild/variable. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Int J Urol. 2008 Mar;15(3):270-1. Sakamoto H, Yajima T, Suzuki K, Ogawa Y
Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation associated with a congenital bilateral absence of vas deferens.
Int J Urol. 2008 Mar;15(3):270-1., [PMID:18304229]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations associated with cystic fibrosis have been reported to be rare in Japanese patients with congenital bilateral absence of vas deferens (CBAVD). A 28-year-old Japanese male was referred for infertility. Vas deferens and epididymis were not palpable bilaterally. Semen analyses showed azoospermia with volumes below 2.0 ml. Serum follicle-stimulating hormone value was slightly elevated. Seminal fructose concentration was also very low. Scrotal ultrasonography showed absence of the bodies and tails of the right and left epididymides. Imaging studies showed cystic dysplasia of the right seminal vesicle and agenesis of the left seminal vesicle. A CFTR gene mutation of I556V was found. Recent studies show that prevalence of CFTR gene mutation in Japanese CBAVD patients may be approximately equal to that of the Caucasian population. Genetic counselling may be recommended for any couple attempting assisted reproduction technology when the man has CBAVD.

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29 In our case, we analyzed exons 10 and 11 in the CFTR gene to evaluate the possibility of transmission of CF to newborn because common mutations such as D508, DI507, 551D, G542X, and R553X associated with CF in Caucasians was frequently identified in these exons.1 I556V found in the present case is a mutation initially reported in a French male who had asthma-like bronchopathy and chronic diarrhea, which was recently identified in 10% to 15% of Asians irrespective of chronic respiratory diseases.7 CBAVD is suggested based on the identification of azoospermia with either normal-sized or slightly smaller testes, a non-palpable vas deferens, characteristic imaging findings and the physical and biological properties of the ejaculate: small volume (<2 mL), low pH (<7), and low fructose concentration.1 Most CBAVD patients have defects in the derivatives of the wolffian duct system presenting as an absence of the distal portion of the epididymides, seminal vesicle atrophy or absence, and the absence of the vas deferens by scrotal and transrectal ultrasonography.4,5,9 However, not all men with CBAVD have extensive abnormalities of the derivatives of the wolffian duct system.5,9 Previous studies showed that seminal vesicle anomalies with either agenesis, hypoplasia, or cystic dysplasia occur in 36% to 92% of men with CBAVD.4-6,9 Jarvi et al. showed that all CBAVD patients with at least one CFTR gene mutation had abnormalities of both the seminal vesicles and ampulla of the vas deferens and that 50% of CBAVD patients with no detectable CFTR gene mutation had a normal ampulla of the vas deferens and seminal vesicles.5 Therefore, the frequency and severity of the wolffian duct malformations in the CBAVD patients may be related directly to the CFTR genotype.5 Moreover, previous studies report that 11% to 21% of CBAVD patients had renal agenesis.6,9 Renal agenesis has been reported to occur predominately in men with a congenital absence of vas deferens (CAVD) without CFTR gene mutations.9 However, Casales et al. showed CFTR gene mutations in five of 16 CAVD patients (bilateral absence in six, and unilateral absence in 10) with renal agenesis.6 In addition, CAVD may also be associated with cryptorchidism and inguinal hernia.6 The prevalence of the CFTR gene mutation carrier in the Japanese population may be approximate to that of the Caucasian.1,2 Moreover, infertile patients with CBAVD can now be treated by assisted reproduction technology.1 Genetic counseling may be recommended for any couple attempting assisted reproduction technology when the man has defects of the vas deferens.1-3,8 References 1 Jarzabek K, Zbucka M, Pepiñski W et al. Cystic fibrosis as a cause of infertility. Login to comment

[hide] N-terminal CFTR missense variants severely affect ... Hum Mutat. 2008 May;29(5):738-49. Gene GG, Llobet A, Larriba S, de Semir D, Martinez I, Escalada A, Solsona C, Casals T, Aran JM
N-terminal CFTR missense variants severely affect the behavior of the CFTR chloride channel.
Hum Mutat. 2008 May;29(5):738-49., [PMID:18306312]

Abstract [show]
Over 1,500 cystic fibrosis transmembrane conductance regulator (CFTR) gene sequence variations have been identified in patients with cystic fibrosis (CF) and related disorders involving an impaired function of the CFTR chloride channel. However, detailed structure-function analyses have only been established for a few of them. This study aimed evaluating the impact of eight N-terminus CFTR natural missense changes on channel behavior. By site-directed mutagenesis, we generated four CFTR variants in the N-terminal cytoplasmic tail (p.P5L, p.S50P, p.E60K, and p.R75Q) and four in the first transmembrane segment of membrane-spanning domain 1 (p.G85E/V, p.Y89C, and p.E92K). Immunoblot analysis revealed that p.S50P, p.E60K, p.G85E/V, and p.E92K produced only core-glycosylated proteins. Immunofluorescence and whole cell patch-clamp confirmed intracellular retention, thus reflecting a defect of CFTR folding and/or trafficking. In contrast, both p.R75Q and p.Y89C had a glycosylation pattern and a subcellular distribution comparable to the wild-type CFTR, while the percentage of mature p.P5L was considerably reduced, suggesting a major biogenesis flaw on this channel. Nevertheless, whole-cell chloride currents were recorded for all three variants. Single-channel patch-clamp analyses revealed that the channel activity of p.R75Q appeared similar to that of the wild-type CFTR, while both p.P5L and p.Y89C channels displayed abnormal gating. Overall, our results predict a major impact of the CFTR missense variants analyzed, except p.R75Q, on the CF phenotype and highlight the importance of the CFTR N-terminus on channel physiology.

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133 Genotype^Phenotype Correlation in the N-Terminal CFTR MissenseVariants Under StudyÃ Missense varianta Phenotype Second allele (number of patients)b p.P5L CF p.F508del (1), p.P205S (1) p.S50P CBAVD p.F508del (1), p.E115del (1) p.E60K CF p.G542X (1), p.I507del (1) p.R75Q HT p.F508del (3), p.E725K (1) B p.R347H (1), p.R75Q (1), n.i. (4) Br c.1584G4A (2), c.1210-7_1210-6delTT (1), n.i.(3) NT p.F508del (1) CP c.1584G4A (1), n.i. (3) MI n.i. (1) CUAVD n.i. (2) OZ n.i. (2) Normal p.R75Q (1), c.2052_2053insA (1), n.i. (1) p.G85E CF p.F508del (8), p.G542X (2), p.I507del (1), c.580-1G4T (1), p.G85E (1), c.1477_ 1478delCA (1) CBAVD p.G576A (1) HT p.L997F (1),WT (1) p.G85V CF p.F508del (2), p.G542X (2), p.Y1092X (1), c.265715G4A (1), p.A1006E, c.1210-7_1210- 6delTT (1), n.i. (1) p.Y89C CF n.i. (1)c p.E92K CF p.F508del (2), p.Q890X (1), p.L206W (1) CBAVD c.1210-7_1210-6delTT (1) ÃThe recommendations for mutation nomenclature (www.hgvs.org/mutnomen/) were used to name CFTR gene sequence variations at both the nucleotide level and the protein level. Login to comment

[hide] Distribution of CFTR mutations in Saguenay- Lac-Sa... Genet Med. 2008 Mar;10(3):201-6. Madore AM, Prevost C, Dorfman R, Taylor C, Durie P, Zielenski J, Laprise C
Distribution of CFTR mutations in Saguenay- Lac-Saint-Jean: proposal of a panel of mutations for population screening.
Genet Med. 2008 Mar;10(3):201-6., [PMID:18344710]

Abstract [show]
PURPOSE: Saguenay-Lac-Saint-Jean is a region located in the northeastern part of the Province of Quebec, Canada, and is characterized by a founder effect. In this region, it has been documented that the incidence of cystic fibrosis reached 1/902 live births between 1975 and 1988, three times higher than the average incidence of 1/2500 live births reported in other Caucasian populations. This corresponds to a carrier rate of 1/15. METHODS: Using genotyping data from the Canadian Consortium for Cystic Fibrosis Genetic Studies, this article describes the cystic fibrosis transmembrane conductance regulator profile of the cystic fibrosis population living in the Saguenay-Lac-Saint-Jean region and compares it with cystic fibrosis populations living in three other regions of the Province of Quebec. RESULTS: Significant differences in allelic frequencies of common mutations (as DeltaF508, 621 + 1G>T and A455E), and in percentage of covered allele with three or six mutations, were found in Saguenay-Lac-Saint-Jean compared to other regions. Based on this result, two mutation panels exceeding 90% sensitivity threshold are now proposed for cystic fibrosis carrier screening in this region. CONCLUSION: The implementation of the proposed carrier screening program could diminish the incidence of this disease in this region and allow future parents to make informed decisions about family planning.

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48 Altogether, the six mutations represent 95.89% of the CFTR allele of CF patients in the SLSJ population, whereas the proportions are 86.85, 85.27, and Table 2 Cystic fibrosis mutations present in the four populations studied Mutationa Allelic frequency (number of alleles [%]) Populationb 1 2 3 4 „F508 106 (62.35) 55 (72.37) 398 (72.36) 67 (57.78) 621 ؉ 1G>T 42 (24.71) 6 (7.89) 30 (5.45) 1 (0.85) A455E 12 (7.06) 2 (2.63) 14 (2.55) 1 (0.85) 3199del6 1 (0.59) 1 (1.32) 7 (1.27) 1 (0.85) 711 ؉ 1G>T 1 (0.59) 1 (1.32) 15 (2.73) 1 (0.85) Y1092X 1 (0.59) 1 (1.32) 5 (0.91) 0 R117C 2 (1.18) 0 0 0 ‚I507 1 (0.59) 2 (2.63) 10 (1.82) 0 L206W 1 (0.59) 1 (1.32) 9 (1.64) 0 R1158X 1 (0.59) 0 0 0 S489X 1 (0.59) 0 1 (0.18) 0 R553X 0 2 (2.63) 2 (0.36) 0 R334W 0 1 (1.32) 2 (0.36) 0 G542X 0 0 10 (1.82) 0 G85E 0 0 6 (1.09) 5 (4.24) N1303K 0 0 5 (0.91) 1 (0.85) IVS8-5T 0 0 4 (0.73) 0 W1282X 0 0 3 (0.55) 7 (5.93) R347P 0 0 1 (0.18) 2 (1.69) V520F 0 0 1 (0.18) 0 I1027T 0 0 1 (0.18) 0 R1066C/IVS 0 0 1 (0.18) 0 Q1313X 0 0 1 (0.18) 0 1898ϩ3GϾA 0 0 1 (0.18) 0 2183AAϾG 0 0 1 (0.18) 0 2951insA 0 0 1 (0.18) 0 G551D 0 0 0 2 (1.69) 1525-iG-A 0 0 0 2 (1.69) Y109C 0 0 0 1 (0.85) S549N 0 0 0 1 (0.85) 3154del1G 0 0 0 1 (0.85) UNKNOWN 1 (0.59) 4 (5.26) 20 (3.82) 25 (21.19) Number of alleles genotypedc 170 (100) 76 (100) 550 (100) 118 (100) a The six mutations included in the panels proposed are in bold. Login to comment

[hide] Comparative analysis of common CFTR polymorphisms ... World J Gastroenterol. 2008 Mar 28;14(12):1925-30. Huang Q, Ding W, Wei MX
Comparative analysis of common CFTR polymorphisms poly-T, TG-repeats and M470V in a healthy Chinese population.
World J Gastroenterol. 2008 Mar 28;14(12):1925-30., 2008-03-28 [PMID:18350634]

Abstract [show]
AIM: To investigate the three important cystic fibrosis transmembrane conductance regulator (CFTR) haplotypes poly-T, TG-repeats and the M470V polymorphisms in the Chinese population, and to compare their distribution with that in Caucasians and other Asian populations. METHODS: Genomic DNA was extracted from blood leukocytes. Exons 9 and 10 of the CFTR gene were obtained through polymerase chain reaction (PCR). Exon 9 DNA sequences were directly detected by an automated sequencer and poly-T and TG-repeats were identified by direct sequence analysis. Pure exon 10 PCR-amplified products were digested by HphI restriction enzyme and the M470V mutation was detected by the AGE photos of digestion products. RESULTS: T7 was the most common (93.6%) haplotype and the (TG)11 frequency of 57.2% and (TG)12 frequency of 40.9% were dominant haplotypes in the junction of intron 8 (IVS-8) and exon 9. The frequency of T5 was 3.8% and all T5 allele tracts (10 alleles) were joined with (TG)12. Four new alleles of T6 (1.5%) were found in three healthy individuals. In exon 10, the V allele (56.1%) was slightly more frequent than the M allele (43.9%), and the M/V (45.5%) was the dominant genotype in these individuals. The three major haplotypes T7-(TG)11-V470, T7-(TG)12-M470 and T7-TG11-M470 were related to nearly 86.0% of the population. CONCLUSION: The polymorphisms of poly-T, TG-repeats, and M470V distribution were similar to those in other East Asians, but they had marked differences in frequency from those single haplotype polymorphisms or linkage haplotypes in Caucasians. Thus, they may be able to explain the low incidence of CF and CF-like diseases in Asians.

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9 However, in Asians, the prevalence of CF is very low, with an incidence of approximately 1 in 100 000, and in particular, the severe mutations, such as ∆F508, G542X and N1303K, are rarely found in Asians. Login to comment

[hide] CFTR mutations in Turkish and North African cystic... Genet Test. 2008 Mar;12(1):25-35. Lakeman P, Gille JJ, Dankert-Roelse JE, Heijerman HG, Munck A, Iron A, Grasemann H, Schuster A, Cornel MC, Ten Kate LP
CFTR mutations in Turkish and North African cystic fibrosis patients in Europe: implications for screening.
Genet Test. 2008 Mar;12(1):25-35., [PMID:18373402]

Abstract [show]
AIMS: To obtain more insight into the variability of the CFTR mutations found in immigrant cystic fibrosis (CF) patients who are living in Europe now, and to estimate the test sensitivity of different frequently used methods of DNA analysis to detect CF carriers or patients among these Turkish or North African immigrants. METHODS: A survey among 373 European CF centers asking which CFTR mutations had been found in Turkish and North African CF patients. RESULTS: 31 and 26 different mutations were reported in Turkish and North African patients, identifying 64.2% (113/176) and 87.4% (118/135) alleles, respectively (p < 0.001). The mean sensitivity (detection rate) of three most common CFTR mutation panels to detect these mutations differed between Turkish and North African people, 44.9% (79/176) versus 69.6% (94/135) (p < 0.001), and can be increased to 57.4% (101/176) and 79.3% (107/135) (p < 0.001), respectively, by expanding these panels with 13 mutations which have been found on two or more alleles. CONCLUSION: 35.8% and 12.6%, respectively, of CF alleles in Turkish and North African patients living in Europe now had not been identified. Among these populations, the test sensitivity of common CFTR mutation panels is insufficient for use in screening programs in Europe, even after expansion with frequent Turkish and North African mutations. This raises questions about whether and how to implement CF carrier and neonatal screening in a multiethnic society.

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113 Identity and Frequency of CFTR Mutations on Unrelated Turkish (Tr) and North African (NA) CF alleles Total number of allelesa Number of CF patients with this mutationb Mutation Exon All Tr NA Homozygote Compound heterozygote: two mutations found Compound heterozygote: one mutation found F508delc 10 73 33 40 27 11 6 N1303K 21 22 12 10 10 5 2 711 þ 1G > T Intron 5 14 - 14 7 2 0 G542X 11 14 6 8 7 1 0 R1162X 19 11 - 11 1 5 2 2183AA > G 13 9 9 - 3 3 1 W1282X 20 7 3 4 2 3 1 2789 þ 5G > A Intron 14b 6 3 3 1 4 1 L227R 6a 4 - 4 3 1 0 1677delTA 10 4 4 - 2 1 1 2184insA 13 4 4 - 1 2 0 R334W 7 4 4 - 1 1 1 G85E 3 4 3 1 1 2 0 R709X 13 3 - 3 2 0 0 L732X 13 3 3 - 2 0 0 2184delA 13 3 3 - 0 3 0 del exon 1-4d 1-4 3 3 - 1 1 0 del exon 19 19 2 2 - 2 0 0 3849 þ 10kbC > T Intron 19 2 - 2 1 0 0 S549N 11 2 1 1 0 1 1 3120 þ G > A Intron 16 2 2 - 1 0 0 3601-2A > G Intron 18 2 2 - 1 0 0 D1152H 18 2 2 - 1 0 0 E1104X 17b 2 - 2 1 0 0 S1159F 19 2 2 - 1 0 0 S977F 16 2 - 2 0 1 0 2347delG 13 2 - 2 1 0 0 4096-3C > G Intron 21 1 1 - 1 0 0 E831X 14a 1 1 - 1 0 0 L619S 13 1 1 - 1 0 0 1525-1G > Ac Intron 9 1 1 - 1 0 0 F1052V 17b 1 1 - 1 0 0 3130delA 17a 1 1 - 1 0 0 R352Q 7 1 - 1 0 1 0 1812-1G > A Intron 11 1 - 1 0 1 0 R553X 11 1 - 1 0 0 1 IVS8-5T Intron 8 1 1 - 0 1 0 R1066C 17b 1 - 1 0 1 0 3129del4 17a 1 - 1 0 1 0 D110H 4 1 1 - 0 1 0 R117H 4 1 - 1 0 1 0 S945L 15 1 - 1 0 1 0 1716G=A 10 1 - 1 0 0 1 711 þ 3A > G Intron 5 1 1 - 0 1 0 R75X 3 1 1 - 0 1 0 R764X 13 1 - 1 0 1 0 S1196X 19 1 1 - 0 1 0 S492F 10 1 - 1 0 1 0 G551D 11 1 - 1 1 0 0 del exon 2 2 1 1 - 1 0 0 Subtotal 231 113 118 - No mutation 80 63 17 - Total 311 176 135 88 60 18 a n ¼ 311 alleles, based on 166 CF patients (332 alleles) with both parents and 22 CF patients (22 alleles) with one parent from Turkey or North Africa, minus 43 alleles of homozygous CF patients with consanguineous parents of whom only one allele was taken into account. Login to comment

[hide] Evaluation and use of a synthetic quality control ... Hum Mutat. 2008 Aug;29(8):1063-70. Berwouts S, Gordon JT, Rundell CA, Barton DE, Dequeker E
Evaluation and use of a synthetic quality control material, included in the European external quality assessment scheme for cystic fibrosis.
Hum Mutat. 2008 Aug;29(8):1063-70., [PMID:18470946]

Abstract [show]
Assuring high quality within the field of genetic testing is fundamental, as the results can have considerable impact on the patient and his or her family. The use of appropriate quality control (QC) samples is therefore essential. Diagnostic laboratories mainly use patient samples as QC material, which of course include a maximum of two mutations per sample. Bearing in mind that some assays (such as for cystic fibrosis [CF] testing) can test for more than 100 mutations, multiplex QC materials including more than two mutations could save valuable time and reagents. Based on this need, synthetic multiplex controls have been developed by Maine Molecular Quality Controls, Inc. (MMQCI) for CF. A synthetic control, containing six homozygous mutations and one polymorphism for CF transmembrane conductance regulator (CFTR), was evaluated by distributing it through the CF external quality assessment (EQA) scheme, along with the EQA samples in 2005. A total of 197 participants returned results of the yearly EQA scheme and 133 laboratories participated in the evaluation of the synthetic sample. Respectively, 76% and 73% of the participants were assigned as successful. This evaluation study revealed that the multiplex QC material performed well in the majority of assays and could be useful in method validation, as a tool to challenge interpretation skills, and as potential proficiency testing (PT) material.

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143 Two of the laboratories that saw this weak signal for wild-type R553X (c.1657C4T, p.Arg553X) also reported weak mutant signals for Q552X (c.1654C4T, p.Gln552X) or G542X (c.1624G4T, p.Gly542X), possibly indicating DNA overload. Login to comment
153 These very faint signals for wild-type R553X (c.1657C4T, p.Arg553X), wild-type R117H (c.350G4A, p.Arg117His), mutant G542X (c.1624G4T, p.Gly542X), and mutant A455E (c.1364C4A, p.Ala455Glu) signal were often not visible to the assessors on the copies of the raw data. Login to comment
157 ErrorTypes for the QCS in More Detail, for the LaboratoriesThat Used Only One Detection AssayÃ Genotype error Genotype Detection assay Number of labs Expected Reported Comment OLA-CFASR v2.0 1 R117 H hom ^ Correct on raw data INNO-LiPA CFTR36 1 R117 H hom R117 H het No signal for wt R117 H visible on copy of the raw data, could be very weak on original raw data INNO-LiPA CFTR36 1 R553X hom R553X het No signal for wt R553X visible on copy of the raw data, could be very weak on original raw dataI507del hom I507del/F508del Sequencing 2 R347 H hom ^ No complete raw data received Sequencing 1 I507del hom ^ No raw data received Additional mutation(s) reported Detection assay Number of labs Additional mutation(s) Comment OLA-CFASR v3.0 US 1 2184delAa hom Software called it INNO-LiPA CFTR36 3 A455E het (3labs), F508del (1lab) No signal for mut A455E visible on copy of the raw data, could be very weak on original raw data ARMS-ElucigeneTM CF29 3 2184delAa (3labs), R347P (3labs), 1717-1G4A (3labs), 3849110kbC4T (2labs) Cross reaction with 2183AA4Gb and R347 H and no full compatibility of MMQCI-CF-P1and ARMS method: no control bands visible ARMS-ElucigeneTM CF29 1CF-HT 1 2184delAa , R347P Cross reaction with 2183AA4Gb and R347H Sequencing 1 W1282X het, N1303 K het No raw data received ASPE-CFTR 4014 Tag-It 1 71111G4T het No raw data received Genotype error 1 additional mutation(s) reported Genotype Detection assay Number of labs Expected Reported Comment Additional mutation(s) Comment OLA-CFASR v3.0 EU 1 R117 H hom ^ No raw data received; probably 2183AA4Gb missed, but 2184delAa reported due to cross reaction 2184delAa hom No raw data received, probably due to cross-reaction with 2183AA4Gb 394delTTc hom 394delTTc het 2183AA4Gb hom ^ INNO-LiPA CFTR36 1 R553X hom I507del hom R553X het I507del/ F508del No signal for wt R553X visible on copy of the raw data, could be very weak on original raw data G542X het A455E het No signal for mut G542X and mut A455E visible on copy of the raw data, could be very weak on original raw data INNO-LiPA CFTR36 1 Italian regional 1 R553X hom R553X het No signal for wt R553X visible on copy of the raw data, could be very weak on original raw data Q552X het Misinterpretation: wt and mut signal for Q552X not visible, but this is a normal reaction pattern when R553X is hom present; the lab reported R553X het ARMS-ElucigeneTM CF29 1 I507del hom ^ No full compatibility of MMQCI- CF-P1 and ARMS method: no control bands R347P Cross-reaction with R347H2183AA4Gb hom ^ ÃIf the zygosity is not mentioned in the table, the laboratory did not report it. Login to comment
191 For example, reporting the weak bands for wild-type R553X (c.1657C4T, p.Arg553X) and R117 H (c.350G4A, p.Arg117His), mutant G542X (c.1624G4T, p.Gly542X) and A455E (c.1364C4A, p.Ala455Glu) seen by some of the laboratories using the INNO-LiPA assay could be explained in this respect. Login to comment

[hide] Tissue transglutaminase activation modulates infla... J Immunol. 2008 Jun 1;180(11):7697-705. Maiuri L, Luciani A, Giardino I, Raia V, Villella VR, D'Apolito M, Pettoello-Mantovani M, Guido S, Ciacci C, Cimmino M, Cexus ON, Londei M, Quaratino S
Tissue transglutaminase activation modulates inflammation in cystic fibrosis via PPARgamma down-regulation.
J Immunol. 2008 Jun 1;180(11):7697-705., 2008-06-01 [PMID:18490773]

Abstract [show]
Cystic fibrosis (CF), the most common life-threatening inherited disease in Caucasians, is due to mutations in the CF transmembrane conductance regulator (CFTR) gene and is characterized by airways chronic inflammation and pulmonary infections. The inflammatory response is not secondary to the pulmonary infections. Indeed, several studies have shown an increased proinflammatory activity in the CF tissues, regardless of bacterial infections, because inflammation is similarly observed in CFTR-defective cell lines kept in sterile conditions. Despite recent studies that have indicated that CF airway epithelial cells can spontaneously initiate the inflammatory cascade, we still do not have a clear insight of the molecular mechanisms involved in this increased inflammatory response. In this study, to understand these mechanisms, we investigated ex vivo cultures of nasal polyp mucosal explants of CF patients and controls, CFTR-defective IB3-1 bronchial epithelial cells, C38 isogenic CFTR corrected, and 16HBE normal bronchial epithelial cell lines. We have shown that a defective CFTR induces a remarkable up-regulation of tissue transglutaminase (TG2) in both tissues and cell lines. The increased TG2 activity leads to functional sequestration of the anti-inflammatory peroxisome proliferator-activated receptor gamma and increase of the classic parameters of inflammation, such as TNF-alpha, tyrosine phosphorylation, and MAPKs. Specific inhibition of TG2 was able to reinstate normal levels of peroxisome proliferator-activated receptor-gamma and dampen down inflammation both in CF tissues and CFTR-defective cells. Our results highlight an unpredicted central role of TG2 in the mechanistic pathway of CF inflammation, also opening a possible new wave of therapies for sufferers of chronic inflammatory diseases.

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35 Materials and Methods Human airway biopsies and ex vivo cultures Nasal polyp explants from 10 CF patients carrying the common CFTR mutations (⌬F508/⌬F508, ⌬F508/W1282X, ⌬F508/N1303K, or ⌬F508/G542X) and 10 non-CF patients with nonallergic idiopathic polyposis were cultured, for 4-24 h (9), with or without specific TG2 inhibitors 1,3-dymethyl-2-[(2-oxopropyl) thio] imidazolium (R283) (250 ␮M) (12) or halo-dihydroisox- azole-derivate transglutaminase inhibitor KCC009 (250 ␮M), reactive oxygen species (ROS) scavenger EUK 134 (50 ␮g/ml; Alexis Biochemical), N-acetylcysteine (NAC, 10 mM; Alexis Biochemical), PPAR␥ antagonist GW9662 (1 ␮M; Alexis Biochemical), or R283 for 24 h, followed by GW9662 (1 ␮M) for 4 h. Informed consent was obtained from all subjects, and the ethical committee of Regione Campania Health Authority approved the study. Login to comment
36 Cell lines and cultures IB3-1 (human CF bronchial epithelial cell line with the common ⌬F508/ W1282X CFTR mutation) and C38 (isogenic stably rescued with functional CFTR) cell lines (LGC Promochem) (2, 7, 10) were stimulated for 6 h with R283 (250 ␮M) or KCC009 (250 ␮M), ionomycin (1 ␮M; Calbiochem), BAPTA-AM (5 ␮M, Calbiochem), EUK 134 (50 ␮g/ml), rosiglitazone (10 ␮␮), NAC (10 mM), proteasome inhibitor MG132 (50 ␮M for 6 h; Calbiochem), or R283 for 24 h, followed by 6-h rosiglitazone. Login to comment

[hide] Diagnosis of cystic fibrosis. Clin Rev Allergy Immunol. 2008 Dec;35(3):100-6. Voter KZ, Ren CL
Diagnosis of cystic fibrosis.
Clin Rev Allergy Immunol. 2008 Dec;35(3):100-6., [PMID:18506640]

Abstract [show]
Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that results in abnormal viscous mucoid secretions in multiple organs and whose main clinical features are pancreatic insufficiency and chronic endobronchial infection. Although it was initially defined and diagnosed based on clinical features and sweat chloride measurement, an in vivo method of assessing CFTR function, the discovery of the CFTR gene in 1989 revealed a broad spectrum of CF phenotypes associated with specific CFTR gene mutations. In this article, we will review the indications for sweat testing, alternative techniques to diagnose CF, and the approach to patients with an ambiguous or indeterminate diagnosis of CF.

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114 Figure reproduced from Ref. [6], with permission Table 7 Classes of CFTR gene mutations associated with CF disease Mutation class Mechanism of action Examples I Absence of protein synthesis because of a stop codon in the gene G542X II Improper folding and processing ΔF508 III Reduced response to regulatory molecules G551D IV Reduce ion conductance R117H V Decreased protein production due to splice defects or promoter mutations 3,849+10 kb C→T VI Decreased protein stability Q1412X 104 measurement of transepithelial ion flow in the nasal mucosa [28-30]. Login to comment

[hide] Genetic determinants and epidemiology of cystic fi... Diabetes Care. 2008 Sep;31(9):1789-94. Epub 2008 Jun 5. Adler AI, Shine BS, Chamnan P, Haworth CS, Bilton D
Genetic determinants and epidemiology of cystic fibrosis-related diabetes: results from a British cohort of children and adults.
Diabetes Care. 2008 Sep;31(9):1789-94. Epub 2008 Jun 5., [PMID:18535191]

Abstract [show]
OBJECTIVE: Longer survival of patients with cystic fibrosis has increased the occurrence of cystic fibrosis-related diabetes (CFRD). In this study we documented the incidence of CFRD and evaluated the association between mutations responsible for cystic fibrosis and incident CFRD, while identifying potential risk factors. RESEARCH DESIGN AND METHODS: This was a population-based longitudinal study of 50 cystic fibrosis speciality clinics in the U.K. Subjects included 8,029 individuals aged 0-64 years enrolled in the U.K. Cystic Fibrosis Registry during 1996-2005. Of these, 5,196 with data and without diabetes were included in analyses of incidence, and 3,275 with complete data were included in analyses of risk factors. Diabetes was defined by physician diagnosis, oral glucose tolerance testing, or treatment with hypoglycemic drugs. RESULTS: A total of 526 individuals developed CFRD over 15,010 person-years. The annual incidence was 3.5%. The incidence was higher in female patients and in patients with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in classes I and II. In a multivariate model of 377 cases of 3,275 patients, CFTR class (relative risk 1.70 [95% CI 1.16-2.49], class I or II versus others), increasing age, female sex, worse pulmonary function, liver dysfunction, pancreatic insufficiency, and corticosteroid use were independently associated with incident diabetes. CONCLUSIONS: The incidence of CFRD is high in Britain. CFTR class I and II mutations increase the risk of diabetes independent of other risk factors including pancreatic exocrine dysfunction.

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54 Genotypes associated with cystic fibrosis were coded into five established classes reflecting CFTR function of defective production, processing, regulation, conductance, and quantity of CFTR protein (12) as follows: I: G542X, R553X, W1282X, R1162X, 621-1G3T, 1717- 1G3 A, 1078⌬T, and 3659⌬C; II: ⌬F508, ⌬I507, N1303K, and S549N; III: G551Dand R560T; IV: R117H, R334W, G85E, and R347P; V: 3849ϩ5G3A, and A455E; and unknown: 711ϩIG3 T, 2184DA, and 1898ϩIG3 A. Login to comment

[hide] Genetic investigations of CFTR mutations in congen... J Androl. 2008 Sep-Oct;29(5):506-13. Epub 2008 Jun 20. Radpour R, Gourabi H, Dizaj AV, Holzgreve W, Zhong XY
Genetic investigations of CFTR mutations in congenital absence of vas deferens, uterus, and vagina as a cause of infertility.
J Androl. 2008 Sep-Oct;29(5):506-13. Epub 2008 Jun 20., [PMID:18567645]

Abstract [show]
A qualitative diagnosis of infertility requires attention to male and female physical abnormalities including endocrine anomalies and genetic conditions that interfere with reproduction. Many genes are likely to be involved in the complex process of reproduction. Congenital bilateral absence of the vas deferens (CBAVD) is a genital form of cystic fibrosis (CF) that is responsible for 2%-6% of male infertility. The incidence of CF varies in different populations; therefore, the incidence of CBAVD will also vary in different populations. The spectrum and distribution of cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations differ between CBAVD and CF patients and are comparable to control individuals. Combinations of particular alleles at several polymorphic loci yield insufficient functional CFTR protein. CFTR mutations are also associated with congenital absence of the uterus and vagina (CAUV). Females with CF are found to be less fertile than normal healthy women. Because of techniques such as intracytoplasmic sperm injection (ICSI), CBAVD patients are now able to father children. Such couples, however, have an increased risk of having a child with cystic fibrosis, and therefore genetic testing and counseling should be provided. Around 10% of obstructive azoospermia is congenital and due to mutations in the CF gene. This review highlights the relationship of mutations in the CFTR gene with CBAVD and CAUV.

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36 Examples include the G542X, G551D, R553X, W1282X, and N1303K mutations. Login to comment

[hide] Molecular analysis of mutations and polymorphisms ... Reprod Biomed Online. 2008 Jul;17(1):27-35. Tamburino L, Guglielmino A, Venti E, Chamayou S
Molecular analysis of mutations and polymorphisms in the CFTR gene in male infertility.
Reprod Biomed Online. 2008 Jul;17(1):27-35., [PMID:18616886]

Abstract [show]
Mutations of the cystic fibrosis transmembrane regulator (CFTR) gene and polymorphisms, such as the (TG)m and Tn polymorphic loci in intron 8 at the splice acceptor site of exon 9, can cause male infertility. The aim of this study was to investigate the frequency of the most prevalent cystic-fibrosis-causing mutations, the IVS8-Tn alleles and IVS8-TG12 variant in the presence of IVS8-5T in patients with altered semen parameters (group I with obstructive azoospermia, group II with secretory azoospermia and group III with severe oligozoospermia) compared with a control group with normozoospermia. CFTR mutations were found in 26.5% and 14.3% of chromosomes of patients of group I and II respectively (P < 0.001, P < 0.05). The frequency of the 5T allele was 23.5% in patients in group I (P < 0.01), and was linked exclusively with TG12 allele. The present study reports for the first time a high proportion of the 5T allele in patients in group III (9.2%, P < 0.05). These results underline the importance of performing molecular analysis of mutations and IVS8-Tn polymorphism in the CFTR gene and appropriate genetic counselling to all couples undergoing assisted reproductive technologies when the partner has azoospermia or severe oligozoospermia.

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136 Cystic fibrosis transmembrane regulator (CFTR) and IV.S8-Poly T genotypes according to semen parameters. Semen analysis Group 1 Total Group !l Total Group m TutuI Group IV Total CFTR genotype AF508/- AF508/- 2789+5G_^A/- N13O3K/- G542X/- - / - - / - - / - AF508/- AF508/R1I7H» NI3Ü3K/- 3849+ 10kbC_T/3849+1 O k b C ^ P - / - -/_ - / - AF5O8/- N1303K/- 3849+!0kbC-+T/- - / - - / - - / - - / - - / - - / - AF508/- N1303K/- 3849+10kbC->T/- I148T/- WI282X/- - / - - / - _/_ - / - - / - -/_ genotype 5T/9T 7T/9T 7T/7T 5T/9T 5T/9T 5T/7T 5T/9T 7T/7T 7T/9T 7T/9T 7T/9T 7T/7T 5T/7T 7T/7T 7T/9T 5T/9T 7r/7T 7T/9T 5T/5T 5T/7T 5T/9T 7T/7T 7T/9T 9T/9T 7T/9T 7T/9T 7T/7T 7T/9T 7T/7T 5T/5T 5T/7r 5T/9T 7T/7T 7T/9T 9T/9T No. Login to comment

[hide] Guidelines for diagnosis of cystic fibrosis in new... J Pediatr. 2008 Aug;153(2):S4-S14. Farrell PM, Rosenstein BJ, White TB, Accurso FJ, Castellani C, Cutting GR, Durie PR, Legrys VA, Massie J, Parad RB, Rock MJ, Campbell PW 3rd
Guidelines for diagnosis of cystic fibrosis in newborns through older adults: Cystic Fibrosis Foundation consensus report.
J Pediatr. 2008 Aug;153(2):S4-S14., [PMID:18639722]

Abstract [show]
Newborn screening (NBS) for cystic fibrosis (CF) is increasingly being implemented and is soon likely to be in use throughout the United States, because early detection permits access to specialized medical care and improves outcomes. The diagnosis of CF is not always straightforward, however. The sweat chloride test remains the gold standard for CF diagnosis but does not always give a clear answer. Genotype analysis also does not always provide clarity; more than 1500 mutations have been identified in the CF transmembrane conductance regulator (CFTR) gene, not all of which result in CF. Harmful mutations in the gene can present as a spectrum of pathology ranging from sinusitis in adulthood to severe lung, pancreatic, or liver disease in infancy. Thus, CF identified postnatally must remain a clinical diagnosis. To provide guidance for the diagnosis of both infants with positive NBS results and older patients presenting with an indistinct clinical picture, the Cystic Fibrosis Foundation convened a meeting of experts in the field of CF diagnosis. Their recommendations, presented herein, involve a combination of clinical presentation, laboratory testing, and genetics to confirm a diagnosis of CF.

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142 Recommended panel of CF-causing mutations Missense, deletion, stop mutations Splicing, frameshift mutations G85E I507del R560T 621ϩ1GϾT 2789ϩ5GϾA R117H F508del R1162X 711ϩ1GϾT 3120ϩ1GϾA R334W G542X W1282X 1717-1GϾA 3659delC R347P G551D N1303K 1898ϩ1GϾA 3849ϩ10kbCϾT A455E R553X 2184delA Revised from the mutation panel for population screening for CF developed by the ACMG.77 Additional or alternative mutations present at significant frequencies in an ethnic population served by an NBS program may be added. Login to comment

[hide] Colonic wall redundancy at CT in patients with cys... Radiology. 2008 Sep;248(3):869-75. Epub 2008 Jul 22. Webb EM, Kleinhenz ME, Coakley FV, Chang CI, Westphalen AC, Yeh BM
Colonic wall redundancy at CT in patients with cystic fibrosis.
Radiology. 2008 Sep;248(3):869-75. Epub 2008 Jul 22., [PMID:18647844]

Abstract [show]
PURPOSE: To describe the appearance, prevalence, and possible associations of colonic wall redundancy in patients with cystic fibrosis (CF). MATERIALS AND METHODS: The institutional review board approved this HIPAA-compliant study. Abdominal computed tomographic (CT) images of 38 consecutive patients with CF and a control group of 38 consecutive potential renal donors were retrospectively identified. Three readers independently recorded presence and location of colonic wall redundancy and wall thickness of the ascending, transverse, and descending colon. Interobserver agreement for colonic wall redundancy was determined with the kappa statistic. Colonic wall thicknesses were compared between patient groups with the Student t test. Proportions of adult and pediatric patients with and those without colonic wall redundancy and prevalence of specific gene mutations were compared between groups with the Fisher exact test. CT findings were compared with radiologic reports and clinical records. RESULTS: Each reviewer found colonic wall redundancy in 11 of 28 adults with CF but in none of the children with CF (P < .05 for each reviewer). There was excellent interobserver agreement for identification of colonic wall redundancy (kappa = 0.91, P < .001). Mean thickness of the wall of the ascending colon was significantly greater in patients with CF who had colonic wall redundancy (4.0 mm) than in those without this finding (1.8 mm, P < .05) or in control patients (1.2 mm, P < .05). Among adult patients with CF, DeltaF508 mutation was the predominant mutant allele in 10 of 13 patients with normal colons at CT, whereas more uncommon non-DeltaF508 mutations were seen in seven of 10 patients with colonic wall redundancy (P < .05). Asymptomatic colonic wall redundancy at CT was prospectively misinterpreted as acute colonic disease in five adult patients. CONCLUSION: Proximal colonic wall redundancy is seen frequently in adults with CF and may be more common in those with non-DeltaF508 CFTR gene mutations. This finding provides a starting point for further investigation of the molecular basis of colonic phenotype in CF.

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86 In particular, the G542X mutation was seen exclusively in patients with colonic wall redundancy (three [30%] of 10 patients, P ϭ .06). Login to comment
96 * Non-⌬F508 gene mutations include G542X, 3905insT, R347P, 711ϩ1GϾT, 3120ϩ1GϾA, W1282X, and 1161delC. Login to comment
126 The next most common mutation, G542X, accounts for only 2.4% of mutations (13). Login to comment

[hide] Best practice guidelines for molecular genetic dia... Eur J Hum Genet. 2009 Jan;17(1):51-65. Epub 2008 Aug 6. Dequeker E, Stuhrmann M, Morris MA, Casals T, Castellani C, Claustres M, Cuppens H, des Georges M, Ferec C, Macek M, Pignatti PF, Scheffer H, Schwartz M, Witt M, Schwarz M, Girodon E
Best practice guidelines for molecular genetic diagnosis of cystic fibrosis and CFTR-related disorders--updated European recommendations.
Eur J Hum Genet. 2009 Jan;17(1):51-65. Epub 2008 Aug 6., [PMID:18685558]

Abstract [show]
The increasing number of laboratories offering molecular genetic analysis of the CFTR gene and the growing use of commercial kits strengthen the need for an update of previous best practice guidelines (published in 2000). The importance of organizing regional or national laboratory networks, to provide both primary and comprehensive CFTR mutation screening, is stressed. Current guidelines focus on strategies for dealing with increasingly complex situations of CFTR testing. Diagnostic flow charts now include testing in CFTR-related disorders and in fetal bowel anomalies. Emphasis is also placed on the need to consider ethnic or geographic origins of patients and individuals, on basic principles of risk calculation and on the importance of providing accurate laboratory reports. Finally, classification of CFTR mutations is reviewed, with regard to their relevance to pathogenicity and to genetic counselling.

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144 A (T)5 variant can either be associated with (TG)11, (TG)12, (TG)13, and rarely (TG)15 repeats.74 When (T)5 is found in diagnostic testing, for example, for CBAVD or atypical presentation, determination of Table 4 Classification of CFTR mutations with regard to their potential for causing disease Mutation group Examples CF-causing F508del Mainly nonsense, frameshift, splicing (invariant dinucleotide): G542X, R553X, W1282X, 2183AA4G, 3659delC, 1717-1G4A, 3120+1G4A Missense that severely affects CFTR synthesis or function: G551D, N1303K, R347P 2789+5G4A, 3849+10kbC4T, 3272-26A4G, L206Wa , D1152Ha , (TG)13(T)5a CFTR-related disorders associated L206Wa , D1152Ha , (TG)13(T)5a [R117H;(T)7], (TG)12(T)5, L997F, V562I, [R668C;G576A;D443Y], [R74W;D1270N] (TG)11(T)5b , S1235Rb No clinical consequences 875+40A4G, M470V (1540A4G), I506V (1648A4G), F508C (1655T4G), 1716G4A, 2694T4G, 4002A4G, 2752-15G4C (TG)11(T)5b , S1235Rb Unproven or uncertain clinical relevance Mainly missense mutations G622D, R170H, V938G, I125T Putative splice mutations: 406-6T4C, 2752-26A4G, 3601-17T4C Only a fraction of mutations and patients have been characterized in detail and, with the exception of frequent mutations, only small numbers of patients have been available for the study of most mutations. Login to comment

[hide] Identification and characterization of CFTR gene m... Ann Hum Genet. 2009 Jan;73(1):26-33. Epub 2008 Sep 8. Sharma N, Singh M, Kaur G, Thapa BR, Prasad R
Identification and characterization of CFTR gene mutations in Indian CF patients.
Ann Hum Genet. 2009 Jan;73(1):26-33. Epub 2008 Sep 8., [PMID:18782298]

Abstract [show]
Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. This study was performed on Indian CF patients (n = 50) to investigate the spectrum of mutations in the CFTR gene and their association with intragenic and extragenic marker haplotypes. We report identification of 14 previously known and eight novel mutations, namely 3986-3987delC, 876-6del4, 1792InsA, L69H, S158N, Q493L, I530L and E1329Q. The frequency of delta F508 was found to be 27%. Absolute linkage between delta F508 and the KM.19-GATT-TUB9-M470V-T854T haplotype (2-2-1-1-1) predicts a relatively recent appearance of delta F508 in Indian CF patients. Low frequency of delta F508 mutation and detection of eight novel and thirteen rare mutations reflect a heterogeneous spectrum of mutations in Indian CF patients. Failure to detect mutations in 34% of alleles indicates the possible presence of gross deletions involving one or more exons or may indicate the location of the molecular defects in either the noncoding parts of the gene or in the promoter region, which warrants analysis of those regions.

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41 Seven other mutations were searched for by either single ARMS PCR (R117H, N1303K, and R553X) or by multiplex ARMS PCR (621 + 1G-T, G542X, G551D, W1282X). Login to comment

[hide] Animal models of chronic lung infection with Pseud... Lab Anim. 2008 Oct;42(4):389-412. Epub 2008 Sep 9. Kukavica-Ibrulj I, Levesque RC
Animal models of chronic lung infection with Pseudomonas aeruginosa: useful tools for cystic fibrosis studies.
Lab Anim. 2008 Oct;42(4):389-412. Epub 2008 Sep 9., [PMID:18782827]

Abstract [show]
Cystic fibrosis (CF) is caused by a defect in the transmembrane conductance regulator (CFTR) protein that functions as a chloride channel. Dysfunction of the CFTR protein results in salty sweat, pancreatic insufficiency, intestinal obstruction, male infertility and severe pulmonary disease. In most patients with CF life expectancy is limited due to a progressive loss of functional lung tissue. Early in life a persistent neutrophylic inflammation can be demonstrated in the airways. The cause of this inflammation, the role of CFTR and the cause of lung morbidity by different CF-specific bacteria, mostly Pseudomonas aeruginosa, are not well understood. The lack of an appropriate animal model with multi-organ pathology having the characteristics of the human form of CF has hampered our understanding of the pathobiology and chronic lung infections of the disease for many years. This review summarizes the main characteristics of CF and focuses on several available animal models that have been frequently used in CF research. A better understanding of the chronic lung infection caused particularly by P. aeruginosa, the pathophysiology of lung inflammation and the pathogenesis of lung disease necessitates animal models to understand CF, and to develop and improve treatment.

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167 Finally, a transgenic KO model expressing a human CFTR with the G542X mutation under the control of the intestinal fatty-acid-binding protein (FABP) gene promoter has been generated and used to study the effect of aminoglycosides on suppression of this CFTR premature stop mutation (Du et al. 2002). Login to comment
170 Table 2 Cystic fibrosis (CF) mouse models CF mice Mutation/molecular strategy Phenotype/limitation CFTR KO CFTRtm1Unc (Snouwaert et al. 1992) Exon 10 replacement, null mutation, inframe stop Severe intestinal phenotype and high mortality; no lung disease CFTRtm1Hgu (Dorin et al. 1992) Exon 10 insertional mutagenesis Intestinal blockage; minor pathology in lungs of one mouse CFTRtm1Cam (Ratcliff et al. 1993) Exon 10 replacement, null mutation Severe intestinal phenotype and high mortality; pathology in lacrimal gland and pancreas of some mice; no lung disease CFTRtm1Bay (O`Neal et al. 1993) Exon 3 insertional duplication, null mutation Severe intestinal phenotype and high mortality; no lung disease CFTRtm1Hsc (Rozmahel et al. 1996) Exon 1 replacement, null mutation Severe intestinal phenotype and high mortality; no lung disease Other mutations CFTRtm1Kth (Zeiher et al. 1995) DF508 by exon 10 replacement High mortality and reduction in size, variable pathology of the gastrointestinal tract, normal lung, pancreas, gallbladder, male reproductive tract, lacrimal gland and submandibular glands CFTRtm1Eur (van Doorninck et al. 1995, French et al. 1996) DF508 by exon 10 'hit and run` Normal survival, growth retarded but no abnormalities or stasis of inspissated mucus in lungs, pancreas, liver bile ducts, vas deferens and salivary glands CFTRtm1G551D (Delaney et al. 1996) G551D by exon 11 replacement Moderate phenotype with reduced incidence of intestinal blockage and 67% survival; no lung disease CFTRtm2Hgu (Dickinson et al. 2000) G480C by exon 10 'hit and run` Normal survival, no reduction in body weight, preserved cAMP-mediated Cl2 response, decreased Ca2þ -related Cl2 response CFTR2/2hCFTR-G542X (Du et al. 2002) CFTR2/2 null mutation that also express a human CFTR-G542X stop mutation under control of the intestinal FABP promoter Suppression of the hCFTR-G542X mutation in vivo by aminoglycosides CFTRtm1Unc -TgN(FABPCFTR) (Zhou et al. 1994) Stop codon in the murine CFTR gene (S489X) but also express human CFTR in the gut epithelium (transgenic introduction of CFTR under FABP promoter) Functional correction of ileal goblet cell and crypt cell hyperplasia and cyclic adenosine monophosphate-stimulated chloride secretion, improved survival Congenic C57BL/6J CFTR2/2 (Durie et al. 2004) Long-lived congenic C57BL/6J CFTR2/2 All organs pathologically affected by the human form of CF Scnn1a-, Scnn1b- and Scnn1c-transgenic mice (Mall et al. 2004) Transgenic mice overexpressing airway-specific ENaC to increase Naþ absorption CF-like lung disease FABP: fatty acid-binding protein, ENaC: epithelial Naþ channels. Login to comment

[hide] Implication of the cystic fibrosis transmembrane c... Biochem Genet. 2008 Dec;46(11-12):847-56. Epub 2008 Sep 23. Sharma N, Singh M, Acharya N, Singh SK, Thapa BR, Kaur G, Prasad R
Implication of the cystic fibrosis transmembrane conductance regulator gene in infertile family members of Indian CF patients.
Biochem Genet. 2008 Dec;46(11-12):847-56. Epub 2008 Sep 23., [PMID:18810634]

Abstract [show]
Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene. Among males with CF, 95% are infertile due to congenital absence of the vas deferens. We investigated the role of family history of infertility among CF subjects and characterized mutations in them. Among 50 CF subjects, four had a family history of infertility. A homozygous c.1521_1523delCTT mutation was detected in one, two had a compound heterozygous genotype (c.1521_1523delCTT/c.3717 + 10 kbC>T), and c.1521_1523delCTT mutation was identified on one allele of fourth CF subject. Genetic analysis of each infertile family members of CF subjects revealed the c.1521_1523delCTT mutation on one allele; however, no mutation could be identified on other allele. Haplotype analysis of the infertile family members showed that at least one of the alleles shared the same haplotype as that of the index case. It is suggested that the CFTR gene is implicated in the infertile members of the CF families. Failure to detect mutations on the other allele by SSCP analysis demands direct gene sequencing to detect mutations in the intronic or promoter region.

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40 1G-T, G542X, G551D, and W1282X by multiplex ARMS PCR (Ferrie et al. 1992). Login to comment

[hide] Localization studies of rare missense mutations in... Hum Mutat. 2008 Nov;29(11):1364-72. Krasnov KV, Tzetis M, Cheng J, Guggino WB, Cutting GR
Localization studies of rare missense mutations in cystic fibrosis transmembrane conductance regulator (CFTR) facilitate interpretation of genotype-phenotype relationships.
Hum Mutat. 2008 Nov;29(11):1364-72., [PMID:18951463]

Abstract [show]
We have been investigating the functional consequences of rare disease-associated amino acid substitutions in the cystic fibrosis transmembrane conductance regulator (CFTR). Mutations of the arginine residue at codon 1070 have been associated with different disease consequences; R1070P and R1070Q with "severe" pancreatic insufficient cystic fibrosis (CF) and R1070W with "mild" pancreatic sufficient CF or congenital bilateral absence of the vas deferens. Intriguingly, CFTR bearing each of these mutations is functional when expressed in nonpolarized cells. To determine whether R1070 mutations cause disease by affecting CFTR localization, we created polarized Madin Darby canine kidney (MDCK) cell lines that express either wild-type or mutant CFTR from the same genomic integration site. Confocal microscopy and biotinylation studies revealed that R1070P was not inserted into the apical membrane, R1070W was inserted at levels reduced from wild-type while R1070Q was present in the apical membrane at levels comparable to wild-type. The abnormal localization of CFTR bearing R1070P and R1070W was consistent with deleterious consequences in patients; however, the profile of CFTR R1070Q was inconsistent with a "severe" phenotype. Reanalysis of 16 patients with the R1070Q mutation revealed that 11 carried an in cis nonsense mutation, S466X. All 11 patients carrying the complex allele R1070Q-S466X had severe disease, while 4 out of 5 patients with R1070Q had "mild" disease, thereby reconciling the apparent discrepancy between the localization studies of R1070Q and the phenotype of patients bearing this mutation. Our results emphasize that localization studies in relevant model systems can greatly assist the interpretation of the disease-causing potential of rare missense mutations.

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140 As for the remaining R1070W patients (8/24 with detailed clinical information), five individuals carried a CFTR mutation associated with pancreatic insufficiency (2869insG (c.2737insG, p.Tyr913fs); G542X (c.1624G4 T, p.Gly542X); R668C-K710X (c. Login to comment

[hide] Clinical and radiological outcome of patients suff... Pancreas. 2008 Nov;37(4):371-6. Frulloni L, Scattolini C, Graziani R, Cavestro GM, Pravadelli C, Amodio A, Manfredi R, Scarpa A, Vantini I
Clinical and radiological outcome of patients suffering from chronic pancreatitis associated with gene mutations.
Pancreas. 2008 Nov;37(4):371-6., [PMID:18953248]

Abstract [show]
OBJECTIVES: Cystic fibrosis transmembrane conductance regulator (CFTR), cationic trypsinogen gene (PRSS1), and serine protease inhibitor kazal type 1 (SPINK1) gene mutations have been associated with chronic pancreatitis (CP). The aim of this study was to compare clinical and radiological findings in sporadic CP with (CPgm) and without (CPwt) gene mutations. METHODS: Data from patients observed between 2001 and 2006 were collected. All patients were tested for 25 CFTR gene mutations, for R122H and N29I on the PRSS1 gene, and for N34S mutation on the SPINK1 gene. RESULTS: We found 34 (17.2%) of 198 patients with CPgm, 23 (11.6%) of them on the CFTR gene, 11 (5.6%) on the SPINK1, and none on the PRSS1 gene. The age at clinical onset was younger in CPgm (36.2 +/- 17.2 years) than in CPwt (44 +/- 12.6 years; P = 0.005). There were more heavy drinkers among CPwt (33%) than among CPgm (9%; P = 0.003), and the same applied to smokers (69% vs 33%, respectively; P < 0.0001). In CPgm group, the onset of pancreatic calcifications was observed more frequently in drinkers and/or smokers. Exocrine and endocrine insufficiency occurred less frequently and later in CPgm than in CPwt patients. CONCLUSIONS: Clinical and radiological outcome differ in CPgm compared with CPwt. Alcohol, even in small quantities, and cigarette smoking influence the onset of pancreatic calcifications.

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31 All patients were tested for 25 CFTR gene mutations ($F508, $I507, R117H, R1162X, 2183AAYG, N1303K, 3849 + 10KbCYT, G542X, G551D, 1717-1GYA, R347P, R352Q, R553X, Q552X, G85E, 711 + 5GYA, W1282X, 3272-26AYG, 3132delTG, R334W, I148T, 3659del_C, 3120 + 1GYA, 1898 + 1GYA, and 2789 + 5GYA), which cover approximately 72% of the cystic fibrosis mutations in the Italian population. Login to comment

[hide] Sweat chloride testing in infants identified as he... J Pediatr. 2008 Dec;153(6):857-9. Soultan ZN, Foster MM, Newman NB, Anbar RD
Sweat chloride testing in infants identified as heterozygote carriers by newborn screening.
J Pediatr. 2008 Dec;153(6):857-9., [PMID:19014821]

Abstract [show]
The reference ranges for sweat [C1(-)] were reevaluated in 300 infants referred to our Center as carriers of at least 1 cystic fibrosis mutation identified through newborn screening. The recommended borderline range of 30 to 59 mmol/L failed to identify all individuals who were compound heterozygotes. Our data support using a borderline range of 24 to 59 mmol/L.

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54 Sweat [Cl- ] and the results of genetic screening of 11 patients with [Cl- ] > 24 mmol/L Patients Sweat [Cl- ] mmol/L Mutations Poly T-TG Repeats 1 89 91 R347P CFTRdel 17a-18 7T/9T 2 85 82 ⌬F508 2622ϩ1 GϾT 9T/9T 3 71 - G542X Y1014del 7T/9T 4 69 65 ⌬F508 c.759AϾG 9T/7T 5 58 49 ⌬F508 L206W 9T/9T 6 44 27 ⌬F508 R352W, P750L - 7 38 41 ⌬F508 - 9T-TG10 5T-TG12 8 24 - ⌬F508 - 9T-TG10 5T-TG12 9 25 27 ⌬F508 - 9T-TG10 5T-TG12 10 24 25 ⌬F508 - 9T-TG12 5T-TG12 11 35 26 ⌬F508 - 9T 9T Soultan et al The Journal of Pediatrics • December 2008 should be followed. Login to comment

[hide] Clinical practice and genetic counseling for cysti... Genet Med. 2008 Dec;10(12):851-68. Moskowitz SM, Chmiel JF, Sternen DL, Cheng E, Gibson RL, Marshall SG, Cutting GR
Clinical practice and genetic counseling for cystic fibrosis and CFTR-related disorders.
Genet Med. 2008 Dec;10(12):851-68., [PMID:19092437]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator-related disorders encompass a disease spectrum from focal male reproductive tract involvement in congenital absence of the vas deferens to multiorgan involvement in classic cystic fibrosis. The reproductive, gastrointestinal, and exocrine manifestations of cystic fibrosis transmembrane conductance regulator deficiency are correlated with CFTR genotype, whereas the respiratory manifestations that are the main cause of morbidity and mortality in cystic fibrosis are less predictable. Molecular genetic testing of CFTR has led to new diagnostic strategies and will enable targeting of molecular therapies now in development. Older diagnostic methods that measure sweat chloride and nasal potential difference nonetheless remain important because of their sensitivity and specificity. In addition, the measurement of immunoreactive trypsinogen and the genotyping of CFTR alleles are key to newborn screening programs because of low cost. The multiorgan nature of cystic fibrosis leads to a heavy burden of care, thus therapeutic regimens are tailored to the specific manifestations present in each patient. The variability of cystic fibrosis lung disease and the variable expressivity of mild CFTR alleles complicate genetic counseling for this autosomal recessive disorder. Widespread implementation of newborn screening programs among populations with significant cystic fibrosis mutation carrier frequencies is expected to result in increasing demands on genetic counseling resources.

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31 Pulmonary disease is the major cause of morbidity and Table 1 Classification scheme for CFTR mutations112 Mutation class Effect on CFTR protein Mechanisms I Reduced or absent synthesis Nonsense, frameshift, or splice junction mutations II Block in protein processing Missense mutations or amino acid deletions III Block in regulation of CFTR chloride channel Missense mutations IV Altered conductance of CFTR chloride channel Missense mutations V Reduced amounts of functioning CFTR protein Missense or splice junction mutations Table 2 Phenotypes of 10 most common CFTR alleles in whites with CF41 Mutation Relative frequency (%)a Functional classb Phenotypec ⌬F508 66.0 II Classic G542X 2.4 I Classic G551D 1.6 III Classic N1303K 1.3 II Classic W1282X 1.2 I Classic R553X 0.7 I Classic 621ϩ1GϾT 0.7 I Classic 1717-1GϾA 0.6 I Classic R117H 0.3 IV Nonclassic R1162X 0.3 Not cleard Classic a Calculated using total CFTR alleles as the denominator. Login to comment
56 Liver disease is second to pulmonary disease (plus organ transplantation complications) as a cause of mortality in CF (1.7% of deaths).26 Table 3 Core mutation panel carrier recommended by the ACMG for routine CF diagnostic testing and carrier screening of the general population7 Intronic mutations Exonic mutations Missense Nonsense In-Frame Deletion 621ϩ1GϾT G85E G542X ⌬I507 711ϩ1GϾT R117H R553X ⌬F508 1717-1GϾA R334W R1162X 1898ϩ1GϾA R347P W1282X 2184delA A455E 2789ϩ5GϾA G551D 3120ϩ1GϾA R560T 3659delC N1303K 3849ϩ10kbCϾT Endocrine manifestations of CF CF-related diabetes mellitus (CFRDM) may present in adolescence. Login to comment

[hide] Poly-L-aspartic acid enhances and prolongs gentami... J Biol Chem. 2009 Mar 13;284(11):6885-92. Epub 2009 Jan 9. Du M, Keeling KM, Fan L, Liu X, Bedwell DM
Poly-L-aspartic acid enhances and prolongs gentamicin-mediated suppression of the CFTR-G542X mutation in a cystic fibrosis mouse model.
J Biol Chem. 2009 Mar 13;284(11):6885-92. Epub 2009 Jan 9., 2009-03-13 [PMID:19136563]

Abstract [show]
Aminoglycosides such as gentamicin have the ability to suppress translation termination at premature stop mutations, leading to a partial restoration of protein expression and function. This observation led to studies showing that this approach may provide a viable treatment for patients with genetic diseases such as cystic fibrosis that are caused by premature stop mutations. Although aminoglycoside treatment is sometimes associated with harmful side effects, several studies have shown that the co-administration of polyanions such as poly-L-aspartic acid (PAA) can both reduce toxicity and increase the intracellular aminoglycoside concentration. In the current study we examined how the co-administration of gentamicin with PAA influenced the readthrough of premature stop codons in cultured cells and a cystic fibrosis mouse model. Using a dual luciferase readthrough reporter system in cultured cells, we found that the co-administration of gentamicin with PAA increased readthrough 20-40% relative to cells treated with the same concentration of gentamicin alone. Using a Cftr-/- hCFTR-G542X mouse model, we found that PAA also increased the in vivo nonsense suppression induced by gentamicin. Following the withdrawal of gentamicin, PAA significantly prolonged the time interval during which readthrough could be detected, as shown by short circuit current measurements and immunofluorescence. Because the use of gentamicin to suppress disease-causing nonsense mutations will require their long term administration, the ability of PAA to reduce toxicity and increase both the level and duration of readthrough has important implications for this promising therapeutic approach.

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35 Printed in the U.S.A. MARCH 13, 2009•VOLUME 284•NUMBER 11 JOURNAL OF BIOLOGICAL CHEMISTRY 6885 an increased and prolonged level of suppression of the CFTR-G542X nonsense mutation in a CF mouse model. Login to comment
55 Mice and Treatment Protocols-The CFTR-G542X mice used in this study contained the Cftrtm1Cam knock-out (20) and expressed a human CFTR transgene with the G542X premature stop mutation (3, 4, 21) (referred to as Cftr-/- hCFTR-G542X mice). Login to comment
57 Transcription of the hCFTR-G542X transgene was driven by the rat intestinal fatty acid-binding protein promoter. Login to comment
114 PAA Enhances Gentamicin-induced Readthrough in Cftr-/- hCFTR-G542X Mice-We previously reported that once daily subcutaneous injections of 5 mg/kg gentamicin or 15 mg/kg amikacin resulted in suppression of the hCFTR-G542X mutation and a partial restoration of CFTR protein and function in Cftr-/- hCFTR-G542X mice (4). Login to comment
117 Because our in vitro results indicated that the co-administration of gentamicin plus PAA enhanced suppression, we next investigated whether PAA could enhance the readthrough of a nonsense mutation by a low dose of gentamicin in Cftr-/- hCFTR-G542X mice. Login to comment
121 To systematically examine the effect of PAA on readthrough of the hCFTR-G542X nonsense mutation, Cftr-/- hCFTR-G542X mice were included in six different treatment groups as shown in Fig. 4A. Treatments consisted of subcutaneous injections of 5 mg/kg gentamicin alone or 5 mg/kg gentamicin plus 70 mg/kg PAA delivered once daily in a hind limb. Login to comment
122 Groups 1-3 contained Cftr-/- hCFTR-G542X mice that were treated as indicated for 14 days. Group 1 contained control mice that were left untreated for 14 days. Group 2 contained mice treated with gentamicin alone for 14 days, whereas group 3 contained Cftr-/- hCFTR-G542X mice that were administered gentamicin plus PAA for 14 days. Login to comment
124 Groups 4-6 contained Cftr-/- hCFTR-G542X mice that were again treated for 14 days as described above, followed by a 4-day chase period before assays were performed. Login to comment
134 PAA Enhances CFTR Nonsense Codon Readthrough 6888 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284•NUMBER 11•MARCH As controls, both wild type mice (Cftrϩ/ϩ , group 8) and Cftr knock-out mice without the hCFTR-G542X transgene (Cftr-/- , group 10) were treated with gentamicin plus PAA for 14 days with continuing administration of PAA for another 4 days. Login to comment
136 The results of short circuit current measurements from the 10 different treatment groups of Cftr-/- hCFTR-G542X, wild type mice, and Cftr-/- mice are shown in Fig. 4B. A summary and statistical analysis of short circuit currents from the 10 different treatment groups are shown in Table 2. Login to comment
137 The results from group 1 (untreated Cftr-/- hCFTR-G542X mice) revealed that 11% of samples (3/28) exhibited cAMP-stimulated short circuit currents, resulting in an average current of only 0.2 ␮A/cm2 . Login to comment
138 The infrequent response observed in untreated Cftr-/- hCFTR-G542X mice may be attributable to a low baseline level of endogenous readthrough of the hCFTR-G542X transgene, because these currents are not observed in Cftr-/- mice that do not carry the transgene, as shown below and discussed in a previous report (21). Login to comment
151 Effect of gentamicin treatment (؎PAA) on cAMP-activated transepithelialchloridecurrentsinintestinaltissuesfromCftr-/- hCFTR-G542X. Login to comment
152 A, diagram of different treatment groups of Cftr-/- hCFTR-G542X mice, as well as wild type (Cftrϩ/ϩ ) and Cftr-/- controls. Login to comment
157 Cftr-/- hCFTR-G542X Cftr؉/؉ Cftr-/- Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9 Group 10 Treatment (14 days) None Gent Gent PAA Gent Gent PAA Gent PAA None Gent PAA None Gent PAA Chase (4 days) No No No Yes Yes Yes ϩPAA No Yes ϩPAA No Yes ϩPAA Positives/total 3/28 9/26 8/18 1/12 6/17 9/18 14/15 13/14 0/8 0/8 Positives (%) 11 35 44 8 35 50 93 93 0 0 Mean current (␮A/cm2 ) 0.2 0.7 1.1 0.13 1.2 2.1 5.0 5.9 0 0 p value (relative to group 1)a Ͻ0.05 Ͻ0.05 0.35 Ͻ0.05 Ͻ0.01 p value (relative to group 4)b Ͻ0.05 Ͻ0.05 Wild type current (%) 4 14 22 3 24 42 100 118 0 0 a p values of mean currents measured in groups 2-6 are relative to the mean current measured in group 1. b p values of mean currents measured in groups 5 and 6 are relative to the mean current measured in group 4. Login to comment
161 When considered together, these results indicate that the co-administration of PAA significantly increases the average cAMP-stimulated short circuit current induced by gentamicin in hCFTR-G542X mice. Login to comment
168 Together, these results confirmed that the CFTR activity that appears following gentamicin (or gentamicin plus PAA) treatment is dependent upon the presence of the hCFTR-G542X transgene. Login to comment
187 Samples from the duodenum of Cftr-/- hCFTR-G542X mice were incubated with either preimmuneserumorCFTR-NBD1serum.Afterincubationofthesamplewithafluorescentsecondaryantibody, the samples were visualized by fluorescence microscopy. Login to comment
197 Our results with Cftr-/- hCFTR-G542X mice suggest that the higher level of accumulated intracellular gentamicin also remains accessible for ribosome binding in vivo, because the co-administration of gentamicin with PAA had a stimulatory effect on readthrough of the hCFTR-G542X mutation. Login to comment
200 These results are consistent with our in vitro readthrough results and strongly suggest that PAA co-administration increases the intracellular aminoglycoside concentration accessible to ribosomes in the intestinal tissues of Cftr-/- hCFTR-G542X mice. Login to comment
202 When Cftr-/- hCFTR-G542X mice were administered gentamicin for 14 days and then left for 4 days without treatment, we found that the CFTR protein and activity was reduced to a negligible level (3% of the mean wild type cAMP-activated short circuit current). Login to comment
204 Because readthrough of the CFTR-G542X mutation probably ceases shortly after the gentamicin treatment is terminated, this loss of CFTR activity probably reflects the turnover of the hCFTR protein synthesized during the treatment period. Login to comment
208 These results demonstrate that the co-administration of gentamicin with PAA not only enhances the suppression of the hCFTR-G542X nonsense mutation in Cftr-/- hCFTR-G542X mice but also extends the time period during which hCFTR activity and protein can be detected following the termination of gentamicin treatment. Login to comment

[hide] Heterogenous spectrum of CFTR gene mutations in In... Hum Reprod. 2009 May;24(5):1229-36. Epub 2009 Jan 30. Sharma N, Acharya N, Singh SK, Singh M, Sharma U, Prasad R
Heterogenous spectrum of CFTR gene mutations in Indian patients with congenital absence of vas deferens.
Hum Reprod. 2009 May;24(5):1229-36. Epub 2009 Jan 30., [PMID:19181743]

Abstract [show]
BACKGROUND: Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene can cause congenital bilateral absence of vas deferens. Yet, the spectrum and frequency of CFTR mutations in Indian males with congenital absence of vas deferens (CAVD) is unknown. METHODS: We investigated 50 Indian males, diagnosed with unilateral or bilateral absence of vas deferens at the PGIMER, Chandigarh, for the presence of the most common CFTR gene mutations as well as unknown mutations by single-strand conformation polymorphism followed by sequence analysis. RESULTS: This study led to the identification of 12 CFTR gene mutations on 48% of 100 Indian CAVD chromosomes. CFTR mutations were identified on both alleles in 11 patients (22%) and on one allele in 26 patients (52%). Novel CFTR mutations identified were L69H, F87I, G126S, F157C, E543A, Y852F and D1270E. The T5 allele (25%) and F508del (11%) were the most common mutations identified. The most common intragenic marker haplotype for F508del was 2111 (GATT, TUB9, M470V and T854T). No mutations could be detected in 13 CAVD patients (26%), including 4 with renal malformations. CONCLUSIONS: This study confirms the molecular heterogeneity of CFTR mutations in CAVD. Although the mutation detection rate is indeed lower in Indian CAVD patients, 74% of the patients tested had at least one CFTR mutation. CAVD alleles with no mutations suggest that other changes may be located at the non-screened sites that require extensive search by direct sequencing. Furthermore, the novel CFTR mutations identified require functional studies in a cell-based system.

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55 We next screened R117H, N1303K and R553X each by single ARMS PCR and 621 þ 1G-T, G542X, G551D and W1282X by multiplex ARMS PCR. Login to comment

[hide] Preliminary evidence for cell membrane amelioratio... PLoS One. 2009;4(3):e4782. Epub 2009 Mar 11. Scambi C, De Franceschi L, Guarini P, Poli F, Siciliano A, Pattini P, Biondani A, La Verde V, Bortolami O, Turrini F, Carta F, D'Orazio C, Assael BM, Faccini G, Bambara LM
Preliminary evidence for cell membrane amelioration in children with cystic fibrosis by 5-MTHF and vitamin B12 supplementation: a single arm trial.
PLoS One. 2009;4(3):e4782. Epub 2009 Mar 11., [PMID:19277125]

Abstract [show]
BACKGROUND: Cystic fibrosis (CF) is one of the most common fatal autosomal recessive disorders in the Caucasian population caused by mutations of gene for the cystic fibrosis transmembrane conductance regulator (CFTR). New experimental therapeutic strategies for CF propose a diet supplementation to affect the plasma membrane fluidity and to modulate amplified inflammatory response. The objective of this study was to evaluate the efficacy of 5-methyltetrahydrofolate (5-MTHF) and vitamin B12 supplementation for ameliorating cell plasma membrane features in pediatric patients with cystic fibrosis. METHODOLOGY AND PRINCIPAL FINDINGS: A single arm trial was conducted from April 2004 to March 2006 in an Italian CF care centre. 31 children with CF aged from 3 to 8 years old were enrolled. Exclusion criteria were diabetes, chronic infections of the airways and regular antibiotics intake. Children with CF were supplemented for 24 weeks with 5-methyltetrahydrofolate (5-MTHF, 7.5 mg /day) and vitamin B12 (0.5 mg/day). Red blood cells (RBCs) were used to investigate plasma membrane, since RBCs share lipid, protein composition and organization with other cell types. We evaluated RBCs membrane lipid composition, membrane protein oxidative damage, cation content, cation transport pathways, plasma and RBCs folate levels and plasma homocysteine levels at baseline and after 24 weeks of 5-MTHF and vitamin B12 supplementation. In CF children, 5-MTHF and vitamin B12 supplementation (i) increased plasma and RBC folate levels; (ii) decreased plasma homocysteine levels; (iii) modified RBC membrane phospholipid fatty acid composition; (iv) increased RBC K(+) content; (v) reduced RBC membrane oxidative damage and HSP70 membrane association. CONCLUSION AND SIGNIFICANCE: 5-MTHF and vitamin B12 supplementation might ameliorate RBC membrane features of children with CF. TRIAL REGISTRATION: ClinicalTrials.gov NCT00730509.

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73 Patient Gender Age (yr) CFTR mutations BMI (kg/m2) FEV1 (%) Pancreatic sufficiency M.o. in sputum Antibiotic treatment 1 M 8 DF508/DF508 17,29 81 no no no 2 F 7 DF508/DF508 21,7 101 no P. aeruginosa C azithromycin p.o. tobramycin neb. 3 F 6 DI507/711+5G A 22,5 91 no no no 4 F 5 DF508/not identified 15,2 NA no no no 5 M 8 DF508/DF508 15,4 92 no no no 6 M 7 N1303K/2789+5G A 19,2 73 no no no 7 F 7 DF508/621+1G T 15,1 82 no S. aureus no 8 F 8 DF508/1717-1G T 18,3 91 no S. aureus no 9 F 8 DF508/not identified 21,2 95 yes no no 10 F 7 DF508/2789+5G A 14,4 93 no no no 11 M 8 DF508/2789+5G A 15,9 106 yes no no 12 F 7 R1162X/R1162X 17,15 78 no S. aureus no 13 M 8 DF508/not identified 15,2 63 no no no 14 M 6 DF508/DF508 17,1 115 no P. aeruginosa I ciprofloxacin p.o. tobramycin neb. 15 F 8 DF508/R1162X 14 49 no P. aeruginosa C azithromycin p.o. tobramycin neb. 16 M 5 G542/1717-1G A 16,5 NA no no No 17 F 5 DF508/not identified 13,3 NA no P. aeruginosa I ciprofloxacin p.o. tobramycin neb. 18 M 4 DF508/G542X 15,7 NA no no no 19 F 7 DI507/R1162X 16,5 94 no no no 20 F 4 DF508/Q552X 13,5 NA no no no 21 M 8 DF508/R1162X 13,8 78 no no no 22 F 6 2183AA G/N1303K 16,8 102 no no no P.: Pseudomonas; S.: Staphylococcus; H.: Haemophilus; C: chronic colonization; I: intermittent colonization; NA: not applicable; p.o.: per os; neb.: nebulized. Login to comment

[hide] A novel approach to CFTR mutation testing by pyros... Clin Chem. 2009 Jun;55(6):1083-91. Epub 2009 Apr 16. Bickmann JK, Kamin W, Wiebel M, Hauser F, Wenzel JJ, Neukirch C, Stuhrmann M, Lackner KJ, Rossmann H
A novel approach to CFTR mutation testing by pyrosequencing-based assay panels adapted to ethnicities.
Clin Chem. 2009 Jun;55(6):1083-91. Epub 2009 Apr 16., [PMID:19372188]

Abstract [show]
BACKGROUND: Cystic fibrosis (CF) is a common autosomal recessive genetic disorder caused by a variety of sequence alterations in the CFTR gene [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)]. Because the relative prevalence of mutations strongly depends on the ethnic background, first-level testing of CF as defined by recent consensus recommendations ought to be adaptable to the ethnicity of patients. METHODS: We therefore developed and implemented a diagnostic approach to first-level testing for CF based on published mutation frequencies and Pyrosequencing (PSQ) technology that we complemented with standard procedures of mutation detection at the second level. RESULTS: The current test system of PSQ assays for 46 target CF mutations [including CFTRdele2,3 (21 kb) and 1342-6 (T)(n) (5T/7T/9T)] permits recombinations of single assays to optimize sensitivities for certain ethnicities. By easy expansion of the original mutation panel, the first-level test sensitivities with other ethnic groups would be increased, provided that the mutation frequencies are known. The test was validated with our local, ethnically mixed, but mainly German population (155 patients). The mutation-detection rate for the 92 patients whose CF was confirmed by the sweat test was 89.0% for the patients of German descent (73 of the 92 patients) and 73.7% for the patients of any other origin (19 of the 92 patients). Ethnicity-adapted testing panels for our foreign CF patients would increase the sensitivities for the respective groups by approximately 5%. CONCLUSIONS: PSQ-based genotyping is a reliable, convenient, highly flexible, and inexpensive alternative to conventional methods for first-level testing of CFTR, facilitating flexible adaptation of the analyzed mutation panel to any local ethnic group.

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62 We had initially focused on CF mutations potentially prevalent in our local, ethnically mixed, but mainly German population: F508del, I507del, 1677delTA, R347P, G542X, G551D, R553X, N1303K, 1717-1GϾA, 3849ϩ10kb CϾT, CFTRdele2,3 (21 kb), R117H, 1342-6 (T)n (5T/7T/9T) (reported by our laboratory only if a R117H allele was present, unless genetic analysis served to investigate a case of CBAVD or atypical mild CF), and the (TG)n region starting at base position 1342-12 of IVS 8 (exclusively tested in the case of a 5T allele) (Fig. 1, boldface text), with an expected sensitivity of 85% among German patients. Login to comment
100 Diagnostic evaluation of the PSQ-based first-level testing of a predominantly German CF population.a Panethnic population Clinical diagnosis All patients Sweat test-confirmed CF Suspected atypical CF Carrier screening Chromosomes, n 310 184 96 30 PSQ screen 168 (54.2%) 158 (85.9%) 5 (5.2%) 5 (33.3%) Conventional sequencing 25 (8.1%) 25 (13.6%) 0 (0%) 0 (0%) Total detected alleles 193 (62.3%) 183 (99.5%) 5 (5.2%) 5 (33.3%) German ethnicity Other ethnicities Clinical diagnosis Sweat test-confirmed CF Sweat test-confirmed CF Chromosomes, n 146 38 PSQ screen F508del 106 (72.6%) 14 (36.8%) I507del 1 (0.7%) 1 (2.6%) 1677delTA 0 (0%) 2 (5.3%) G551D 6 (4.1%) 0 (0%) R553X 2 (1.4%) 0 (0%) Q552X 1 (0.7%) 0 (0%) G542X 2 (1.4%) 1 (2.6%) S549N 0 (0%) 2 (5.3%) W1282X 1 (0.7%) 3 (7.9%) R117H 1 (0.7%) 0 (0%) 1342-12 (TG)11-5T 0 (0%) 0 (0%) R347P 2 (1.4%) 1 (2.6%) 3849ϩ10kb CϾT 2 (1.4%) 0 (0%) N1303K 3 (2.1%) 3 (7.9%) 1717-1 GϾA 1 (0.7%) 0 (0%) CFTRdele2,3 (21 kb) 2 (1.4%) 1 (2.6%) Sum 130 (89.0%) 28 (73.7%) Conventional sequencing 16 (11.0%) 9 (23.7%) Total detected alleles 146 (100%) 37 (97.4%) a Data are presented as the number of chromosomes (percent). Login to comment
118 An example of the integration of a mutation that was not primarily targeted (S549N) into a PSQ assay for G542X. Login to comment
119 The G542X (1756 GϾT) assay had originally been designed to include adjacent but rare mutations G544S (1762 GϾA) and G544V (1763 GϾT), with the sequence to analyze reaching as far as base 1783. Login to comment
123 See Fig. 1 in the online Data Supplement for example pyrograms of a wild-type sample and heterozygous samples for G542X and S549N. Login to comment
153 The fact that simultaneously detecting some mutations (e.g.: F508del, 1677delTA, and I507del; G542X and S549N; or G551D, R553X, and Q552X) within a single assay improves the sensitivity of each PSQ run underlines even further the advantages that arise from detecting neighboring mutations as well as the target mutation within one assay (Table 2). Login to comment

[hide] Substance P stimulates human airway submucosal gla... J Clin Invest. 2009 May;119(5):1189-200. doi: 10.1172/JCI37284. Epub 2009 Apr 20. Choi JY, Khansaheb M, Joo NS, Krouse ME, Robbins RC, Weill D, Wine JJ
Substance P stimulates human airway submucosal gland secretion mainly via a CFTR-dependent process.
J Clin Invest. 2009 May;119(5):1189-200. doi: 10.1172/JCI37284. Epub 2009 Apr 20., [PMID:19381016]

Abstract [show]
Chronic bacterial airway infections are the major cause of mortality in cystic fibrosis (CF). Normal airway defenses include reflex stimulation of submucosal gland mucus secretion by sensory neurons that release substance P (SubP). CFTR is an anion channel involved in fluid secretion and mutated in CF; the role of CFTR in secretions stimulated by SubP is unknown. We used optical methods to measure SubP-mediated secretion from human submucosal glands in lung transplant tissue. Glands from control but not CF subjects responded to mucosal chili oil. Similarly, serosal SubP stimulated secretion in more than 60% of control glands but only 4% of CF glands. Secretion triggered by SubP was synergistic with vasoactive intestinal peptide and/or forskolin but not with carbachol; synergy was absent in CF glands. Pig glands demonstrated a nearly 10-fold greater response to SubP. In 10 of 11 control glands isolated by fine dissection, SubP caused cell volume loss, lumen expansion, and mucus flow, but in 3 of 4 CF glands, it induced lumen narrowing. Thus, in CF, the reduced ability of mucosal irritants to stimulate airway gland secretion via SubP may be another factor that predisposes the airways to infections.

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279 Genotypes were available for 8 of the CF subjects; 5 were ΔF508 homozygous, with 1 of each of the following: ΔF508/N1303K, G542X/W1282X, and 406-1 G->A/H119Y. Login to comment

[hide] IL1B polymorphisms modulate cystic fibrosis lung d... Pediatr Pulmonol. 2009 Jun;44(6):580-93. Levy H, Murphy A, Zou F, Gerard C, Klanderman B, Schuemann B, Lazarus R, Garcia KC, Celedon JC, Drumm M, Dahmer M, Quasney M, Schneck K, Reske M, Knowles MR, Pier GB, Lange C, Weiss ST
IL1B polymorphisms modulate cystic fibrosis lung disease.
Pediatr Pulmonol. 2009 Jun;44(6):580-93., [PMID:19431193]

Abstract [show]
RATIONALE: Variability in pulmonary disease severity is found in patients with cystic fibrosis (CF) who have identical mutations in the CF transmembrane conductance regulator (CFTR) gene. We hypothesized that one factor accounting for heterogeneity in pulmonary disease severity is variation in the family of genes affecting the biology of interleukin-1 (IL-1), which impacts acquisition and maintenance of Pseudomonas aeruginosa infection in animal models of chronic infection. METHODS: We genotyped 58 single nucleotide polymorphisms (SNPs) in the IL-1 gene cluster in 808 CF subjects from the University of North Carolina and Case Western Reserve University (UNC/CWRU) joint cohort. All were homozygous for DeltaF508, and categories of "severe" (cases) or "mild" (control subjects) lung disease were defined by the lowest or highest quartile of forced expired volume (FEV(1)) for age in the CF population. After adjustment for age and gender, genotypic data were tested for association with lung disease severity. Odds ratios (ORs) comparing severe versus mild CF were also calculated for each genotype (with the homozygote major allele as the reference group) for all 58 SNPs. From these analyses, nine SNPs with a moderate effect size, OR < or =0.5 or >1.5, were selected for further testing. To replicate the case-control study results, we genotyped the same nine SNPs in a second population of CF parent-offspring trios (recruited from Children's Hospital Boston), in which the offspring had similar pulmonary phenotypes. For the trio analysis, both family-based and population-based associations were performed. RESULTS: SNPs rs1143634 and rs1143639 in the IL1B gene demonstrated a consistent association with lung disease severity categories (P < 0.10) and longitudinal analysis of lung disease severity (P < 0.10) in CF in both the case-control and family-based studies. In females, there was a consistent association (false discovery rate adjusted joint P-value <0.06 for both SNPs) in both the analysis of lung disease severity in the UNC/CWRU cohort and the family-based analysis of affection status. CONCLUSION: Our findings suggest that IL1beta is a clinically relevant modulator of CF lung disease.

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111 The other 12% of the CFTR mutations included G551D, G542X, G85E, W1282X, and N1303K. Login to comment

[hide] Three novel CFTR polymorphic repeats improve segre... Clin Chem. 2009 Jul;55(7):1372-9. Epub 2009 May 14. Elce A, Boccia A, Cardillo G, Giordano S, Tomaiuolo R, Paolella G, Castaldo G
Three novel CFTR polymorphic repeats improve segregation analysis for cystic fibrosis.
Clin Chem. 2009 Jul;55(7):1372-9. Epub 2009 May 14., [PMID:19443567]

Abstract [show]
BACKGROUND: Molecular diagnosis for cystic fibrosis (CF) is based on the direct identification of mutations in the CFTR gene [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] (detection rate about 90% with scanning procedures) and on segregation analysis of intragenic polymorphisms for carrier and prenatal diagnosis in about 20% of CF families in which 1 or both causal mutations are unknown. METHODS: We identified 3 novel intragenic polymorphic repeats (IVS3polyA, IVS4polyA, and IVS10CA repeats) in the CFTR gene and developed and validated a procedure based on the PCR followed by capillary electrophoresis for large-scale analysis of these polymorphisms and the 4 previously identified microsatellites (IVS1CA, IVS8CA, IVS17bTA, and IVS17bCA repeats) in a single run. We validated the procedure for both single- and 2-cell samples (for a possible use in preimplantation diagnosis), and on a large number of CF patients bearing different genotypes and non-CF controls. RESULTS: The allelic distribution and heterozygosity results suggest that the 3 novel polymorphisms strongly contribute to carrier and prenatal diagnosis of CF in families in which 1 or both causal mutations have not been identified. At least 1 of the 4 previously identified microsatellites was informative in 78 of 100 unrelated CF families; at least 1 of all 7 polymorphisms was informative in 98 of the families. Finally, the analysis of haplotypes for the 7 polymorphisms revealed that most CF mutations are associated with different haplotypes, suggesting multiple slippage events but a single origin for most CFTR mutations. CONCLUSIONS: The analysis of the 7 polymorphisms is a rapid and efficient tool for routine carrier, prenatal, and preimplantation diagnosis of CF.

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105 The 98 chromosomes from non-CF individuals were associated with 59 differ- enthaplotypes.Similarly,mostfrequentCFTRmutations are associated with more haplotypes (26 different haplotypes for the 90 chromosomes bearing the F508del mutation, 13 alleles for the 20 chromosomes bearing the N1303K mutation, and 7 haplotypes for the 10 chromosomes bearing the G542X mutation). Login to comment

[hide] SUMOylation of tissue transglutaminase as link bet... J Immunol. 2009 Aug 15;183(4):2775-84. Epub 2009 Jul 22. Luciani A, Villella VR, Vasaturo A, Giardino I, Raia V, Pettoello-Mantovani M, D'Apolito M, Guido S, Leal T, Quaratino S, Maiuri L
SUMOylation of tissue transglutaminase as link between oxidative stress and inflammation.
J Immunol. 2009 Aug 15;183(4):2775-84. Epub 2009 Jul 22., 2009-08-15 [PMID:19625650]

Abstract [show]
Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. CF is characterized by chronic bacterial lung infections and inflammation, and we have previously reported that tissue transglutaminase (TG2), a multifunctional enzyme critical to several diseases, is constitutively up-regulated in CF airways and drives chronic inflammation. Here, we demonstrate that the generation of an oxidative stress induced by CFTR-defective function leads to protein inhibitor of activated STAT (PIAS)y-mediated TG2 SUMOylation and inhibits TG2 ubiquitination and proteasome degradation, leading to sustained TG2 activation. This prevents peroxisome proliferator-activated receptor (PPAR)gamma and IkBalpha SUMOylation, leading to NF-kappaB activation and to an uncontrolled inflammatory response. Cellular homeostasis can be restored by small ubiquitin-like modifier (SUMO)-1 or PIASy gene silencing, which induce TG2 ubiquitination and proteasome degradation, restore PPARgamma SUMOylation, and prevent IkBalpha cross-linking and degradation, thus switching off inflammation. Manganese superoxide dismutase overexpression as well as the treatment with the synthetic superoxide dismutase mimetic EUK-134 control PIASy-TG2 interaction and TG2 SUMOylation. TG2 inhibition switches off inflammation in vitro as well as in vivo in a homozygous F508del-CFTR mouse model. Thus, TG2 may function as a link between oxidative stress and inflammation by driving the decision as to whether a protein should undergo SUMO-mediated regulation or degradation. Targeting TG2-SUMO interactions might represent a new option to control disease evolution in CF patients as well as in other chronic inflammatory diseases, neurodegenerative pathologies, and cancer.

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77 Patients and ex vivo cultures of nasal polyp mucosal biopsies Seven consecutive CF patients carrying the common CFTR mutations (F508del/F508del, F508del/W1282X, F508del/N1303K, or F508del/ G542X) (mean age, 19 years; range, 13-29 years) with chronic sinusitis and nasal polyposis and seven consecutive non-CF patients (mean age, 21 years; range, 16-32 years) with idiopathic polyposis underwent surgical treatment. Login to comment
78 Patients and ex vivo cultures of nasal polyp mucosal biopsies Seven consecutive CF patients carrying the common CFTR mutations (F508del/F508del, F508del/W1282X, F508del/N1303K, or F508del/ G542X) (mean age, 19 years; range, 13-29 years) with chronic sinusitis and nasal polyposis and seven consecutive non-CF patients (mean age, 21 years; range, 16-32 years) with idiopathic polyposis underwent surgical treatment. Login to comment

[hide] Cystic fibrosis. Pediatr Rev. 2009 Aug;30(8):302-9; quiz 310. Montgomery GS, Howenstine M
Cystic fibrosis.
Pediatr Rev. 2009 Aug;30(8):302-9; quiz 310., [PMID:19648261]

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31 Twelve of the most common mutations account for 85% of CF genotypes in North American patients and include deltaF508, G542X, G551D, W1282X, W1303K, and R553X. Login to comment

[hide] Sweat gland bioelectrics differ in cystic fibrosis... Thorax. 2009 Nov;64(11):932-8. Epub 2009 Sep 3. Gonska T, Ip W, Turner D, Han WS, Rose J, Durie P, Quinton P
Sweat gland bioelectrics differ in cystic fibrosis: a new concept for potential diagnosis and assessment of CFTR function in cystic fibrosis.
Thorax. 2009 Nov;64(11):932-8. Epub 2009 Sep 3., [PMID:19734129]

Abstract [show]
BACKGROUND: For nearly 50 years the diagnosis of cystic fibrosis (CF) has depended on measurements of sweat chloride concentration. While the validity of this test is universally accepted, increasing diagnostic challenges and the search for adequate biomarker assays to support curative-orientated clinical drug trials have created a new demand for accurate, reliable and more practical CF tests. A novel concept is proposed that may provide a more efficient real-time method for assessing CFTR function in vivo. METHODS: Cholinergic and beta-adrenergic agonists were iontophoresed to stimulate sweating. The bioelectric potential from stimulated sweat glands (SPD) was measured in vivo using a standard ECG electrode applied to the skin surface. SPD and sweat chloride concentrations were compared in cohorts predicted to express a range of CFTR function as presented by healthy controls (HC), heterozygotes (Hz), pancreatic sufficient (CFPS) and pancreatic insufficient patients with CF (CFPI). RESULTS: The median SPD was hyperpolarized in patients with CF compared with control subjects (-47.4 mV vs -14.5 mV, p<0.001). In distinguishing between control and CF subjects, SPD (area under receiver operator curve (AUC) = 0.997) was similar to sweat chloride concentration (AUC = 0.986). Sequential cholinergic/beta-adrenergic sweat stimulation dramatically depolarised the SPD in patients with CF (p<0.001) but had no effect in control subjects (p = 0.6) or on the sweat chloride concentration in either group (p>0.5). Furthermore, the positive SPD response was larger in CFPI than in CFPS subjects (p = 0.04). CONCLUSION: These results support the concept that skin surface voltages arising from stimulated sweat glands can be exploited to assess expressed CFTR function in vivo and may prove to be a useful diagnostic tool.

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68 Table 1 Summary of study subjects ID Category Sex Age Genotype ID Category Sex Age Genotype 1 HC F 49 +/+ 21 CFPS M 46 deltaF508/P67L 2 HC F 39 +/+ 22 CFPS F 41 deltaF508/R117C 3 HC M 32 +/+ 23 CFPS F 57 G542X/D1152H 4 HC M 23 +/+ 24 CFPS M 34 deltaF508/M1101K 5 HC F 28 +/+ 25 CFPS F 29 deltaF508/L1335P 6 HC M 26 +/+ 26 CFPS F 48 deltaF508/+ 7 HC M 26 R75Q/+ 27 CFPS M 26 deltaF508/R117H 8 HC M 30 +/+ 28 CFPS M 44 deltaF508/3272_26A.G 9 HC M 22 +/+ 29 CFPS M 46 deltaF508/R117H 5T 10 HC M 22 +/+ 30 CFPS M 48 R347P/2753-2A.G 11 Hz F 26 deltaF508/+ 31 CFPI M 29 deltaF508/deltaF508 12 Hz F 54 deltaF508/+ 32 CFPI M 29 deltaF508/2194inA 13 Hz F 24 deltaF508/+ 33 CFPI F 40 G551D/621+1 G.T 14 Hz F 33 deltaF508/+ 34 CFPI M 33 deltaF508/deltaF508 15 Hz M 25 deltaF508/+ 35 CFPI M 27 deltaF508/deltaF508 16 Hz F 37 deltaF508/+ 36 CFPI M 25 deltaF508/deltaF508 17 Hz F 49 deltaF508/+ 37 CFPI M 27 deltaF508/deltaF508 18 Hz M 49 deltaF508/+ 38 CFPI M 29 deltaF508/deltaF508 19 Hz F 55 deltaF508/+ 20 Hz M 61 deltaF508/+ CFPI, pancreatic-insufficient CF patients; CFPS, pancreatic-sufficient CF patients; HC, healthy controls; Hz, heterozygotes. Login to comment

[hide] Characterization of a disease-associated mutation ... J Biol Chem. 2009 Oct 30;284(44):30024-31. Epub 2009 Sep 15. Faa V, Incani F, Meloni A, Corda D, Masala M, Baffico AM, Seia M, Cao A, Rosatelli MC
Characterization of a disease-associated mutation affecting a putative splicing regulatory element in intron 6b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.
J Biol Chem. 2009 Oct 30;284(44):30024-31. Epub 2009 Sep 15., 2009-10-30 [PMID:19759008]

Abstract [show]
Cystic fibrosis (CF) is a common recessive disorder caused by >1600 mutations in the CF transmembrane conductance regulator (CFTR) gene. About 13% of CFTR mutations are classified as "splicing mutations," but for almost 40% of these, their role in affecting the pre-mRNA splicing of the gene is not yet defined. In this work, we describe a new splicing mutation detected in three unrelated Italian CF patients. By DNA analyses and mRNA studies, we identified the c.1002-1110_1113delTAAG mutation localized in intron 6b of the CFTR gene. At the mRNA level, this mutation creates an aberrant inclusion of a sequence of 101 nucleotides between exons 6b and 7. This sequence corresponds to a portion of intron 6b and resembles a cryptic exon because it is characterized by an upstream ag and a downstream gt sequence, which are most probably recognized as 5'- and 3'-splice sites by the spliceosome. Through functional analysis of this splicing defect, we show that this mutation abolishes the interaction of the splicing regulatory protein heterogeneous nuclear ribonucleoprotein A2/B1 with an intronic splicing regulatory element and creates a new recognition motif for the SRp75 splicing factor, causing activation of the cryptic exon. Our results show that the c.1002-1110_1113delTAAG mutation creates a new intronic splicing regulatory element in intron 6b of the CFTR gene exclusively recognized by SRp75.

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39 The CF genotype of the three patients was F508del/unknown, G542X/unknown, and exon 2 deletion (c.545811_164ϩ2186del8108ins182)/unknown (18). Login to comment

[hide] Genetics of cystic fibrosis. Semin Respir Crit Care Med. 2009 Oct;30(5):531-8. Epub 2009 Sep 16. Lommatzsch ST, Aris R
Genetics of cystic fibrosis.
Semin Respir Crit Care Med. 2009 Oct;30(5):531-8. Epub 2009 Sep 16., [PMID:19760540]

Abstract [show]
Cystic fibrosis (CF) is a complicated disease involving many organ systems. Identification of the cystic fibrosis transmembrane regulator (CFTR) genetic code has not only enhanced our understanding of the mechanism of CF pathology but has also provided explanations for phenotypic variation. Additionally, genetic testing has refined our ability to identify patients with CF and CF-related illnesses. Genetic mutations may be grouped by class (I-VI) and are directly related to the quantity of CFTR protein produced. This has direct implications regarding the severity of disease and has suggested organ-specific sensitivity to the presence of normally functioning CFTR. Further, it has improved understanding of the mechanism behind seemingly organ-specific manifestations of CF, such as congenital bilateral absence of the vas deferens (CBVAD).

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63 The most common muta- Table 1 Most Common Genotype Based on Ethnicity Ethnicity Genotype Chromosome Frequency (%) Caucasian (N. European) ~F508 70-80 Caucasian (S. European) ~F508 50-55 African Americans ~F508 48 3120 þ 1 G-to-A 12 Ashkenazi Jews W1282X 48 ~F508 30 Hispanic (U.S.) ~F508 46 G542X 5.4 Adapted from Knowles et al.13 534 SEMINARS IN RESPIRATORY AND CRITICAL CARE MEDICINE/VOLUME 30, tion in this group is ~F508, which results in >99% of the protein being degraded as opposed to $75% in the normal individual.8 This mutation causes a three-base pair deletion in exon 10 specifically affecting the integral tertiary interaction between the N-terminus of NBD-1 and C-terminus of transmembrane segment-4 regulating CFTR channel gating.5,6,25 Interestingly, the ~F508 CFTR has been found to be a ''temperature sensitive`` mutant given that, in vitro, it can be delivered by gene therapy vectors to the apical membrane if cells are incubated at 23 to 308C.9,13 Class III and IV mutations typically confer more mild disease. Login to comment

[hide] Clinical and molecular characterization of S1118F-... Pediatr Pulmonol. 2009 Oct;44(10):1003-9. Penmatsa H, Frederick CA, Nekkalapu S, Conoley VG, Zhang W, Li C, Kappes J, Stokes DC, Naren AP
Clinical and molecular characterization of S1118F-CFTR.
Pediatr Pulmonol. 2009 Oct;44(10):1003-9., [PMID:19774621]

Abstract [show]
BACKGROUND: Cystic fibrosis is a lethal autosomal recessive disorder usually associated with lung disease, pancreatic insufficiency and high sweat chloride levels. CLINICAL CASE: A patient admitted to Le Bonheur Children's Medical Center (LBCMC, Memphis, TN) showed symptoms of meconium ileus which required exploratory laparotomy, bowel resection and ileostomy. Genotyping showed DeltaF508/I1027T on one chromosome and S1118F on the other. Sweat testing on three different occasions gave negative and intermediate results (22.7, 24.6 mmol/L; 55.1, 58.6 mmol/L and 55.1, 58 mmol/L) and pancreatic elastase testing showed normal levels. OBJECTIVE: To characterize S1118F-CFTR mutation at a molecular level to help understand the associated CF-phenotype. METHODS: Molecular characterization of S1118F-CFTR mutant was studied in HEK-293 cells at 37 degrees C. Various biochemical methods such as Western blotting, real-time PCR, Pulse chase labeling and iodide efflux assay were employed. RESULTS: S1118F-CFTR makes less than 10-15% of mature CFTR (band C) compared to WT-CFTR. The mRNA levels of S1118F-CFTR and WT-CFTR are comparable. S1118F-CFTR is functional but shows about 10-15% of WT-CFTR activity. S1118F-CFTR shows impaired maturation and CF-correctors can increase the amount of mature and functional CFTR by three- to fourfold. CONCLUSION: S1118F-CFTR shows impaired maturation and an individual with S1118F-CFTR paired with DeltaF508-CFTR exhibits atypical CF symptoms with intermediate sweat chloride level and meconium ileus despite documented pancreatic sufficiency.

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21 The Class I mutations constitute nonsense, splice and frame shift mutants that encode truncated forms of CFTR (e.g., G542X and W1242X). Login to comment

[hide] Population-based carrier screening for cystic fibr... Aust N Z J Obstet Gynaecol. 2009 Oct;49(5):484-9. Massie J, Petrou V, Forbes R, Curnow L, Ioannou L, Dusart D, Bankier A, Delatycki M
Population-based carrier screening for cystic fibrosis in Victoria: the first three years experience.
Aust N Z J Obstet Gynaecol. 2009 Oct;49(5):484-9., [PMID:19780730]

Abstract [show]
BACKGROUND: Cystic fibrosis (CF) is the most common inherited, life-shortening condition affecting Australian children. The carrier frequency is one per 25 and most babies with CF are born to parents with no family history. Carrier testing is possible before a couple has an affected infant. AIMS: To report the outcomes of a carrier screening program for CF. METHOD: Carrier screening was offered to women and couples planning a pregnancy, or in early pregnancy, through obstetricians and general practitioners in Victoria, Australia. Samples were collected by cheek swab and posted to the laboratory. Twelve CFTR gene mutations were tested. Carriers were offered genetic counselling and partner testing. Carrier couples were offered prenatal testing by chorionic villous sampling (CVS) if pregnant. The number of people tested, carriers detected and pregnancy outcomes were recorded from January 2006 to December 2008. RESULTS: A total of 3200 individuals were screened (3000 females). One hundred and six carriers were identified (one per 30, 95% confidence interval one per 25, one per 36). All carrier partners were screened, and nine carrier couples identified (total carriers 115). Ninety-six individuals (83%) were carriers of the p.508del mutation. Of the nine carrier couples, six were pregnant at the time of screening (five natural conception and one in vitro fertilisation) and all had CVS (mean gestation 12.5 weeks). Two fetuses were affected, three were carriers and one was not a carrier. Termination of pregnancy was undertaken for the affected fetuses. CONCLUSION: Carrier screening for CF by obstetricians and general practitioners by cheek swab sample can be successfully undertaken prior to pregnancy or in the early stages of pregnancy.

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103 Although we have promoted the uptake of CF carrier screening to both partners in the relationship it is evident Table 1 Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations identified in 2006-2008 CFTR gene mutation n p.508del 96 W1282X 5 c.3718-2477C > T 5 p.G551D 3 p.G542X 1 p.N1303K 1 p.507del 1 p.R560T 1 p.R553X 1 c.489+1G > T 1 p.V520F 0 c.1585-1G > A 0 Total 115 Carrierscreeningforcysticfibrosis (c)2009TheAuthors487 Journalcompilation(c)2009TheRoyalAustralianandNewZealandCollegeofObstetriciansandGynaecologists;49:484-489 Table 2 Carrier couples detected by cystic fibrosis population screening program, Victoria 2006-2008 Subjects Timing of CF carrier test (gestation) Conception Parents genotype Counselling Prenatal diagnosis Status of pregnancy Future plans 1 Pre-pregnancy Natural Both Genetic counsellor and CF physician CVS 12 weeks Termination of pregnancy 2008: Second pregnancy: CVS: carrier p508delp.508del Affected (p.508del/p.508del) 2 10 weeks Natural Both Genetic counsellor and CF physician CVS 12 weeks Continued p.508del Unaffected (no mutations) 3 11 weeks Natural Both Genetic counsellor CVS 13 weeks Continued p.508del Carrier (p.508del/-) 4 10 weeks Natural Both Genetic counsellor CVS 13 weeks Continued p.508del Carrier (p.508del/-) 5 11 weeks Natural Both Genetic counsellor CVS 13 weeks Continued p.508del Unaffected (no mutations) 6* 9 weeks IVF Both Genetic counsellor and CF physician CVS 12 weeks Termination of pregnancy Currently undergoing IVF conception with PGD.p.508del Affected (p.508del/p.508del) 7 Pre-pregnancy Not applicable Both Genetic counsellor and CF physician CVS 12 weeks Continued Did not attend PGD, established natural pregnancy 2 months after seen by genetic counsellor and respiratory physician p.508del Carrier p.508del 8** Pre-pregnancy Not applicable Both Genetic counsellor Not applicable Not applicable Likely to pursue PGD p.508del 9*** Pre-pregnancy Not applicable c.3718-2477C > T, Genetic counsellor and CF physician Not applicable Not applicable Likely to pursue PGD p.W1282X *This couple had an IVF pregnancy but were not offered carrier screening until nine weeks gestation. Login to comment
38 The following 12 mutations were screened using a polymerase chain reaction multiplex: p.508del, p.G551D, p.G542X, p.N1303K, c.1585-1G > A, p.I507del, p.R560T, p.W1282X, p.V520F, c.489+1G > T, p.R553X and c.3718-2477C > T. Login to comment

[hide] Non-classic cystic fibrosis associated with D1152H... Clin Genet. 2010 Apr;77(4):355-64. Epub 2009 Oct 15. Burgel PR, Fajac I, Hubert D, Grenet D, Stremler N, Roussey M, Siret D, Languepin J, Mely L, Fanton A, Labbe A, Domblides P, Vic P, Dagorne M, Reynaud-Gaubert M, Counil F, Varaigne F, Bienvenu T, Bellis G, Dusser D
Non-classic cystic fibrosis associated with D1152H CFTR mutation.
Clin Genet. 2010 Apr;77(4):355-64. Epub 2009 Oct 15., [PMID:19843100]

Abstract [show]
BACKGROUND: Limited knowledge exists on phenotypes associated with the D1152H cystic fibrosis transmembrane conductance regulator (CFTR) mutation. METHODS: Subjects with a D1152H allele in trans with another CFTR mutation were identified using the French Cystic Fibrosis Registry. Phenotypic characteristics were compared with those of pancreatic insufficient (PI) and pancreatic sufficient (PS) cystic fibrosis (CF) subjects in the Registry (CF cohort). RESULTS: Forty-two subjects with D1152H alleles were identified. Features leading to diagnosis included chronic sinopulmonary disease (n = 25), congenital absence of the vas deferens (n = 11), systematic neonatal screening (n = 4), and genetic counseling (n = 2). Median age at diagnosis was 33 [interquartile range (IQR, 24-41)] years in D1152H subjects. Median sweat chloride concentrations were 43.5 (39-63) mmol/l in D1152H subjects and were markedly lower than in PI and PS CF subjects (p < 0.05). Bronchiectasis was present in 67% of D1152H subjects, but Pseudomonas aeruginosa colonization and pancreatic insufficiency were present in <30% of subjects. Estimated rates of decline in forced expiratory volume in 1 s (FEV(1)) were lower in D1152H subjects vs PI CF subjects (p < 0.05). None of the D1152H subjects identified since 1999 had died or required lung transplantation. CONCLUSIONS: When present in trans with a CF-causing mutation, D1152H causes significant pulmonary disease, but all subjects had prolonged survival.

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42 The CF genetic analysis panel used in France seeks for 32 mutations: G85E, 394delTT, 621+1G>T, 711+1G>T, R334W, R347P, R347H, 1078delT, 5T/7T/9T, A455E, F508del, I507del, V520F, 1717-1G>A, G542X, G551D, R553X, R560T, S549R (T>G), S549N, 1898+1G>A, 2183AA>G, 2184delA, 2789+5G>A, 3120+1G>A, R1162X, 3659delC, 3849+10kbC>T, W1282X, 3905insT, 3876delA, N1303K. Login to comment
98 Diagnostic features in 42 D1152H subjects according to the other CFTR mutation class Subject Sex (M/F) Other CFTR mutation Sweat Cl- mean (mmol/l) Age at diagnosis (years) Presentation at diagnosis Class I mutations 1 F W1282X 58 4 Pneumonia recurrent bronchitis 2 F W1282X 25 74 Bronchiectasis 3 M W1282X 43 33 CBAVD 4 M G542X 48 39 CBAVD 5 M G542X 72 27 CBAVD 6 F S1206X 18 13 Recurrent bronchitis+ diarrhea 7 F 394delTT 19 41 Bronchiectasis 8 F 394delTT 25 18 Bronchiectasis 9 F Q552X 56 43 Bronchiectasis Class II mutations 10 F F508del 13 42 Bronchiectasis 11 F F508del 40 32 Bronchiectasis 12 F F508del 52 23 Bronchiectasis 13 M F508del 51 15 Bronchiectasis 14 F F508del 100 24 Bronchiectasis 15 M F508del 79 26 Bronchiectasis 16 F F508del - 43 Bronchiectasis 17 M F508del - 23 Bronchiectasis 18 F F508del 19 55 Bronchiectasis 19 F F508del 25 33 Bronchiectasis 20 F F508del 78 15 Bronchiectasis 21 M F508del 90 40 Bronchiectasis 22 F F508del 44 42 Bronchiectasis 23 M F508del 88 11 Bronchiectasis 24 F F508del 63 47 Bronchiectasis 25 F F508del 43 33 Bronchiectasis 26 M F508 del 62 49 Bronchiectasis 27 M F508del 20 - CBAVD 28 M F508del - 27 CBAVD 29 M F508del 42 36 CBAVD 30 M F508del 36 34 CBAVD 31 M F508del 40 36 CBAVD 32 M F508del 41 30 CBAVD 33 M F508del 82 9 Asymptomatic genetic counseling 34 M F508del - 0 Neonatal screening 35 F F508del 53 0 Neonatal screening 36 F F508del 35 0 Neonatal screening 37 M F508del 35 0 Neonatal screening Class III mutation 38 F S549N 75 31 Bronchiectasis Class IV mutations 39 M E116K 80 41 ABPA+ diarrhea 40 M D1152H 34 34 CBAVD 41 M R1070Q 56 38 CBAVD Class V mutation 42 M 3849+10kbC>T 31 40 Asymptomatic genetic counseling ABPA, allergic bronchopulmonary aspergillosis; CBAVD, congenital bilateral absence of the vas deferens. Login to comment
120 Nasal epithelial physiologic properties in eight CF subjects carrying the D1152H mutation Nasal bioelectric properties Subject Other CFTR mutation Sweat Cl- mean (mmol/l) Maximal PD (mV) Amil (mV) Cl-/amil (mV) Iso/Cl- (mV) Wilchanski`s indexa Class I mutations 2 W1282X 25 -44 38 -2 -14 0.7 3 W1282X 43 -34 9 -1 1 1.0 4 G542X 48 -16 12 -3 -1 0.7 6 S1206X 18 -44 28 -11 0 0.7 7 394delTT 19 -25 25 -15 -15 0.3 8 394delTT 25 -42 23 -4 -4 0.7 Class II mutation 18 F508del 19 -19 9 0 0 1.0 Class V mutation 42 3849+10 kb C>T 31 -19 9 1 3 1.6 Cl- , chloride; PD, potential difference; Amil, amiloride; Iso, isoproterenol. Login to comment

[hide] CFTR mutations in cystic fibrosis patients from Mu... Clin Genet. 2009 Dec;76(6):577-9. Epub 2009 Oct 21. Moya-Quiles MR, Mondejar-Lopez P, Pastor-Vivero MD, Gonzalez-Gallego I, Juan-Fita MJ, Egea-Mellado JM, Carbonell P, Casals T, Fernandez-Sanchez A, Sanchez-Solis M, Glover G
CFTR mutations in cystic fibrosis patients from Murcia region (southeastern Spain): implications for genetic testing.
Clin Genet. 2009 Dec;76(6):577-9. Epub 2009 Oct 21., [PMID:19845690]

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19 By contrast, G542X showed a frequency higher than that for the entire Spanish population (7.7%) (4), although closer to that reported for the Andalucia Region (11.4%) (10). Login to comment

[hide] A 10-year large-scale cystic fibrosis carrier scre... J Cyst Fibros. 2010 Jan;9(1):29-35. Epub 2009 Nov 7. Picci L, Cameran M, Marangon O, Marzenta D, Ferrari S, Frigo AC, Scarpa M
A 10-year large-scale cystic fibrosis carrier screening in the Italian population.
J Cyst Fibros. 2010 Jan;9(1):29-35. Epub 2009 Nov 7., [PMID:19897426]

Abstract [show]
BACKGROUND: Cystic Fibrosis (CF) is one of the most common autosomal recessive genetic disorders, with the majority of patients born to couples unaware of their carrier status. Carrier screenings might help reducing the incidence of CF. METHODS: We used a semi-automated reverse-dot blot assay identifying the 47 most common CFTR gene mutations followed by DGGE/dHPLC analysis. RESULTS: Results of a 10-year (1996-2006) CF carrier screening on 57,999 individuals with no prior family history of CF are reported. Of these, 25,104 were couples and 7791 singles, with 77.9% from the Italian Veneto region. CFTR mutations were found in 1879 carriers (frequency 1/31), with DeltaF508 being the most common (42.6%). Subjects undergoing medically assisted reproduction (MAR) had significantly (p<0.0001) higher CF carrier frequency (1/22 vs 1/32) compared to non-MAR subjects. CONCLUSIONS: If coupled to counselling programmes, CF carrier screening tests might help reducing the CF incidence.

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48 Forty-seven different CFTR mutations/gene alterations were chosen and analysed: ΔF508, G85E, 541delC, D110H, R117H, 621+1G→T, 711+5G→A, R334W, R334Q, T338I, R347H, R347P, R352Q, S466X, ΔI507, E527G, 1717-1G→A, 1717-8G→A, G542X, S549N, S549R A→C, G551D, Q552X, R553X, D579G, 1874insT, E585X, 1898+3A→G, 2183AA→G, 2184delA, R709X, 2789+5G→A, 3132delTG, 3199del6, 3272-26A→G, L1077P, L1065P, R1066H, M1101K, D1152H, R1158X, R1162X, 3849+10KbC→T, G1244E, W1282X, N1303K and 4016insT. Login to comment
89 Mutations found in the homozygous (n=2) and heterozygous (n=20) diagnosed foetuses are the following: ΔF508/ΔF508 (n=1), 711+5G→A/711+5G→A (n=1), ΔF508/P5L (n=1), 2183AA→G/S42F (n=1), ΔF508/ D1445N (n=1), 711+5G→A/ΔF508 (n=1), G542X/E527G (n=1), N1303K/1717-1 G→A (n=1), R117H/E527G (n=1), ΔF508/2183AA→G (n=1), ΔF508/D1152H (n=1), R347H/ ΔF508 (n=1), ΔF508/G542X (n=2), ΔF508/N1303K (n=2), R1162X/ΔF508 (n=3), N1303K/D1152H (n=3). Login to comment
97 CF mutation General adult population MAR population n=1879 n=236 ΔF508 42.6 45.7 2183AA→G 5.9 5.9 R1162X 5.7 8.2 N1303K 5.4 5.9 G542X 4.2 3.7 D1152H 3.9 5.0 R553X 3.7 3.1 R117H 3.3 1.8 711+5G→A 2.8 4.1 Q552X 2.8 0.4 2789+5G→A 2.2 3.1 1717-1G→A 2.6 2.8 E527G 2.4 - G85E 2.4 0.9 R334Q 0.9 0.4 W1282X 0.7 0.9 R334W 0.6 - 1898+3A→G 0.5 0.4 R1158X 0.4 - R1066H 0.4 0.4 T338I 0.4 1.8 3849+10Kb C→T 0.4 1.3 3272-26 A→G - 0.9 3132delTG - 0.9 3659 del C - 0.4 4016 ins T - 0.4 1717-8G→A - 0.4 R347H - 0.4 ΔI507 - 0.4 R1070Q - 0.4 Other (16) 5.4 - Table 2a List of CFTR compound heterozygotes in the adult general population. Mutation Health status Disorder Gender Age (years) Notes and refs ΔF508/A238V Infertile CBAVD M 36 (A) ΔF508/R352W Infertile CBAVD M 45 (A) R553X/R334Q M 38 ΔF508/R347H M 53 [17] S42F/D372E (1251T→G) M 39 (A) (B) ΔF508/D110H Infertile M 38 ΔF508/L1414S (4373T→C) Infertile CBAVD M 44 (A) (B) ΔF508/V201M, D1270N & R74W Infertile CBAVD M 44 (A) [18,19] 2183AA→G/L206W Infertile CBAVD M 40 (A) 711+5G→A/ L206W Infertile CBAVD M 40 (A) Table 2b List of CFTR compound heterozygotes in the population enrolled for medically assisted reproduction. Login to comment
98 Mutation Disorder Gender Age (years) Notes and refs ΔF508/R117H M 47 (C) [20,21] ΔF508/R117H F 36 (C) [20,21] ΔF508/R117H M 43 (C) [20,21] G542X/D1152H M 40 (C) R1162X/2789+5G→A CBAVD M 44 (C) R117H/2789+5G→A CBAVD M 42 (C) N1303K/D110H CBAVD M 32 (C) N1303K/D1152H M 40 (C) 2789+5G→A/R1066H M 40 (C) Abbreviations: CBAVD: Congenital Bilateral Absence of the Vas Deference; M: Male; F: Female. Login to comment
105 Among the subjects tested, 9 resulted to be compound heterozygotes: ΔF508/R117H (n=3), G542X/D1152H (n=1), R1162X/2789+5G→A (n=1), R117H/2789 + 5G→A (n = 1), N1303K/D110H (n = 1), N1303K/D1152H (n = 1), 2789 + 5G→A/R1066H (n = 1) (Table 2b). Login to comment
139 In our study a rare mutation such as S1235R was found to be moderately frequent (1/77) and despite being normally classified as "mild", association with a second CFTR gene mutation (G542X) can lead to idiopathic chronic pancreatitis [23]. Login to comment

[hide] Cystic fibrosis: exploiting its genetic basis in t... Pharmacol Ther. 2010 Feb;125(2):219-29. Epub 2009 Nov 10. Kreindler JL
Cystic fibrosis: exploiting its genetic basis in the hunt for new therapies.
Pharmacol Ther. 2010 Feb;125(2):219-29. Epub 2009 Nov 10., [PMID:19903491]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel expressed in epithelial cells throughout the body. In the lungs, absence or dysfunction of CFTR results in altered epithelial salt and water transport eventuating in impaired mucociliary clearance, chronic infection and inflammation, and tissue damage. CF lung disease is the major cause of morbidity and mortality in CF despite the many therapies aimed at reducing it. However, recent technological advances combined with two decades of research driven by the discovery of the CFTR gene have resulted in the development and clinical testing of novel therapies aimed at the principal underlying defect in CF, thereby ushering in a new age of therapy for CF.

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527 It restored translation of dystrophin with premature stop mutations in human muscle cells in vitro and mdx mice in vivo (Welch et al., 2007), and restored translation of human G542X CFTR in a transgenic CFTR -/- mouse (Du et al., 2008). Login to comment

[hide] Aquagenic wrinkling of the palms in cystic fibrosi... Arch Dermatol. 2009 Nov;145(11):1296-9. Berk DR, Ciliberto HM, Sweet SC, Ferkol TW, Bayliss SJ
Aquagenic wrinkling of the palms in cystic fibrosis: comparison with controls and genotype-phenotype correlations.
Arch Dermatol. 2009 Nov;145(11):1296-9., [PMID:19917960]

Abstract [show]
OBJECTIVE: To determine the prevalence of aquagenic wrinkling of the palms (AWP) in patients with cystic fibrosis (CF) compared with control patients, and evaluate for genotype-phenotype correlations. Since its first description over 30 years ago, AWP has frequently been anecdotally associated with CF, but this association has not been confirmed in a rigorous prospective case-control study. DESIGN: Blinded comparison. SETTING: The CF and dermatology clinics at St Louis Children's Hospital. PARTICIPANTS: Forty-four individuals with CF from a CF clinic and 26 controls from a dermatology clinic. Intervention Participants were tested for AWP using 3 minutes of water immersion with room-temperature tap water. Main Outcome Measure The degree of AWP was scored from 0 (no wrinkling) to 4 (severe wrinkling) by 3 blinded physicians. For genotype-phenotype correlations, patients with CF were divided into those homozygous for the DeltaF508 mutation and those with other genotypes. RESULTS: The mean AWP score of the CF group was significantly higher than the mean score of the control group (1.5 vs 0.6; P < .001). Patients with CF who were homozygous for the DeltaF508 mutation (n = 27) had significantly higher scores than patients with CF who were not homozygous for the DeltaF508 mutation (n = 17) (1.7 vs 1.1; P = .02). The 17 patients with CF who were not homozygous for the DeltaF508 mutation still had higher scores than the control group (1.1 vs 0.6; P = .03). There was no correlation between sweat chloride concentrations measured at the time of diagnosis and AWP score. CONCLUSIONS: Our results confirm the association between AWP and CF. Among patients with CF, greater AWP occurs in those who are homozygous for the DeltaF508 mutation.

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57 (%) 12 (46) 23 (52) Mean age, y 9.3 11.5 CFTR genotype NA ⌬F508/⌬F508 27 ⌬F508/unidentified 4 ⌬F508/R553X 2 ⌬F508/1898 ϩ 1G ~ A 2 ⌬F508/G542X 1 ⌬F508/G551D 1 ⌬F508/W1282X 1 ⌬F508/1717 ϩ 1G ~ A 1 ⌬F508/3120 ϩ 1G ~ A 1 ⌬F508/3849 ϩ 10KBC→T 1 ⌬F508/S1251N 1 R560T/unidentified 1 2184insA/4357 2A→G 1 Abbreviations: CF, cystic fibrosis; NA, not applicable. Login to comment
59 (%) 12 (46) 23 (52) Mean age, y 9.3 11.5 CFTR genotype NA ⌬F508/⌬F508 27 ⌬F508/unidentified 4 ⌬F508/R553X 2 ⌬F508/1898 ϩ 1G ~ A 2 ⌬F508/G542X 1 ⌬F508/G551D 1 ⌬F508/W1282X 1 ⌬F508/1717 ϩ 1G ~ A 1 ⌬F508/3120 ϩ 1G ~ A 1 ⌬F508/3849 ϩ 10KBC→T 1 ⌬F508/S1251N 1 R560T/unidentified 1 2184insA/4357 2A→G 1 Abbreviations: CF, cystic fibrosis; NA, not applicable. Login to comment

[hide] CFTR allelic heterogeneity in Brazil: historical a... J Hum Genet. 2010 Feb;55(2):71-6. Epub 2009 Nov 27. Faucz FR, Souza DA, Olandoski M, Raskin S
CFTR allelic heterogeneity in Brazil: historical and geographical perspectives and implications for screening and counseling for cystic fibrosis in this country.
J Hum Genet. 2010 Feb;55(2):71-6. Epub 2009 Nov 27., [PMID:19942933]

Abstract [show]
The goal of the present study was to provide a complete and updated spectrum of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene mutations in the Brazilian population combining all available in silico data for patients with CF in Brazil, including founder background and migration flow that consisted of the actual genetic pool of the Brazilian population. Information sources in international databases (PUBMED and SCIELO) were searched. The Brazilian population shows a wide variation in the frequency of CFTR mutations in states Rio Grande do Sul (RS), Santa Catarina (SC), Parana (PR), Sao Paulo (SP), Rio de Janeiro (RJ), Minas Gerais (MG), Para (PA) and Bahia (BA); this variation includes the most common mutation p.F508del. Apparently, this frequency variation is because of the different ethnic compositions. States such as SC and PR have a greater European admixture with almost 90% of CF alleles identified. In other states, such as BA, higher frequency of alleles that are common among African populations is seen. Overall, the CFTR mutational spectrum indicates the presence of European, African and Amerindian ethnic groups in the contemporary Brazilian CF patients. Here, we present an analysis of the CFTR allelic heterogeneity and discuss the origin of its genetic composition, in an attempt to provide improved perspective for the CF population screening in Brazil and genetic counseling.

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174 Prevalence of deltaF508, G551D, G542X, and R553X mutations among cystic fibrosis patients in the North of Brazil. Login to comment
11 We previously reported mutation heterogeneity in Brazilian CF patients by direct analysis of the p.F508del mutation and other common sequence alterations (p.G542X, p.N1303 K, p.G551D and p.R553X). Login to comment
20 We previously studied the p.F508del and other four mutations (p.G542X, p.N1303 K, p.G551D and p.R553X) that are common worldwide in the Brazilian population. Login to comment
42 Table 1 Frequencies of some mutations in different regions from Brazil South Southeast North Northeast Mutation PR and SC19 PR and SC11 RS35 SP34 RJ36 MG11 PA37 BA38 p.F508del 45.54% (51/112) 46.94% (92/196) 48.7% (75/154) 50.00% (96/192) 28.42% (54/190) 47.37% (54/114) 22.73% (15/66) 8.68% (25/288) p.G542X 6.25% (7/112) 7.65% (15/196) 3.25% (5/154) 4.17% (8/192) 2.10% (4/190) 7.02% (8/114) 0.00% (0/66) nt p.N1303K 4.46% (5/112) 5.10% (10/196) 0.00% (0/154) 2.08% (4/192) nt 0.00% (0/114) nt nt p.G85E 3.57% (4/112) 2.04% (4/196) nt nt 4.73% (9/190) 3.51% (4/114) nt nt p.R334W 3.57% (4/112) 3.06% (6/196) 1.30% (2/154) nt 2.63% (5/190) 3.51% (4/114) nt nt p.R1162X 3.57% (4/112) 5.61% (11/196) 0.00% (0/154) nt 0.53% (1/190) 3.51% (4/114) nt nt c.2183AA4G 2.68% (3/112) 1.53% (3/196) nt nt 0.00% (0/190) 0.00% (0/114) nt nt p.W1282X 2.68% (3/112) 2.55% (5/196) 0.65% (1/154) 0.52% (1/192) 0.00% (0/190) 0.88% (1/114) nt nt p.R553X 1.78% (2/112) 1.02% (2/196) 0.65% (1/154) 0.52% (1/192) 0.00% (0/190) 0.00% (0/114) 0.00% (0/66) nt p.G551D 0.00% (0/112) 0.00% (0/196) 0.00% (0/154) 1.04% (2/192) 0.53% (1/190) 0.00% (0/114) 4.55% (3/66) nt Othera 25.89% (29/112) 24.49% (48/196) 45.45% (70/154) 56.25% (108/192) 61.05% (116/190) 65.79% (54/114) 72.73% (48/66) 91.32% (263/288) P¼0.9226b P¼0.0007c Abbreviations: BA, Bahia state; MG, Minas Gerais state; nt, not tested; PA, Para´ state; PR, Parana´ state; RJ, Rio de Janeiro state; RS, Rio Grande do Sul state; SC, Santa Catarina state; SP, Sa˜o Paulo state. Login to comment
53 This can be showed when we compare the occurrence of the eight most frequent mutations in Italy (which consists of B70% of all mutations in this country) with those of other populations (Table 3).14,53,54 Faucz et al.19 found nine mutations with a frequency higher than 1% (p.F508del: 45.5%; p.G542X: 6.3%; p.N1303K: 4.5%; p.G85E, p.R334W and p.R1162X: total of 3.6%; c.2183AA4G and p.W1282X: 2.7%; and p.R553X: 1.8%) in CF patients from PR and SC (south of Brazil). Login to comment
58 Table 3 The eight more frequent cystic fibrosis mutations in Italy and the comparison between the frequency of these mutations in south of Brazil with the frequency in Italy, Portugal, Germany and Europe Mutation South of Brazil11,19 Italy53 Portugal14 Germany54 Europe14 p.F508del 46.43% (143/308) 48.92% (745/1 523) 44.49% (202/454) 68.39% (4 199/6 140) 66.78% (18 149/27 177) p.G542X 7.14% (22/308) 5.91% (90/1 523) 1.32% (6/454) 1.51% (93/6 140) 2.64% (717/27 177) p.N1303K 4.87% (15/308) 5.91% (90/1 523) 0.66% (3/454) 1.32% (81/6 140) 1.64% (446/27 177) p.R1162X 4.87% (15/308) 1.58% (24/1 523) 0.22% (1/454) 0.07% (4/6 140) 0.51% (139/27 177) p.W1282X 2.60% (8/308) 1.77% (27/1 523) 0.00% (0/454) 0.24% (15/6 140) 1.00% (272/27 177) c.2183AA4G 1.95% (6/308) 2.63% (40/1 523) 0.00% (0/454) 0.00% (0/6 140) 0.36% (99/27 177) p.R553X 1.30% (4/308) 1.38% (21/1 523) 0.00% (0/454) 1.61% (99/6 140) 0.75% (204/27 177) c.1717-1G4A 0.97% (3/308) 1.77% (27/1 523) 0.00% (0/454) 0.50% (31/6 140) 0.83% (226/27 177) Others 29.87% (92/308) 30.14% (459/1 523) 53.30% (242/454) 26.35% (1 618/6 140) 25.48% (6925/27 177) P¼0.6401a Po0.0001b Po0.0001b Po0.0001b Numbers of chromosomes with the mutation/number of analyzed chromosomes are given in parentheses. Login to comment
64 The p.G542X mutation is found more frequently in SP (19.6%) and MG (13.8%) than in other states, such as, RJ (2.1%), RS (4.9%), SC (6.2%) and PR (8.7%).18,36 Cabello et al.36 found the c.3120+1G4A mutation in 3.7% of the alleles in RJ; they explained that this higher frequency was most probably the result of the ethnic composition of the RJ population, which generally has a higher proportion of African Brazilians (see Table 1 to compare other mutations found in Brazil). Login to comment

[hide] The variable phenotype of the p.A16V mutation of c... Gut. 2010 Mar;59(3):357-63. Epub 2009 Dec 1. Grocock CJ, Rebours V, Delhaye MN, Andren-Sandberg A, Weiss FU, Mountford R, Harcus MJ, Niemczyck E, Vitone LJ, Dodd S, Jorgensen MT, Ammann RW, Schaffalitzky de Muckadell O, Butler JV, Burgess P, Kerr B, Charnley R, Sutton R, Raraty MG, Deviere J, Whitcomb DC, Neoptolemos JP, Levy P, Lerch MM, Greenhalf W
The variable phenotype of the p.A16V mutation of cationic trypsinogen (PRSS1) in pancreatitis families.
Gut. 2010 Mar;59(3):357-63. Epub 2009 Dec 1., [PMID:19951905]

Abstract [show]
OBJECTIVE: To characterise the phenotypes associated with the p.A16V mutation of PRSS1. DESIGN: Clinical and epidemiological data were collected for any family in which a p.A16V mutation was identified, either referred directly to the European Registry of Hereditary Pancreatitis and Familial Pancreatic Cancer or via a collaborator. DNA samples were tested for mutations in PRSS1, SPINK1, CFTR and CTRC. PATIENTS: Participants were recruited on the basis of either family history of pancreatitis (acute or chronic) or the results of genetic testing. Families were categorised as having hereditary pancreatitis (HP), idiopathic disease or pancreatitis in a single generation. HP was defined as >or=2 cases in >or=2 generations. Main outcome measures Onset of painful episodes of pancreatitis, death from pancreatic cancer, diagnosis of diabetes mellitus and exocrine pancreatic failure. RESULTS: Ten families with p.A16V mutations were identified (22 affected individuals): six HP families, three with idiopathic disease and one with only a single generation affected. The median age of onset, ignoring non-penetrants, was 10 years (95% CI 5 to 25). There were eight confirmed cases of exocrine failure, four of whom also had diabetes mellitus. There were three pancreatic cancer cases. Two of these were confirmed as p.A16V carriers, only one of whom was affected by pancreatitis. Those with p.A16V pancreatitis were compared to affected individuals with p.R122H, p.N29I and no PRSS1 mutation. No significant differences were proven using logrank or Mann-Whitney U tests. CONCLUSIONS: Penetrance of p.A16V is highly variable and family dependent, suggesting it contributes to multigenic inheritance of a predisposition to pancreatitis.

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47 Exon 3 of SPINK1 was sequenced to identify any possible p.N34S mutations and CFTR was tested in all cases for p.DF508, p.G542X, p.N1303K, p. R117H, 621+1 G-T, 1898+1GA, p.W1282X and p.G551D and in some cases with an additional 24 markers according to the recommendations of the American College of Medical Genetics (ACMG) and the American College of Obstetricians and Gynaecologists (ACOG).15 In this study affected p.A16V carriers were also tested for mutations in CTRC exons 2, 3 and 7. Login to comment

[hide] Cystic fibrosis genotype and assessing rates of de... Radiology. 2009 Dec;253(3):813-21. Cleveland RH, Zurakowski D, Slattery D, Colin AA
Cystic fibrosis genotype and assessing rates of decline in pulmonary status.
Radiology. 2009 Dec;253(3):813-21., [PMID:19952026]

Abstract [show]
PURPOSE: To evaluate the hierarchical phenotypic expression of cystic fibrosis transmembrane conductance regulator (CFTR) genotypes in the respiratory system as has been documented in the pancreas. MATERIALS AND METHODS: This study was institutional review board approved; informed consent was not required. HIPAA guidelines were followed. Genotype effects were assessed by using chest radiographic and pulmonary function test (PFT) results in 93 patients. Serial chest radiographic and PFT (percentage of predicted forced expiratory volume in 1 second [FEV(1)], percentage of predicted forced vital capacity [FVC]) results were compared by using analysis of variance with repeated measures. By using CFTR class of mutations, two groups were created: group S (severe disease) and group M (mild disease). Within group S, three subgroups were created: A consisted of patients with two class I alleles; B, class I allele and class II or III allele; C, class II allele and class II or III allele. Group M consisted of patients with at least one allele from class IV-VI. RESULTS: Within group S, subgroup A had a faster deterioration than B or C according to radiographic data (A vs B, P = .014; A vs C, P = .009), with only a borderline difference in FEV(1) for subgroups A versus C (P = .031). Otherwise, PFTs were not sensitive for distinguishing subgroups. Only radiographic results identified that subgroup B had faster progression than C (P = .003); all parameters had trends of decline in the same direction. Group S had a faster decline than group M (radiography, P = .005; FVC, P = .011; FEV(1), P = .529). CONCLUSION: Disease progressed more rapidly with gene class hierarchical correlations seen in pancreatic disease. Radiography was more sensitive for identifying differences.

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56 Measurement Tools All chest radiographic, FEV1, and FVC studies were performed at the study institution during the observed life spans Table 2 Patients according to CF Genotype Group Parameter Genotype Class Pancreatic Exocrine Status* No. of Patients Group S (severe pancreatic and pulmonary phenotypes) Subgroup A (class I and class I) 5 G542X/W1282X I/I PI 2 W1282X/W1282X I/I PI 1 621ϩ1G-T/Y1092X I/I PI 1 3120ϩ1G-A/3120ϩ1G-A I/I PI 1 Subgroup B (class I and class II or III) 16 G542X/⌬F508 I/II PI 6 W1282X/⌬F508 I/II PI 3 Q493X/⌬F508 I/II PI 2 R553X/⌬F508 I/II PI 2 1717-1G/⌬F508 I/II PI 1 621ϩ1G-T/⌬F508 I/II PI 1 2184delA/G551D I/III PI 1 Subgroup C (class II and class II or III) 68 D1507/⌬F508 II/II PI 3 N1303K/⌬F508 II/II PI 2 ⌬F508/⌬F508 II/II PI 57 G551D/⌬F508 II/III PI 6 Group M (mild pancreatic and pulmonary phenotypes) Miscellaneous severe and miscellaneous mild 4 ⌬F508/G85E II/IV PS 2 ⌬F508/R117H II/IV PS 1 D1507/R352Q II/IV PS 1 Miscellaneous mild and miscellaneous mild . Login to comment

[hide] Association of cystic fibrosis genetic modifiers w... Fertil Steril. 2010 Nov;94(6):2122-7. Epub 2010 Jan 25. Havasi V, Rowe SM, Kolettis PN, Dayangac D, Sahin A, Grangeia A, Carvalho F, Barros A, Sousa M, Bassas L, Casals T, Sorscher EJ
Association of cystic fibrosis genetic modifiers with congenital bilateral absence of the vas deferens.
Fertil Steril. 2010 Nov;94(6):2122-7. Epub 2010 Jan 25., [PMID:20100616]

Abstract [show]
OBJECTIVE: To investigate whether genetic modifiers of cystic fibrosis (CF) lung disease also predispose to congenital bilateral absence of the vas deferens (CBAVD) in association with cystic fibrosis transmembrane conductance regulator (CFTR) mutations. We tested the hypothesis that polymorphisms of transforming growth factor (TGF)-beta1 (rs 1982073, rs 1800471) and endothelin receptor type A (EDNRA) (rs 5335, rs 1801708) are associated with the CBAVD phenotype. DESIGN: Genotyping of subjects with clinical CBAVD. SETTING: Outpatient and hospital-based clinical evaluation. PATIENT(S): DNA samples from 80 subjects with CBAVD and 51 healthy male controls from various regions of Europe. This is one of the largest genetic studies of this disease to date. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Genotype analysis. RESULT(S): For single nucleotide polymorphism (SNP) rs 5335, we found increased frequency of the CC genotype among subjects with CBAVD. The difference was significant among Turkish patients versus controls (45.2% vs. 19.4%), and between all cases versus controls (36% vs. 15.7%). No associations between CBAVD penetrance and polymorphisms rs 1982073, rs 1800471, or rs 1801708 were observed. CONCLUSION(S): Our findings indicate that endothelin receptor type A polymorphism rs 5335 may be associated with CBAVD penetrance. To our knowledge, this is the first study to investigate genetic modifiers relevant to CBAVD.

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68 Portuguese CFTR alleles Spanish CFTR alleles Turkish CFTR alleles 5T 22 F508del 11 5T 20 F508del 14 5T 9 D1152H 14 R334W 5 D443Ya 3 D110H 3 R117H 3 G576Aa 3 F508del 2 S1235R 3 R668Ca 3 3041-11del7 2 N1303K 2 G542X 2 1767del6 2 P205S 2 R117H 2 2789þ5G>A 2 D614G 2 V232D 2 CFTRdele2(ins186) 2 G542X 1 L997F 1 3120þ1G>A 1 L206W 1 H609R 1 G1130A 1 V562I 1 N1303H 1 M952I 1 I507del 1 L206W 1 365insT 1 3272-26A>G 1 3272-26A/G 1 E585X 1 2789þ5G>A 1 L15P 1 2752-15C>G 1 G576Aa 1 R347H 1 R334Q 1 R668Ca 1 2689insG 1 R347H 1 CFTRdele2,3 1 R1070W 1 E831X 1 L1227S 1 I 1027T 1 R1070W 1 E831X 1 3272-26A>G 1 L997F 1 I853F 1 A349V 1 6T 1 Note: CFTR ¼ cystic fibrosis transmembrane conductance regulator. Login to comment

[hide] Incidence, prevalence, etiology, and prognosis of ... Dig Dis Sci. 2010 Oct;55(10):2988-98. Epub 2010 Jan 28. Joergensen M, Brusgaard K, Cruger DG, Gerdes AM, de Muckadell OB
Incidence, prevalence, etiology, and prognosis of first-time chronic pancreatitis in young patients: a nationwide cohort study.
Dig Dis Sci. 2010 Oct;55(10):2988-98. Epub 2010 Jan 28., [PMID:20108119]

Abstract [show]
BACKGROUND/AIMS: Publications on etiology of chronic pancreatitis (CP) are infrequent. Etiologies today encompass genetic disorders. We wanted to describe etiologies of today and identify patients with genetic disorders like hereditary pancreatitis (HP), mutations in Serine Protease Inhibitor Kazal type1 (SPINK1), and the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) among patients formerly considered to have idiopathic CP. METHODS: Data on patients diagnosed with first-time CP < 30 years of age in Denmark identified in the Danish National Registry of Patients were retrieved. Patients previously considered to have idiopathic pancreatitis were offered genetic counseling and evaluation for HP, SPINK1, and CFTR mutations. RESULTS: In the period 1980-2004, 580 patients < 30 years of age presented with CP, the standardized prevalence ratio of CP increased from 11.7 per 100,000 person years in 1980-1984 to 17.0 per 100,000 in 2000-2004 (p < 0.001). The odds ratio (OR) having gallstone-related CP increased in the latter time period, especially in women, that of alcohol-induced CP decreased over time. OR having idiopathic CP increased in the latter period; 50% of patients with idiopathic pancreatitis accepted genetic reevaluation; 28 patients had a genetic mutation that totally or partly could explain their pancreatitis, nine of these had two, and 11 patients had HP. CONCLUSION: The prevalence of CP, especially in women, increased over time. Genetic causes that partly or totally could explain the CP were found in 54.90% (95% CI (40.45-68.62)) of those with idiopathic CP, as a minimum estimation 1.9% (95% CI (1.00-3.47)) of the total cohort had HP.

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48 1G [ T, R1162X, 1717-1G [ A, 3659delC, G542X, 2183A [ G, W1282X, 1078delT, 711 ? Login to comment

[hide] Pharmacological correction of a defect in PPAR-gam... Nat Med. 2010 Mar;16(3):313-8. Epub 2010 Feb 14. Harmon GS, Dumlao DS, Ng DT, Barrett KE, Dennis EA, Dong H, Glass CK
Pharmacological correction of a defect in PPAR-gamma signaling ameliorates disease severity in Cftr-deficient mice.
Nat Med. 2010 Mar;16(3):313-8. Epub 2010 Feb 14., [PMID:20154695]

Abstract [show]
Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (encoded by Cftr) that impair its role as an apical chloride channel that supports bicarbonate transport. Individuals with cystic fibrosis show retained, thickened mucus that plugs airways and obstructs luminal organs as well as numerous other abnormalities that include inflammation of affected organs, alterations in lipid metabolism and insulin resistance. Here we show that colonic epithelial cells and whole lung tissue from Cftr-deficient mice show a defect in peroxisome proliferator-activated receptor-gamma (PPAR-gamma, encoded by Pparg) function that contributes to a pathological program of gene expression. Lipidomic analysis of colonic epithelial cells suggests that this defect results in part from reduced amounts of the endogenous PPAR-gamma ligand 15-keto-prostaglandin E(2) (15-keto-PGE(2)). Treatment of Cftr-deficient mice with the synthetic PPAR-gamma ligand rosiglitazone partially normalizes the altered gene expression pattern associated with Cftr deficiency and reduces disease severity. Rosiglitazone has no effect on chloride secretion in the colon, but it increases expression of the genes encoding carbonic anhydrases 4 and 2 (Car4 and Car2), increases bicarbonate secretion and reduces mucus retention. These studies reveal a reversible defect in PPAR-gamma signaling in Cftr-deficient cells that can be pharmacologically corrected to ameliorate the severity of the cystic fibrosis phenotype in mice.

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204 Hamosh, A., Rosenstein, B.J. & Cutting, G.R. CFTR nonsense mutations G542X and W1282X associated with severe reduction of CFTR mRNA in nasal epithelial cells. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Am J Respir Crit Care Med. 2010 May 15;181(10):1078-84. Epub 2010 Feb 18. Bienvenu T, Sermet-Gaudelus I, Burgel PR, Hubert D, Crestani B, Bassinet L, Dusser D, Fajac I
Cystic fibrosis transmembrane conductance regulator channel dysfunction in non-cystic fibrosis bronchiectasis.
Am J Respir Crit Care Med. 2010 May 15;181(10):1078-84. Epub 2010 Feb 18., 2010-05-15 [PMID:20167849]

Abstract [show]
RATIONALE: Although in patients with diffuse bronchiectasis (DB) and a normal sweat test the presence of one mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is frequently observed, its pathogenic role in the development of DB remains unclear. OBJECTIVES: To evaluate the association between CFTR heterozygosity and CFTR protein dysfunction in the airways of patients with DB. METHODS: Nasal potential difference was measured in 122 patients with DB of unknown origin and with a normal sweat test (Cl(-) < 60 mmol/L). They were classified according to the presence of CFTR mutations: zero (85 patients), one (22 patients), or two mutations (15 patients). Control groups comprised 26 healthy subjects, 38 obligate heterozygotes for CFTR, and 92 patients with classic cystic fibrosis (CF) with an abnormal sweat test (Cl(-) > or = 60 mmol/L). Patients classified as mild-CF were carrying at least one mild mutation and patients classified as severe-CF were homozygous for the F508del mutation. MEASUREMENTS AND MAIN RESULTS: There was a continuum of airway CFTR dysfunction in the study population as shown by nasal potential difference measurements, ranging from normal values in healthy subjects, to intermediate values in subjects with DB, to highly abnormal values in subjects classified as severe-CF. This continuum of airway CFTR dysfunction was thus strongly associated with defects in the CFTR gene. Moreover, among patients with DB, a similar continuum in intermediate nasal potential difference was identified that was associated with the bearing of zero, one, or two CFTR mutations. These electrophysiological phenotypes and CFTR genotypes were also associated with the clinical phenotype, as shown by the frequency of Staphylococcus aureus and Pseudomonas aeruginosa bronchial colonization. CONCLUSIONS: Our study supports the hypothesis that a unique CFTR mutation may have pathogenic consequences in patients with DB.

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58 In DB-1, 12 patients carried a severe loss-of-function mutation: 3 patients carried a class 1 mutation (G542X, 2183AA.G, and W1282X), and 9 patients carried the F508del class 2 mutation; 10 patients carried a mild mutation predicted to retain some residual CFTR function: 7 patients carried the IVS8-5T class 5 mutation, and 3 patients carried a class 4 mutation (S1235R, R347P-I148T, and R117H-7T) (Table 1). Login to comment
79 GENOTYPE AND PHENOTYPE OF PATIENTS WITH DIFFUSE BRONCHIECTASIS BEARING ONE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR MUTATION Patient No. Age (yr) Sex (M/F) CFTR Mutation Sweat Cl2 (mmol/L) Basal PD (mV) NPD Index Age at Onset (yr) FEV1 (% pred) Bacterial Colonization 1 46 F F508del/2 10 215 0.44 20 124 Pa 2 51 M S1235R/2 8 219 0.56 10 40 Sa/Pa 3 19 F R347P-I148T/2 13 219 0.48 10 91 None 4 31 F F508del/2 35 220 0.20 2 76 None 5 34 M IVS8-5T/2 10 221 0.51 2 27 None 6 49 F IVS8-5T/2 15 222 0.30 40 92 None 7 20 F IVS8-5T/2 13 223 0.42 16 90 None 8 38 M F508del/2 9 224 0.85 20 ND None 9 65 M F508del/2 21 224 0.88 60 99 None 10 52 F F508del/2 20 226 0.37 5 91 Pa 11 72 F G542X/2 15 226 0.37 40 68 None 12 67 F IVS8-5T/2 26 226 0.82 40 97 None 13 51 F W1282X/2 17 228 0.12 29 27 Pa 14 59 M R117H-7T/2 31 229 0.88 49 89 None 15 56 F F508del/2 17 230 0.41 40 75 None 16 49 F F508del/2 21 232 0.58 45 67 None 17 46 F 2183AA.G/2 23 233 0.26 45 132 None 18 19 F IVS8-5T/2 19 234 0.45 5 82 None 19 70 M IVS8-5T/2 20 238 0.34 50 64 None 20 22 F F508del/2 25 241 0.86 20 82 Sa 21 77 M IVS8-5T/2 26 242 1.00 65 86 None 22 73 M F508del/2 21 245 0.91 25 70 Pa Definition of abbreviations: Cl2 5 chloride; F 5 female; M 5 male; ND 5 not determined; NPD index 5 nasal potential difference index 5 e(response to øCl2 and iso/response to amil); a cut off . Login to comment
82 GENOTYPE AND PHENOTYPE OF PATIENTS WITH DIFFUSE BRONCHIECTASIS BEARING TWO CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR MUTATIONS Patient No. Age (yr) Sex (M/F) CFTR Mutations Sweat Cl2 (mmol/L) Basal PD (mV) NPD Index Age at Onset (yr) FEV1 (% pred) Bacteria Colonization 1 55 F F508del/D1152H 19 219 1.00 54 99 Sa 2 71 F F508del/G576A-R668C 29 223 0.44 70 114 None 3 24 M G542X/3849110kbCT 52 224 1.22 10 78 Pa 4 41 F 394delTT/D1152H 19 225 0.30 41 89 Sa 5 31 M 3849110kbC.T/3849110kbC.T 35 230 0.64 2 30 Sa/Pa 6 74 F G542X/S912L 40 233 0.19 60 106 None 7 50 M W1282X/D1152H 35 236 1.00 10 32 Pa 8 42 F F508del/D1152H 13 240 0.68 30 32 Pa 9 56 F F508del/IVS8-5T 30 242 0.70 10 70 None 10 45 F 394delTT/D1152H 25 242 0.71 18 62 Sa/Pa 11 74 F W1282X/D1152H 25 244 0.66 12 56 Pa 12 23 F S1206X/D1152H 19 244 0.68 13 107 None 13 41 F R553X/R851L-T351S 31 248 0.50 35 72 Pa 14 58 M F508del/R117H-7T 46 251 0.61 45 35 Sa/Pa 15 53 F F508del/R347H 49 258 0.63 40 77 Pa Definition of abbreviations: Cl2 5 chloride; F 5 female; M 5 male; NPD index 5 nasal potential difference index 5 e(response to øCl2 and iso/response to amil); a cut off . Login to comment

[hide] Novel cftr gene sequence variation in Serbian pati... Fetal Pediatr Pathol. 2010 Jan;29(2):95-8. Nikolic A, Milosevic K, Divac A, Ljujic M, Grkovic S, Nestorovic B
Novel cftr gene sequence variation in Serbian patient with idiopathic disseminated bronchiectasis.
Fetal Pediatr Pathol. 2010 Jan;29(2):95-8., [PMID:20334484]

Abstract [show]
This paper reports a novel Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene variant, 1811+1G-->T, detected in a 5-year-old girl diagnosed with idiopathic disseminated bronchiectasis and negative sweat chloride test (17 mmol/L). The performed CFTR gene mutation analysis included detection of the F508del mutation, analysis of Tn polymorphism and screening of CFTR exons 3, 10 and 11. The CFTR gene screening has shown the altered band pattern in exon 11. The DNA sequencing of CFTR exon 11 revealed the presence of the novel sequence variation 1811+1G-->T in heterozygous state. This sequence variation was not found in any of 100 control alleles, analyzed by polymerase chain reaction - restriction fragment length polymorphism method. The novel sequence variation 1811+1G-->T is located at the splicing site at the boundary of exon 11 and intron 11 and might be either a sequence variation or a splicing site defect.

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28 Detection of novel CFTR gene sequence variation 1811+1G→T: a) Analysis of CFTR exon 11 on DGGE gel: 1 - wt/wt, 2 - wt/wt, 3 - G542X/wt, 4 - 1811+1G→T/wt; b) Results of automated DNA sequencing of CFTR exon 11. Login to comment

[hide] All azoospermic males should be screened for cysti... Fertil Steril. 2010 Nov;94(6):2448-50. Epub 2010 Apr 9. Mocanu E, Shattock R, Barton D, Rogers M, Conroy R, Sheils O, Collins C, Martin C, Harrison R, O'Leary J
All azoospermic males should be screened for cystic fibrosis mutations before intracytoplasmic sperm injection.
Fertil Steril. 2010 Nov;94(6):2448-50. Epub 2010 Apr 9., [PMID:20381036]

Abstract [show]
We assessed the frequency of CFTR mutations in groups with varying degrees of sub-fertility and compared these groups to a fertile male group with proven paternity. Screening for CFTR mutations should be routine for all azoospermic males, irrespective of obstructive or non-obstructive etiology, prior to proposing ICSI treatment. CFTR testing has no value in the investigation of non-azoospermic infertile males.

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50 Of these, 83% were F508del, 3.7% R117H, G551D, and 621þ1G>T, and 1.85% R560T, G542X, and I507del. Login to comment

[hide] Clinical and genetic characteristics of meconium i... J Pediatr Gastroenterol Nutr. 2010 May;50(5):569-72. Gorter RR, Karimi A, Sleeboom C, Kneepkens CM, Heij HA
Clinical and genetic characteristics of meconium ileus in newborns with and without cystic fibrosis.
J Pediatr Gastroenterol Nutr. 2010 May;50(5):569-72., [PMID:20386322]

Abstract [show]
The present study compares the clinical presentation and diagnostic features of meconium ileus (MI) in newborns with and without cystic fibrosis (CF). A retrospective study of 43 patients treated in the Pediatric Surgical Center of Amsterdam was performed. Twenty-three of the patients (53.5%) were diagnosed as having CF. Complex MI was significantly more frequent in patients without CF, and these patients had lower gestational ages and birth weights than patients with CF. All of the patients with complex MI had homozygous DF508 mutations, whereas the patients with simple MI also had other mutations. None of the patients with other mutations had complex MI. Therefore, we conclude that the clinical entity of MI represents a spectrum of underlying pathologies.

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25 The mutations tested for include the most common mutations DF508, F508C, G542X, R553X, N1303K, R1162X, and E60X, which represent 94% to 98% of the known mutations in the CFTR gene and are found in more than 99% of the Dutch population with CF. Login to comment
65 One child was G542X homozygous. Login to comment
104 It has been reported that in newborns carrying DF508 or G542X, a higher incidence of MI is found, whereas G551D and R117H are associated with a decreased MI incidence (19,21,22). Login to comment

[hide] Impact of gene patents and licensing practices on ... Genet Med. 2010 Apr;12(4 Suppl):S194-211. Chandrasekharan S, Heaney C, James T, Conover C, Cook-Deegan R
Impact of gene patents and licensing practices on access to genetic testing for cystic fibrosis.
Genet Med. 2010 Apr;12(4 Suppl):S194-211., [PMID:20393308]

Abstract [show]
Cystic fibrosis is one of the most commonly tested autosomal recessive disorders in the United States. Clinical cystic fibrosis is associated with mutations in the CFTR gene, of which the most common mutation among Caucasians, DeltaF508, was identified in 1989. The University of Michigan, Johns Hopkins University, and the Hospital for Sick Children, where much of the initial research occurred, hold key patents on cystic fibrosis genetic sequences, mutations, and methods for detecting them. Several patents, including the one that covers detection of the DeltaF508 mutation, are jointly held by the University of Michigan and the Hospital for Sick Children in Toronto, with Michigan administering patent licensing in the United States. The University of Michigan broadly licenses the DeltaF508 patent for genetic testing with >60 providers of genetic testing to date. Genetic testing is now used in newborn screening, diagnosis, and for carrier screening. Interviews with key researchers and intellectual property managers, a survey of laboratories' prices for cystic fibrosis genetic testing, a review of literature on cystic fibrosis tests' cost-effectiveness, and a review of the developing market for cystic fibrosis testing provide no evidence that patents have significantly hindered access to genetic tests for cystic fibrosis or prevented financially cost-effective screening. Current licensing practices for cystic fibrosis genetic testing seem to facilitate both academic research and commercial testing. More than 1000 different CFTR mutations have been identified, and research continues to determine their clinical significance. Patents have been nonexclusively licensed for diagnostic use and have been variably licensed for gene transfer and other therapeutic applications. The Cystic Fibrosis Foundation has been engaged in licensing decisions, making cystic fibrosis a model of collaborative and cooperative patenting and licensing practice.

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182 The ACMG specifically indicated that "Asian-Americans and Native Americans without significant Caucasian admixture should be informed of Table 1 Recommended core mutation panel for cystic fibrosis carrier screening in the general population Standard mutation panel R560T, ⌬F508a , R553Xb , R1162X, ⌬I507, 2184delA, G542X, G551Db , W1282X, N1303K, 621ϩ1G⌬T, R117H, 1717-1G⌬A, A455E, G85E, R334W, R347P, 711ϩ1G⌬T, 1898ϩ1G⌬A, 3849ϩ10kbC⌬T, 2789ϩ5G⌬A, 3659delC, and 3120ϩ1G⌬A Additional testable mutations I506Vc , I507Vc , F508Cc , and 5T/ 7T/9Td a University of Michigan/HSC Patent No. US 5,776,677. b Johns Hopkins University, Patent No. US 5,407,796. c Benign variants. Login to comment

[hide] Genetic testing in pancreatitis. Gastroenterology. 2010 Jun;138(7):2202-6, 2206.e1. Epub 2010 Apr 20. Ooi CY, Gonska T, Durie PR, Freedman SD
Genetic testing in pancreatitis.
Gastroenterology. 2010 Jun;138(7):2202-6, 2206.e1. Epub 2010 Apr 20., [PMID:20416310]

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53 Interpretation of Mutations Requires an Understanding of Their Functional Consequences Mutation group Reported mutations Complex allele: These mutations are recognized to occur on a single allele R117H ϩ T G576A ϩ R668C F508del ϩ I1027T Benign sequence alterations: These mutations have no known clinical consequence R74Q R297Q R74W 621 * 25 AϾG 3500-19 CϾT T164S C855I I1139V CFTR-related disorder associated: These mutations have been described in individuals with CF-like single organ disease (such as pancreatitis, sinopulmonary disease, or obstructive azoospermia), but do not fulfill the diagnostic criteria for CF 5T R117H D1270N L320V Q1352H 1818-18 GϾA S1235R CF causing F508del Q1476X R553X K710X G542X G551D F311L 2789-5 GϾA 2183AAϾG 711ϩ3 AϾG 3849ϩ10kb CϾT 1341ϩ1GϾA D1152Ha F1074La R553X Unknown clinical consequence F575Y L1260P G194R G1069R L997F K598E F834L R785Q To illustrate this point, mutations identified by extensive mutation testing in a cohort of patients with recurrent acute or chronic pancre- atitis14 are listed according to their clinical consequences (based on current consensus guidelines13 and functional and/or clinical reports; available: http://www.genet.sickkids.on.ca). Login to comment

[hide] The NF-kappaB signaling in cystic fibrosis lung di... Discov Med. 2010 Apr;9(47):346-56. Bodas M, Vij N
The NF-kappaB signaling in cystic fibrosis lung disease: pathophysiology and therapeutic potential.
Discov Med. 2010 Apr;9(47):346-56., [PMID:20423679]

Abstract [show]
Lung disease is the major cause of morbidity and mortality of cystic fibrosis (CF), an autosomal recessive disease caused by mutations in CF transmembrane-conductance regulator (CFTR) gene. In CF, elevated levels of interleukin-8 (IL-8) signaling mediated by the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) result in chronic infection, neutrophilic inflammation, and progressive airway destruction. The most frequent mutation in the CFTR gene is the deletion of phenylalanine 508 (DeltaF508), which results in its endoplasmic reticulum associated degradation (ERAD) by the ubiquitin-proteasome system. The inability of DeltaF508-CFTR to reach cell surface leads to inherently high levels of NF-kappaB. Severity of CF lung disease depends on the levels of functional CFTR on cell surface that control its chloride transport and NF-kappaB mediated innate immune response functions. NF-kappaB mediated chronic inflammation is a prominent feature of CF lung disease and the mechanism(s) by which CFTR regulates these inflammatory signaling pathways is becoming apparent. Recent data suggest that CFTR localization to lipid-rafts is critical for regulating NF-kappaB mediated innate immune response and chronic CF lung disease. We anticipate that targeting the pathways, which facilitates CFTR's rescue to the cell surface and lipid-rafts, will not only restore CFTR channel function but also control NF-kappaB mediated chronic inflammation, although the level of correction may be a critical factor for therapeutic efficacy. We discuss here the mechanisms of NF-kappaB induction in CF, pathogenesis of CF lung disease, and novel therapeutic strategies that may help in reversing the chronic CF lung disease.

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102 Recent data suggest that the higher inflammation in ΔF508 CF could be a consequence of fewer CFTR molecules at the membrane, as would be predicted also with other rare CF stop mutations such as G542X (McCormick et al., 2002). Login to comment

[hide] Carrier screening for cystic fibrosis. Obstet Gynecol Clin North Am. 2010 Mar;37(1):47-59, Table of Contents. Dungan JS
Carrier screening for cystic fibrosis.
Obstet Gynecol Clin North Am. 2010 Mar;37(1):47-59, Table of Contents., [PMID:20494257]

Abstract [show]
Cystic fibrosis is the first genetic disorder for which universal screening of preconceptional or prenatal patients became a component of standard prenatal care. The molecular genetics and mutation profile of the CFTR gene are complex, with a wide range of phenotypic consequences. Carrier screening can facilitate risk assessment for prospective parents to have an affected offspring, although there remains a small residual risk for carrying a mutation even with a negative screening result. There are ethnic differences with respect to disease incidence and effectiveness of carrier testing, which may complicate counseling.

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102 However, in instances of a positive family history of affected individuals, but with no known mutation, further Table 2 Mutation panel recommended by ACOG and ACMG (listed in order of decreasing frequency in non-Hispanic Caucasian population) F508 del delI507 R347P R1162X G542X R553X 71111G>T 2184delA G551D R117H R560T 189811G>A 62111G>T 3849110kbC>T 3569delC R334W W1282X 1717À1G>T A455E 312011G>T N1303K 278915G>A G85E Data from Watson MS, Cutting GR, Desnick RJ, et al. Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel. Login to comment

[hide] Notable contribution of large CFTR gene rearrangem... Eur J Hum Genet. 2010 Oct;18(10):1166-9. Epub 2010 May 26. de Becdelievre A, Costa C, LeFloch A, Legendre M, Jouannic JM, Vigneron J, Bresson JL, Gobin S, Martin J, Goossens M, Girodon E
Notable contribution of large CFTR gene rearrangements to the diagnosis of cystic fibrosis in fetuses with bowel anomalies.
Eur J Hum Genet. 2010 Oct;18(10):1166-9. Epub 2010 May 26., [PMID:20512161]

Abstract [show]
Grade III fetal bowel hyperechogenicity and/or loop dilatation observed at the second trimester of pregnancy can be due to several disease conditions, including cystic fibrosis (CF). Screening for frequent CF mutations is performed as a first step and, in certain situations, such as when a frequent CF mutation is found in the fetus, the increased risk of CF justifies an in-depth study of the second allele. To determine the contribution of large CFTR gene rearrangements in such cases, detected using a semiquantitative fluorescent multiplex PCR (QFM-PCR) assay, we collated data on 669 referrals related to suspicion of CF in fetuses from 1998 to 2009. Deletions were found in 5/70 cases in which QFM-PCR was applied, dele19, dele22_23, dele2_6b, dele14b_15 and dele6a_6b, of which the last three remain undescribed. In 3/5 cases, hyperechogenicity was associated with dilatation and/or gallbladder anomalies. Of the total cases of CF recognized in the subgroup of first-hand referrals, deletions represent 16.7% of CF alleles. Our study thus strengthens the need to consider large CFTR gene rearrangements in the diagnosis strategy of fetal bowel anomalies, in particular in the presence of multiple anomalies.

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48 Table 1 Reasons of screening for large rearrangements In group 1 (first-hand referrals): 17/450 First step of the study: one CF mutation identified (n¼8) F508del (n¼6), 394delTT (n¼1), Q1352H (n¼1) Abnormal AF-DE (n¼4) Consanguinity in the couple (n¼1) Very suggestive ultrasound signsa (n¼4) In group 2 (second-hand referrals): 53/219 First step of the study: one CF mutation identified in another laboratory (n¼45) F508del (n¼36), N1303K (n¼3), G542X (n¼2), G551D, R553X, W1282X, 3849+10kbC4T (n¼1 for each) Abnormal AF-DE (n¼1) Consanguinity in the couple and presence of the [R74W;V201M;D1270N] complex allele (n¼1) Very suggestive ultrasound signsa (n¼6) aVery suggestive ultrasound signs mean that several abnormal signs were associated and/or clinicians insisted on a comprehensive study of the CFTR gene. Login to comment

[hide] Cystic fibrosis newborn screening: using experienc... J Inherit Metab Dis. 2010 Oct;33(Suppl 2):S255-61. Epub 2010 Jun 3. Hale JE, Parad RB, Dorkin HL, Gerstle R, Lapey A, O'Sullivan BP, Spencer T, Yee W, Comeau AM
Cystic fibrosis newborn screening: using experience to optimize the screening algorithm.
J Inherit Metab Dis. 2010 Oct;33(Suppl 2):S255-61. Epub 2010 Jun 3., [PMID:20521170]

Abstract [show]
Newborn screening (NBS) for cystic fibrosis (CF) offers the opportunity for early diagnosis and improved outcomes in patients with CF and has been universally available in the state of Massachusetts since 1999 using an immunoreactive trypsinogen (IRT)-DNA algorithm. Ideally, CF NBS is incorporated as part of an integrated NBS system that allows for comprehensive and coordinated education, laboratory screening, clinical follow-up, and evaluation so that evidence-based data can be used to maximize quality improvements and optimize the screening algorithm. The New England Newborn Screening Program (NENSP) retrospectively analyzed Massachusetts's CF newborn screening data that yielded decisions to eliminate a screen-positive category, maintain the IRT cutoff value that prompts the second tier DNA testing, and communicate CF relative risk to primary care providers (PCPs) based on categorization of positive CF NBS results.

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47 Extensive follow-up Table 1 Children who are followed at a cystic fibrosis (CF) center who were not identified by CF newborn screening (NBS) Presentation Status at last update NBS IRT%, age at dx Genotype Sweat [Cl- ] (MEq/L)a Five CF infants with false-negative CF NBS results FTT, upper respiratory infections, chronic cough Pancreatic sufficient, sinus disease, positive cultures for Staph. aureus and H. flu 84.2%, 3 months DF508/R117H 67 Meconium ileus 93.9%, birth G542X / unknown 57.7, 67.4 FTT, recurrent pneumonia, asthma 62.3%, 4 years D828G / 3271+18 C or T 62 Asthma 78.6%, 3 years D1270N / R74W 86.5 Chronic cough and sinusitis 74.1%, 4 years R75Q / unknown (second mutation not identified by sequencing) 82, 68 Four additional infants followed at CF center who do not (yet) carry a CF diagnosis Chronic cough Pancreatic sufficient, asthma, moderate Staph. aureus and H. flu 39.7%, 5 years DF508 / unknown 39 Chronic cough; sweat-tested and genotyped after parents found to be carriers during pregnancy with younger sibling Does not carry CF diagnosis, pancreatic sufficient, exercise-induced asthma, normal PFTs, cultures Staph. aureus 94.6%, 3 years DF508/R117H 56 Two siblings who are well; genotyped for family history Positive cultures for Staph. aureus and H.flu 21.3%, 71.2% (sib) DF508 / R117H 20, not done IRT Immunoreactive trypsinogen, FTT failure to thrive, PFT pulmonary function test a Value(s) reported from independent visits of infants with positive CF NBS results has allowed the MA CF NBS program to incorporate communication of relative risk of CF following a positive NBS result that is based upon combined consideration (multi-analyte profiling) of both the IRT concentration and the screening-genotype results. Login to comment

[hide] The CFTR Met 470 allele is associated with lower b... PLoS Genet. 2010 Jun 3;6(6):e1000974. Kosova G, Pickrell JK, Kelley JL, McArdle PF, Shuldiner AR, Abney M, Ober C
The CFTR Met 470 allele is associated with lower birth rates in fertile men from a population isolate.
PLoS Genet. 2010 Jun 3;6(6):e1000974., [PMID:20532200]

Abstract [show]
Although little is known about the role of the cystic fibrosis transmembrane regulator (CFTR) gene in reproductive physiology, numerous variants in this gene have been implicated in etiology of male infertility due to congenital bilateral absence of the vas deferens (CBAVD). Here, we studied the fertility effects of three CBAVD-associated CFTR polymorphisms, the (TG)m and polyT repeat polymorphisms in intron 8 and Met470Val in exon 10, in healthy men of European descent. Homozygosity for the Met470 allele was associated with lower birth rates, defined as the number of births per year of marriage (P = 0.0029). The Met470Val locus explained 4.36% of the phenotypic variance in birth rate, and men homozygous for the Met470 allele had 0.56 fewer children on average compared to Val470 carrier men. The derived Val470 allele occurs at high frequencies in non-African populations (allele frequency = 0.51 in HapMap CEU), whereas it is very rare in African population (Fst = 0.43 between HapMap CEU and YRI). In addition, haplotypes bearing Val470 show a lack of genetic diversity and are thus longer than haplotypes bearing Met470 (measured by an integrated haplotype score [iHS] of -1.93 in HapMap CEU). The fraction of SNPs in the HapMap Phase2 data set with more extreme Fst and iHS measures is 0.003, consistent with a selective sweep outside of Africa. The fertility advantage conferred by Val470 relative to Met470 may provide a selective mechanism for these population genetic observations.

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135 The most common CF causing mutations in Europeans (i.e. DF508, G542X, N1303K, W1282X) and the most common mutation in the Hutterites, M1101K [16], all reside on haplotypes carrying the ancestral, Met470 allele in exon 10 [29], the 9T allele at the polyT locus, and (by inference) the TG10 or TG11 alleles at the (TG)m locus in intron 8 [5]. Login to comment

[hide] Clinical phenotype and genotype of children with b... Am J Respir Crit Care Med. 2010 Oct 1;182(7):929-36. Epub 2010 Jun 10. Sermet-Gaudelus I, Girodon E, Sands D, Stremmler N, Vavrova V, Deneuville E, Reix P, Bui S, Huet F, Lebourgeois M, Munck A, Iron A, Skalicka V, Bienvenu T, Roussel D, Lenoir G, Bellon G, Sarles J, Macek M, Roussey M, Fajac I, Edelman A
Clinical phenotype and genotype of children with borderline sweat test and abnormal nasal epithelial chloride transport.
Am J Respir Crit Care Med. 2010 Oct 1;182(7):929-36. Epub 2010 Jun 10., 2010-10-01 [PMID:20538955]

Abstract [show]
RATIONALE: The diagnosis of cystic fibrosis (CF) is based on a characteristic clinical picture in association with a sweat chloride (Cl(-)) concentration greater than 60 mmol/L or the identification of two CF-causing mutations. A challenging problem is the significant number of children for whom no definitive diagnosis is possible because they present with symptoms suggestive of CF, a sweat chloride level in the intermediate range between 30 and 60 mmol/L, and only one or no identified CF-causing mutation. OBJECTIVES: To investigate the function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in the airways of children with intermediate sweat tests and inconclusive genetic findings in correlation with clinical phenotype and genotype. METHODS: We developed a composite nasal potential difference (NPD) diagnostic score to discriminate patients with CF from non-CF patients. We tested NPD in 50 children (age, 6 mo to 18 yr) with equivocal diagnoses and correlated the NPD diagnostic score with clinical phenotypes and genotypes. MEASUREMENTS AND MAIN RESULTS: Fifteen of the 50 children had NPD scores in the CF range. Eight of the 15 carried two CFTR mutations compared with only 5 of the 35 children with normal NPD scores (P = 0.01). They were significantly younger at evaluation and had recurrent lower respiratory tract infections, chronic productive coughs, and chronic Staphylococcus aureus colonization significantly more often than the 35 children with normal NPD results. CONCLUSIONS: Evaluation of CFTR function in the nasal epithelium of children with inconclusive CF diagnoses can be a useful diagnostic tool and help clinicians to individualize therapeutic strategy.

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162 CLINICAL CHARACTERISTICS OF CHILDREN WITH EQUIVOCAL DIAGNOSES AND NASAL POTENTIAL DIFFERENCE DIAGNOSTIC SCORE <0.27 Pt Mutation Age (yr) NPD Score Sweat Cl2 Chronic CF Pulmonary Disease CF Pathogens Airway Obstruction CF Lung Imaging FEV1 (%) BMI Others 1 F508del/S977F A-D 8 0.181 43 RLRTI, chronic productive cough S. aureus No Bronchiectasis 80 14.5 No Bronchial thickening Atelectasis 2 0/0 4 0.121 43 No S. aureus Yes Air trapping NA 13 Pancreatic extracts 0-0 Bronchial thickening 3 0/0 15 20.032 46 RLRTI S. aureus, P. aeruginosa Yes Air trapping 74 14 Polyposis 0-0 Bronchiectasis 4 F508del/0 2 20.249 57 RLRTI P. aeruginosa Yes Air trapping NA 16 No A-0 5 N1303K/(TG12)T5 11.8 20.263 47 RLRTI S. aureus, P. aeruginosa No Bronchial thickening ND 20 No A-B 6 F508del/L206W 5.9 20.278 40 RLRTI S. aureus No Bronchial thickening 115 22 Chronic pancreatitis A-AB 7 R668C/0 15 20.403 40 RLRTI None Yes Bronchiectasis 112 20 No B-0 Air trapping 8 F508del/L997F A-B 1 20.594 38 RLRTI, chronic productive cough P. aeruginosa No Bronchial thickening NA 16 CF hepatopathy 9 G576A;R668C/S1235R 8 20.659 31 0 None Wheezing Normal 100 20 No B-B 10 G542X/0 5 20.718 49 RLRTI, chronic productive cough S. aureus No Bronchial thickening NA 18 No A-0 11 0/0 7 20.742 37 RLRTI None No Normal 106 18 No 0-0 12 F508del/D110E 16 20.777 50 No S. aureus No No 100 21 No A-AB 13 F508del/R1070W 7 21.006 40 RLRTI S. aureus Wheezing Bronchial thickening 110 14 No A-AB 14 F508del-L467F/0 12 21.897 55 RLRTI, chronic productive cough S. aureus No Bronchiectasis 109 17 Pansinusitis A-0 15 F508del/H1054D 9 22.327 59 RLRTI, chronic productive cough S. aureus No Bronchial thickening 100 20 DIOS A-D Definition of abbreviations: A, B, AB, and D: A 5 CF-causing mutation; B 5 mutation that results in a CFTR-RD (clinical entities associated with CFTR mutations that do not meet the current diagnostic criteria for CF); AB 5 wide-spectrum mutation that may belong to either group A or group B; D 5 mutation of uncertain clinical relevance; BMI 5 body mass index; CF 5 cystic fibrosis; CFTR 5 gene encoding cystic fibrosis transmembrane conductance regulator; DIOS 5 distal intestinal obstructive syndrome; NA 5 not applicable; ND 5 not determined; NPD 5 nasal potential difference; P. aeruginosa 5 Pseudomonas aeruginosa; Pt 5 patient; RLRTI 5 recurrent lower respiratory tract infection; S. aureus 5 Staphylococcus aureus. Login to comment

[hide] Ataluren (PTC124) induces cystic fibrosis transmem... Am J Respir Crit Care Med. 2010 Nov 15;182(10):1262-72. Epub 2010 Jul 9. Sermet-Gaudelus I, Boeck KD, Casimir GJ, Vermeulen F, Leal T, Mogenet A, Roussel D, Fritsch J, Hanssens L, Hirawat S, Miller NL, Constantine S, Reha A, Ajayi T, Elfring GL, Miller LL
Ataluren (PTC124) induces cystic fibrosis transmembrane conductance regulator protein expression and activity in children with nonsense mutation cystic fibrosis.
Am J Respir Crit Care Med. 2010 Nov 15;182(10):1262-72. Epub 2010 Jul 9., 2010-11-15 [PMID:20622033]

Abstract [show]
RATIONALE: Nonsense (premature stop codon) mutations in mRNA for the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF) in approximately 10% of patients. Ataluren (PTC124) is an oral drug that permits ribosomes to readthrough premature stop codons in mRNA to produce functional protein. OBJECTIVES: To evaluate ataluren activity, safety, and pharmacokinetics in children with nonsense mutation CF. METHODS: Patients were assessed in two 28-day cycles, comprising 14 days on and 14 days off ataluren. Patients took ataluren three times per day (morning, midday, and evening) with randomization to the order of receiving a lower dose (4, 4, and 8 mg/kg) and a higher dose (10, 10, and 20 mg/kg) in the two cycles. MEASUREMENTS AND MAIN RESULTS: The study enrolled 30 patients (16 male and 14 female, ages 6 through 18 yr) with a nonsense mutation in at least one allele of the CFTR gene, a classical CF phenotype, and abnormal baseline nasal epithelial chloride transport. Ataluren induced a nasal chloride transport response (at least a -5-mV improvement) or hyperpolarization (value more electrically negative than -5 mV) in 50% and 47% of patients, respectively, with more hyperpolarizations at the higher dose. Improvements were seen in seven of nine nonsense mutation genotypes represented. Ataluren significantly increased the proportion of nasal epithelial cells expressing apical full-length CFTR protein. Adverse events and laboratory abnormalities were infrequent and usually mild. Ataluren pharmacokinetics were similar to those in adults. CONCLUSIONS: In children with nonsense mutation CF, ataluren can induce functional CFTR production and is well tolerated.

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136 Patients with end-of-treatment total chloride transport hyperpolarization in either cycle were more likely to show both intrinsic and stimulated chloride transport changes; such changes are demonstrated in an example of pretreatment and end-of-treatment nasal TEPD tracings from a 14-year-old female patient with a G542X/DF508 genotype and a UGA premature stop codon type who received the higher dose of ataluren in Cycle 1 (Figure 2). Login to comment
138 Of these genotypes, G542X was the most common; in the 14 patients with this genotype, 6 (43%) met the criterion for a total chloride transport response and 7 (50%) showed hyperpolarization of total chloride transport. Login to comment
139 An examination of the data in the two patients who were homozygous for stop mutations in both alleles (one with G542X/G542X and one with R1162X/ R1162X) did not suggest that these patients were more likely to have an improvement in total chloride transport than patients who were heterozygous for a nonsense mutation in only one allele. Login to comment
159 k Two patients were homozygous for nonsense mutations (G542X/G542X, n 5 1; R1162X/R1162X, n 5 1). Login to comment
189 TOTAL CHLORIDE TRANSPORT RESPONSE AND HYPERPOLARIZATION BY NONSENSE MUTATION TYPE Nonsense Mutation Type Responses* n/N† % Response Rate Hyperpolarizations‡ n/N† % Hyperpolarization Rate Q493X (UAG) 1/3 33 1/3 33 G542X (UGA) 8/14 57 7/14 50 R553X (UGA) 1/2 50 1/2 50 W846X (UGA) 0/1 0 0/1 0 W882X (UAG) 1/1 100 1/1 100 E1104X (UGA) 1/2 50 0/2 0 R1162X (UGA) 1/2 50 2/2 100 W1282X (UGA) 2/4 50 2/4 50 Q1313X (UAA) 0/1 0 0/1 0 * At least a 25 mV total chloride transport improvement in either cycle. Login to comment
190 † Patients with nonsense mutations in both cystic fibrosis transmembrane conductance regulator alleles are counted only once (G542X/G542X, n 5 1; R1162X/R1162X, n 5 1). Login to comment
234 However, our data do extend the range of nonsense mutations studied beyond the three responsive nonsense mutation genotypes (G542X, W1282X, and 3849110 kB C/T) evaluated in adults with CF (29). Login to comment
235 Our findings indicate that multiple genotypes (Q493X, G542X, R553X, W882X, E1104X, R1162X, and W1282X) can be responsive to ataluren therapy. Login to comment

[hide] An update on cystic fibrosis screening. Clin Lab Med. 2010 Sep;30(3):533-43. Goetzinger KR, Cahill AG
An update on cystic fibrosis screening.
Clin Lab Med. 2010 Sep;30(3):533-43., [PMID:20638569]

Abstract [show]
Cystic fibrosis (CF) is a monogenic, autosomal recessive disorder, which ultimately leads to multisystem organ dysfunction and a subsequent decrease in life expectancy. Because of the sizeable number of disease causing mutations (>1000) and expansive ethnic and racial distribution, CF has presented a challenge for prenatal diagnosis. This article aims to review the genetics of CF, its spectrum of genotypic-phenotypic variations, current prenatal carrier screening and diagnostic recommendations, ultrasonographic markers of CF, and available reproductive options for carrier couples.

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12 The DF508 mutation causes a protein misfold that inhibits migration of the CFTR protein from the endoplasmic reticulum to the cell membrane.1,7 Other common mutations include G542X, R553X, W1282X, N1303K, 62111 G-to-T, 1717-1 G-to-A, and R117H.8 These result in a spectrum of protein dysfunction ranging from the production of unstable RNA to CFTR cell surface instability.7 PHENOTYPIC VARIATION IN CFTR MUTATIONS Although 70% of CF patients are either homozygotes or compound heterozygotes for these 8 common mutations, there is tremendous variation in their phenotype. Login to comment

[hide] Expression of wild-type CFTR suppresses NF-kappaB-... PLoS One. 2010 Jul 14;5(7):e11598. Hunter MJ, Treharne KJ, Winter AK, Cassidy DM, Land S, Mehta A
Expression of wild-type CFTR suppresses NF-kappaB-driven inflammatory signalling.
PLoS One. 2010 Jul 14;5(7):e11598., [PMID:20644644]

Abstract [show]
BACKGROUND: Mutation of the cystic fibrosis transmembrane-conductance regulator (CFTR) causes cystic fibrosis (CF) but not all CF aspects can easily be explained by deficient ion transport. CF-inflammation provides one example but its pathogenesis remains controversial. Here, we tested the simple but fundamental hypothesis that wild-type CFTR is needed to suppress NF-kappaB activity. METHODOLOGY/PRINCIPAL FINDINGS: In lung epithelial (H441) and engineered (H57) cell lines; we report that inflammatory markers are significantly suppressed by wild-type CFTR. Transient-transfection of wild-type CFTR into CFTR-naive H441 cells, dose-dependently down-regulates both basal and Tumour Necrosis Factor-alpha evoked NF-kappaB activity when compared to transfection with empty vector alone (p<0.01, n>5). This effect was also observed in CFTR-naive H57-HeLa cells which stably express a reporter of NF-kappaB activity, confirming that the CFTR-mediated repression of inflammation was not due to variable reporter gene transfection efficiency. In contrast, H57 cells transfected with a control cyano-fluorescent protein show a significantly elevated basal level of NF-kappaB activity above control. Initial cell seeding density may be a critical factor in mediating the suppressive effects of CFTR on inflammation as only at a certain density (1x10(5) cells/well) did we observe the reduction in NF-kappaB activity. CFTR channel activity may be necessary for this suppression because the CFTR specific inhibitor CFTR(inh172) significantly stimulates NF-kappaB activity by approximately 30% in CFTR expressing 16HBE14o- cells whereas pharmacological elevation of cyclic-AMP depresses activity by approximately 25% below baseline. CONCLUSIONS/SIGNIFICANCE: These data indicate that CFTR has inherent anti-inflammatory properties. We propose that the hyper-inflammation found in CF may arise as a consequence of disrupted repression of NF-kappaB signalling which is normally mediated by CFTR. Our data therefore concur with in vivo and in vitro data from Vij and colleagues which highlights CFTR as a suppressor of basal inflammation acting through NF-kappaB, a central hub in inflammatory signalling.

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138 Our data predict that the higher inflammation in CF cells could be a consequence of fewer wt-CFTR molecules at the membrane, as would be predicted to occur with stop mutants such as G542X that occur in a minority of CF patients and the clinical data supports the view that the presence of CFTR mutants such as G551D-CFTR that traffic normally but fail to transport chloride at all, nevertheless reduce lung damage to a degree [36]. Login to comment

[hide] Identification of the second CFTR mutation in pati... Asian J Androl. 2010 Nov;12(6):819-26. Epub 2010 Jul 26. Giuliani R, Antonucci I, Torrente I, Grammatico P, Palka G, Stuppia L
Identification of the second CFTR mutation in patients with congenital bilateral absence of vas deferens undergoing ART protocols.
Asian J Androl. 2010 Nov;12(6):819-26. Epub 2010 Jul 26., [PMID:20657600]

Abstract [show]
Congenital bilateral absence of vas deferens (CBAVD) is a manifestation of the mildest form of cystic fibrosis (CF) and is characterized by obstructive azoospermia in otherwise healthy patients. Owing to the availability of assisted reproductive technology, CBAVD patients can father children. These fathers are at risk of transmitting a mutated allele of the CF transmembrane conductance regulator (CFTR) gene, responsible for CF, to their offspring. The identification of mutations in both CFTR alleles in CBAVD patients is a crucial requirement for calculating the risk of producing a child with full-blown CF if the female partner is a healthy CF carrier. However, in the majority of CBAVD patients, conventional mutation screening is not able to detect mutations in both CFTR alleles, and this difficulty hampers the execution of correct genetic counselling. To obtain information about the most represented CFTR mutations in CBAVD patients, we analysed 23 CBAVD patients, 15 of whom had a single CFTR mutation after screening for 36 mutations and the 5T allele. The search for the second CFTR mutation in these cases was performed by using a triplex approach: (i) first, a reverse dot-blot analysis was performed to detect mutations with regional impact; (ii) next, multiple ligation-dependent probe amplification assays were conducted to search for large rearrangements; and (iii) finally, denaturing high-performance liquid chromatography was used to search for point mutations in the entire coding region. Using these approaches, the second CFTR mutation was detected in six patients, which increased the final detection rate to 60.8%.

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58 INNO-LiPA CFTR19 INNO-LiPA CFTR17 INNO-LiPA CFTR Italian regional [delta]F508 621+1G>T 1259insA G542X 3849+10kbC>T 4016insT N1303K 2183AA>G 4382delA W1282X 394delTT 852del22 G551D 2789+5G> A R1162X D579G 1717-1G>A 3659delC G1244E R553X R117H G1349D CFTRdele2,3 (21 kb) R334W I502T [delta]I507 R347P L1065P 711+1G>T G85E R1158X 3272-26A>G 3905insT 1078delT T338I R560T A455E S549R(A>C) 1898+1G>A S1251N 2143delA 711+5G>A 991del5 I148T E60X D1152H 3199del6 3120+1G>A 2184delA 1898+3A>G, R1070Q Q552X Poli-T tract variations R1066H R347H 621+3A>G R334Q E217G Abbreviation: CFTR, cystic fibrosis transmembrane conductance regulator. Login to comment
64 The detected genotypes consisted of [delta] F508/5T in five cases (pats. 1-5), G542X/5T in two cases (pats. 6 and 7) and W1282X/5T in the last patient (pat. 8). Login to comment
81 Patient First-level CFTR screening CFTR Italian MLPA analysis DHPLC analysis Final genotype (36 mutations + 5T allele) regional kit 1 [delta]F508/5T - - - [delta]F508/5T 2 [delta]F508/5T - - - [delta]F508 /5T 3 [delta]F508/5T - - - [delta]F508/5T 4 [delta]F508/5T - - - [delta]F508/5T 5 [delta]F508 /5T - - - [delta]F508/5T 6 G542X/5T - - - [delta]F508/5T 7 G542X/5T - - - [delta]F508/5T 8 W1282X/5T - - - W1282X/5T 9 [delta]F508/wt [delta]F508/T338I - - [delta]F508/T338I 10 [delta]F508/wt [delta]F508/wt [delta]F508/wt [delta]F508/wt [delta]F508/wt 11 5T/wt 5T/T338I - - 5T/T338I 12 5T/wt 5T/wt 5T/del ex1 - 5T/del ex1 13 5T/wt 5T/wt 5T/del ex19 - 5T/del ex19 14 5T/wt 5T/wt 5T/wt 5T/2811G/T 5T/2811G/T 15 5T/wt 5T/wt 5T/wt 5T/I105N 5T/I105N 16 5T/wt 5T/wt 5T/wt 5T/wt 5T/wt 17 5T/wt 5T/wt 5T/wt 5T/wt 5T/wt 18 5T/wt 5T/wt 5T/wt 5T/wt 5T/wt 19 5T/wt 5T/wt 5T/wt 5T/wt 5T/wt 20 5T/wt 5T/wt 5T/wt 5T/wt 5T/wt 21 5T/wt 5T/wt 5T/wt 5T/wt 5T/wt 22 5T/wt 5T/wt 5T/wt 5T/wt 5T/wt 23 5T/wt 5T/wt 5T/wt 5T/wt 5T/wt Detection rate 8/23 (34.8%) 2/15 (13.3%) 2/13 (15.3%) 2/11 (18.1%) 14/23 (60.8%) Abbreviations: CFTR, cystic fibrosis transmembrane conductance regulator; DHPLC, denaturing high-performance liquid chromatography; MLPA, multiple ligation-dependent probe amplification; wt, wildtype. Login to comment
117 This mutation has also been detected in two CBAVD patients in compound heterozygosity with the G542X mutation [8]. Login to comment

[hide] Defective CFTR induces aggresome formation and lun... Nat Cell Biol. 2010 Sep;12(9):863-75. Epub 2010 Aug 15. Luciani A, Villella VR, Esposito S, Brunetti-Pierri N, Medina D, Settembre C, Gavina M, Pulze L, Giardino I, Pettoello-Mantovani M, D'Apolito M, Guido S, Masliah E, Spencer B, Quaratino S, Raia V, Ballabio A, Maiuri L
Defective CFTR induces aggresome formation and lung inflammation in cystic fibrosis through ROS-mediated autophagy inhibition.
Nat Cell Biol. 2010 Sep;12(9):863-75. Epub 2010 Aug 15., [PMID:20711182]

Abstract [show]
Accumulation of unwanted/misfolded proteins in aggregates has been observed in airways of patients with cystic fibrosis (CF), a life-threatening genetic disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). Here we show how the defective CFTR results in defective autophagy and decreases the clearance of aggresomes. Defective CFTR-induced upregulation of reactive oxygen species (ROS) and tissue transglutaminase (TG2) drive the crosslinking of beclin 1, leading to sequestration of phosphatidylinositol-3-kinase (PI(3)K) complex III and accumulation of p62, which regulates aggresome formation. Both CFTR knockdown and the overexpression of green fluorescent protein (GFP)-tagged-CFTR(F508del) induce beclin 1 downregulation and defective autophagy in non-CF airway epithelia through the ROS-TG2 pathway. Restoration of beclin 1 and autophagy by either beclin 1 overexpression, cystamine or antioxidants rescues the localization of the beclin 1 interactome to the endoplasmic reticulum and reverts the CF airway phenotype in vitro, in vivo in Scnn1b-transgenic and Cftr(F508del) homozygous mice, and in human CF nasal biopsies. Restoring beclin 1 or knocking down p62 rescued the trafficking of CFTR(F508del) to the cell surface. These data link the CFTR defect to autophagy deficiency, leading to the accumulation of protein aggregates and to lung inflammation.

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918 Patients # 1 2 3 4 5 6 7 8 9 10 Sex; Age* F 13, 2 M 14, 3 F 13,4 M 13, 1 F 13, 6 M 13,3 M 29 F 19 M 11 F 24 Age at diagnosis* 0, 6 0, 3 0, 8 2, 3 11, 0 1,5 7,2 2,0 0,4 7,0 Genotype F508del/ F508del F508del/ W1282X F508del/ N1303K F508del/ G542X F508del/ F508del F508del/ F508del F508del/ G542X F508del/ W1282X F508del/ F508del F508del/ W1282X Pancreatic insufficiency Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Chronic respiratory infection (PA) Yes No No No Yes Yes No Yes Yes No Mean FEV1, % predicted 69 78 73 80 69 81 72 64 72 75 # patient's number; *, (years,months) (c) 2010 Macmillan Publishers Limited. Login to comment

[hide] Initial evaluation of a biochemical cystic fibrosi... J Inherit Metab Dis. 2010 Oct;33(Suppl 2):S263-71. Epub 2010 Aug 17. Sommerburg O, Lindner M, Muckenthaler M, Kohlmueller D, Leible S, Feneberg R, Kulozik AE, Mall MA, Hoffmann GF
Initial evaluation of a biochemical cystic fibrosis newborn screening by sequential analysis of immunoreactive trypsinogen and pancreatitis-associated protein (IRT/PAP) as a strategy that does not involve DNA testing in a Northern European population.
J Inherit Metab Dis. 2010 Oct;33(Suppl 2):S263-71. Epub 2010 Aug 17., [PMID:20714932]

Abstract [show]
BACKGROUND: Ethical concerns and disadvantages of newborn screening (NBS) for cystic fibrosis (CF) related to genetic testing have raised controversies and impeded implementation of CF NBS in some countries. In the present study, we used a prospective and sequential immunoreactive trypsinogene (IRT)/pancreatitis-associated protein (PAP) strategy, with IRT as first and PAP as second tier, and validated this biochemical approach against the widely used IRT/DNA protocol in a population-based NBS study in southwest Germany. METHODS: Prospective quantitation of PAP and genetic analysis for the presence of four mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene most prevalent in southwest Germany (F508del, R553X, G551D, G542X) were performed in all newborns with IRT > 99.0th percentile. NBS was rated positive when either PAP was >/=1.0 ng/mL and/or at least one CFTR mutation was detected. In addition, IRT > 99.9th percentile was also considered a positive rating. Positive rating led to referral to a CF centre for testing of sweat Cl(-) concentration. FINDINGS: Out of 73,759 newborns tested, 98 (0.13%) were positive with IRT/PAP and 56 (0.08%) with IRT/DNA. After sweat testing of 135 CF NBS-positive infants, 13 were diagnosed with CF. Detection rates were similar for both IRT/PAP and IRT/DNA. One of the 13 diagnosed CF newborns had a PAP concentration <1.0 ng/mL. CONCLUSIONS: Sequential measurement of IRT/PAP provides good sensitivity and specificity and allows reliable and cost-effective CF NBS which circumvents the necessity of genetic testing with its inherent ethical problems.

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2 Methods Prospective quantitation of PAP and genetic analysis for the presence of four mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene most prevalent in southwest Germany (F508del, R553X, G551D, G542X) were performed in all newborns with IRT> 99.0th percentile. Login to comment
58 DNA analysis was performed for the four most common CFTR mutations in southwest Germany (F508del, R553X, G551D, G542X) (Lindner et al. 1992; Tummler et al. 1996) in parallel with biochemical analysis from the same dried blood spot. Login to comment
65 IRT testing Mutation analysis F508del, R553X, G551D, G542X CF NBS positive, Recall for sweat testing NBS negative IRT > 99.0 P. no 1. Login to comment
77 These newborns were included in the study, and their samples were further analysed by measurements of PAP and CFTR mutation analysis. Out of these 632 IRT-positive newborns, 98 (0.13% of all newborns screened for CF) showed elevated PAP values, and 56 (0.08% of all newborns screened for CF) were positive in the genetic analysis. Out of these 56, homozygosity for F508del was detected in 6 cases, and 1 case was compound heterozygous for F508del /G542X and F508del /G551D. Login to comment
78 In the remaining 48 newborns, one mutation was found in 39 (36 F508del, 2 G551D, and 1 G542X) and 9 newborns had initial results which were inconclusive because PCR amplification of DNA from dried blood spots on Guthrie cards failed. Login to comment
110 In the second column, the results for both screening strategies are given CF patient True result for PAP/DNA Meconium ileus IRT (ng/ml) PAP (ng/ml) initial DNA result Age at referral (weeks) Mean of sweat Cl- measures (mmol/l) Age at diagnosis (weeks) Subsequent investigation Further DNA analysis 1 FN/FN No 36.0 n.d. n.d. 10 84 12 No special F508del/S1251N 2 TP/TP No 95.5 2.56 F508del/G542X 5 84 6 No special n.d. 3 TP/TP No 132.5 5.81 F508del/ - 4 95 5 No special n.d.a 4 TP/FN No 152.5 2.70 - / - 8b 44 10 ICMc CFTRdele2,3/ - c 5 TP/TP No 204.0 1.00 F508del/G551D 6 95 6 No special n.d. 6 TP/TP Yes 245.0 1.00 F508del/F508del - n.d.d 1 No special n.d. 7 TP/TP No 220.5 1.70 F508del/F508del 8b 82 10 No special n.d. 8 FN/FN No 139.0 0.95 - / - 15b 93 16 No special N1303K/R709X 9 TP/TP Yes 197.5 1.20 F508del/F508del - n.d.d 1 No special n.d. 10 TP/TP Yes 143.5 1.10 F508del/F508del - 92 1 No special n.d. 11 TP/TP No 114.0 1.45 F508del/ - 7b 116 7 No special F508del/p.Q552X 12 TP/TP No 174.5 2.60 F508del/F508del 4 88 5 No special n.d. 13 TP/TP Yes 81 1.30 F508del/F508del 1 n.d.d 1 No special n.d. 14 TP/FN No 198.5 9.45 - / - 8b 103 8 No special CFTRdele2,3/ E664X PAP IRT/PAP strategy, DNA IRT/DNA strategy, TP true positive, FN false negative a Further DNA analysis was not performed in the local CF centre after the health insurance of the patient refused to pay for further DNA analysis. Login to comment
117 Values for IRT/PAP are given for the cut-off value of ≥1.0 ng/mL used in this study and extrapolated for cut-off value of >0.9 ng/mL Patient group Number Newborns screened for CF (April 2008- November 2009) 73,759 CF patients detected (including failsafe protocol) 13 CF patients not detected (missed by all protocols) 1 Prevalence 1: 5268 (2,857-9,493) Screening strategy IRT/DNA (p.F508del, p.G551D, p.R553X, p.G542X) IRT/PAP (cut-off ≥1.0 ng/mL) IRT/PAP (suggested cut-off >0.9 ng/mL) Detected CF patients 10 12 13 False negative results 4 2 1 False positive results 46 86 99 Sensitivity 0.714 (0.478-0.951) 0.857 (0.674-0.999) 0.928 (0.794-0.999) Specificity 0.999 (0.999-0.999) 0.999 (0.998-0.999) 0.999 (0.998-0.999) Positive predictive value 0.179 (0.078-0.278) 0.122 (0.058-0.187) 0.116 (0.057-0.175) Negative predictive value 0.999 (0.999-0.999) 0.999 (0.999-0.999) 0.999 (0.999-0.999) 50 100 150 200 250 300 0 1 2 3 4 5 6 7 8 9 10 Non-CF CF Cut-off 1.0 ng/ml a IRT [ng/ml] PAP[ng/ml] Non-CF CF 0 1 2 3 b 9 10 11 p < 0.02 p < 0.0001 Non-CF carriers PAP[ng/ml] Fig. 3a, b Relationship between IRT and PAP concentrations and summary of PAP concentrations in non-CF and CF newborns. Login to comment

[hide] Mutational spectrum of cystic fibrosis in the Leba... J Cyst Fibros. 2010 Dec;9(6):406-10. Epub 2010 Aug 25. Farra C, Menassa R, Awwad J, Morel Y, Salameh P, Yazbeck N, Majdalani M, Wakim R, Yunis K, Mroueh S, Cabet F
Mutational spectrum of cystic fibrosis in the Lebanese population.
J Cyst Fibros. 2010 Dec;9(6):406-10. Epub 2010 Aug 25., [PMID:20797923]

Abstract [show]
BACKGROUND: Cystic fibrosis (CF) is the most common autosomal recessive disease in Caucasians; it is however, considered to be rare in the Arab populations. Reports of the cystic fibrosis transmembrane regulator (CFTR) mutations from Arabs, especially from the Lebanese population, are limited. METHODS: Twenty-two unrelated Lebanese families, with at least one child with CF, were studied. DNA extracts from blood samples of patients and parents were screened for CFTR gene mutations. RESULTS: Eleven different mutations were identified. Of the 44 alleles studied, the most common mutations were: F508del (34%), N1303K (27%), W1282X (7%), and S4X (7%). Five mutations - not previously reported in the Lebanese population - were identified; these are: S549N, G542X, 2043delG, 4016insG, and R117H-7T. CONCLUSIONS: The most common CFTR mutations in addition to five mutations not previously described in the Lebanese population were identified. Identification of CFTR mutations in the Lebanese population is important for molecular investigations and genetic counseling.

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6 Five mutations - not previously reported in the Lebanese population - were identified; these are: S549N, G542X, 2043delG, 4016insG, and R117H-7T. Login to comment
27 Family Origin Community CM Sex CF mutations Age at diagnosis CP Sweat test 1 Bekaa Maronite No M W1282X Mount Lebanon Maronite F 4010del4 M W1282X/4010del4 1 year Pu Positive 2 North Sunnite Yes M N1303K North Sunnite F N1303K M N1303K/N1303K 7 months Pu, Gi, GR Positive 3 South Shiite Yes M F508del Shiite F F508del M F508del/F508del 2 years Pu, Gi, GR Positive 4 Mount Lebanon Greek-orthodox Yes M F508del Mount Lebanon Maronite F W1282X M F508del/W1282X 2 weeks Gi Positive 5 North Sunnite No M S549N North Sunnite F G542X M S549N/G542X 19 years Pu, Gi Positive 6 Bekaa Sunnite Yes M N1303K Bekaa Sunnite F N1303K M N1303K/N1303K 8 months Gi, GR Positive 7 Beirut Maronite No M 2789+5GNA Beirut Greek-Orthodox F F508del M F508del/2789+5GNA 6 months Pu, Gi, GR Positive 8 North Sunnite Yes M 2043delG North Sunnite F 2043delG M 2043delG/2043delG 6 weeks Gi No data 9 North Sunnite Yes M R117H-7T North Sunnite F R117H-7T M R117H-7T/R117H-7T 3 months Pu Positive 10 South Sunnite Yes M 4016insG South Sunnite F 4016insG M 4016insG/4016insG 3 months Pu Positive M 4016insG/4016insG 5 months Pu Positive 11 Mount Lebanon Maronite No M N1303K Mount Lebanon Greek-Catholic F N1303K M N1303K/N1303K 3 months Pu, Gi Positive 12 North Maronite No M S4X Mount Lebanon Maronite F N1303K M N1303K/S4X 1 month Pu, Gi, Gr Positive 13 Bekaa Greek-Catholic No M F508del No data Maronite F 4010del4 F F508del/4010del4 11 months Pu, Gi Positive 14 No data Greek-Catholic No M W1282X No data Maronite F F508del F W1282X/F508del 2 years No data Positive 15 Mount Lebanon Baptist No M F508del Mount Lebanon Maronite F N1303K F F508del/N1303K 3 years Pu, Gi, Gr Positive 16 North Sunnite Yes M F508del North Sunnite F F508del F F508del/F508del 9 months Pu, Gi, Gr Negative 17 North Sunnite Yes M N1303K Sunnite F N1303K F N1303K./N1303K 2 years Pu, Gr Positive 18 North Sunnite No M F508del North Sunnite F F508del F F508del/F508del 7 months Pu, Gi, Gr Positive 19 North Maronite No M F508del No data Maronite F F508del M F508del/F508del 7 years Pu, Gi, Gr Positive 20 Beirut Maronite No data M S4X No data Maronite F S4X M S4X/S4X 9 months Pu, Gi No data (continued on next page) diagnosis was based mainly on the clinical picture according to the consensus criteria [16] and was confirmed when possible by a positive sweat chloride test. Login to comment
39 These mutations were W1282X, 4010del4, N1303K, F508del, S549N, G542X, 2043delG, R117H-7T, 2789 + 5G NA, 4016insG, and S4X. Login to comment
42 Five mutations that were not previously reported in the Lebanese population were identified, these are: S549N, G542X, 2043delG, 4016insG and R117H-7T. Login to comment
51 Common CFTR mutations in the Lebanese population Frequency of CF alleles (%) Lebanona Palestine [17] Jordan [24] Syria [29] Saudi Arabia, United Arab Emirates, Oman, Qatar, Kuwait, and Jordan [1] Saudi Arabia [3,25] Bahrain [27] F508 del 36.3 23.5 7.4 1 patient 12 15 7.7 W1282X 13.8 10.6 N1303K 20 21 1.5 3-14 4010del4 7.5 2.3 S4X 6.3 2789+5GNA 2.5 2043delG 2.5 7 30.8 4016insG 2.5 R117H-7T 2.5 G542X 1.3 1.2 4096-28G→A 1.3 E672del 1.3 M952I 1.3 S549N 1.3 a Mutations in a total of 80 identified CF alleles in the Lebanese population from this study combined with Desgeorges et al. (1997) [2]. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Curr Opin Pulm Med. 2010 Nov;16(6):591-7. Sloane PA, Rowe SM
Cystic fibrosis transmembrane conductance regulator protein repair as a therapeutic strategy in cystic fibrosis.
Curr Opin Pulm Med. 2010 Nov;16(6):591-7., [PMID:20829696]

Abstract [show]
PURPOSE OF REVIEW: Recent progress in understanding the production, processing, and function of the cystic fibrosis gene product, the cystic fibrosis transmembrane conductance regulator (CFTR), has revealed new therapeutic targets to repair the mutant protein. Classification of CFTR mutations and new treatment strategies to address each will be described here. RECENT FINDINGS: High-throughput screening and other drug discovery efforts have identified small molecules that restore activity to mutant CFTR. Compounds such as VX-770 that potentiate CFTR have demonstrated exciting results in recent clinical trials and demonstrate robust effects across several CFTR mutation classes in the laboratory. A number of novel F508del CFTR processing correctors restore protein to the cell surface and improve ion channel function in vitro and are augmented by coadministration of CFTR potentiators. Ongoing discovery efforts that target protein folding, CFTR trafficking, and cell stress have also indicated promising results. Aminoglycosides and the novel small molecule ataluren induce translational readthrough of nonsense mutations in CFTR and other genetic diseases in vitro and in vivo and have shown activity in proof of concept trials, and ataluren is now being studied in confirmatory trials. SUMMARY: An improved understanding of the molecular mechanisms underlying the basic genetic defect in cystic fibrosis have led to new treatment strategies to repair the mutant protein.

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100 Surface localized full-length CFTR was substantially improved in cross-sections of intestinal tissues following administration to CF mice carrying the G542X mutation and restored CFTR function by Ussing chamber analysis in intestinal samples of mice after 2 weeks of treatment with ataluren. Login to comment

[hide] Cystic fibrosis and survival to 40 years: a study ... Eur Respir J. 2011 May;37(5):1076-82. Epub 2010 Sep 16. Simmonds NJ, D'Souza L, Roughton M, Alton EW, Davies JC, Hodson ME
Cystic fibrosis and survival to 40 years: a study of cystic fibrosis transmembrane conductance regulator function.
Eur Respir J. 2011 May;37(5):1076-82. Epub 2010 Sep 16., [PMID:20847077]

Abstract [show]
Significant survival heterogeneity exists in cystic fibrosis. Our aim was to determine whether residual function of the cystic fibrosis transmembrane conductance regulator (CFTR) is present in long-term survivors with severe mutations. Nasal potential difference (PD) and sweat chloride were measured in 34 long-term survivors (aged >/= 40 yrs) and compared with young patients (18-23 yrs) with severe (n = 30) and mild (n = 31) lung disease. Baseline PD was not significantly different across the three groups (long-term survivors, -42.8 (range -71.0- -20.5) mV; young/mild, -40.5 (-58.8- -19.5) mV; young/severe,-46.3 (-74.0- -20.0) mV). Response to amiloride (DeltaAmil) was significantly different across the three groups (p = 0.01); long-term survivors had values (27.8 (range 8.5-46) mV) which were not different to either young group, but the young/severe group had significantly higher values (29.5 (11-47) mV) than those in the young/mild group (22.0 (7-39) mV; p<0.01). Baseline PD and DeltaAmil were associated with forced expiratory volume in 1 s (FEV) (co-efficient (95% CI) -0.13 (-0.23- -0.03); p = 0.009 and -0.12 (-0.20- -0.04); p = 0.003, respectively). Sweat chloride was lowest (p <0.05) in the young/severe group (93.5 (74-111) mmol.L(1) versus 98.8 (76.5-116.0) mmol.L(1); long-term survivors; and 99.5 (80.0-113.5) mmol.L(1); young/mild). Delta Amil is associated with FEV but our findings indicate that long-term survival cannot be explained by residual CFTR function when measurements are taken in later life.

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57 76 (80.9%) patients were homozygous DF508 and the remainder were DF508 compound heterozygotes (with genotype G551D (n55), G542X (n53), N1303K (n53), 1717-1GRA (n52), 621-1GRT (n51), R1162X (n51), 2789+3delG (n51), 3659delC (n51) and D1507 (n51)); no significant difference between the groups for any genotype was present. Login to comment

[hide] Type of CFTR mutation determines risk of pancreati... Gastroenterology. 2011 Jan;140(1):153-61. Epub 2010 Nov 9. Ooi CY, Dorfman R, Cipolli M, Gonska T, Castellani C, Keenan K, Freedman SD, Zielenski J, Berthiaume Y, Corey M, Schibli S, Tullis E, Durie PR
Type of CFTR mutation determines risk of pancreatitis in patients with cystic fibrosis.
Gastroenterology. 2011 Jan;140(1):153-61. Epub 2010 Nov 9., [PMID:20923678]

Abstract [show]
BACKGROUND & AIMS: Different mutations in the cystic fibrosis gene (CFTR) are associated with different functional status of the exocrine pancreas. We investigated whether CFTR genotypes determine the risk of pancreatitis in patients with cystic fibrosis (CF). METHODS: Patients with pancreatic-sufficient CF were identified from 2 CF population-based databases (N = 277; 62 with pancreatitis and 215 without pancreatitis); patients' genotypes and clinical characteristics were analyzed. The loss of pancreatic function associated with each CFTR genotype was determined based on the pancreatic insufficiency prevalence (PIP) score. RESULTS: Patients with pancreatitis were more likely to have genotypes associated with mild (70%) than moderate-severe (30%) PIP scores (P = .004). The cumulative proportion of patients who developed pancreatitis through to the age of 50 years was significantly greater for genotypes associated with mild (50%) than moderate-severe (27%) PIP scores (P = .006). The genotype associated with mild PIP scores had a hazard ratio of 2.4 for pancreatitis (95% confidence interval, 1.3-4.5; P = .006). Patients with pancreatitis were diagnosed with CF at an older median age than those without pancreatitis (14.9 years [interquartile range, 9.5-27.7] vs 9.3 years [interquartile range, 1.5-21.4]; P = .003) and had lower mean levels of sweat chloride than patients without pancreatitis (74.5 +/- 26.2 mmol/L vs 82.8 +/- 25.2 mmol/L; P = .03). CONCLUSIONS: Specific CFTR genotypes are significantly associated with pancreatitis. Patients with genotypes associated with mild phenotypic effects have a greater risk of developing pancreatitis than patients with genotypes associated with moderate-severe phenotypes. This observation provides further insight into the complex pathogenesis of pancreatitis.

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55 PIP Scores for Common, Well-Defined CFTR Mutations Mutation Canadian Consortium for CF Genetic Studies Verona CF Centre Mutation classTotal PI Total PIϩPS PIP score Total PI Total PIϩPS PIP score 621ϩ1GϾT 96 96 1.00 4 4 1.00 I-III 711ϩ1GϾT 36 36 1.00 1 1 1.00 I-III R553X 24 24 1.00 9 9 1.00 I-III I507del 11 11 1.00 12 12 1.00 I-III G542X 74 75 0.99 22 22 1.00 I-III F508del 1276 1324 0.96 181 188 0.96 I-III 1717-1GϾA 20 21 0.95 23 24 0.96 I-III W1282X 19 20 0.95 2 2 1.00 I-III N1303K 45 48 0.94 30 31 0.97 I-III R1162X 12 13 0.92 21 22 0.95 I-III G551D 59 67 0.88 0 0 - I-III G85E 16 22 0.73 4 5 0.80 I-III A455E 18 37 0.49 0 0 - IV-V 2789ϩ5GϾA 6 16 0.38 3 11 0.27 IV-V R334W 1 10 0.10 0 0 - IV-V 3849ϩ10kbCϾT 2 22 0.09 0 1 0.00 IV-V R117H 1 25 0.04 0 0 - IV-V NOTE. Login to comment

[hide] Mutations that permit residual CFTR function delay... Respir Res. 2010 Oct 8;11:140. Green DM, McDougal KE, Blackman SM, Sosnay PR, Henderson LB, Naughton KM, Collaco JM, Cutting GR
Mutations that permit residual CFTR function delay acquisition of multiple respiratory pathogens in CF patients.
Respir Res. 2010 Oct 8;11:140., [PMID:20932301]

Abstract [show]
BACKGROUND: Lung infection by various organisms is a characteristic feature of cystic fibrosis (CF). CFTR genotype effects acquisition of Pseudomonas aeruginosa (Pa), however the effect on acquisition of other infectious organisms that frequently precede Pa is relatively unknown. Understanding the role of CFTR in the acquisition of organisms first detected in patients may help guide symptomatic and molecular-based treatment for CF. METHODS: Lung infection, defined as a single positive respiratory tract culture, was assessed for 13 organisms in 1,381 individuals with CF. Subjects were divided by predicted CFTR function: 'Residual': carrying at least one partial function CFTR mutation (class IV or V) and 'Minimal' those who do not carry a partial function mutation. Kaplan-Meier estimates were created to assess CFTR effect on age of acquisition for each organism. Cox proportional hazard models were performed to control for possible cofactors. A separate Cox regression was used to determine whether defining infection with Pa, mucoid Pa or Aspergillus (Asp) using alternative criteria affected the results. The influence of severity of lung disease at the time of acquisition was evaluated using stratified Cox regression methods by lung disease categories. RESULTS: Subjects with 'Minimal' CFTR function had a higher hazard than patients with 'Residual' function for acquisition of 9 of 13 organisms studied (HR ranging from 1.7 to 3.78 based on the organism studied). Subjects with minimal CFTR function acquired infection at a younger age than those with residual function for 12 of 13 organisms (p-values ranging: < 0.001 to 0.017). Minimal CFTR function also associated with younger age of infection when 3 alternative definitions of infection with Pa, mucoid Pa or Asp were employed. Risk of infection is correlated with CFTR function for 8 of 9 organisms in patients with good lung function (>90%ile) but only 1 of 9 organisms in those with poorer lung function (<50%ile). CONCLUSIONS: Residual CFTR function correlates with later onset of respiratory tract infection by a wide spectrum of organisms frequently cultured from CF patients. The protective effect conferred by residual CFTR function is diminished in CF patients with more advanced lung disease.

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74 For Pa, the hazard ratio Table 1 Classification of CFTR alleles Category Mutation Specific mutations Class I Defective Protein Synthesis (nonsense, frameshift, aberrant splicing) 1078delT, 1154 insTC, 1525-2A > G, 1717-1G > A, 1898+1G > A, 2184delA, 2184 insA, 3007delG, 3120+1G > A, 3659delC, 3876delA, 3905insT, 394delTT, 4010del4, 4016insT, 4326delTC, 4374+1G > T, 441delA, 556delA, 621+1G > T, 621-1G > T, 711+1G > T, 875+1G > C, E1104X, E585X, E60X, E822X, G542X, G551D/R553X, Q493X, Q552X, Q814X, R1066C, R1162X, R553X, V520F, W1282X, Y1092X Class II Abnormal Processing and Trafficking A559T, D979A, ΔF508, ΔI507, G480C, G85E, N1303K, S549I, S549N, S549R Class III Defective Channel Regulation/Gating G1244E, G1349D, G551D, G551S, G85E, H199R, I1072T, I48T, L1077P, R560T, S1255P, S549 (R75Q) Class IV Decreased Channel Conductance A800G, D1152H, D1154G, D614G, delM1140, E822K, G314E, G576A, G622D, G85E, H620Q, I1139V, I1234V, L1335P, M1137V, P67L, R117C, R117P, R117H, R334W, R347H, R347P, R347P/ R347H, R792G, S1251N, V232D Class V Reduced Synthesis and/or Trafficking 2789+5G > A, 3120G > A, 3272-26A > G, 3849+10kbC > T, 5T variant, 621+3A > G, 711+3A > G, A445E, A455E, IVS8 poly T, P574H was increased 3 fold for those with 'Minimal` function when compared to those with 'Residual` function. Login to comment

[hide] Effect of VX-770 in persons with cystic fibrosis a... N Engl J Med. 2010 Nov 18;363(21):1991-2003. Accurso FJ, Rowe SM, Clancy JP, Boyle MP, Dunitz JM, Durie PR, Sagel SD, Hornick DB, Konstan MW, Donaldson SH, Moss RB, Pilewski JM, Rubenstein RC, Uluer AZ, Aitken ML, Freedman SD, Rose LM, Mayer-Hamblett N, Dong Q, Zha J, Stone AJ, Olson ER, Ordonez CL, Campbell PW, Ashlock MA, Ramsey BW
Effect of VX-770 in persons with cystic fibrosis and the G551D-CFTR mutation.
N Engl J Med. 2010 Nov 18;363(21):1991-2003., 2010-11-18 [PMID:21083385]

Abstract [show]
BACKGROUND: A new approach in the treatment of cystic fibrosis involves improving the function of mutant cystic fibrosis transmembrane conductance regulator (CFTR). VX-770, a CFTR potentiator, has been shown to increase the activity of wild-type and defective cell-surface CFTR in vitro. METHODS: We randomly assigned 39 adults with cystic fibrosis and at least one G551D-CFTR allele to receive oral VX-770 every 12 hours at a dose of 25, 75, or 150 mg or placebo for 14 days (in part 1 of the study) or VX-770 every 12 hours at a dose of 150 or 250 mg or placebo for 28 days (in part 2 of the study). RESULTS: At day 28, in the group of subjects who received 150 mg of VX-770, the median change in the nasal potential difference (in response to the administration of a chloride-free isoproterenol solution) from baseline was -3.5 mV (range, -8.3 to 0.5; P=0.02 for the within-subject comparison, P=0.13 vs. placebo), and the median change in the level of sweat chloride was -59.5 mmol per liter (range, -66.0 to -19.0; P=0.008 within-subject, P=0.02 vs. placebo). The median change from baseline in the percent of predicted forced expiratory volume in 1 second was 8.7% (range, 2.3 to 31.3; P=0.008 for the within-subject comparison, P=0.56 vs. placebo). None of the subjects withdrew from the study. Six severe adverse events occurred in two subjects (diffuse macular rash in one subject and five incidents of elevated blood and urine glucose levels in one subject with diabetes). All severe adverse events resolved without the discontinuation of VX-770. CONCLUSIONS: This study to evaluate the safety and adverse-event profile of VX-770 showed that VX-770 was associated with within-subject improvements in CFTR and lung function. These findings provide support for further studies of pharmacologic potentiation of CFTR as a means to treat cystic fibrosis. (Funded by Vertex Pharmaceuticals and others; ClinicalTrials.gov number, NCT00457821.).

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70 (%)‡4(100)4(100)4(100)4(100)4(100)20(100)4(100)8(100)7(100)19(100) Age-yr Median36314126213024232121 Range19to4822to5122to5019to3419to3319to5118to4218to4020to3818to42 Body-massindex Median23232420212322222322 Range22to2920to2419to2719to2417to2617to2921to2320to2320to2520to25 CFTRgenotype G551D/F508del3(75)4(100)4(100)2(50)3(75)16(80)4(100)7(88)5(71)16(84) G551D/1078delT1(25)----1(5)---- G551D/G551D----1(25)1(5)---- G551D/N1303K---1(25)-1(5)---- G551D/R553X---1(25)-1(5)---- G551D/3849+10kbC→T--------1(14)1(5) G551D/621+1G→T-------1(12)-1(5) G551D/G542X--------1(14)1(5) FEV1 Median%ofpredictedvalue57665663495677657669 Range%ofpredictedvalue48to9744to10942to6546to10242to5842to10953to11242to12240to10640to122 40to<70%ofpredictedvalue -no. Login to comment
65 (%)‡4(100)4(100)4(100)4(100)4(100)20(100)4(100)8(100)7(100)19(100) Age-yr Median36314126213024232121 Range19to4822to5122to5019to3419to3319to5118to4218to4020to3818to42 Body-massindex Median23232420212322222322 Range22to2920to2419to2719to2417to2617to2921to2320to2320to2520to25 CFTRgenotype G551D/F508del3(75)4(100)4(100)2(50)3(75)16(80)4(100)7(88)5(71)16(84) G551D/1078delT1(25)----1(5)---- G551D/G551D----1(25)1(5)---- G551D/N1303K---1(25)-1(5)---- G551D/R553X---1(25)-1(5)---- G551D/3849+10kbC→T--------1(14)1(5) G551D/621+1G→T-------1(12)-1(5) G551D/G542X--------1(14)1(5) FEV1 Median%ofpredictedvalue57665663495677657669 Range%ofpredictedvalue48to9744to10942to6546to10242to5842to10953to11242to12240to10640to122 40to<70%ofpredictedvalue -no. Login to comment

[hide] Comprehensive description of CFTR genotypes and ul... Hum Genet. 2011 Apr;129(4):387-96. Epub 2010 Dec 24. de Becdelievre A, Costa C, Jouannic JM, LeFloch A, Giurgea I, Martin J, Medina R, Boissier B, Gameiro C, Muller F, Goossens M, Alberti C, Girodon E
Comprehensive description of CFTR genotypes and ultrasound patterns in 694 cases of fetal bowel anomalies: a revised strategy.
Hum Genet. 2011 Apr;129(4):387-96. Epub 2010 Dec 24., [PMID:21184098]

Abstract [show]
Fetal bowel anomalies may reveal cystic fibrosis (CF) and the search for CF transmembrane conductance regulator (CFTR) gene mutations is part of the diagnostic investigations in such pregnancies, according to European recommendations. We report on our 18-year experience to document comprehensive CFTR genotypes and correlations with ultrasound patterns in a series of 694 cases of fetal bowel anomalies. CFTR gene analysis was performed in a multistep process, including search for frequent mutations in the parents and subsequent in-depth search for rare mutations, depending on the context. Ultrasound patterns were correlated with the genotypes. Cases were distinguished according to whether they had been referred directly to our laboratory or after an initial testing in another laboratory. A total of 30 CF fetuses and 8 cases compatible with CFTR-related disorders were identified. CFTR rearrangements were found in 5/30 CF fetuses. 21.2% of fetuses carrying a frequent mutation had a second rare mutation, indicative of CF. The frequency of CF among fetuses with no frequent mutation was 0.43%. Correlation with ultrasound patterns revealed a significant frequency of multiple bowel anomalies in CF fetuses. The results emphasize the need to search for rearrangements in the diagnosis strategy of fetal bowel anomalies. The diagnostic value of ultrasound patterns combining hyperechogenic bowel, loop dilatation and/or non-visualized gallbladder reveals a need to revise current strategies and to offer extensive CFTR gene testing when the triad is diagnosed, even when no frequent mutation is found in the first-step analysis.

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138 [2125C[T] 28 - - MP Born, CF, MI [G542X]? Login to comment

[hide] Low abundance of sweat duct Cl- channel CFTR in bo... Am J Physiol Regul Integr Comp Physiol. 2011 Mar;300(3):R605-15. Epub 2011 Jan 12. Brown MB, Haack KK, Pollack BP, Millard-Stafford M, McCarty NA
Low abundance of sweat duct Cl- channel CFTR in both healthy and cystic fibrosis athletes with exceptionally salty sweat during exercise.
Am J Physiol Regul Integr Comp Physiol. 2011 Mar;300(3):R605-15. Epub 2011 Jan 12., [PMID:21228336]

Abstract [show]
To understand potential mechanisms explaining interindividual variability observed in human sweat sodium concentration ([Na(+)]), we investigated the relationship among [Na(+)] of thermoregulatory sweat, plasma membrane expression of Na(+) and Cl(-) transport proteins in biopsied human eccrine sweat ducts, and basal levels of vasopressin (AVP) and aldosterone. Lower ductal luminal membrane expression of the Cl(-) channel cystic fibrosis transmembrane conductance regulator (CFTR) was observed in immunofluorescent staining of sweat glands from healthy young adults identified as exceptionally "salty sweaters" (SS) (n = 6, P < 0.05) and from patients with cystic fibrosis (CF) (n = 6, P < 0.005) compared with ducts from healthy young adults with "typical" sweat [Na(+)] (control, n = 6). Genetic testing of healthy subjects did not reveal any heterozygotes ("carriers") for any of the 39 most common disease-causing CFTR mutations in the United States. SS had higher baseline plasma [AVP] compared with control (P = 0.029). Immunostaining to investigate a potential relationship between higher plasma [AVP] (and sweat [Na(+)]) and ductal membrane aquaporin-5 revealed for all groups a relatively sparse and location-dependent ductal expression of the water channel with localization primarily to the secretory coil. Availability of CFTR for NaCl transport across the ductal membrane appears related to the significant physiological variability observed in sweat salt concentration in apparently healthy humans. At present, a heritable link between healthy salty sweaters and the most prevalent disease-causing CFTR mutations cannot be established.

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114 Mutations tested in this panel were ⌬F508, R334W, S549N, 3659delC, ⌬I507, I347P, A559T, S1255X, 1898ϩ1GϾA, R347H, N1303K, 1898ϩ5GϾT, 3876delA, A455E, 394delTT, 2183GGϾA, 3905insT, 3120ϩ1GϾA, V520F, 2184delA, G85E, Y1092X, 711ϩ1GϾT, 2307insA, Y122X, S549R, M1101K, 1078delT, 2789ϩ5GϾA, G551D, G542X, 621ϩ1GϾT, R560T, W1282X, 1717-1 GϾA, 3849 ϩ 10KbCϾT, R553X, R117H, and R1162X. Login to comment
119 Mutations tested in this panel were ⌬F508, R334W, S549N, 3659delC, ⌬I507, I347P, A559T, S1255X, 1898ϩ1GϾA, R347H, N1303K, 1898ϩ5GϾT, 3876delA, A455E, 394delTT, 2183GGϾA, 3905insT, 3120ϩ1GϾA, V520F, 2184delA, G85E, Y1092X, 711ϩ1GϾT, 2307insA, Y122X, S549R, M1101K, 1078delT, 2789ϩ5GϾA, G551D, G542X, 621ϩ1GϾT, R560T, W1282X, 1717-1 GϾA, 3849 ϩ 10KbCϾT, R553X, R117H, and R1162X. Login to comment

[hide] Chronic ataluren (PTC124) treatment of nonsense mu... Eur Respir J. 2011 Jul;38(1):59-69. Epub 2011 Jan 13. Wilschanski M, Miller LL, Shoseyov D, Blau H, Rivlin J, Aviram M, Cohen M, Armoni S, Yaakov Y, Pugatch T, Cohen-Cymberknoh M, Miller NL, Reha A, Northcutt VJ, Hirawat S, Donnelly K, Elfring GL, Ajayi T, Kerem E
Chronic ataluren (PTC124) treatment of nonsense mutation cystic fibrosis.
Eur Respir J. 2011 Jul;38(1):59-69. Epub 2011 Jan 13., [PMID:21233271]

Abstract [show]
In a subset of patients with cystic fibrosis (CF), nonsense mutations (premature stop codons) disrupt production of full-length, functional CF transmembrane conductance regulator (CFTR). Ataluren (PTC124) allows ribosomal readthrough of premature stop codons in mRNA. We evaluated drug activity and safety in patients with nonsense mutation CF who took ataluren three times daily (morning, midday and evening) for 12 weeks at either a lower dose (4, 4 and 8 mg.kg(-1)) or higher dose (10, 10 and 20 mg.kg(-1)). The study enrolled 19 patients (10 males and nine females aged 19-57 yrs; dose: lower 12, higher seven) with a classic CF phenotype, at least one CFTR nonsense mutation allele, and an abnormal nasal total chloride transport. Both ataluren doses were similarly active, improving total chloride transport with a combined mean change of -5.4 mV (p<0.001), and on-treatment responses (at least -5 mV improvement) and hyperpolarisations (values more electrically negative than -5 mV) in 61% (p<0.001) and 56% (p = 0.002) of patients. CFTR function was greater with time and was accompanied by trends toward improvements in pulmonary function and CF-related coughing. Adverse clinical and laboratory findings were uncommon and usually mild. Chronic ataluren administration produced time-dependent improvements in CFTR activity and clinical parameters with generally good tolerability.

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104 Ataluren treatment was associated with an on-treatment total chloride response in both patients with the G542X and W1282X mutations. Login to comment
110 Changes in total chloride transport were TABLE 1 Baseline patient characteristics Ataluren dose level Low# High" Combined Age yrs 21 (19-57) 27 (19-45) 26 (19-57) Sex Male 8 2 10 Female 4 5 9 BMI 22.3 (15.4-27.8) 21.8 (19.0-26.6) 21.8 (15.4-27.8) Sweat chloride+ mEq?L-1 88 (47-109) 89 (49-106) 88 (47-109) Nasal total chloride transport1 mV 1.6 (-1.6-9.3) -0.3 (-2.3-1.9) 0.7 (-2.3-9.3) Pulmonary function % prede FEV1 64 (44-106) 65 (46-92) 65 (44-106) FVC 82 (63-109) 77 (57-101) 78 (57-109) Pathological lung infection 12 6 18 Pseudomonas aeruginosa 11 6 17 Mycobacterium abscessus 2 0 2 Methicillin-resistant Staphylococcus aureus 1 0 1 None 0 1 1 Pancreatic insufficiency Exocrine 11 6 17 Endocrine 2 5 7 Abnormalities of liver-related serum parameters 3 2 5 ALT 0 2 2 AST 1 0 1 Alkaline phosphatase 2 1 3 GGT 0 0 0 Bilirubin 1 0 1 LDH 1 0 1 Genotype allele 1/allele 2 G542X (UGA)/DF508 2 0 2 G542X (UGA)/W1282X (UGA) 0 1 1 G542X (UGA)/N1303K 0 1 1 W1282X (UGA)/DF508 8 2 10 W1282X (UGA)/W1282X (UGA) 1 2 3 W1282X (UGA)/3849+10kB CRT## (UAA) 0 1 1 3849+10kB CRT## (UAA)/DF508 1 0 1 Time from last ataluren dose in prior study months 10.5 (9.3-12.2) 10.5 (8.5-11.5) 10.5 (8.5-12.2) Data are presented as n or median (range). Login to comment
121 Rather than using the mean NPD values obtained from both nostrils (as in the primary analyses), we also analysed outcome 5 0 Response -5 -15 -10 Changefrombaseline totalchloridetransportmV CFTR mutation type G542X/ΔF508 W1282X/ΔF508 W1282X/ΔF508 W1282X/ΔF508 W1282X/ΔF508 W1282X/ΔF508 W1282X/ΔF508 W1282X/ΔF508 W1282X/ΔF508 W1282X/ΔF508 W1282X/G542X W1282X/W1282X W1282X/W1282X W1282X/W1282X G542X/N1303K G542X/ΔF508 W1282X/ΔF508 W1282X/3849+10KBC→T 4, 4 and 8 mg·kg-1 (n=11) 10, 10 and 20 mg·kg-1 (n=7) FIGURE 1. Login to comment
191 Total chloride transport improvements were noted in patients with both G542X and W1282X premature stop codon mutations, confirming data from our prior study [20] and from other ataluren trials [42], indicating that multiple nonsense mutation genotypes can be responsive to ataluren therapy. Login to comment

[hide] Combating Cystic Fibrosis: In Search for CF Transm... ChemMedChem. 2011 Jan 14. Noy E, Senderowitz H
Combating Cystic Fibrosis: In Search for CF Transmembrane Conductance Regulator (CFTR) Modulators.
ChemMedChem. 2011 Jan 14., 2011-01-14 [PMID:21240931]

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19 Class I mutations are found in ~10% of CF patients, and the most common ones are G542X and W1282X, the latter being particularly common in Ashkenazi Jews. Login to comment

[hide] An overview of international literature from cysti... J Cyst Fibros. 2011 Mar;10(2):71-85. Epub 2011 Jan 22. Salvatore D, Buzzetti R, Baldo E, Forneris MP, Lucidi V, Manunza D, Marinelli I, Messore B, Neri AS, Raia V, Furnari ML, Mastella G
An overview of international literature from cystic fibrosis registries. Part 3. Disease incidence, genotype/phenotype correlation, microbiology, pregnancy, clinical complications, lung transplantation, and miscellanea.
J Cyst Fibros. 2011 Mar;10(2):71-85. Epub 2011 Jan 22., [PMID:21257352]

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This is the third article related to a review of the literature based on data from national cystic fibrosis (CF) patient registries up to June 2008 and covering a total of 115 published studies. It focuses on several topics: CF incidence, genotype/phenotype correlation, microbiology, pregnancy/paternity, clinical complications, lung transplantation, and others. Seventy seven papers meeting the inclusion criteria were found to be related to the topics listed above. Another seven studies, already evaluated in previous papers of this series, were recalled for specific topics. Incidence is described by several studies, results being quite different from one country to another and quite inhomogeneous among regions within the same country. Studies on genetics address the genotype/phenotype correlation and look for a predictive value of CFTR mutations in terms of clinical outcome, with controversial results. Papers on microbiology describe the clinical relevance of different pathogens and their role in the progress of CF lung disease. A few articles give information on the features of CF women undergoing a pregnancy and try to identify the ones associated with a better outcome. Studies on clinical complications discuss prevalence and the role of haemoptysis, pneumothorax, CF related diabetes, ABPA and cancer. Papers on lung transplantation focus on models able to improve the selection criteria for transplantation candidates and the factors linked to post transplantation survival. Finally, several studies deal with a number of interesting topics related to CF epidemiology: clinical trial methodology, quality of care comparison among countries and centers, relationship between diagnosis and age/gender, and evaluation of pharmacological therapy. On the whole, CF Registries have already contributed to important advances in the knowledge of the natural history of CF, establishing the foundations for future improvement in CF research and care.

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1264 The 3 most common mutations associated with MI were F508del, G542X, and W1282X. Login to comment

[hide] Combating cystic fibrosis: in search for CF transm... ChemMedChem. 2011 Feb 7;6(2):243-51. doi: 10.1002/cmdc.201000488. Epub 2011 Jan 14. Noy E, Senderowitz H
Combating cystic fibrosis: in search for CF transmembrane conductance regulator (CFTR) modulators.
ChemMedChem. 2011 Feb 7;6(2):243-51. doi: 10.1002/cmdc.201000488. Epub 2011 Jan 14., 2011-02-07 [PMID:21275046]

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19 Class I mutations are found in ~10% of CF patients, and the most common ones are G542X and W1282X, the latter being particularly common in Ashkenazi Jews. Login to comment

[hide] Distribution of CFTR mutations in Eastern Hungaria... J Cyst Fibros. 2011 May;10(3):217-20. doi: 10.1016/j.jcf.2010.12.009. Epub 2011 Feb 4. Ivady G, Madar L, Nagy B, Gonczi F, Ajzner E, Dzsudzsak E, Dvorakova L, Gombos E, Kappelmayer J, Macek M Jr, Balogh I
Distribution of CFTR mutations in Eastern Hungarians: relevance to genetic testing and to the introduction of newborn screening for cystic fibrosis.
J Cyst Fibros. 2011 May;10(3):217-20. doi: 10.1016/j.jcf.2010.12.009. Epub 2011 Feb 4., [PMID:21296036]

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BACKGROUND: The aim of this study was characterization of an updated distribution of CFTR mutations in a representative cohort of 40 CF patients with the classical form of the disease drawn from Eastern Hungary. Due to the homogeneity of the Hungarian population our data are generally applicable to other regions of the country, including the sizeable diaspora. METHODS: We utilized the recommended "cascade" CFTR mutation screening approach, initially using a commercial assay, followed by examination of the common "Slavic" deletion CFTRdele2,3(21kb). Subsequently, the entire CFTR coding region of the CFTR gene was sequenced in patients with yet unidentified mutations. RESULTS: The Elucigene CF29(Tm) v2 assay detected 81.25% of all CF causing mutations. An addition of the CFTRdele2,3(21kb) increased the mutation detection rate to 86.25%. DNA sequencing enabled us to identify mutations on 79/80 CF alleles. Mutations [CFTRdele2,3(21kb), p.Gln685ThrfsX4 (2184insA) were found at an unusually high frequency, each comprising 5.00% of all CF alleles. CONCLUSION: We have identified common CF causing mutations in the Hungarian population with the most common mutations (p.Phe508del, p.Asn1303Lys, CFTRdele2,3(21kb), 2184insA, p.Gly542X, and p.Leu101X), comprising over 93.75% of all CF alleles. Obtained data are applicable to the improvement of DNA diagnostics in Hungary and beyond, and are the necessary prerequisite for the introduction of a nationwide "two tier" CF newborn screening program.

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77 CFTR mutation Germany 1994 Romania 2008 Austria 1997 Slovakia 2008 Hungary 1992 This study deltaF508 (c.1521_1523 delCTT) 72.0% 56.3% 74.6% 38.2% 64.3% 70.0% G551D (c.1652 GNA) 1.0% N/F 1.6% N/F N/F N/F R553X (c.1657 CNT) 2.3% N/F N/F 1.2% 2.4% N/F G542X (c.1624 GNT) 1.4% 3.9% 2.4% 2.4% 1.2% 3.75% 621+1 GNT (c.489+1 GNT) 0.1% 0.8% N/F N/F N/F N/F 1717-1 GNA (c.1585-1 GNA) 0.9% N/F 0.8% 0.6% 1.2% 1.25% W1282X (c.3846 GNA) 0.7% 2.3% N/F N/F 1.2% N/F N1303K (c.3909 CNG) 2.3% 0.8% N/F 1.2% 1.2% 5.0% R347P (c.1040 GNC) 1.6% N/F 1.6% 1.2% N/A 1.25% CFTRdele2,3(21 kb) 1.5%a 1.6% 2.6%a 1.1%a N/A 5.0% 2184insA (c.2052_2053 insA) 0.6% N/F N/F 2.4% N/A 5.0% L101X (c.302 TNG) N/F N/F N/F N/F N/A 2.5% Q220X (c.658 CNT) N/F N/F N/F N/F N/A 1.25% S466X (c.1397 CNG) N/F N/F N/F N/F N/A 1.25% E831X (c.2491 GNT) N/F N/F N/F 0.6% N/A 1.25% Y1092X (c.3276 CNA) 0.3% N/F N/F N/F N/A 1.25% Legend: data for Germany [8], Romania [9], Austria [10], Slovakia [11] and Hungary [3]; N/A: not analyzed; N/F: not found, a frequencies reported by Dork et al. in 2000 [6], mutations included in the Elucigene CF29 v2 assay are formatted in italics; the original "legacy name" is followed by the recommended mutation nomenclature [17]. Login to comment

[hide] Association between genotype and pulmonary phenoty... J Cyst Fibros. 2011 May;10(3):187-92. doi: 10.1016/j.jcf.2011.01.005. Epub 2011 Feb 26. Geborek A, Hjelte L
Association between genotype and pulmonary phenotype in cystic fibrosis patients with severe mutations.
J Cyst Fibros. 2011 May;10(3):187-92. doi: 10.1016/j.jcf.2011.01.005. Epub 2011 Feb 26., [PMID:21354377]

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BACKGROUND: Despite numerous studies a clear relationship between genotype and pulmonary phenotype has not been established within the group pancreatic insufficient cystic fibrosis (CF) patients. We studied the relationship between class I and class II mutations and pulmonary function in Swedish patients with known CFTR functional classification. METHODS: 170 CF patients with two class II mutations, 18 with two class I mutations and 78 with a combination of class I and II mutations were included in the study. Spirometry was performed when patients were in an optimal clinical condition. RESULTS: Patients with two class I mutations had lower lung function (FEV(1) and FVC) compared to the group with either a combination of class I and II mutations or two class II mutations. CONCLUSION: CF patients carrying two class I mutations risk developing more severe lung disease compared to patients with at least one class II mutation.

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95 Class I/class I Class I/class II Class II/class II 1717-1 G-NA/1717-1 G-NA n=1 3659delC/S945L n=1 F508del/F508del n=165 3659delC/3659delC n=5 3659delC/F508del n=23 F508del/S945L n=5 3659delC/394delTT n=7 394delTT/F508del n=38 394delTT/394delTT n=4 621+1 G-NT/F508del n=6 R553X/E60X n=1 E60X/F508del n=4 G542X/F508del n=1 R553X/F508del n=2 W79R/F508del n=1 W1282X/F508del n=1 1717-1 G-NA/F508del n=1 Total 18 78 170 The other class combinations are not shown. Login to comment
98 Class I Class II Class III Class IV Class V 1717-1 G-NA F508del G551D 297 C-NA 2789+5 G-NA 3659delC S945L R560T R117C 3849+10 kb CNT 394delTT R347P A455E R553X T 3381 3849+10 kb C-T 621+1 G-NT E60X G542X W79R W1282X decline of pulmonary function was more rapid in patients with pancreatic insufficiency, mainly class II mutations, compared to CF patients with normal pancreatic function [4]. Login to comment

[hide] Optimal DNA tier for the IRT/DNA algorithm determi... J Cyst Fibros. 2011 Jul;10(4):278-81. Epub 2011 Mar 8. Baker MW, Groose M, Hoffman G, Rock M, Levy H, Farrell PM
Optimal DNA tier for the IRT/DNA algorithm determined by CFTR mutation results over 14 years of newborn screening.
J Cyst Fibros. 2011 Jul;10(4):278-81. Epub 2011 Mar 8., [PMID:21388895]

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BACKGROUND: There has been great variation and uncertainty about how many and what CFTR mutations to include in cystic fibrosis (CF) newborn screening algorithms, and very little research on this topic using large populations of newborns. METHODS: We reviewed Wisconsin screening results for 1994-2008 to identify an ideal panel. RESULTS: Upon analyzing approximately 1 million screening results, we found it optimal to use a 23 CFTR mutation panel as a second tier when an immunoreactive trypsinogen (IRT)/DNA algorithm was applied for CF screening. This panel in association with a 96th percentile IRT cutoff gave a sensitivity of 97.3%, but restricting the DNA tier to F508del was associated with 90% (P<.0001). CONCLUSIONS: Although CFTR panel selection has been challenging, our data show that a 23 mutation method optimizes sensitivity and is advantageous. The IRT cutoff value, however, is actually more critical than DNA in determining CF newborn screening sensitivity.

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36 In 6 non-Caucasian cases (three African American, two Native American and one Hispanic), five had at least one F508del allele, and one was compound heterozygote of G542X/1812-1 GNA. There were 8 screening false negative cases, including 5 in period 1, and 3 in period 2. Login to comment
75 CFTR mutationa Proportion of allele Frequency of allele (%) Cumulative detection (%)b F508del 137/214 64.02 92.52 3849+10KbCNT 6/214 2.80 92.52c G542X 5/214 2.34 94.39 N1303K 4/214 1.87 98.13 R117H 4/214 1.87 99.07 R553X 3/214 1.40 99.07 1717-1GNA 2/214 0.93 99.07 G551D 1/214 0.47 100 R347P 1/214 0.47 100 A455E 1/214 0.47 100 W1282X 1/214 0.47 100 621+1GNT 1/214 0.47 100 a The other 11 mutations in ACMG 23 mutation panel are G85E, 711+1GNT, R334W, I507del, R560T, 1898+1GNA, 2184delA, 2789+5GNA, 3120+1GNA, R1162X and 3659delC. Login to comment

[hide] Preconceptional identification of cystic fibrosis ... J Cyst Fibros. 2011 May;10(3):207-11. doi: 10.1016/j.jcf.2011.02.006. Epub 2011 Mar 22. Coiana A, Faa' V, Carta D, Puddu R, Cao A, Rosatelli MC
Preconceptional identification of cystic fibrosis carriers in the Sardinian population: A pilot screening program.
J Cyst Fibros. 2011 May;10(3):207-11. doi: 10.1016/j.jcf.2011.02.006. Epub 2011 Mar 22., [PMID:21429822]

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BACKGROUND: In Sardinia the mutational spectrum of CFTR gene is well defined. A mutation detection rate of 94% can be achieved by screening for 15 CFTR mutations with a frequency higher than 0.5%. The efficiency of this molecular test suggests that Sardinians may represent a suitable population for a preconceptional screening. METHODS: Five hundred couples of Sardinia descent were screened for 38 mutations using a semi-automated reverse-dot blot and PCR-gel electrophoresis assays. This mutation panel included the 15 most frequent CF alleles in Sardinia. RESULTS: We identified 38 CF carriers, revealing an overall frequency of 1/25 (4%). The most common CF allele was the p.Thr338Ile (T338I) (65%), followed by the p.Phe508del (F508del) (22.5%). We also identified one couple at risk and an asymptomatic female homozygote for the p.Thr338Ile allele. CONCLUSIONS: In spite of the low number of the couples tested, the results herein reported demonstrate the efficacy and efficiency of the preconceptional screening program and the high participation rate of the Sardinian population (99%).

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88 Mutation nomenclaturea Alleles (%) T338I (p.Thr338Ile) 26 (65.0) F508del (p.Phe508del) 9 (22.5) N1303K (p.Asn1303Lys) 1 (2.5) 2183AANG (c.2051_2052delAAinsG) 1 (2.5) 621+1GNT (c.489+1GNT) 1 (2.5) exon 2 del (c.54-5811_164+2187del8108ins182) 1 (2.5) R347P (p.Arg347Pro) 1 (2.5) The 3849+10kbCNT (c.3717+12191CNT), G85E (p.Gly85Glu), 2789+5GNA (c.2657+5GNA), W1282X (p.Trp1282X), G1244E (p.Gly1244Glu), 711+5GNA (c.579+5GNA), 711+1GNT (c.579+1GNA), 4016insT (p.Ser1297PhefsX5), G542X (p.Gly542X), 1717-1GNA (c.1585-1GNA), R553X (p.Arg553X), Q552X (p.Gln552X), G551D (p.Gly551Asp), S549R (ANC) (p.Ser549Arg), I507del (p.Ile507del), F508C (p.Phe508Cys), I502T (p.Ile502Thr), 1706del17 (p.Gln525LeufsX37), 1677delTA (p.Tyr515X), R117H (p.Arg117His), D1152H (p.Asp1152His), L1065P (p.Leu1065Pro), R1066H (p.Arg1066His), L1077P (p.Leu1077Pro), 4382delA (p.Glu1418ArgfsX14), R1162X (p.Arg1162X), R1158X (p.Arg1158X), 1259 insA (p.Gln378AlafsX4), 852del22 (p.Gly241GlufsX13), S912X (p.Ser912X), and 991del5bp (p.Asn287LysfsX19) mutations included in the CF panel were not detected in the population tested. Login to comment

[hide] Cystic fibrosis carrier testing in an ethnically d... Clin Chem. 2011 Jun;57(6):841-8. Epub 2011 Apr 7. Rohlfs EM, Zhou Z, Heim RA, Nagan N, Rosenblum LS, Flynn K, Scholl T, Akmaev VR, Sirko-Osadsa DA, Allitto BA, Sugarman EA
Cystic fibrosis carrier testing in an ethnically diverse US population.
Clin Chem. 2011 Jun;57(6):841-8. Epub 2011 Apr 7., [PMID:21474639]

Abstract [show]
BACKGROUND: The incidence of cystic fibrosis (CF) and the frequency of specific disease-causing mutations vary among populations. Affected individuals experience a range of serious clinical consequences, notably lung and pancreatic disease, which are only partially dependent on genotype. METHODS: An allele-specific primer-extension reaction, liquid-phase hybridization to a bead array, and subsequent fluorescence detection were used in testing for carriers of 98 CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] mutations among 364 890 referred individuals with no family history of CF. RESULTS: One in 38 individuals carried one of the 98 CFTR mutations included in this panel. Of the 87 different mutations detected, 18 were limited to a single ethnic group. African American, Hispanic, and Asian individuals accounted for 33% of the individuals tested. The mutation frequency distribution of Caucasians was significantly different from that of each of these ethnic groups (P < 1 x 10(1)). CONCLUSIONS: Carrier testing using a broad mutation panel detects differences in the distribution of mutations among ethnic groups in the US.

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65 The median fluorescent intensity was determined, and the presence or absence of mutant and wild-type alleles was evaluated from the ratio of the mutant signal to the wild-type signal for the following mutations: c.1155_1156dupTA, c.2657ϩ5GϾA, c.3717ϩ12191CϾT, p.A455E, p.D1152H, p.F508del, p.G542X, p.G551D, p.I507del, p.L206W, p.N1303K, p.R117H, p.W1282X, and c.54-5940_ 273ϩ10250del21kb. Login to comment
123 CFTR mutationsa Individuals, n p.F508del/p.R117H 16 5T/9T 1 7T/9T 15 p.F508del/p.D1152H 3 p.R117H/p.R117H, 7T/7T 2 p.D1152H/p.D1152H 2 p.W1282X/p.D1152H 2 p.D1152H/p.G551D 1 c.3717ϩ12191CϾT/p.R352Q 1 c.3717ϩ12191CϾT/c.3717ϩ12191CϾT 1 p.F508del/c.3717ϩ12191CϾT 1 p.F508del/p.L206W 1 p.F508del/p.R117C 1 p.F508del/p.R347H 1 p.F508del/p.R347P 1 p.R117H/p.W1282X, 7T/7T 1 p.R117H/p.G551D, 7T/7T 1 p.R117H/p.G542X, 7T/9T 1 a Human Genome Variation Society nomenclature [Ogino et al. (23)]. Login to comment

[hide] Implementation of the first worldwide quality assu... Clin Chim Acta. 2011 Jul 15;412(15-16):1376-81. Epub 2011 Apr 14. Earley MC, Laxova A, Farrell PM, Driscoll-Dunn R, Cordovado S, Mogayzel PJ Jr, Konstan MW, Hannon WH
Implementation of the first worldwide quality assurance program for cystic fibrosis multiple mutation detection in population-based screening.
Clin Chim Acta. 2011 Jul 15;412(15-16):1376-81. Epub 2011 Apr 14., 2011-07-15 [PMID:21514289]

Abstract [show]
BACKGROUND: CDC's Newborn Screening Quality Assurance Program collaborated with several U.S. Cystic Fibrosis Care Centers to collect specimens for development of a molecular CFTR proficiency testing program using dried-blood spots for newborn screening laboratories. METHODS: Adult and adolescent patients or carriers donated whole blood that was aliquoted onto filter paper cards. Five blind-coded specimens were sent to participating newborn screening laboratories quarterly. Proficiency testing results were evaluated based on presumptive clinical assessment. Individual evaluations and summary reports were sent to each participating laboratory and technical consultations were offered if incorrect assessments were reported. RESULTS: The current CDC repository contains specimens with 39 different CFTR mutations. Up to 45 laboratories have participated in the program. Three years of data showed that correct assessments were reported 97.7% of the time overall when both mutations could be determined. Incorrect assessments that could have lead to a missed case occurred 0.9% of the time, and no information was reported 1.1% of the time due to sample failure. CONCLUSIONS: Results show that laboratories using molecular assays to detect CFTR mutations are performing satisfactorily. The programmatic results presented demonstrate the importance and complexity of providing proficiency testing for DNA-based assays.

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129 Allele Allele Allele Allele p.Gly85Glu G85E (0.26) p.Arg117His R117H (0.54) c.489+1 GNT 621+1 GNT (1.3) p.Phe508del F508del (66.31) p.Arg347Pro R347P (0.36) p.lle507del I507del (0.90) p.Gly551Asp G551D (1.93) c.2052delA 2184delA (0.15) c.1585-1 GNA 1717-1 GNA (0.44) p.Gly542X G542X (2.64) c.3528delC 3659delC (0.28) p.Asn1303Lys N1303K (1.27) p.Arg553X R553X (1.21) p.Arg560Thr R560T (0.30) p.Arg1162X R1162X (0.30) c.2657+5 GNA 2789+5 GNA (0.38) c.3717+12191 CNT 3849+10kbCNT (0.85) c.2988+1 GNA 3120+1 GNA (0.86) p.Trp1282X W1282X (2.20) p.Ala455Glu A455E (0.26) c.1766+1 GNA 1898+1 GNA (0.13) c.579+1 GNT 711+1 GNT (0.35) p.Arg334Trp R334W (0.37) c.54-5940 _273+10250del21kb CFTR dele2,3 p.Ser549Asn S549N (0.14) c.1584 GNA 1716 G→A c.2051_2052delAAinsG 2183AANG (0.1) c.3140-26ANG 3272-26ANG c.262_263delTT 394delTT p.Arg1066Cys R1066C (0.03) p.Arg1066His R1066H c.1022_1023insTC 1154insTC c.2989-1 GNA 3121-1 GNA c.(?_2989)_(3139_? Login to comment

[hide] Clinical outcomes in infants with cystic fibrosis ... Pediatr Pulmonol. 2011 Apr 29. doi: 10.1002/ppul.21475. Ren CL, Desai H, Platt M, Dixon M
Clinical outcomes in infants with cystic fibrosis transmembrane conductance regulator (CFTR) related metabolic syndrome.
Pediatr Pulmonol. 2011 Apr 29. doi: 10.1002/ppul.21475., 2011-04-29 [PMID:21538969]

Abstract [show]
An unavoidable outcome of cystic fibrosis newborn screening (CF NBS) programs is the detection of infants with an indeterminate diagnosis. The United States CF Foundation recently proposed the term cystic fibrosis transmembrane conductance regulator related metabolic syndrome (CRMS) to describe infants with elevated immunoreactive trypsinogen (IRT) on NBS who do not meet diagnostic criteria for CF. The objective of this study was to describe the clinical outcomes of infants with CRMS identified through an IRT/DNA algorithm. We reviewed the records of all infants with CRMS diagnosed at our CF Center from 2002 to 2010. We identified 12 infants, and compared them to 27 infants diagnosed with CF by NBS. Compared to CF patients, CRMS patients were more likely to be pancreatic sufficient as assessed by fecal elastase measurement (100% vs. 8%, P < 0.01). Their weight for age percentile was normal from birth. A positive oropharyngeal (OP) culture for Pseudomonas aeruginosa (Pa) was found in 25% of CRMS patients. One patient with the F508del/R117H/7T genotype was reassigned the diagnosis of CF after he had a positive OP culture for Pa, and his follow up sweat Cl at 1 year of life was 73 mmol/L. CF patients were more likely to receive oral antibiotics and be hospitalized for pulmonary symptoms. Our results indicate that CRMS patients can develop signs of CF disease, but have a milder clinical course than CF infants. Close initial monitoring of these patients is warranted. Pediatr. Pulmonol. (c) 2011 Wiley-Liss, Inc.

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60 Infants in both groups received treatment with inhaled tobramycin if they had a positive Pa OP culture, and treatment in both groups was associated with eradication of TABLE 1-CFTR Gene Mutation Panel Used by New York CF NBS Program F508del I50e7del G542X G551D W1282X N1303K R553X 621þ1G>T R117H 1717-1G>A A455E R560T R1162X G85E R334W R347P 711þ1G>T 1898þ1G>A 2184delA 1078delT 3849þ10kbC>T 2789þ5G>A 3659delC I148T 3120þ1G>A 3876delA V520F S549R S549N 3849þ4 A-G 3905insT R347H Reflex testing for 5T polymorphism is performed if R117H is detected. Login to comment
78 TABLE 3- Clinical Features and Outcomes of the CRMS Infants Patient number Gender Race/ ethnicity Mean age at 1st sweat (weeks) Mean sweat chloride (range) CFTR gene mutations identified Follow up time (months) Fecal elastase (mcg/gm stool) History of Pa infection History of hospitalization 1 Male Caucasian 4 46 (38-54) F508del/R117H/7T 36 303 Yes No 2 Male Caucasian 5 40 (40-43) F508del/R117H/7T 60 500 Yes No 3 Female Caucasian 3 29 (27-31) F508del/R117H/7T 60 488 No No 4 Male Caucasian 3 34 (33-38) F508del/none 26 383 No No 5 Male Caucasian 3 45 (40-50) F508del/none 72 424 No No 6 Female Caucasian 3 35 (32-38) F508del/none 9 454 No No 7 Male Caucasian 3 41 (36-46) F508del/none 39 462 No No 8 Female Caucasian 5 50 (46-52) F508del/none 72 440 No Yes 9 Male Caucasian 4 43 (41-45) F508del/Y1032C 14 401 No No 10 Male Caucasian 3 52 (50-54) G542X/none 21 500 No No 11 Female Caucasian 8 34 (30-38) R560T/none 9 433 No No 12 Female Hispanic 6 36 (32-40) R334W/R117H/7T 24 500 Yes No Mean sweat chloride levels represent the mean of all tests performed in the neonatal period. Login to comment
61 Infants in both groups received treatment with inhaled tobramycin if they had a positive Pa OP culture, and treatment in both groups was associated with eradication of TABLE 1- CFTR Gene Mutation Panel Used by New York CF NBS Program F508del I50e7del G542X G551D W1282X N1303K R553X 621þ1G>T R117H 1717-1G>A A455E R560T R1162X G85E R334W R347P 711þ1G>T 1898þ1G>A 2184delA 1078delT 3849þ10kbC>T 2789þ5G>A 3659delC I148T 3120þ1G>A 3876delA V520F S549R S549N 3849þ4 A-G 3905insT R347H Reflex testing for 5T polymorphism is performed if R117H is detected. Login to comment
79 TABLE 3- Clinical Features and Outcomes of the CRMS Infants Patient number Gender Race/ ethnicity Mean age at 1st sweat (weeks) Mean sweat chloride (range) CFTR gene mutations identified Follow up time (months) Fecal elastase (mcg/gm stool) History of Pa infection History of hospitalization 1 Male Caucasian 4 46 (38-54) F508del/R117H/7T 36 303 Yes No 2 Male Caucasian 5 40 (40-43) F508del/R117H/7T 60 500 Yes No 3 Female Caucasian 3 29 (27-31) F508del/R117H/7T 60 488 No No 4 Male Caucasian 3 34 (33-38) F508del/none 26 383 No No 5 Male Caucasian 3 45 (40-50) F508del/none 72 424 No No 6 Female Caucasian 3 35 (32-38) F508del/none 9 454 No No 7 Male Caucasian 3 41 (36-46) F508del/none 39 462 No No 8 Female Caucasian 5 50 (46-52) F508del/none 72 440 No Yes 9 Male Caucasian 4 43 (41-45) F508del/Y1032C 14 401 No No 10 Male Caucasian 3 52 (50-54) G542X/none 21 500 No No 11 Female Caucasian 8 34 (30-38) R560T/none 9 433 No No 12 Female Hispanic 6 36 (32-40) R334W/R117H/7T 24 500 Yes No Mean sweat chloride levels represent the mean of all tests performed in the neonatal period. Login to comment

[hide] Nonsense-mediated mRNA decay and cystic fibrosis. Methods Mol Biol. 2011;741:137-54. Linde L, Kerem B
Nonsense-mediated mRNA decay and cystic fibrosis.
Methods Mol Biol. 2011;741:137-54., [PMID:21594783]

Abstract [show]
Approximately one-third of the alleles causing genetic diseases carry premature termination codons (PTCs). Therapeutic approaches for mutations generating in-frame PTCs are aimed at promoting translational readthrough of the PTC, to enable the synthesis and expression of full-length functional proteins. Interestingly, readthrough studies in tissue culture cells, mouse models, and clinical trials revealed a wide variability in the response to the readthrough treatments. The molecular basis for this variability includes the identity of the PTC and its sequence context, the chemical composition of the readthrough drug, and, as we showed recently, the level of PTC-bearing transcripts. One post-transcriptional mechanism that specifically regulates the level of PTC-bearing transcripts is nonsense-mediated mRNA decay (NMD). We have previously shown a role for NMD in regulating the response of CF patients carrying CFTR PTCs to readthrough treatment. Here we describe all the protocols for analyzing CFTR nonsense transcript levels and for investigating the role of NMD in the response to readthrough treatment. This includes inhibition of the NMD mechanism, quantification of CFTR nonsense transcripts and physiologic NMD substrates, and analysis of the CFTR function.

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234 2. For quantification of CFTR W1282X transcript levels in HeLa and MCF7 cells, transfected with the CFTR constructs RNA is extracted according to the manufacturer`s protocol and RT reactions are performed to obtain cDNA. Real-time PCR is performed using the LightCycler with the Patients RelativeRNAlevel 0 0.1 0.2 0.3 0.4 0.5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 W /3849 W /G542X W /W W /ΔF508 W /ΔF508 W /W W /ΔF508W /ΔF508W /ΔF508W /ΔF508W /ΔF508W /ΔF508 W /ΔF508W /ΔF508W /ΔF508 Response to readthrough treatment No response Fig. 10.1. Login to comment

[hide] Understanding protein kinase CK2 mis-regulation up... Naunyn Schmiedebergs Arch Pharmacol. 2011 May 24. Venerando A, Pagano MA, Tosoni K, Meggio F, Cassidy D, Stobbart M, Pinna LA, Mehta A
Understanding protein kinase CK2 mis-regulation upon F508del CFTR expression.
Naunyn Schmiedebergs Arch Pharmacol. 2011 May 24., 2011-05-24 [PMID:21607646]

Abstract [show]
We review areas of overlap between nucleoside diphosphate kinase (NDPK; nm23) and two proteins manifesting an equivalent diversity of action, each with many thousands of publications. The first is a constitutively active protein kinase, CK2 (formerly casein kinase 2), that includes NDPK amongst its hundreds of targets. The second is an enigmatic member of the ATP-binding cassette (ABC) family of membrane pumps that normally hydrolyse ATP to transport substrates. Yet our unusual family member (ABCC7) is not a pump but, uniquely, acts as a regulated anion channel. ABCC7 is the cystic fibrosis transmembrane conductance regulator (CFTR), and we discuss the highly prevalent CFTR mutation (F508del CFTR) in terms of the uncertainties surrounding the molecular basis of cystic fibrosis that cloud approaches to corrective therapy. Using lysates from cells stably expressing either wild-type or F508del CFTR, incubated with the CK2 substrate GTP, we show that the phosphoproteome of F508del CFTR-expressing cells both differs from wild-type CFTR-expressing cells and is significantly enhanced in intensity by approximately 1.5-fold (p < 0.05, paired t test with Bonferroni correction, n = 4). Phosphorylation is about 50% attenuated with a specific CK2 inhibitor. We propose that a new function may exist for the CFTR region that is commonly mutated, noting that its sequence (PGTIKENIIF(508)GVSYDEYRYR) is not only highly conserved within the C sub-family of ABC proteins but also a related sequence is found in NDPK. We conclude that a latent path may exist between mutation of this conserved sequence, CK2 hyperactivity and disease pathogenesis that might also explain the heterozygote advantage for the common F508del CFTR mutant .

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79 This drug is claimed to restore full-length CFTR bearing stop mutations that normally induce nonsense-mediated CFTR decay (for example, the G542X stop mutation in CFTR and others like it that together account for about 10% of CFTR mutants). Login to comment
80 At the very least, this drug, which was developed to create read-through at single stop codons such as G542X, should reverse the above 'gold standard` sweat test, but it absolutely fails to do so, thus adding further doubt to our ability to fully understand the path from CFTR defect to disease. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Chest. 2011 Jun;139(6):1480-90. Rogan MP, Stoltz DA, Hornick DB
Cystic fibrosis transmembrane conductance regulator intracellular processing, trafficking, and opportunities for mutation-specific treatment.
Chest. 2011 Jun;139(6):1480-90., [PMID:21652558]

Abstract [show]
Recent advances in basic science have greatly expanded our understanding of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the chloride and bicarbonate channel that is encoded by the gene, which is mutated in patients with CF. We review the structure, function, biosynthetic processing, and intracellular trafficking of CFTR and discuss the five classes of mutations and their impact on the CF phenotype. The therapeutic discussion is focused on the significant progress toward CFTR mutation-specific therapies. We review the results of encouraging clinical trials examining orally administered therapeutics, including agents that promote read-through of class I mutations (premature termination codons); correctors, which overcome the CFTR misfolding that characterizes the common class II mutation F508del; and potentiators, which enhance the function of class III or IV mutated CFTR at the plasma membrane. Long-term outcomes from successful mutation-specific treatments could finally answer the question that has been lingering since and even before the CFTR gene discovery: Will therapies that specifically restore CFTR-mediated chloride secretion slow or arrest the deleterious cascade of events leading to chronic infection, bronchiectasis, and end-stage lung disease?

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65 Five Classes of Defective CFTR Protein Processing Based on Gene Mutation Although .1,500 mutations of CFTR have been identified, only four specific mutations besides F508del reach a frequency of 1% to 3%: G551D, W1282X, G542X, and N1303K. Login to comment
61 Five Classes of Defective CFTR Protein Processing Based on Gene Mutation Although .1,500 mutations of CFTR have been identified, only four specific mutations besides F508del reach a frequency of 1% to 3%: G551D, W1282X, G542X, and N1303K. Login to comment

[hide] Recommendations for the classification of diseases... J Cyst Fibros. 2011 Jun;10 Suppl 2:S86-102. Bombieri C, Claustres M, De Boeck K, Derichs N, Dodge J, Girodon E, Sermet I, Schwarz M, Tzetis M, Wilschanski M, Bareil C, Bilton D, Castellani C, Cuppens H, Cutting GR, Drevinek P, Farrell P, Elborn JS, Jarvi K, Kerem B, Kerem E, Knowles M, Macek M Jr, Munck A, Radojkovic D, Seia M, Sheppard DN, Southern KW, Stuhrmann M, Tullis E, Zielenski J, Pignatti PF, Ferec C
Recommendations for the classification of diseases as CFTR-related disorders.
J Cyst Fibros. 2011 Jun;10 Suppl 2:S86-102., [PMID:21658649]

Abstract [show]
Several diseases have been clinically or genetically related to cystic fibrosis (CF), but a consensus definition is lacking. Here, we present a proposal for consensus guidelines on cystic fibrosis transmembrane conductance regulator (CFTR)-related disorders (CFTR-RDs), reached after expert discussion and two dedicated workshops. A CFTR-RD may be defined as "a clinical entity associated with CFTR dysfunction that does not fulfil diagnostic criteria for CF". The utility of sweat testing, mutation analysis, nasal potential difference, and/or intestinal current measurement for the differential diagnosis of CF and CFTR-RD is discussed. Algorithms which use genetic and functional diagnostic tests to distinguish CF and CFTR-RDs are presented. According to present knowledge, congenital bilateral absence of vas deferens (CBAVD), acute recurrent or chronic pancreatitis and disseminated bronchiectasis, all with CFTR dysfunction, are CFTR-RDs.

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217 In one study [123], three CFTR mutations (p.F508del, p.G542X and c.579+1G>T (previously named 711+1G>T)) were detected in 8.9% of 449 ACP patients, although the mutation detection rate was not significantly different from that observed in patients with alcoholic liver disease (3.0%) nor that expected in the geographical area under investigation (3.2%). Login to comment

[hide] Screening of DeltaF508 mutation and IVS8-poly T po... Andrologia. 2011 Jul 18. doi: 10.1111/j.1439-0272.2011.01193.x. Ghorbel M, Baklouti-Gargouri S, Keskes R, Sellami-Ben Hamida A, Feki-Chakroun N, Bahloul A, Fakhfakh F, Ammar-Keskes L
Screening of DeltaF508 mutation and IVS8-poly T polymorphism in CFTR gene in Tunisian infertile men without CBAVD.
Andrologia. 2011 Jul 18. doi: 10.1111/j.1439-0272.2011.01193.x., 2011-07-18 [PMID:21762191]

Abstract [show]
It is well established that cystic fibrosis transmembrane conductance regulator gene (CFTR) mutations are involved in congenital bilateral absence of the vas deferens (CBAVD), causing obstructive azoospermia and male infertility. Also, several studies reported a relatively high prevalence of CFTR gene mutations in healthy men presenting reduced sperm quality. In this study, we investigate DeltaF508 mutation and IVS8-polyT polymorphism in CFTR gene in Tunisian infertile men without CBAVD. Genetic analyses were performed in 148 infertile patients and 126 fertile individuals. The polymorphic IVS8-polyT tract in CFTR gene was analysed in only 129 infertile patients and 54 individuals of control group. As well, we screened for Y chromosome microdeletions in all infertile patients. No DeltaF508 mutation was diagnosed either in infertile patients or in control group. 5T allele of IVS8-polyT tract was found in both infertile men (4.26%) and fertile individuals (8.33%). 5T/5T genotype was observed only in two azoospermic patients without Y microdeletions. The most frequent genotype of IVS8-polyT tract in infertile men and controls was 7T/7T (69.75% and 59.25% respectively). There was no association between IVS8-polyT polymorphism and reduced semen quality. Neither DeltaF508 mutation nor 5T allele is involved in pathogenesis of male infertility in Tunisian infertile patients without CBAVD.

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91 Table 2 Genotype frequencies of IVS8-polyT tract variants in infertile men and controls IVS8-polyT genotype frequencies (%) 5T/5T 7T/7T 9T/9T All infertile patients (n = 129) 1.55 69.76 4.65 Azoospermia (n = 57) 3.50 73.68 5.26 Oligospermia (n = 39) 0 71.79 5.12 Normospermia (n = 33) 0 60.60 3.03 Controls (n = 54) 0 59.25 3.70 Table 3 Microdeleted STSs and IVS8-polyT genotype in patients with Y chromosome microdeletions (n = 7) Number of microdeleted patients Y chromosome microdeleted AZF loci and STSs IVS8-polyT genotype in CFTR gene Azoospermia (n = 57) 1 AZFa (SY86) 9T/9T 1 AZFa (SY84) 7T/7T Oligospermia (n = 48) 1 AZFa (SY86) 9T/9T 1 AZFa (SY86) 7T/7T 1 AZFa (SY84) 7T/7T 1 AZFa + b (SY86 + SY134) 7T/7T Normospermia (n = 43) 1 AZFc (SY254) 7T/7T patients (Boucher et al., 1999); it also agrees with data reported by Tuerlings et al. (1998) showing no increase in 5T allele and in three frequent CFTR mutations (deltaF508, A455E and G542X) among patients with oligozoospermia. Login to comment

[hide] Suppression of CFTR premature termination codons a... J Mol Med (Berl). 2011 Jul 22. Rowe SM, Sloane P, Tang LP, Backer K, Mazur M, Buckley-Lanier J, Nudelman I, Belakhov V, Bebok Z, Schwiebert E, Baasov T, Bedwell DM
Suppression of CFTR premature termination codons and rescue of CFTR protein and function by the synthetic aminoglycoside NB54.
J Mol Med (Berl). 2011 Jul 22., 2011-07-22 [PMID:21779978]

Abstract [show]
Certain aminoglycosides are capable of inducing "translational readthrough" of premature termination codons (PTCs). However, toxicity and relative lack of efficacy deter treatment with clinically available aminoglycosides for genetic diseases caused by PTCs, including cystic fibrosis (CF). Using a structure-based approach, the novel aminoglycoside NB54 was developed that exhibits reduced toxicity and enhanced suppression of PTCs in cell-based reporter assays relative to gentamicin. We examined whether NB54 administration rescued CFTR protein and function in clinically relevant CF models. In a fluorescence-based halide efflux assay, NB54 partially restored halide efflux in a CF bronchial epithelial cell line (CFTR genotype W1282X/F508del), but not in a CF epithelial cell line lacking a PTC (F508del/F508del). In polarized airway epithelial cells expressing either a CFTR-W1282X or -G542X cDNA, treatment with NB54 increased stimulated short-circuit current (I (SC)) with greater efficiency than gentamicin. NB54 and gentamicin induced comparable increases in forskolin-stimulated I (SC) in primary airway epithelial cells derived from a G542X/F508del CF donor. Systemic administration of NB54 to Cftr-/- mice expressing a human CFTR-G542X transgene restored 15-17% of the average stimulated transepithelial chloride currents observed in wild-type (Cftr+/+) mice, comparable to gentamicin. NB54 exhibited reduced cellular toxicity in vitro and was tolerated at higher concentrations than gentamicin in vivo. These results provide evidence that synthetic aminoglycosides are capable of PTC suppression in relevant human CF cells and a CF animal model and support further development of these compounds as a treatment modality for genetic diseases caused by PTCs.

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5 In polarized airway epithelial cells expressing either a CFTR-W1282X or - G542X cDNA, treatment with NB54 increased stimulated short-circuit current (ISC) with greater efficiency than gentamicin. Login to comment
6 NB54 and gentamicin induced comparable increases in forskolin-stimulated ISC in primary airway epithelial cells derived from a G542X/F508del CF donor. Login to comment
7 Systemic administration of NB54 to Cftr-/- mice expressing a human CFTR-G542X transgene restored 15-17% of the average stimulated transepithelial chloride currents Electronic supplementary material The online version of this article (doi:10.1007/s00109-011-0787-6) contains supplementary material, which is available to authorized users. Login to comment
32 We also show that NB54 restores a comparable level of CFTR expression and function as gentamicin in transgenic mice expressing a human CFTR-G542X cDNA. Login to comment
43 Primary human airway epithelial cells were derived from lung explants of CF subjects who provided written informed consent and were confirmed to harbor two severe CFTR mutations (including the G542X premature termination codon) by methods described previously [24, 25]. Login to comment
65 Mouse lines and treatment protocols The CFTR-G542X mice used in this study contained the Cftrtm1Cam knockout [30] and expressed a human CFTR transgene with the G542X premature stop mutation [17, 31, 32] (referred to as Cftr-/- hCFTR-G542X mice). Login to comment
107 To test the more common CFTR-G542X allele and examine dose dependence, CFBE41o- human airway cells transduced with an adenovirus vector expressing a CFTR-G542X cDNA were tested following treatment with gentamicin or NB54. Login to comment
116 We obtained primary HBE cells from a compound heterozygote subject (G542X/F508del) following a lung Fig. 3 Increased W1282X-CFTR dependent short-circuit current following incubation of synthetic aminoglycosides. Login to comment
122 *P<0.05; **P<0.005; ±SEM, n=16 Fig. 4 Short-circuit current assay of CFBE41o-cells transduced with an adenovirus expressing CFTR-G542X following treatment with gentamicin, NB30, or NB54. Login to comment
123 a Representative short-circuit current tracings of CFBE41o-cells were grown on air-liquid interface, transduced with adenovirus encoding CFTR-G542X, allowed to recover 48 h, and exposed to aminoglycosides (500 μg/ml) for 24 h. Login to comment
127 b Mean stimulated short-circuit currents measured in CFBE41o-cells transduced with adenovirus CFTR-G542X following treatment with the indicated doses of gentamicin or NB54. Login to comment
142 Readthrough of CFTR-G542X in vivo induced by the synthetic aminoglycoside NB54 To examine whether NB54 could suppress a CFTR PTC in vivo, we next utilized a transgenic human CFTR-G542X (hCFTR-G542X) mouse line [17, 31-33]. Login to comment
143 In this mouse model, the hCFTR-G542X transgene is expressed from the rat intestinal FABP promoter in a mouse Cftr-/- background. Login to comment
144 Samples from the ileum of untreated Cftr+/+ littermates carrying the transgene were used as positive controls (Fig. 6a), while corresponding samples from untreated Cftr-/- mice carrying the hCFTR-G542X transgene were used as negative controls (Fig. 6b). Login to comment
146 Fully differentiated primary airway cells derived from a CF (G542X/ F508Del) donor were grown at air-liquid interface until terminally differentiated (e.g., 90% ciliated), and then treated with NB54, gentamicin, or vehicle (500 μg/ml) for the times indicated, then mounted in modified Ussing chambers under voltage clamp conditions. Login to comment
150 Representative short-circuit current tracings are shown from a untreated Cftr+/+ hCFTR-G542X mice (WT control), b untreated Cftr-/- hCFTR-G542X (negative control), c gentamicin-treated Cftr-/- hCFTR-G542X (60 mg/kg), and d NB54-treated Cftr-/- hCFTR-G542X (120 mg/kg). Login to comment
151 e Scatter plot of the total ISC data from Cftr-/- hCFTR-G542X mice (untreated, gentamicin treated, and NB54 treated) that combines the forskolin/IBMX and carbachol responses. Login to comment
156 A scatter plot of the total ISC (ΔISC following forskolin/ IBMX + carbachol) from all Cftr-/- hCFTR-G542X mice (untreated, gentamicin treated, and NB54 treated) is presented in Fig. 6e, while an analysis of that data is represented in Fig. 6f. In Cftr+/+ hCFTR-G542X mice, ileal tissues yielded a mean cAMP-stimulated ISC of 161 μA/cm2 10 min after forskolin/IBMX addition, and a further increase in ISC of 209 μA/cm2 following the addition of carbachol (a muscarinic agonist that transiently enhances the cAMP-dependent chloride secretory response in intestinal tissues by activating Ca2+ -dependent potassium channels in the basolateral plasma membrane [37]). Login to comment
158 In contrast, stimulated responses were effectively absent in untreated Cftr-/- hCFTR-G542X control mice. Login to comment
161 We initially compared the relative efficacy of gentamicin and NB54 to restore activated ISC, a measure of full-length CFTR protein function resulting from suppression of the hCFTR-G542X mutation. Login to comment
169 Next, we examined the ability of NB54 to induce readthrough and restore CFTR activity in the Cftr-/- hCFTR-G542X transgenic mouse model. Login to comment
176 Only background staining was observed in untreated Cftr-/- hCFTR-G542X mice (Fig. 7a). Login to comment
193 We tested the ability of these compounds to suppress two CFTR PTCs (G542X and W1282X) in a variety of CF cellular models, including heterologous CFTR expression systems, immortalized CF cell lines, and primary HBE cells isolated from a CF lung. Login to comment
194 In addition, we examined the ability of these compounds to suppress the human CFTR-G542X mutation in a transgenic mouse model. Login to comment
199 The maximal ISC measured in CFBE41o-airway epithelial monolayers stably transduced with a lentivirus vector expressing CFTR-W1282X or an adenovirus vector expressing CFTR-G542X was consistently higher with NB54 than gentamicin, and NB54 was also nontoxic at these doses (Figs. Login to comment
201 NB54 also induced a cAMP-stimulated ISC that was comparable to gentamicin in primary HBE cells carrying the CFTR-G542X mutation, and the induction of that response occurred sooner than we observed with gentamicin treatment (Fig. 5). Login to comment
208 a Samples from untreated Cftr-/- hCFTR-G542X, b samples from Cftr-/- hCFTR-G542X mice treated with 60 mg/kg gentamicin, and c samples from Cftr-/- hCFTR-G542X mice treated with 120 mg/kg NB54. Login to comment
213 As a further test of the ability of NB54 to suppress CF-related PTCs, we examined the propensity of NB54 to restore human CFTR expression in Cftr-/- hCFTR-G542X transgenic mice (Fig. 6). Login to comment
214 We found that NB54 induced significant and dose-dependent increases in stimulated ISC in Cftr-/- hCFTR-G542X transgenic mice, resulting in 15.8% of the current measured in WT mice at a dose of 120 mg/kg. Login to comment
218 Since it was demonstrated in a previous study that gentamicin and PTC124 (ataluren) restored a comparable level of CFTR activity in the same hCFTR-G542X Cftr-/- mouse model [17], the level of CFTR function restored by NB54 should be similar to PTC124 as well. Login to comment
261 Am J Respir Cell Mol Biol 37:57-66 17. Du M, Liu X, Welch EM, Hirawat S, Peltz SW, Bedwell DM (2008) PTC124 is an orally bioavailable compound that promotes suppression of the human CFTR-G542X nonsense allele in a CF mouse model. Login to comment
285 Nat Genet 4:35-41 31. Du M, Jones JR, Lanier J, Keeling KM, Lindsey RJ, Tousson A, Bebok Z, Whitsett JA, Dey CR, Colledge WH et al (2002) Aminoglycoside suppression of a premature stop mutation in a Cftr-/- mouse carrying a human CFTR-G542X transgene. Login to comment
286 J Mol Med 80:595-604 32. Du M, Keeling KM, Fan L, Liu X, Kovacs T, Sorscher E, Bedwell DM (2006) Clinical doses of amikacin provide more effective suppression of the human CFTR-G542X stop mutation than gentamicin in a transgenic CF mouse model. J Mol Med 84:573-582 33. Du M, Keeling KM, Fan L, Liu X, Bedwell DM (2009) Poly-L-aspartic acid enhances and prolongs gentamicin-mediated suppression of the CFTR-G542X mutation in a cystic fibrosis mouse model. J Biol Chem 284:6885-6892 34. Login to comment

[hide] A recurrent deep-intronic splicing CF mutation emp... J Cyst Fibros. 2011 Jul 21. Costa C, Pruliere-Escabasse V, de Becdelievre A, Gameiro C, Golmard L, Guittard C, Bassinet L, Bienvenu T, Georges MD, Epaud R, Bieth E, Giurgea I, Aissat A, Hinzpeter A, Costes B, Fanen P, Goossens M, Claustres M, Coste A, Girodon E
A recurrent deep-intronic splicing CF mutation emphasizes the importance of mRNA studies in clinical practice.
J Cyst Fibros. 2011 Jul 21., 2011-07-21 [PMID:21783433]

Abstract [show]
BACKGROUND: The identification by CFTR mRNA studies of a new deep-intronic splicing mutation, c.870-1113_1110delGAAT, in one patient of our series with mild CF symptoms and in three CF patients of an Italian study, led us to evaluate the mutation frequency and phenotype/genotype correlations. METHODS: 266 patients with CF and related disorders and having at least one undetected mutation, were tested at the gDNA level in three French reference laboratories. RESULTS: In total, the mutation was found in 13 unrelated patients (5% of those already carrying a mutation) plus 4 siblings, including one homozygote and 12 heterozygotes having a severe CF mutation. The sweat test was positive in 10/14 documented cases, the diagnosis was delayed after 20years in 9/15 and pancreatic insufficiency was present in 5/16. CONCLUSION: c.870-1113_1110delGAAT should be considered as CF-causing with phenotype variability and overall delayed diagnosis. Its frequency highlights the potential of mRNA studies.

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51 The mutations in trans of c.870-1113_1110delGAAT were mostly frequent severe mutations: p.Phe508del (n=5 families); c.1624GNT, p.Gly542X (G542X) (n = 3 families); and c.1657CNT, p.Arg553X (R553X) (n=1 family). Login to comment

[hide] Defective CFTR expression and function are detecta... PLoS One. 2011;6(7):e22212. Epub 2011 Jul 21. Sorio C, Buffelli M, Angiari C, Ettorre M, Johansson J, Vezzalini M, Viviani L, Ricciardi M, Verze G, Assael BM, Melotti P
Defective CFTR expression and function are detectable in blood monocytes: development of a new blood test for cystic fibrosis.
PLoS One. 2011;6(7):e22212. Epub 2011 Jul 21., [PMID:21811577]

Abstract [show]
BACKGROUND: Evaluation of cystic fibrosis transmembrane conductance regulator (CFTR) functional activity to assess new therapies and define diagnosis of cystic fibrosis (CF) is cumbersome. It is known that leukocytes express detectable levels of CFTR but the molecule has not been characterized in these cells. In this study we aim at setting up and validating a blood test to evaluate CFTR expression and function in leukocytes. DESCRIPTION: Western blot, PCR, immunofluorescence and cell membrane depolarization analysis by single-cell fluorescence imaging, using the potential-sensitive DiSBAC(2)(3) probe were utilized. Expression of PKA phosphorylated, cell membrane-localized CFTR was detected in non-CF monocytes, being undetectable or present in truncated form in monocytes derived from CF patients presenting with nonsense mutations. CFTR agonist administration induced membrane depolarization in monocytes isolated from non-CF donors (31 subjects) and, to a lesser extent, obligate CFTR heterozygous carriers (HTZ: 15 subjects), but it failed in monocytes from CF patients (44 subjects). We propose an index, which values in CF patients are significantly (p<0.001) lower than in the other two groups. Nasal Potential Difference, measured in selected subjects had concordant results with monocytes assay (Kappa statistic 0.93, 95%CI: 0.80-1.00). RESULTS AND SIGNIFICANCE: CFTR is detectable and is functional in human monocytes. We also showed that CFTR-associated activity can be evaluated in 5 ml of peripheral blood and devise an index potentially applicable for diagnostic purposes and both basic and translational research: from drug development to evaluation of functional outcomes in clinical trials.

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202 Case Gender Age at diagnosis (years) CFTR genotype* Age (years) Sweat Cl- mEq/L** FEV1 % mean values 2009 Pa PI NPD results*** CF-index 1 F 0 3132delTG 1497delGG 34 129 75 yes yes nd 222,10 2 F 0 R1162X R1162X 43 144 52 yes yes nd 229,65 3 M 0 R1162X R1162X 10 102 59 no yes 1,02 210,18 4 M 0 R1162X R1162X 25 115 81 no yes 1,07 267,11 5 M 7 G542X 711+5 G.A 24 105 59 yes yes nd 25,84 6 M 1 CFTRdele1 G542X 36 107 22 yes yes nd 2113,92 7 M 0 G542X G542X 16 110 71 yes yes 0,97 280,20 8 F 1 Q552X CFTRdele17a-18 35 99 72 yes yes 2,08 2219,81 9 M 16 R1162X 3849+10 Kb C.T 42 74 43 yes no 1,02 271,47 10 M 0 R1162X R1162X 32 105 45 yes yes 1,43 2114,67 11 M 1 F508del F508del 16 86 71 no yes nd 260,04 12 F 0 F508del F508del 16 88 118 no yes nd 248,20 13 M 0 F508del F508del 33 118 51 yes yes nd 265,49 14 M 7 F508del F508del 37 89 37 yes yes nd 2359,82 15 F 0 F508del F508del 27 118 71 yes yes nd 267,26 16 F 8 1717-1 G.A F508del 38 140 74 yes yes nd 2136,80 17 F 0 R1158X F508del 32 95 60 yes yes 1,77 228,31 18 M 7 G542X F508del 39 110 46 yes yes nd 247,52 19 M 0 Q39X F508del 17 101 79 no yes 1,11 264,20 20 F 1 R1162X F508del 41 188 60 no yes 0,94 296,73 21 M 13 3849+10 Kb C.T F508del 24 76 78 yes no 4,67 26,33 22 M 0 W1282X 621+1G.T 33 119 77 yes yes 1,27 242,74 23 F 4 R553X 2789+5 G.A 31 92 44 yes no 7,4 260,94 24 F 11 F508del R553X 39 116 55 yes yes nd 2113,67 25 M 12 F508del 3849+10 Kb C.T 27 51 71 yes no 1,12 298,84 26 F 0 F508del G542X 19 109 109 yes yes nd 2173,24 27 F 0 F508del R1162X 32 94 86 yes yes 1,34 270,16 28 F 0 F508del W57X (TAG) 27 99 78 yes yes 1,21 269,33 29 M 0 F508del Q552X 24 94 41 yes yes 1,50 272,75 30 M 20 F508del 3849+10 Kb C.T 43 58 60 no no 1,13 2112,56 31 M 0 F508del R1162X 12 99 65 no yes 2,14 280,92 32 M 4 F508del 3849+10 Kb C.T 17 60 100 no no nd 2121,31 33 F 1 F508del 1717-1 G.A 26 105 73 yes yes 2,05 255,66 34 F 11 F508del 3849+10 Kb C.T 40 85 59 yes no nd 2152,23 35 F 4 F508del 1717-1 G.A 44 130 97 yes yes nd 2116,56 36 M 13 F508del 3849+10 Kb C.T 43 70 65 yes no CF 265,10 37 F 19 F508del unknown 29 95 100 no no nd 240,53 38 M 6 F508del unknown 15 92 87 yes no nd 270,17 39 F 0 G542X N1303K 34 108 97 yes yes nd 296,14 40 M 50 G1249R IVS8 T5TG12 50 61 74 no no nd 2199,15 41 F 10 2183 AA.G IVS8 T5TG15/T7TG10 45 79 29 yes no 1,9 286,27 42 F 1 G85E unknown 43 120 107 yes no nd 249,21 43 F 0 3272-26 A.G I507del 21 113 88 no no nd 236,79 44 M 8 F508del D1152H 10 77 107 no no nd 210,85 *Cystic Fibrosis mutation database reference: http://www3.genet.sickkids.on.ca/cftr/app. Login to comment

[hide] Buccal cell DNA mutation analysis for diagnosis of... Pediatrics. 1998 May;101(5):851-5. Parad RB
Buccal cell DNA mutation analysis for diagnosis of cystic fibrosis in newborns and infants inaccessible to sweat chloride measurement.
Pediatrics. 1998 May;101(5):851-5., [PMID:9565413]

Abstract [show]
OBJECTIVES: To assess the application of DNA-based cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation analysis as a primary cystic fibrosis (CF) diagnostic test in preterm and term newborns and infants for whom the quantitative pilocarpine iontophoresis test (QPIT) cannot be used. DESIGN: Retrospective survey. SETTING: DNA Diagnostic Laboratory, Children's Hospital, Boston, Massachusetts. Buccal cell DNA samples were received from inpatients, outpatients, and three neonatal intensive care units. OUTCOME MEASURE: Detection of at least 1 of 12 CFTR mutations. PATIENTS: Between November 1, 1992, and April 30, 1994, 28 newborns and infants under 12 months of age at risk for CF had CFTR DNA mutation analysis performed because a sweat chloride (SC) value could not be obtained. QPIT was either not performed (infant weight <2 kg, QPIT not available at site of hospitalization, or infant not accessible to QPIT laboratory) or was inconclusive (sweat volume <75 mg or indeterminate SC [>/=40, <60 mEq/L]). The postnatal age at time of testing ranged from 1 day to 11 months, and gestational age at birth from 25 to 40 weeks. RESULTS: Six (21%) of 28 infants with unobtainable or indeterminate QPIT had 1 or 2 CFTR mutations detected. Immediate CF diagnosis by direct detection of 2 CFTR mutations was made in 5 of these 6 patients. Definitive CF diagnosis in the infant with 1 CFTR mutation was delayed until an elevation in SC could be documented. The patients with no CFTR mutations detected had a low likelihood of CF. CONCLUSIONS: For infants in whom CF is suspected but QPIT cannot be obtained, buccal cell DNA-based CFTR mutation analysis can be used as a rapid, noninvasive primary diagnostic test. This simple mode of DNA collection may aid in the diagnosis of other inherited disorders in newborns.

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58 The brushes were then discarded and 60 ␮L 1 M Tris, pH 8.0, was added to the tubes.7 CFTR mutation analysis was performed for 12 mutations (⌬F508, G551D, G542X, 621ϩ1G3T, ⌬I507, 1717-1G3A, R117H, N1303K, W1282X, R560T, R553X, and 3849ϩ10kb C3T). Login to comment
107 Genotypes and Sweat Chloride Confirmation by Diagnostic Category Diagnostic Category Number of Patients (%) Primary SC Before Genotype Genotype Confirmatory SC After Genotyping (mEq/L) Affected 6 (21) 1 inadequate volume 2 ⌬F508/⌬F508 2 ND* 2 G542X/W1282X 2 ND* 1 ⌬F508/N Ն60 1 W1282X/W1282X Ն60 Unaffected 19 (68) Confirmed (SC Ͻ40) 1 inadequate volume 9 N/N 9 Ͻ40 Presumed (no SC*) 10 N/N 10 ND* Unknown (indeterminate) 3 (11) 3 Ն 40, Ͻ60 mEq/L 3 N/N 3 ND* Total 28 (100) * ND, not done. Login to comment
110 Genotype GI* 25 (89) Newborn bowel obstruction Meconium plug 10 N/N Meconium ileus 1 ⌬F508/⌬F508 1 W1282X/W1282X 3 N/N Atresia or other obstruction 4 N/N In utero dilated bowel 2 ⌬F508/G542X In utero abdominal calcification 1 N/N Neonatal idiopathic cholestasis 1 N/N Failure to thrive 1 N/N Respiratory 3 (11) CLD ϩ P aeruginosa in trachea 1 N/N Chronic cough 1 N/N Bronchiolitis 1 ⌬F508/⌬F508 Total 28 (100) * Secondary reasons: 4/25 positive family history, 3/25 CLD, 3/22 in utero abdominal abnormality. Login to comment
142 Genotype Ͻ2 kg Premature or intrauterine growth restriction 14 (50) 2 Transient in utero dilated bowel (twins), affected sibling 2 G542X/W1282X* 1 In utero abdominal calcifications 1 N/N 10 Meconium plug (3 with CLD, 1 in utero dilated bowel) 10 N/N 1 CLD/P aeruginosa in tracheal aspirate 1 N/N Ͼ2 kg Inadequate sweat amount or inconclusive SC 5 (18) 1 Meconium ileus, affected sibling 1 ⌬F508/⌬F508* 1 Scrotal swelling, in utero abdominal calcifications 1 N/N 1 Chronic cough 1 N/N 1 Affected relative 1 N/N 1 Failure to thrive, affected uncle 1 N/N Sweat lab inaccessible: 9 (32) 1 Bronchiolitis 1 ⌬F508/⌬F508* Patient intensive care unit- bound or no laboratory in hospital 8 Bowel obstruction 5 Meconium ileus 1 ⌬F508/N* 1 W1282X/W1282X* 3 N/N 3 Small bowel atresia 3 N/N Total 28 (100) * Affectd. Login to comment

[hide] Alpha1-antitrypsin deficiency alleles and the Taq-... Eur Respir J. 1998 Apr;11(4):873-9. Mahadeva R, Westerbeek RC, Perry DJ, Lovegrove JU, Whitehouse DB, Carroll NR, Ross-Russell RI, Webb AK, Bilton D, Lomas DA
Alpha1-antitrypsin deficiency alleles and the Taq-I G-->A allele in cystic fibrosis lung disease.
Eur Respir J. 1998 Apr;11(4):873-9., [PMID:9623690]

Abstract [show]
Cystic fibrosis (CF) is characterized by progressive and ultimately fatal pulmonary disease although there are notable variations in clinical features. This heterogeneity is thought to lie outside the cystic fibrosis transmembrane regulator (CFTR) gene locus and may stem from deficiencies in the antiproteinase screen that protects the lung from proteolytic attack. One hundred and fifty seven patients were recruited from two UK CF centres. The serum concentrations of alpha1-antitrypsin, alpha1-antichymotrypsin and C-reactive protein (CRP) were determined and patients were screened for the common S and Z deficiency alleles of alpha1-antitrypsin and the G-->A mutation in the 3' noncoding region of the alpha1-antitrypsin gene (Taq-I G-->A allele). Alpha1-antitrypsin deficiency phenotypes were detected in 20 (16 MS, 1 S and 3 MZ) out of 147 unrelated tested CF patients and were, surprisingly, associated with significantly better lung function (adjusted mean forced expiratory volume in one second (FEV1) 62.5% of predicted for deficient group and 51.1% pred for normal alleles; p=0.043). The Taq-I G-->A allele was found in 21 out of 150 unrelated patients and had no significant effect on CF lung disease or on levels of alpha1-antitrypsin during the inflammatory response. We show here that, contrary to current thinking, common mutations of alpha1-antitrypsin that are associated with mild to moderate deficiency of the protein predict a subgroup of cystic fibrosis patients with less severe pulmonary disease. Moreover, the Taq-I G-->A allele has no effect on serum levels of alpha1-antitrypsin in the inflammatory response, which suggests that the previously reported association of the Taq-I G-->A allele with chronic obstructive pulmonary disease is not mediated by its effect on the serum level of alpha1-antitrypsin.

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51 The 39 "other" CF mutations in the normal α1-AT phenotype 508/other group were: six patients G551D, three R117H, three 621+1G→T, two R1162X, two G542X and one each had P67L, 1078delT, 2711delT, 1717-1G→A, V520F, 1898+1G→T, W1310X and N1303K in addition to the ∆F508 mutation. Login to comment

[hide] Mutation detection and single-molecule counting us... Nat Genet. 1998 Jul;19(3):225-32. Lizardi PM, Huang X, Zhu Z, Bray-Ward P, Thomas DC, Ward DC
Mutation detection and single-molecule counting using isothermal rolling-circle amplification.
Nat Genet. 1998 Jul;19(3):225-32., [PMID:9662393]

Abstract [show]
Rolling-circle amplification (RCA) driven by DNA polymerase can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions. In the presence of two primers, one hybridizing to the + strand, and the other, to the - strand of DNA, a complex pattern of DNA strand displacement ensues that generates 10(9) or more copies of each circle in 90 minutes, enabling detection of point mutations in human genomic DNA. Using a single primer, RCA generates hundreds of tandemly linked copies of a covalently closed circle in a few minutes. If matrix-associated, the DNA product remains bound at the site of synthesis, where it may be tagged, condensed and imaged as a point light source. Linear oligonucleotide probes bound covalently on a glass surface can generate RCA signals, the colour of which indicates the allele status of the target, depending on the outcome of specific, target-directed ligation events. As RCA permits millions of individual probe molecules to be counted and sorted using colour codes, it is particularly amenable for the analysis of rare somatic mutations. RCA also shows promise for the detection of padlock probes bound to single-copy genes in cytological preparations.

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23 We designed an allele-discriminating probe for a 46-nt target sequence in the CFTR G542X gene locus (Fig. 2a). Login to comment
35 Allele discrimination using HRCA and genomic DNA An alternative probe design for a 46-nt target sequence in the CFTR G542X gene locus (Fig. 2c) was used in this experiment. Login to comment
51 a, Sequence of the hybridizing arms of a circularizable probe, and two alternative 8-base gap probes designed for the CFTR G542X locus. Login to comment
64 c, Sequence of the hybridizing arms of a circularizable gap-fill probe designed for the CFTR G542X mutation locus. Login to comment
80 Human lymphocytes that were either wild-type, homozygous or heterozygous for the G542X locus were used as a DNA source. Login to comment
81 An assay was performed where G542X probes of the gap-fill design (Fig. 2c) were extended and ligated using genomic DNA targets, and amplified using HRCA. Login to comment
91 We prepared slides containing an oligonucleotide probe (P1) specific for a 39-base sequence adjacent to the G542X locus of the CFTR gene. Login to comment
118 We performed an assay to measure the ratio of mutant to wild type strands at the G542X locus in genomic DNA samples that had been constructed to simulate the presence of rare somatic mutations. Login to comment
122 a, Design of primers used to amplify an 89-base probe that had been extended by DNA polymerase copying of 7 nt (shown in small type) from the G542X target region, and circularized by DNA ligase. Login to comment
126 Lanes 1-7 were seeded, respectively, with 2×105, 2×104, 2×103, 2×102, 2×101, 2×101 (repeat), or zero molecules of G542X circularized mutant probe. Login to comment
128 c, Detection of the G542X point mutation. Login to comment
129 DNA samples from homozygous wild type, heterozygous or homozygous mutant cultured lymphocytes were denatured by heating for 4 min at 96 °C, and mixed with the gap-fill open circle probe for the G542X locus shown in 6A. Login to comment
142 P1 is designed to form 39 bp with the G542X target, and the 5´ terminus of P1 contains a 5´-phosphate to permit ligation. Login to comment
163 To test the feasibility of padlock probe detection on deproteinized DNA, we ligated the G542X padlock probe (Fig. 2a) on a sample of wild-type human genomic DNA which had been immobilized and denatured on a polylysine coated glass slide, at a density of approximately 480 haploid genomes per square mm. Login to comment
167 The same padlock probe, with a gap oligonucleotide specific for the wild-type G542X locus, was ligated on salt-extracted nuclei prepared by the 'halo` method11,12. Login to comment
179 a, Observation of non-condensed rolling circle DNA product from a padlock probe specific for the CFTR G542X wt locus, labelled with BUDR as a hapten, lying on the surface of a polylysine-coated slide. Login to comment
180 b,c, Observation of partly condensed (b) and fully condensed (c) rolling-circle amplification signals generated by CFTR G542X wt padlock probes that had been hybridized to nuclear 'halo` cytological preparations (see text for details). Login to comment
182 The frequency of nuclei displaying one or two RCA signals in halo preparations of wild-type cells was 197/2182 for the G542X locus wt probes, 1936/2573 for deltaF508 locus wt probes. Login to comment
236 The sequences are: CFTR G542X phosphorylated open circle probe (85 bases) for the gap oligo method, 5´-AAGAACTATATTGTCTTTCATTCTTGCATG- GTCACACGTCGTTCTAGTACGCTTCTAGTACGCTTTTCCACTCAGTG- TGATTCCA-3´; G542X wild type phosphorylated gap probe, 5´- CCTTCTCC-3´; G542X mutant phosphorylated gap probe, 5´- CCTTCTCA-3´; primer for rolling circle reaction, 542X.P1.18, 5´- ACGACGTGTGACCATGCA-3´. Login to comment
238 Primers for the HRCA reactions performed with gap-fill probe were: first primer, 542X.P1-23, 5´- CTAAAGCTGAGACATGACGAGTC-3´; alternative second primers for allele discrimination, 542X.P2-23-C, 5´-CTCAGTGTGATTCCACCTTC-3´ or 542X.P2-23-A, 5´-CTCAGTGTGATTCCACCTTCACA-3´; G542X artificial wild type target, 5´-TTGCAGAGAAAGACAATATAGTTCTTGGAGAAG- GTGGAATCACACTGAGTGGA-3´; G542X artificial mutant target, 5´- TTGCAGAGAAAGACAATATAGTTCTTTGAGAAGGTGGAATCACACTG- AGTGGA-3´. Login to comment
242 Oligonucleotides for the RCA-CACHET method, carbon spacers in parenthesis: phosphorylated P1-G542X, 5´-GAGAAGGTGGAATCACACTGAGTG- GAGGTCAACGAGCAATTTTTTTTTTT-(C7-NH2)-3´; P2wt, 3´-GTT- CTTGATATAACAGAAAGTTTT-(C18)-TTTTTATGATCACAGCTGAGGA- TAGGACATGCGA-3´; P2mu, 3´-TTTCTTGATATAACAGAAAGTTTT- (C18)-TTTTTACGTCGTCCGTGCTAGAAGGAAACACGCA-3´; pre-made amplification circles with cognate detector tags, Cwt, 5´-CGCATGTCCTAT- CCTCAGCTGTGATCATCAGAACTCACCTGTTAGACGCCACCAGCTCC- AACTGTGAAGATCGCTTAT-3´; detector tag Fl-det1c-dinitrophenol (DNP), FITC-TCAGAACTCACCTGTTAG-3´-DNP; detector tag Fl-det1d- DNP, FITC-ACTGTGAAGATCGCTTAT-3´-DNP; Cmu, 5´-GCGTGTTTC- CTTCTAGCACGGACGACGTATATGATGGTACCGCAGCCAGCATCACC- AGACTGAGTATCTCCTATCACT-3´; detector tag Cy3-det2b-DNP, Cy3- TATATGATGGTACCGCAG-3´-DNP; detector tag Cy3-det2c-DNP, Cy3- TGAGTATCTCCTATCACT-3´-DNP. Login to comment
249 Cell lines of known genotypes for the G542X locus (GM07828[+/+], GM11497B[+/-], GM11496[-/-]) were purchased from Corriell Cell Repositories. Login to comment
255 The circularizable G542X probe and an equimolar mixture of the two allele-specific gap oligonucleotides were ligated in the presence of the mutant sequence artificial DNA target. Login to comment
256 Circular DNA resulting from the ligation of G542X gapped probes were amplified with ø29 DNA polymerase, and the amplified ssDNA was cloned and sequenced. Login to comment
257 The resulting sequence contained only T at the position of the G542X mutation in 20 independent clones, indicating that only the mutant gap oligonucleotide had been ligated and amplified. Login to comment
258 With the wild-type template, the RCA product contained only G at the G542X site in 20 clones (data not shown). Login to comment
272 The oligonucleotide P1-G542X was immobilized over an area of 1 mm in diameter on the surface of a glass slide activated with reactive groups. Login to comment
274 Covalent coupling was obtained by reaction of a primary amine attached to the 3´ end of the P1-G542X oligonucleotide. Login to comment
275 Genomic DNA preparations obtained from human cell lines that were either wild type or homozygous mutant at the CFTR-G542X locus were mixed in different pre-determined ratios (mutant/wild type equal to 1:0, 1:1, 1:25 and 1:100) and then amplified by PCR as described17. Login to comment
305 Padlock probe ligationin situ was performed for 2 h at 52 °C by incubating 18 µl of reaction mixture under a cover slip sealed with rubber cement as follows: 150 nM phosphorylated G542X 89-mer probe, 1200 nM wild type G542X gap probe, in 20 mM Tris•HCl (pH 8.3), 50 mM KCl, 0.5 mM NAD, 10 mM MgCl2, 150 µg/ml acetylated BSA, 0.01% Triton X-100 and 0.7 U/µl Ampligase DNA ligase. Login to comment

[hide] Partial restoration of cAMP-stimulated CFTR chlori... Am J Physiol. 1998 Jul;275(1 Pt 1):C171-8. Jiang C, Fang SL, Xiao YF, O'Connor SP, Nadler SG, Lee DW, Jefferson DM, Kaplan JM, Smith AE, Cheng SH
Partial restoration of cAMP-stimulated CFTR chloride channel activity in DeltaF508 cells by deoxyspergualin.
Am J Physiol. 1998 Jul;275(1 Pt 1):C171-8., [PMID:9688848]

Abstract [show]
Deletion of the codon encoding phenylalanine 508 (DeltaF508) is the most common mutation in cystic fibrosis (CF) and results in a trafficking defect. Mutant DeltaF508-CF transmembrane conductance regulator (CFTR) protein retains functional activity, but the nascent protein is recognized as abnormal and, in consequence, is retained in the endoplasmic reticulum (ER) and degraded. It has been proposed that this retention in the ER is mediated, at least in part, by the cellular chaperones heat shock protein (HSP) 70 and calnexin. We have investigated the ability of deoxyspergualin (DSG), a compound known to compete effectively for binding with HSP70 and HSP90, to promote trafficking of DeltaF508-CFTR to the cell membrane. We show that DSG treatment of immortalized human CF epithelial cells (DeltaF508) and cells expressing recombinant DeltaF508-CFTR partially restored cAMP-stimulated CFTR Cl- channel activity at the plasma membrane. Although there are several possible explanations for these results, one simple interpretation is that DSG may have altered the interaction between DeltaF508-CFTR and its associated chaperones. If this is correct, agents capable of altering the normal functioning of cellular chaperones may provide yet another means of restoring CFTR Cl- channel activity to CF subjects harboring this class of mutations.

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139 The ability of DSG to influence the presence of endogenous mutant ⌬F508-CFTR at the plasma membrane was also assessed in IBE-1 cells, an immortalized human CF intrahepatic biliary epithelial cell line that harbors the ⌬F508 and G542X (premature stop mutation at residue 542) mutations (8). Login to comment

[hide] Pathology of pancreatic and intestinal disorders i... J R Soc Med. 1998;91 Suppl 34:40-9. Wilschanski M, Durie PR
Pathology of pancreatic and intestinal disorders in cystic fibrosis.
J R Soc Med. 1998;91 Suppl 34:40-9., [PMID:9709387]

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84 The next most common mutations are G542X, G551D, N1303K and W1282X, each of which account for 1% to 2.5% of CF chromosomes throughout the world. Login to comment
85 Two are missense mutations (G55 ID, N1303K) resulting in an amino acid substitution of the protein product, while G542X and W1 282X are nonsense mutations which give rise to a premature stop codon, leading to the expression of no CFTR or a non-functional truncated protein. Login to comment
97 Molecular consequences of cftr gene mutations Several classification systems have been developed in an attempt to define the large number of cftr gene mutations on 43 I =ON 4o0 o 00 4O0 40 40 I II 111 IV V Normal NOmAfl Missnso G542X Missmnse Missense Missense A455E Fremeshift AA dIeIIOn 05510 R117H Allerndve 39soTT AF508 spiRlng Spl$oeJunculon 3849t10kbC4T1717-1G-4A Figure 4 Molecular consequences of cystic fibrosis transmembrane conductance regulator mutations. Login to comment
152 A small number of more Table 1 Classification of cystic fibrosis gene mutation as severe, mild or indeterminate with respect to pancreatic function Severe Mild Variable (classes 1, I/ or 111) (classes IV or V) (classes IV or V) AF508 R117H G85E 1148T R334W 2789+5G-*A G480C R347P G551D A455E R560T P574H N1303K 3849+1 Okb C-+T G542X G551S W1282X P5748 621 +1 G-T R352Q 1717-1G-T T3381 556delA Adapted from Ref 20 with permission recently described mutations [G85E and 278+5G-÷AI are less clearly determinant with respect to the pancreatic sufficient and pancreatic insufficient phenotypes. Login to comment
168 An early study suggested that the nonsense mutation G542X is associated with an increased risk of meconium ileus, but this association has not been confirmed in a study of larger patient cohort. Login to comment

[hide] Cystic fibrosis screening: a fetus with hyperechog... J Med Genet. 1998 Aug;35(8):657-60. Muller F, Dommergues M, Simon-Bouy B, Ferec C, Oury JF, Aubry MC, Bessis R, Vuillard E, Denamur E, Bienvenu T, Serre JL
Cystic fibrosis screening: a fetus with hyperechogenic bowel may be the index case.
J Med Genet. 1998 Aug;35(8):657-60., [PMID:9719372]

Abstract [show]
BACKGROUND: The potential of hyperechogenic fetal bowel to act as a hallmark for prenatal cystic fibrosis screening in the general population is controversial. METHODS: Our goal was to evaluate the incidence of cystic fibrosis in 209 fetuses with hyperechogenic bowel diagnosed at routine ultrasonography and with no family history of cystic fibrosis. The diagnosis of cystic fibrosis was based on prenatal screening for the eight mutations most frequently observed in France (deltaF508, deltaI507, 1717-1G-->A, G542X, G551D, R553X, W1282X, N1303K) and at postnatal follow up. RESULTS: The overall incidence of cystic fibrosis was 7/209 (3.3%) which is 84 times the estimated risk of CF in the general population (112500). Of these seven cases, six were diagnosed prenatally based on DNA analysis (deltaF508/deltaF508, n=5; deltaF508/G542X, n=1). One case in which only one mutation had been recognised was diagnosed clinically after birth (deltaF508/unidentified mutation). Of the seven cases, none was diagnosed at 16-19 weeks, four at 16-24 weeks, and three after this. The incidence of heterozygous fetuses (15/209, 7%) was not significantly higher than the 5% expected in the general population. The mutations involved in these heterozygous cases were deltaF508 (n=13), G542X (n=1), and G551D (n=1). CONCLUSIONS: Screening for cystic fibrosis should be offered to families in which fetal hyperechogenic bowel is diagnosed at routine ultrasonography. This underlines the need to review genetic counselling in this situation where the fetus is the index case for a genetic disease.

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28 In all cases screening covered at least the eight mutations most frequently observed in France and North America, that is, AF508, AI507, 1717-1G--*A, G542X, G551D, R553X, W1282X, and N1303K. Login to comment
31 Other mutations were shown by ASO (allele Table 1 Fetal andparental mutations ofcysticfibrosis and pregnancy outcome in 209 cases ofhyperechogenicfetal bowel Prenatal genetic screening No CFTR mutation detected (n= 188) * Heterozygous CFTR mutation detected (n= 15) Fetus AF508/x (n=13) Fetus G55 1 D/x, father x/x, mother G55 1D/x (n= 1) Fetus G542X/x, father G542X/x, mother x/x (n=1) Homozygous CFTR mutation detected (n=6) Fetus AF508/AF508, father AF508/x, mother AF508/x (n=5) Fetus AF508/G542X, father G542X/x, mother AF508/x (n=1) Outcome Normal infant (n= 148) IUD or miscarriage (n= 14) Trisomy 21, TOP (n=3) Tetrasomy 12p, TOP (n=1) CMV/toxoplasmosis infection, TOP (n=7) Multiple malformations, TOP (n=2) Neonatal death unrelated to CF (n=3) Bowel atresia (n=8) Neonatal gastric haemorrhage (n= 1) Sudden infant death syndrome (n= 1) Normal infant (n=5) IUD (n=3) Digestive atresia, surgical treatment (n=3) TOP, fetal ascites (n= 1) Cystic fibrosis with meconium ileus at birth (n= 1) Normal infant (n= 1) Normal infant (n= 1) CF affected: TOP (n=2) CF affected: IUD (n= 1) CF affected: one neonatal death, one survivor (n=2) CF affected: TOP (n=1) CF=cystic fibrosis; x/x=no CFTR mutation detected; CFTR=CF transmembrane regulator; TOP=termination of pregnancy; IUD=intrauterine death; CMV=cytomegalovirus infection. Login to comment
49 Six fetuses were homozygous for CFTR mutations (five AF508/AF508, one compound heterozygous AF508/G542X). Login to comment
51 Fifteen fetuses were found to be heterozygous for one of the CFTR mutations tested: 13 AF508, one G542X, and one G551D. Login to comment

[hide] Mutations of the cystic fibrosis gene in patients ... N Engl J Med. 1998 Sep 3;339(10):645-52. Sharer N, Schwarz M, Malone G, Howarth A, Painter J, Super M, Braganza J
Mutations of the cystic fibrosis gene in patients with chronic pancreatitis.
N Engl J Med. 1998 Sep 3;339(10):645-52., 1998-09-03 [PMID:9725921]

Abstract [show]
BACKGROUND: The pancreatic lesions of cystic fibrosis develop in utero and closely resemble those of chronic pancreatitis. Therefore, we hypothesized that mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene may be more common than expected among patients with chronic pancreatitis. METHODS: We studied 134 consecutive patients with chronic pancreatitis (alcohol-related disease in 71, hyperparathyroidism in 2, hypertriglyceridemia in 1, and idiopathic disease in 60). We examined DNA for 22 mutations of the CFTR gene that together account for 95 percent of all mutations in patients with cystic fibrosis in the northwest of England. We also determined the length of the noncoding sequence of thymidines in intron 8, since the shorter the sequence, the lower the proportion of normal CFTR messenger RNA. RESULTS: The 94 male and 40 female patients ranged in age from 16 to 86 years. None had a mutation on both copies of the CFTR gene. Eighteen patients (13.4 percent), including 12 without alcoholism, had a CFTR mutation on one chromosome, as compared with a frequency of 5.3 percent among 600 local unrelated partners of persons with a family history of cystic fibrosis (P<0.001). A total of 10.4 percent of the patients had the 5T allele in intron 8 (14 of 134), which is twice the expected frequency (P=0.008). Four patients were heterozygous for both a CFTR mutation and the 5T allele. Patients with a CFTR mutation were younger than those with no mutations (P=0.03). None had the combination of sinopulmonary disease, high sweat electrolyte concentrations, and low nasal potential-difference values that are diagnostic of cystic fibrosis. CONCLUSIONS: Mutations of the CFTR gene and the 5T genotype are associated with chronic pancreatitis.

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32 DNA Studies We extracted DNA from buccal cells obtained by having the patients rinse their mouths with 10 ml of 4 percent sucrose.19 The CFTR locus was examined for the 22 mutations that together account for 95 percent of all such mutations in patients with cystic fibrosis in the northwest of England.20 The amplification- refractory mutation system Elucigene CF(4)m kit (Zeneca Diagnostics, Macclesfield, United Kingdom) was used to detect the four most common mutations: ∆F508, G551D, G542X, and 621+1(G→T)21; the polymerase chain reaction, restriction-enzyme analysis, and allele-specific oligonucleotide hybridization facilitated the detection of R560T, R117H, 1898+1(G→A), R553X, S549N, 1717¡1(G→A), N1303K, W1282X, E60X, 1154insTC, R347P, 3659delC, Q493X, V520F, R334W, ∆I507, 3849+10Kb(C→T), and 1078delT. Login to comment

[hide] Relation between mutations of the cystic fibrosis ... N Engl J Med. 1998 Sep 3;339(10):653-8. Cohn JA, Friedman KJ, Noone PG, Knowles MR, Silverman LM, Jowell PS
Relation between mutations of the cystic fibrosis gene and idiopathic pancreatitis.
N Engl J Med. 1998 Sep 3;339(10):653-8., 1998-09-03 [PMID:9725922]

Abstract [show]
BACKGROUND: It is unknown whether genetic factors predispose patients to idiopathic pancreatitis. In patients with cystic fibrosis, mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene typically cause pulmonary and pancreatic insufficiency while rarely causing pancreatitis. We examined whether idiopathic pancreatitis is associated with CFTR mutations in persons who do not have lung disease of cystic fibrosis. METHODS: We studied 27 patients (mean age at diagnosis, 36 years), 22 of whom were female, who had been referred for an evaluation of idiopathic pancreatitis. DNA was tested for 17 CFTR mutations and for the 5T allele in intron 8 of the CFTR gene. The 5T allele reduces the level of functional CFTR and is associated with an inherited form of infertility in males. Patients with two abnormal CFTR alleles were further evaluated for unrecognized cystic fibrosis-related lung disease, and both base-line and CFTR-mediated ion transport were measured in the nasal mucosa. RESULTS: Ten patients with idiopathic chronic pancreatitis (37 percent) had at least one abnormal CFTR allele. Eight CFTR mutations were detected (prevalence ratio, 11:1; 95 percent confidence interval, 5 to 23; P<0.001). In three patients both alleles were affected (prevalence ratio, 80:1; 95 percent confidence interval, 17 to 379; P<0.001). These three patients did not have lung disease typical of cystic fibrosis on the basis of sweat testing, spirometry, or base-line nasal potential-difference measurements. Nonetheless, each had abnormal nasal cyclic AMP-mediated chloride transport. CONCLUSION: In a group of patients referred for evaluation of idiopathic pancreatitis, there was a strong association between mutations in the CFTR gene and pancreatitis. The abnormal CFTR genotypes in these patients with pancreatitis resemble those associated with male infertility.

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34 Pancreatograms were assessed for the severity of chronic pancreatitis according to published criteria by a reviewer who was unaware of the patients` histories (Table 1).19 DNA Studies We extracted DNA from blood samples20 and tested for 16 CFTR mutations - ∆F508, W1282X, R117H, 621+1(G→T), R334W, R347P, A455E, ∆I507, 1717¡1(G→A), G542X, S549N, G551D, R553X, R560T, N1303K, and 3849+10Kb(C→T) - using reverse dot blot strips (Roche Molecular Systems, Alameda, Calif.). Login to comment

[hide] Pancreatitis and mutations of the cystic fibrosis ... N Engl J Med. 1998 Sep 3;339(10):687-8. Durie PR
Pancreatitis and mutations of the cystic fibrosis gene.
N Engl J Med. 1998 Sep 3;339(10):687-8., 1998-09-03 [PMID:9725928]

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12 The next most common mutations, G542X, G551D, N1303K, and W128X, each account for only 1 to 2.5 percent of known cystic fibrosis chromosomes. Login to comment

[hide] Development and validation of a screening test for... Eur Respir J. 1998 Aug;12(2):477-82. Robertson NH, Weston SL, Kelly SJ, Duxbury NJ, Pearce SR, Elsmore P, Webb MB, Newton CR, Little S
Development and validation of a screening test for 12 common mutations of the cystic fibrosis CFTR gene.
Eur Respir J. 1998 Aug;12(2):477-82., [PMID:9727805]

Abstract [show]
The results obtained using deoxyribonucleic acid (DNA) amplification-based tests must be accurate and reproducible. One such test that simultaneously detects any of 12 of the most common mutations of the cystic fibrosis transmembrane conductance regulator gene is presented in this report. An investigation was conducted into how changes of primer, DNA template and Taq DNA polymerase concentrations and of polymerase chain reaction annealing temperatures affect the test. A total of 383 DNA samples obtained from different laboratories was then examined. The preliminary studies defined the conditions under which accurate results are obtained even if the test is performed under suboptimal conditions. Subsequently, 377 (98.4%) of the DNA samples analysed were in full agreement with DNA typing results derived by other methods. The remaining 1.6% of samples were not mistyped, rather they were not scored owing to failure to detect control DNA sequences. These were also archival DNA preparations rather than freshly prepared samples from venous blood. Careful primer design and optimization of reaction conditions are important in the development of multiplex deoxyribonucleic acid amplification-based diagnostic tests. Providing the recommended protocols are followed, the test described here is simple to carry out, gives accurate results and works well if performed within defined operational windows for each reaction variable.

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12 The CFTR gene mutations that are detected by the test are 1717-1G>A, G542X, W1282X, N1303K, ∆F508, 3849+ 10kbC>T, 621+1G>T, R553X, G551D, R117H, R1162X and R334W, which are described by KAZAZIAN [10] and papers cited therein. Login to comment
48 The A-tube contains ARMS primers specific for the 1717-1G>A, G542X, W1282X, N1303K, ∆F508 and 3849+10kb C>T mutations. Login to comment
73 - Analysis of the 754 chromosomes tested Mutation Independent typing method* Totals 1717-1G>A G542X W1282X N1303K ∆F508 3849+10kbC>T 621+1G>T R553X G551D R117H R1162X R334W Other/none Number of samples Total number of chromosomes ASO ASO ASO ASO Electrophoresis Digest (HphI) Digest (MseI) Digest (HincII) Digest (NdeI) ASO Digest (DdeI) Digest (MspI) 16 10 16 12 89 11 7 15 16 13 11 6 532 377 754 *: Confirmatory typing as detailed in references cited within [10]. Login to comment
75 (97) (130) (160) (212) (240) (279) (329) (487) (487) (383) (325) (285) (243) (200) (160) (140) (97) (100) (150) (200) (250) (300) (350) (400) (450) (500) (550) apoB apoB ∆F508(N) ODCODC 3849+10kbC>T 1717-1G>A G542X W1282X N1303K ∆F508(M) R334W R1162X R117H G551D R553X 621+1G>T A-tube B-tube Marker Fig. 1. Login to comment
84 Where rare non-∆F508 compound heterozygotes have been obtained (3849+10kbC>T/W1282X; 3849+10kb C>T/G542X; G542X/N1303K; G542X/W1282X; G551D/ R553X; N1303K/1717-1G>A; G542X/17171G>A; N1303K/ W1282X; R553X/R334W) and analysed, both mutations were correctly identified. Login to comment
99 1: 1717-1G>A/+; 2: G542X/+; 3: W1282X/+; 4: N1303K/+; 5: ∆F508/+; 6: 3849+10kbC>T/+; 7: +/+; 8: +/+; 9: ∆F508/∆F508; 10: 621+1G>T/+; 11: R553X/+; 12: G551D/+; 13: R117H/+; 14: R1162X/ +; 15: R334W/+; 16: +/+; 17: +/+; 18: ∆F508/∆F508; 19: ∆F508/+. Login to comment
102 1: +/+; 2: 1717-1G>A/+; 3: G542X/+; 4: W1282X/+; 5: N1303K/+; 6: ∆F508/+; 7: 3849+10kbC>T/+; 8: 621+1G>T/+; 9: R553X/+; 10: G551D/+; 11: R117H/+; 12: R1162X/+; 13: ∆F508/∆F508; 14: R334W/+. Login to comment
104 15: +/+; 16: +/+; 17: R553X/+; 18: +/+; 19: ∆F508/+; 20: +/+; 21: +/+; 22: R117H/∆F508; 23: ∆F508/∆F508; 24: +/+: 25: G542X/N1303K; 26: no deoxyribonucleic acid (DNA) control. Login to comment
107 The CF(12)m test screens for the CF mutations 1717-1G>A, G542X, W1282X, N1303K, ∆F508, 3849+10kbC>T, 621+ 1G>T, R553X, G551D, R117H, R1162X and R334W, the most common CF mutations in Caucasians and Ashkenazi Jews. Login to comment
119 Furthermore, the finding from another study [15], which compared multiplex ARMS screening for the ∆F508, G551D, G542X and 621+1G>T alleles [8] with alternative routine procedures for the same alleles, was that multiplex ARMS was the preferred method. Login to comment

[hide] Nasal potential difference in congenital bilateral... Am J Respir Crit Care Med. 1998 Sep;158(3):896-901. Pradal U, Castellani C, Delmarco A, Mastella G
Nasal potential difference in congenital bilateral absence of the vas deferens.
Am J Respir Crit Care Med. 1998 Sep;158(3):896-901., [PMID:9731023]

Abstract [show]
Congenital bilateral absence of the vas deferens (CBAVD) is supposed to be due to defective activity of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) in the genital tract. With the aim of studying CFTR activity in vivo we measured nasal potential difference (NPD) in a group of CBAVD subjects, who were then compared with normal control subjects and CF patients. Sodium transport, measured under basal conditions and after amiloride superinfusion, was normal in almost all CBAVD patients, who had NPD values similar to those of normal control subjects. Chloride transport was studied by measuring NPD during perfusion with a chloride-free solution and isoproterenol. Under these circumstances CBAVD patients as a whole showed normal chloride secretion. However, three subjects with CBAVD had abnormal NPD values. They had either elevated sweat chloride concentrations together with symptoms of mild CF, or compound heterozygosity (DeltaF508/R117H). In conclusion the group of CBAVD patients as a whole presented normal bioelectric properties of nasal epithelium, suggesting normal CFTR activity. In a small subgroup NPD was abnormal, suggesting a diagnosis of CF, later confirmed by elevated sweat chloride concentrations or positive DNA testing. We suggest that CBAVD patients with altered NPD should undergo further clinical follow-up in order to detect possible late complications of CF.

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39 ⌬F508, R117H, R1162X, 2183AA→G, N1303K, 3849 ϩ 10KbC→T, G542X, 1717-1G→A, R553X, Q552X, G85E, 711 ϩ 5G→A, 3132delTG and 2789 ϩ 5G→A were tested using for R117H two specifically designed primers which create a CFoI restriction site when the mutation is absent, and for all the other mutations a reverse dot blot assay (19). Login to comment
64 Age (yr) Sweat Cl- (mmol/L) Sweat Naϩ (mmol/L) CFTR Mutation PolyT Variant NPD* (mV) Main Anamnestical and Clinical Data 1 37 35 51 G542X/- 7/9 -16.2 SA† and HI‡ in sputum culture 2 24 62 72 ⌬F508/- 7/9 -12.3 Sinusitis 3 32 88 86 ⌬F508/- 9/9 -16.5 Relation of a CF patient, sinusitis, previous tuberculous lymphadenitis, 4 39 16 39 -/- 7/7 -10.3 chronic cough, diabetes 5 40 8 22 -/- 7/9 -12.8 - 6 37 6 12 -/- 5/7 -14.7 Asthma 7 29 34 37 ⌬F508/- 7/9 -10.1 Primary tuberculosis infection 8 32 44 55 ⌬F508/- 9/9 -13.7 HI in sputum culture 9 44 40 43 ⌬F508/- 5/9 -16.9 Nasal polyposis, biliary stones, chronic sinusitis 10 40 39 52 -ր- 7/7 -11.7 SA sputum culture, bilateral inguinal hernia 11 36 32 50 ⌬F508/R117H 7/9 -29.1 Funnel chest, pulmonary hyperinflation 12 32 65 70 ⌬F508/- 7/9 -46.7 SA sputum culture * Basal nasal potential difference. Login to comment
69 CFTR gene mutations were found in eight patients (seven ⌬F508 and one G542X), the 5T variant in two. Login to comment

[hide] Testicular CFTR splice variants in patients with c... Hum Mol Genet. 1998 Oct;7(11):1739-43. Larriba S, Bassas L, Gimenez J, Ramos MD, Segura A, Nunes V, Estivill X, Casals T
Testicular CFTR splice variants in patients with congenital absence of the vas deferens.
Hum Mol Genet. 1998 Oct;7(11):1739-43., [PMID:9736775]

Abstract [show]
The involvement of the five thymidine (5T) variant in intron 8 of the cystic fibrosis membrane regulator (CFTR) gene in congenital bilateral absence of the vas deferens (CBAVD) phenotype has been extensively demonstrated. This variant leads to alternative splicing of the CFTR gene which results in a wild-type transcript and one without exon 9. Little is known about expression of the CFTR gene in the testis. We analysed the level of the aberrantly spliced transcripts in testicular biopsies and correlated it with disease expression. Quantitative RT-PCR analysis in testicular biopsies from control and CBAVD patients showed a correlation between the length of the IVS8-6(T) n tract and the level of alternatively spliced transcripts. Results from histological analysis also suggest an involvement of the alternative transcript in the spermatogenic status of patients, leading to a decreased number of mature sperm forms in the tubule.

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18 RESULTS CFTR analysis Eight different mutations (R117H, L206W, V232D, ∆F508, G542X, 711+1G→T, D1270N and 2789+5G→A) were found in nine of the 12 CBAVD patients, yielding a CFTR mutation frequencyof75%.ThreepatientspresentedtwoCFTRmutations, with one of them homozygous for the V232D mutation. Login to comment
26 CFTR genotype, IVS8-6 poly(T) allele and proportion of exon 9+ (E9+) and exon 9- (E9-) CFTR transcripts in testicular and epididymal biopsies Sample Phenotype CF mutation IVS8-6(T) Testis Epididymis n E9+ (%) E9- (%) n E9+ (%) E9- (%) 1 Non-CBAVD N/N 9T/9T 5 99 ± 0 1 ± 0 2 Non-CBAVD N/N 7T/7T 2 96 ± 2 4 ± 2 3 Non-CBAVD N/N 7T/7T 3 98 ± 0 2 ± 0 4 Non-CBAVD N/N 7T/7T 3 97 ± 1.5 3 ± 1.5 5 Non-CBAVD R334W/N 7T/7T 3 94 ± 1 6 ± 1 6 Non-CBAVD N/N 7T/7T 2 95 ± 1 5 ± 1 7 CBAVD V232D/V232D 9T/9T 4 96 ± 1.5 4 ± 1.5 8 CBAVD ∆F508/N 9T/9T 2 99 ± 0 1 ± 0 9 CBAVD ∆F508/D1270N 7T/9T 2 98 ± 1 2 ± 1 10 CBAVD G542X/2789+5G→A 7T/9T 2 96 ± 1 4 ± 1 11 CBAVD N/N 7T/7T 3 96 ± 2 4 ± 2 2 90 ± 3 10 ± 3 12 CBAVD N/N 7T/7T 2 94 ± 2 6 ± 2 5 78 ± 5 22 ± 5 13 CBAVD R117H/N 7T/7T 2 99 ± 0 1 ± 0 4 95 ± 2 5 ± 2 14 CBAVD G542X/5T 5T/9T 3 30 ± 2 70 ± 2 15 CBAVD ∆F508/5T 5T/9T 2 80 ± 5 20 ± 5 16 CBAVD L206W/5T 5T/9T 2 58 ± 2 42 ± 2 17 CBAVD 711+1G→T/5T 5T/7T 3 77 ± 4 23 ± 4 18 CBAVD 5T/N 5T/7T 5 71 ± 2 29 ± 2 The mean proportion of E9+ and E9- CFTR transcripts is calculated as the mean of the proportions found for each sample. Login to comment
112 Each sample of genomic DNA was first studied for the most common CF mutations in the Spanish population, ∆F508 and G542X. Login to comment

[hide] Characterization of 19 disease-associated missense... Hum Mol Genet. 1998 Oct;7(11):1761-9. Vankeerberghen A, Wei L, Jaspers M, Cassiman JJ, Nilius B, Cuppens H
Characterization of 19 disease-associated missense mutations in the regulatory domain of the cystic fibrosis transmembrane conductance regulator.
Hum Mol Genet. 1998 Oct;7(11):1761-9., [PMID:9736778]

Abstract [show]
In order to gain a better insight into the structure and function of the regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, 19 RD missense mutations that had been identified in patients were functionally characterized. Nine of these (I601F, L610S, A613T, D614G, I618T, L619S, H620P, G628R and L633P) resulted in aberrant processing. No or a very small number of functional CFTR proteins will therefore appear at the cell membrane in cells expressing these mutants. These mutations were clustered in the N-terminal part of the RD, suggesting that this subdomain has a folding pattern that is very sensitive to amino acid changes. Mutations that caused no aberrant processing were further characterized at the electrophysiological level. First, they were studied at the whole cell level in Xenopus laevis oocytes. Mutants that induced a whole cell current that was significantly different from wild-type CFTR were subsequently analysed at the single channel level in COS1 cells transiently expressing the different mutant and wild-type proteins. Three mutant chloride channels, G622D, R792G and E822K CFTR, were characterized by significantly lower intrinsic chloride channel activities compared with wild-type CFTR. Two mutations, H620Q and A800G, resulted in increased intrinsic chloride transport activities. Finally, T665S and E826K CFTR had single channel properties not significantly different from wild-type CFTR.

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87 Maturation pattern of RD mutations and their associated phenotype found in patients with the indicated genotype (when the mutation is associated with CF, only the pancreas status is given) Mutation A-form B-form C-form Clinical data Genotype Phenotype Reference I601F + + - I601F/G542X PS M. Schwarz, personal communication L610S + + - Unknown Unknown A613T + + - Unknown Unknown D614G + + - D614G/unknown PI 14 I618T + + - I618T/dF508 PS G.R. Cutting, personal communication L619S + + - L619S/unknown PI B. Tümmler, personal communication H620P + + - H620P/R1158X PS M. Schwarz, personal communication H620Q + + + H620Q/dF508 PI T. Dörk, personal communication G622D + + + G622D/unknown Oligospermia J. Zielenski, personal communication G628R + + - Unknown Unknown L633P + + - L633P/3659delC M. Schwarz, personal communication D648V + + + D648V/3849+10kb C/T PI C. Ferec, personal communication T665S + + + Unknown Unknown F693L + + + F693L/W1282X Healthy C. Ferec; CF Genetic Analysis Consortium R766M + + + R766M/R792G CBAVD D. Glavac, personal communication R792G + + + R766M/R792G CBAVD D. Glavac, personal communication A800G + + + A800G/unknown CBAVD 34 I807M + + + I807M/unknown CBAVD Our observation E822K + + + E822K/unknown PI 35 E826K + + + E826K/unknown Thoracic sarcoidosis C. Bombieri, personal communication +, the protein matures up to that form; -, the protein does not reach the respective maturation step. Login to comment

[hide] Molecular basis of cystic fibrosis in the Republic... Clin Genet. 1998 Sep;54(3):203-9. Petreska L, Koceva S, Plaseska D, Chernick M, Gordova-Muratovska A, Fustic S, Nestorov R, Efremov GD
Molecular basis of cystic fibrosis in the Republic of Macedonia.
Clin Genet. 1998 Sep;54(3):203-9., [PMID:9788722]

Abstract [show]
Eighty-three cystic fibrosis (CF) patients and their families, belonging to various ethnic groups living in the Republic of Macedonia were studied for molecular defects in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and for the associated extragenic marker loci XV-2c and KM19. The DNA methodology used included characterization of CFTR mutations in 19 exons (and flanking sequences) of the gene and analysis of distribution of the XV-2c/KM19 haplotypes among normal (N) and CF chromosomes by polymerase chain reaction (PCR) amplification followed by dot blot hybridization, restriction digestion, single-strand conformational polymorphism, constant denaturing gel electrophoresis, denaturing gradient gel electrophoresis, and sequencing. We identified 58.4% (97/166) of the CF chromosomes. Nine different CFTR gene mutations, including three novel ones, were found. Eight known and one new CFTR intragene polymorphisms were also characterized. The haplotype analysis of the XV-2c/TaqI and KM19/PstI polymorphic loci have shown that haplotype C is the most frequently found haplotype among the non-deltaF508 CF chromosomes from Macedonia (36.5%). The results demonstrate the broad heterogeneity of CF origin in this part of the Balkan Peninsula.

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40 The screening procedures of 17 other known CF mutations included detection of mutations in the PCR products of positive controls and samples by: a) direct analysis on PAGE for A1507 and 1677delTA, simultaneously to AF508; b) hybridization with ASOs for mutation R117H (21), 1717-1GdA (22), G542X (22), N1303K (23), and W1316X (24), and c) restriction digestion `followed by agarose or polyacrylamide gel electrophoresis (exon 3 PCR product digested with HinfI for CUE, exon 4 with HinfI for 444delA, exon 5 with RsaI for 711 + 5G --*A,exon 7 with HhaI for R347H or with RsaI for Q359K/T360, exon 11 with HincII for both G551D and R553X, exon 19 with DdeI for R1162X or with HphI for 3849G+A, a 175 bp PCR fragment of exon 13 with HaeIII for 2556insAT) (4). Login to comment
65 Only G542X and N1303K were found among the 17 more common mutations that, besides AF508, were screened by direct detection. Login to comment
70 Frequency of mutations identified in CF chromosomesfrom the Republic of Macedonia Mutation (location) Number of chromosomeswith mutation Ethnic group (fraction%) Method of detection XV-2clKM19 haplo- References (96) type AF508 (exon 10) 79 (47.6) All groups 12% PAGE BD (3) G542X (exon 11) 6 (3.6) All groups Dot blot B (22) N1303K (exon 21) 3 (1.8) MKa(2.8%) Dot blot B (23) 621t l G + T 2 (1.2) MK (1.9%) SSCP B (30) fintron 4) (intron 5) (exon 19) (exon 4) (intron 11) 711t3A-rG 2 (1.2) ALb (3.8%) SSCP A (31)c 384% +A 2 (1.2) MK (1.9%) SSCP C (32) 457TAT+G 1 (0.6) MK (0.9%) SSCP B (33) 1811+1G-.C l(0.6) MK (0.9%) DGGE 8 Hphl dig. Login to comment
72 Clinical feaiures of the CF patients from Macedonia Genotype Number of CF patients (%) PI PS Shwachman scorea Age of onset GI- (mmljl) NDb AF508/AF508 AF508/G542X AF508/N1303K AF508/711+3A-.G AF508/711+W+G AF508/621+lG+T AF508/621+1G+T AF508/1811+1G-*C AF508/457TAT-*G AF508/3849G+A G542X/3849G+A N1303K/ND V1397E/ND AF508/ND NDiND Total 26 (31.3) 5 (6.0) 2 (2.4) 2 (2.4) 2 (2.4) 1 (1.2) 1 (1.2) 1 (1.2) 1 (1.2) 1 (1.2) 1 (1.2) 13 (15.7) 27 (32.5) 83 (100.0) 26 5 2 1 1 1 1 1 1 1 1 1 7 4 1 15 7 63 13 25-85 30-60 38 84 82 35 78 80 83 50 82 83 I 30-60 20-90 ~~~~~ 1-6 months 1 month 3 months 1 month 1 month 2 months 2.5 years 2 months 2 months 2 months 1 month 6 years 3 weeks Variable Vanable ~ ~ ~~~ 80-210 116-166 180-200 80 120 170 156 240 150 98 190 65 65 65-2300 2 65-130 (ps) 58-230 (PO 5 65-130 (PS) 7 a Shown is the most recent Shwachman score. Login to comment
79 Genotype-phenotype data Nine different genotypes were observed in 41/83 (49.4%) unrelated families: AF508/AF508 (n =26), AF508/457TAT-,G (n = l), AF508/621+1G+T (n = 2), AF508/G542X (n = 5), AF508/N1303K (n =2), AF508/38496-,A (n= I), G542X/ 384963 A (n = l), AF508/711 +3A G (n = 2), AF508/1811+1G-C (n = 1). Login to comment
98 They include the 'oldest` G542X (found in different ethnic groups) and N1303K mutations (found only among Macedonians), and one additional mutation, 621 +lG+T, that is among the world`s most common CF alleles. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Biochemistry. 1998 Oct 27;37(43):15222-30. Clancy JP, Hong JS, Bebok Z, King SA, Demolombe S, Bedwell DM, Sorscher EJ
Cystic fibrosis transmembrane conductance regulator (CFTR) nucleotide-binding domain 1 (NBD-1) and CFTR truncated within NBD-1 target to the epithelial plasma membrane and increase anion permeability.
Biochemistry. 1998 Oct 27;37(43):15222-30., 1998-10-27 [PMID:9790686]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the traffic ATPase family that includes multiple proteins characterized by (1) ATP binding, (2) conserved transmembrane (TM) motifs and nucleotide binding domains (NBDs), and (3) molecular transport of small molecules across the cell membrane. While CFTR NBD-1 mediates ATP binding and hydrolysis, the membrane topology and function of this domain in living eukaryotic cells remains uncertain. In these studies, we have expressed wild-type CFTR NBD-1 (amino acids 433-586) or NBD-1 containing the DeltaF508 mutation transiently in COS-7 cells and established that the domain is situated across the plasma membrane by four independent assays; namely, extracellular chymotrypsin digestion, surface protein biotinylation, confocal immunofluorescent microscopy, and functional measurements of cell membrane anion permeability. Functional studies indicate that basal halide permeability is enhanced above control conditions following wild-type or DeltaF508 NBD-1 expression in three different epithelial cell lines. Furthermore, when clinically relevant CFTR proteins truncated within NBD-1 (R553X or G542X) are expressed, surface localization and enhanced halide permeability are again established. Together, these findings suggest that isolated CFTR NBD-1 (with or without the DeltaF508 mutation) is capable of targeting the epithelial cell membrane and enhancing cellular halide permeability. Furthermore, CFTR truncated at position 553 or 542 and possessing the majority of NBD-1 demonstrates surface localization and also confers increased halide permeability. These findings indicate that targeting to the plasma membrane and assumption of a transmembrane configuration are innate properties of the CFTR NBD-1. The results also support the notion that components of the halide-selective pore of CFTR reside within NBD-1.

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4 Furthermore, when clinically relevant CFTR proteins truncated within NBD-1 (R553X or G542X) are expressed, surface localization and enhanced halide permeability are again established. Login to comment
32 Expression of CFTR containing the clinically relevant mutations R553X or G542X, which each contain >70% of the NBD-1 motif, produce polypeptides that also target the cell membrane and enhance halide permeability in a manner similar to isolated NBD-1. Login to comment
45 Recombinant vaccinia viruses containing wtCFTR and CFTR containing a premature stop codon at position 542 (G542X) or 553 (R553X) under the regulatory control of the T7 promoter was generated from constructs in the pTM1 vector (15) using standard techniques (16). Login to comment
75 Immunoprecipitation of NBD-1, G542X, R553X, and wtCFTR. Login to comment
87 Digital Confocal Immunofluorescent Microscopy of COS-7 Cells Expressing wt or ∆F508 CFTR, G542X, or R553X. Login to comment
157 Expression of R553X and G542X CFTR in Mammalian Epithelial Cell Lines. Login to comment
160 We chose to study two clinically relevant mutant CFTR molecules possessing premature stop codons, G542X and R553X. Login to comment
161 These mutations are found in approximately 5% of the CF population (34) and include 72% (G542X) to 79% (R553X) of the NBD-1 (aa 432-586). Login to comment
162 Figure 6 shows detection of 35 S-labeled truncated proteins [~53 kDa (G542X); ~55 kDa (R553X)] from COS-7 cell lysates following vaccinia-based expression. Login to comment
176 This antibody recognizes an epitope in the first predicted extracellular loop of CFTR TM-1, which is shared by wtCFTR, R553X, and G542X. Login to comment
179 Figure 7 indicates a plasma membrane staining pattern in cells without detergent permeabilization following wtCFTR, R553X, and G542X expression, but not ∆F508 CFTR. Login to comment
181 These studies provide evidence of plasma membrane targeting by CFTR possessing either the G542X or R553X mutations. Login to comment
182 To determine whether either R553X or G542X CFTR had positive effects on basal cellular halide permeability, we performed SPQ analysis in COS-7 cells expressing these mutant cDNAs (Figure 8). Login to comment
185 The effects of R553X, G542X, and NBD-1 on COS-7 cell basal halide permeability were qualitatively and quantitatively similar (compare with Figure 5B). Login to comment
199 FIGURE 6: Immunoblot of wt, R553X, and G542X CFTR in COS-7 cells. Cells were labeled with translabel for 20 min at 12 h after infection and immunoprecipitated with anti-NBD-1 antibody as described in the Experimental Procedures. Lane A ) wtCFTR, lane B ) G542X, lane C ) R553X. Login to comment
202 Expression of truncated CFTR (R553X CFTR and G542X CFTR, Figures 6, 7, and 8) produces a protein which also targets the eukaryotic plasma membrane, enhancing cellular basal halide permeability. Login to comment
203 Neither R553X nor G542X produces cAMP-regulated halide permeability, and no full-length CFTR could be detected by immunoprecipitation, indicating that suppression of the premature termination codons is not responsible for the ion transport results shown here (39, 40). Login to comment
204 The enhanced basal halide permeability conferred by R553X and G542X is qualitatively and quantitatively similar to that produced by NBD-1 (Figure 5). Login to comment
205 We suggest that the truncated CFTR proteins G542X and R553X contain adequate domains and the appropriate cellular signals to fold into functional peptides and localize to the cell membrane. Login to comment
206 The common halide permeability enhancing effects produced by G542X, R553X, and isolated NBD-1 suggest that these three polypeptides share common amino acids (422-542 of the complete CFTR molecule) which may be able to activate halide permeability in several epithelial cell types. Login to comment
207 On the basis of the findings that R553X and G542X retain surface localizing and residual halide transport function, we recently studied five patients possessing at least one of these alleles, but found no evidence of residual Cl-secretion during a nasal potential difference protocol (25). Login to comment
211 In either case, our results clearly establish that, unlike class II CFTR mutations (34), R553X and G542X CFTR are capable of escaping intracellular degradation, targetting the plasma membrane, and forming functional proteins that maintain some residual halide transport function. Login to comment
213 FIGURE 7: Localization of wtCFTR, R553X, and G542X to the cell surface in COS-7 cells by confocal immunocytochemistry. Login to comment
217 Lower panels from left to right are (D) G542X without permeabilization, (E) R553X without permeabilization, and (F) vT7 controls with permeabilization. Login to comment
218 FIGURE 8: R553X or G542X expression increases halide permeability in COS-7 cells. Cells grown on glass coverslips were studied by SPQ as in Figure 5. Login to comment
221 (A) Dequench of the G542X expressing cells was significantly elevated above control cells (P < 0.0001 compared with vT7 or LacZ conditions). Login to comment

[hide] Detection of more than 91% cystic fibrosis mutatio... Clin Genet. 1998 Nov;54(5):437-9. Cartault F, Steffann J, Vidaud D, Bousquet S, Lesure F, Renouil M, McDonell N, Feingold J, Beldjord C, Bienvenu T
Detection of more than 91% cystic fibrosis mutations in a sample of the population from Reunion Island and identification of two novel mutations (A309G, S1255L) and one novel polymorphism (L49L)
Clin Genet. 1998 Nov;54(5):437-9., [PMID:9842999]

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30 Ten CFTR mutations identified in 69 CF families from Reunion Island Mutationa Exonlintron CF alleles Percentage Ama E.10 72 52 Y122X E.4 33 24 A455E E.9 3 2.2 G551D E.11 2 1.4 1717-1G-+A i.10 1 0.7 G542X E.ll 1 0.7 116ldelC E.7 1 0.7 A3G9G E.7 1 0.7 zag+ 5~-+A i.14b 1 0.7 3120tlG-A i.16 11 a Unknown mutations 12 8.7 aCystic Fibrosis Genetic Analysis Consortium: Web site: http // w.genet.sickkids.on.ca/cftr/ CFTR represents the missense mutation A309G (Fig. 1A). Login to comment

[hide] Chronic pancreatitis and mutations of the cystic f... Gut. 1999 Jan;44(1):8-9. Taylor CJ
Chronic pancreatitis and mutations of the cystic fibrosis gene.
Gut. 1999 Jan;44(1):8-9., [PMID:9862818]

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29 To date, more than 700 mutations have been identified in the CF gene; most, with the exception of G551D, G542X and 621+1 (G-A) are rare, aVecting <1% of CF chromosomes. Login to comment
61 Reproduced with permission from Wilschanski et al.2 V Missense A455E Alternative splicing 3849+10kbC (f) IV Missense R117H (e) III Missense G551D (d) II Missense AA deletion ∆F508 (c) I Nonsense G542X Frameshift 394delTT Splice junction 1717-1G (b) Normal (a) T A Chronic pancreatitis and mutations of the cyctic fibrosis gene group.bmj.comon August 8, 2011 - Published bygut.bmj.comDownloaded from doi: 10.1136/gut.44.1.8 1999 44: 8-9Gut C J TAYLOR cystic fibrosis gene Chronic pancreatitis and mutations of the http://gut.bmj.com/content/44/1/8.full.html Updated information and services can be found at: These include: References http://gut.bmj.com/content/44/1/8.full.html#related-urls Article cited in: http://gut.bmj.com/content/44/1/8.full.html#ref-list-1 This article cites 4 articles, 2 of which can be accessed free at: service Email alerting box at the top right corner of the online article. Login to comment

[hide] Relationships between nasal potential difference a... Eur Respir J. 1998 Dec;12(6):1295-300. Fajac I, Hubert D, Bienvenu T, Richaud-Thiriez B, Matran R, Kaplan JC, Dall'Ava-Santucci J, Dusser DJ
Relationships between nasal potential difference and respiratory function in adults with cystic fibrosis.
Eur Respir J. 1998 Dec;12(6):1295-300., [PMID:9877480]

Abstract [show]
This study investigated the relations between nasal transepithelial electric potential difference (PD) and the phenotype and genotype of cystic fibrosis (CF) adult patients. Basal nasal PD was measured in 95 adult CF patients who were classified into three groups of nasal PD (expressed as absolute values) according to the 10th and the 90th percentiles (28.3 and 49.2 mV, respectively), which defined group 1 (nasal PD < or =28.3 mV), group 2 (nasal PD 28.3-49.2 mV) and group 3 (nasal PD > or =49.2 mV). Patients from group 1 had a higher forced vital capacity (FVC) than patients from groups 2 and 3 (76.5+/-22.4 versus 57.4+/-21.2 and 55.7+/-21.1% predicted, respectively, p<0.05) and a higher forced expiratory volume in one second (FEV1) (69.3+/-24.0 versus 42.5+/-22.4 and 42.2+/-21.4% pred, respectively, p<0.01). Among patients with severe mutations (deltaF508 homozygotes, or one deltaF508 mutation plus another "severe" mutation, or two "severe" mutations), patients from group 1 had a higher FVC, FEV1 and arterial oxygen tension than patients from groups 2 and 3 (p<0.05 for each comparison). The results show that in adult cystic fibrosis patients a normal basal nasal potential difference is related to milder respiratory disease, irrespective of the severity of the genotype.

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34 Only three patients had a normal sweat test: one was homozygotic for the ∆F508 mutation and two patients were compound heterozygotic for the G542X and 3849+10 kb cytosine (C) → thymine (T) mutations and for the R1070Q and D1152H mutations, respectively. Login to comment

[hide] Validation of double gradient denaturing gradient ... Clin Chem. 1999 Jan;45(1):35-40. Cremonesi L, Carrera P, Fumagalli A, Lucchiari S, Cardillo E, Ferrari M, Righetti SC, Zunino F, Righetti PG, Gelfi C
Validation of double gradient denaturing gradient gel electrophoresis through multigenic retrospective analysis.
Clin Chem. 1999 Jan;45(1):35-40., [PMID:9895335]

Abstract [show]
Among established techniques for the identification of either known or new mutations, denaturing gradient gel electrophoresis (DGGE) is one of the most effective. However, conventional DGGE is affected by major drawbacks that limit its routine application: the different denaturant gradient ranges and migration times required for different DNA fragments. We developed a modified version of DGGE for high-throughput mutational analysis, double gradient DGGE (DG-DGGE), by superimposing a porous gradient over the denaturant gradient, which maintains the zone-sharpening effect even during lengthy analyses. Because of this innovation, DG-DGGE achieves the double goals of retaining full effectiveness in the detection of mutations while allowing identical run time conditions for all fragments analyzed. Here we use retrospective analysis of a large number of well-characterized mutations and polymorphisms, spanning all predicted melting domains and the whole genomic sequence of three different genes--the cystic fibrosis transmembrane conductance regulator (CFTR), the beta-globin, and the p53 genes--to demonstrate that DG-DGGE may be applied to the rapid scanning of any sequence variation.

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31 Mutations and polymorphisms analyzed in the CFTR gene. Position Denaturant gradient Mutation Exon 1 40-90% 125G/Ca,b M1V (A3G at 133) 175insT 182delT Exon 3 10-60% W57G (T3G at 301) 356G/Aa G85E (G3A at 386) Exon 4 20-70% R117H (G3A at 482) 541delC 621ϩ1G3T I148T (T3C at 575) Exon 5 20-70% E193K (G3A at 709) Intron 5 20-70% 711ϩ3A3G Exon 7 20-70% 1078delT R334W (C3T at 1132) T338I (C3T at 1145) R347P (G3C at 1172)b R347H (G3A at 1172) R352Q (G3A at 1187) Exon 10 20-70% M470V (1540A/G)a ⌬F508 (del 3 bp at 1652) Intron 10 10-60% 1717-1G3A Exon 11 10-60% G542X (G3T at 1756) 1784delG R553X (C3T at 1789) Exon 12 10-60% D579G (A3G at 1868) E585X (G3T at 1885) Intron 12 10-60% 1898ϩ3A3G Exon 13 30-80% 2183AA3G E730X (G3T at 2320) L732X (T3G at 2327) 2347delG Exon 14a 10-60% T854T (2694T/G)a V868V (2736G/A)a Intron 14b 30-80% 2789ϩ5G3A Exon 15 20-70% M952I (G3C at 2988)b Exon 17a 20-70% L997F (G3C at 3123)b Exon 17b 20-70% F1052V (T3G at 3286) R1066C (C3T at 3328) R1066H (G3A at 3329) A1067T (G3A at 3331) Exon 18 20-70% D1152H (G3C at 3586)b Exon 19 30-80% R1158X (C3T at 3604) Exon 20 20-70% S1251N (G3A at 3384) W1282X (G3A at 3978) Exon 21 20-70% N1303K (C3G at 4041)b Exon 22 30-80% G1349D (G3A at 4178) 4382delA Exon 24 30-80% Y1424Y (4404C/T)a a Polymorphism. Login to comment

[hide] Clinical and genetic risk factors for cystic fibro... Pediatrics. 1999 Jan;103(1):52-7. Wilschanski M, Rivlin J, Cohen S, Augarten A, Blau H, Aviram M, Bentur L, Springer C, Vila Y, Branski D, Kerem B, Kerem E
Clinical and genetic risk factors for cystic fibrosis-related liver disease.
Pediatrics. 1999 Jan;103(1):52-7., [PMID:9917439]

Abstract [show]
OBJECTIVE: The aim of this study was to define the role of possible risk factors for the development of cystic fibrosis (CF)-related liver disease and to analyze the association between liver disease and the different genotypes present in the Israeli CF patient population. PATIENTS AND METHODS: All patients followed at the seven CF centers in Israel were included in this study. Liver disease was determined by persistently elevated serum liver enzymes and/or bilirubin, and/or significant ultrasonographic changes suggestive of chronic liver disease. The following clinical parameters were evaluated: ethnic origin, age at assessment of liver function, sex, history of meconium ileus, pancreatic function, history of distal intestinal obstruction syndrome, pulmonary function, and cystic fibrosis transmembrane conductance regulator mutation analysis. RESULTS: Of the 288 patients screened, 80 (28%) had liver disease. Of the 256 patients with pancreatic insufficiency, 80 (31%) had liver disease compared with none of the 32 patients with pancreatic sufficiency. Genotype-phenotype correlation was performed on 207 patients carrying identified mutations that were previously classified according to phenotype severity. Liver disease was found in 56 (32%) of 173 patients carrying mutations associated with a severe phenotype and in 6 (38%) of 16 patients carrying at least one mutation associated with a variable genotype (G85E and/or 5T allele). None of the 18 patients carrying the 3849+10kb C->T mutation had liver disease. Prevalence of liver disease increased with age. No correlation was found between liver disease and severity of lung disease, nutritional status, history of meconium ileus, or distal intestinal obstruction syndrome. CONCLUSION: CF patients who have pancreatic insufficiency and carry mutations associated with a severe or a variable genotype are at increased risk to develop liver disease.

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32 The correlation between liver disease and CF genotype was studied in seven mutations associated with the severe phenotype: ⌬F508, R553X, 1717-1G-ϾA, G542X, W1282X, N1303K, and G551D.2,14 No significant cor- From the *Department of Pediatrics, Cystic Fibrosis Center, Shaare Zedek Medical Center, Hebrew University, Jerusalem; ‡Cystic Fibrosis Center, Carmel Medical Center, Haifa; §Cystic Fibrosis Center, Sheba Medical Center, Tel Hashomer; ࿣Cystic Fibrosis Center, Schneider Children`s Medical Center, Petah Tikva; ¶Cystic Fibrosis Center, Soroka Medical Center, Ben Gurion University, Beer Sheba; #Cystic Fibrosis Center, Rambam Medical Center, Haifa; **Cystic Fibrosis Center, Hadassah University Hospital, Jerusalem; ‡‡Department of Medical Statistics, Ichilov Medical Center, Tel Aviv; and §§Department of Genetics, Life Sciences Institute, Hebrew University, Jerusalem, Israel. Login to comment
50 Forced expiratory volume in 1 second, and forced vital capacity, were measured and expressed as a percentage of predicted values for height and sex, using previously described standardized pulmonary equations.17 Current height and weight percentiles were computed using the tables of Tanner.18 Mutation Analysis All the patients were screened for all of the CFTR mutations previously identified in the Israeli CF population.15,16 Patients were classified according to severity of genotype: patients with severe genotype were homozygous or compound heterozygous to ⌬F508, W1282X, G542X, N1303K, 405ϩ1G3A, delTATT 4010, 1717-1G-ϾA. Login to comment
117 Classification of Identified Genotype According to Severity of Disease Severe n Milder n Variable n Unclassified n ⌬F508/⌬F508 52 3849 ϩ 10kbC 3 T/⌬F508 7 ⌬F508/G85E 1 S549R/S549R 1 W1282X/W1282X 30 3849 ϩ 10kbC 3 T/405 ϩ 1G3A 3 G85E/G85E 5 S549R/G542X 2 ⌬F508/W1282X 39 3849 ϩ 10 kbC 3 T/W1282X 7 G85E/5T 1 S549R/W1282X 1 ⌬F508/G542X 10 3849 ϩ 10kbC 3 T/G85E 1 ⌬F508/5T 1 ⌬F508/W1089X 1 W1282X/G542X 12 W1282X/5T 2 Y1092X/Y1092X 1 W1282X/N1303K 7 W1282X/5T 1 Q359K-T360K/? Login to comment
121 12 N1303K/T4010 2 G542X/? Login to comment
122 3 N1303K/G542X 3 405 ϩ 1G3A/? Login to comment
123 1 N1303K/N1303K 6 Q359K-T360K/ 4 Q359K-T360K ⌬F508/405 ϩ 1G3A 5 W1282X/1717-1G 3 A 1 G542X/G542X 1 N1303K/1717-1G 3 A 1 Total 173 18 16 36 ARTICLES high prevalence of liver disease. Login to comment

[hide] CFTR mutations in patients from Colombia: implicat... Hum Mutat. 2003 Sep;22(3):259. Keyeux G, Rodas C, Bienvenu T, Garavito P, Vidaud D, Sanchez D, Kaplan JC, Aristizabal G
CFTR mutations in patients from Colombia: implications for local and regional molecular diagnosis programs.
Hum Mutat. 2003 Sep;22(3):259., [PMID:12938099]

Abstract [show]
Cystic Fibrosis is a worldwide distributed hereditary disease. The incidence of the main (p.F508del) and other frequent mutations has been determined in a great number of countries and ethnic groups, but its incidence in most Latin American countries has remained unknown until recently. It is now beginning to be recognized as a frequent cause of infant mortality, and some countries report the incidence of their mutations. Colombia started several years ago a concerted program of molecular study of patients which were clinically diagnosed as probable cystic fibrosis. We screened the whole CFTR (ABCC7) coding sequence in 92 patients from 6 different geographic regions, using conventional PAGE analyses and DGGE followed by sequencing. Additionally, we established the frequency of the p.F508del mutation in 130 unrelated healthy controls. The results of this pilot study in Colombian patients from various ethnic admixture show six main mutations: p.F508del (41.8%), c.1811+1.6kbA>G (6.5%), p.G542X (3.8%), p.S549R (2.2%), p.W1282X (1.1%) and p.R1162X (1.1%). Thirteen other rare mutations, including three novel, were detected, accounting for a total of 63.6% known mutations. There is a great variability between the geographic regions, both in the frequency of the p.F508del mutation and non-p.F508del CF chromosomes. Our results point to a varied origin of the disease genes. These results also show that careful scrutiny of the mutations is needed in each part of Latin America in order to establish priority-screening protocols adapted to each country and region.

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8 The results of this pilot study in Colombian patients from various ethnic admixture show six main mutations: p.F508del (41.8%), c.1811+1.6kbA>G (6.5%), p.G542X (3.8%), p.S549R (2.2%), p.W1282X (1.1%) and p.R1162X (1.1%). Login to comment
38 The second and third mutations in frequency in Colombian patients are c.1811+1.6kbA>G (6.5%) and p.G542X (3.8%), respectively. The distribution of these mutations over the country is heterogeneous, since, for instance, most c.1811+1.6kbA>G chromosomes are found in Bogotá (10.5%), whereas some Departments do not exhibit this mutation. Login to comment
50 CFTR Mutation Frequencies in Colombian Cystic Fibrosis Patients MUTATION ANTIOQUIA BOGOTA BOLIVAR CALDAS VALLE OTHER COLOMBIA n=34 n=76 n=20 n=10 n=24 n=20 n=184 N (%) N (%) N (%) N (%) N (%) N (%) N (%) p.F508del 16 (47.1) 31 (40.8) 5 (25) 6 (60.0) 10 (41.7) 9 (45.0) 77 (41.8) c.1811+1.6KbA>G 0 8 (10.5) 2 (10.0) 0 1 (4.2) 1 (5.0) 12 (6.5) p.G542X 0 4 (5.3) 0 0 2 (8.3) 1 (5.0) 7 (3.8) p.S549R 1 (2.9) 3 (3.9) 0 0 0 0 4 (2.2) p.W1282X 0 1 (1.3) 0 0 1 (4.2) 0 2 (1.1) p.R1162X 0 0 2 (10.0) 0 0 0 2 (1.1) p.A559T 1 (2.9) 0 0 0 0 0 1 (0.5) p.Y1092X 0 0 1 (5.0) 0 0 0 1 (0.5) p.R334W 0 0 0 0 1 (4.2) 0 1 (0.5) c.1215delG 0 1 (1.3) 0 0 0 0 1 (0.5) c.2185_2186insC 0 0 0 0 0 1 (5.0) 1 (0.5) c.2789+5G>A 0 0 0 0 1 (4.2) 0 1 (0.5) c.3120+1G>A 0 0 1 (5.0) 0 0 0 1 (0.5) c.3849+1G>A 0 1 (1.3) 0 0 0 0 1 (0.5) p.R1066C 0 1 (1.3) 0 0 0 0 1 (0.5) p.N1303K 1 (2.9) 0 0 0 0 0 1 (0.5) c.3500-2A>G* 1 (2.9) 0 0 0 0 0 1 (0.5) c.1323_1324insA* 0 0 1 (5.0) 0 0 0 1 (0.5) p.H609R* 0 0 0 0 0 1 (5.0) 1 (0.5) Unidentified 14 (41.2) 26 (34.2) 8 (40.0) 4 (40.0) 8 (33.3) 7 (35) 67 (36.4) The regions of the country where few patients were studied are grouped as other. Login to comment
66 As shown in Table 2, the five worldwide main mutations (p.F508del, p.G542X, p.R1162X, p.W1282X and p.N1303K) account for 48% to 66% of the mutations in IberoAmerican countries: Colombia (48.3%) (present study) and Mexico (49%) (Orozco et al. 2000) are very similar and have the lowest frequencies, whereas Brazil (61.7%) (CFGAC, 1999) and Argentina (66.2%) (Chertkoff et al., 1997) have the highest frequencies and are much closer to the values observed in Spanish CF patients (66.4%) (Estivill et al., 1997). Login to comment
69 Comparison of the Spectrum of CFTR Mutations in Colombia and Other Ibero-American Countries COLOMBIA1 SPAIN2 MEXICO3 ARGENTINA4 BRAZIL5 MUTATION n=92 n=1356 n=194 n=228 n=272 % % % % % p.F508del 41.8 54.42 40.72 57 45.6 p.G542X 3.8 7.7 6.18 3.94 6.6 p.W1282X 1.1 0.5 0 3.07 2.2 p.R1162X 1.1 1.3 0 0.43 4.4 p.N1303K 0.5 2.5 2.06 1.75 2.9 c.1811+1.6KbA>G 6.5 1.5 0 0.43 0 p.S549R 2.2 0.07 0 0 0 p.A559T 0.5 0 0 0 0 p.Y1092X 0.5 0.01 0.51 0 0 p.R334W 0.5 0.9 0 0 2.9 c.1215delG 0.5 0 0 0 0 c.2185_2186insC 0.5 0 0 0 0 c.2789+5G>A 0.5 0.7 0 0.43 0 c.3120+1G>A 0.5 0 0 0 0 c.3849+1G>A 0.5 0 0 0 0 p.R1066C 0.5 0.7 0 0.43 0 c.3500-2A>G (novel) 0.5 0 0 0 0 c.1323_1324insA (novel) 0.5 0 0 0 0 p.H609R (novel) 0.5 0 0 0 0 Other a (# mutations) - (32) 1.8 (30) 5.28 (9) 4.89 (8) 6.98 Unknown 36.4 17.9 25.25 27.63 28.3 a The frequencies of the other rare mutations found in Spain, Mexico, Argentina and Brazil are pooled together, and the number of different mutations is given in parenthesis. Login to comment

[hide] Detection of CFTR mutations using temporal tempera... Electrophoresis. 2004 Aug;25(15):2593-601. Wong LJ, Alper OM
Detection of CFTR mutations using temporal temperature gradient gel electrophoresis.
Electrophoresis. 2004 Aug;25(15):2593-601., [PMID:15300780]

Abstract [show]
Cystic fibrosis (CF), caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, is one of the most common autosomal recessive diseases with variable incidences and mutation spectra among different ethnic groups. Current commercially available mutation panels designed for the analysis of known recurrent mutations have a detection rate between 38 to 95%, depending upon the ethnic background of the patient. We describe the application of a novel mutation detection method, temporal temperature gradient gel electrophoresis (TTGE), to the study of the molecular genetics of Hispanic CF patients. TTGE effectively identified numerous rare and novel mutations and polymorphisms. One interesting observation is that the majority of the novel mutations are splice site, frame shift, or nonsense mutations that cause severe clinical phenotypes. Our data demonstrate that screening of the 27 exons and intron/exon junctions of the CFTR gene by TTGE greatly improves the molecular diagnosis of Hispanic CF patients.

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89 For example, the p.Q98X and p.Q98R mutations in exon 4; and p.S466X and p.S492F mutations in exon 10, were detected in the temperature range of 52-607C and 51- 577C, respectively. The p.G542X, p.R553X, p.S549N, and p.A559T in exon 11; p.A561E, c.189811G.A, and c.189813A.G in exon 12; and p.W1204X in exon 19; were detected in the temperature range of 51 to 567C. Login to comment
96 Detection of known mutations and polymorphisms by TTGE Base substitution Mutation Exon or intron Homozygote or heterozygote Polymorphism or mutation # Alleles detected 1 c.386G.A p.G85E 3 Heterozygote Mutation 2 2 c.575T.C p.I148T 4 Heterozygote Mutation 2 3 c.406-1G.A Splice Int 4 Heterozygote Mutation 9 4 c.71111G.T Splice Int 5 Heterozygote Mutation 1 5 c.1059_1069del 3bp p.F311del 7 Heterozygote Mutation 2 6 c.1132C.T p.R334W 7 Heterozygote Mutation 2 7 c.1652_1655del 3bp p.F508del 10 Heterozygote Mutation 94 8 Homozygote Mutation 12 c.1540A/G p.M470V 10 Heterozygote Polymorphism 15 9 Homozygote Polymorphism 4 c.1756G.T p.G542X 11 Heterozygote Mutation 13 10 c.1784G.A p.G551D 11 Heterozygote Mutation 1 11 c.1778G.A p.S549N 11 Heterozygote Mutation 4 12 c.1789C.T p.R553X 11 Homozygote Mutation 2 13 c.1807G.A p.A559T 11 Heterozygote Mutation 2 14 c.189811G.A Splice Int 12 Heterozygote Mutation 1 15 c.1949del84bp Frameshift 13 Heterozygote Mutation 3 16 c.278915G.A Splice Int 14b Heterozygote Mutation 2 17 c.312011G.A Splice Int 16 Heterozygote Mutation 9 18 c.3171delC Frameshift 17a Heterozygote Mutation 1 19 c.3398G.A p.W1089X 17b Heterozygote Mutation 1 20 c.3425G.A p.W1098X 17b Heterozygote Mutation 1 21 c.3616C.T p.R1162X 19 Heterozygote Mutation 2 22 c.3791delC Frameshift 19 Heterozygote Mutation 1 23 c.3821delT Frameshift 19 Heterozygote Mutation 1 24 c.3876delA Frameshift 20 Heterozygote Mutation 4 25 c.3905insT Frameshift 20 Heterozygote Mutation 1 26 c.4041C.G p.N1303K 21 Heterozygote Mutation 2 Total 194 The translation starts at c.133 of CFTR CDNA sequence in GenBank Acc. Login to comment

[hide] Increased risk of idiopathic chronic pancreatitis ... Hum Mutat. 2005 Oct;26(4):303-7. Cohn JA, Neoptolemos JP, Feng J, Yan J, Jiang Z, Greenhalf W, McFaul C, Mountford R, Sommer SS
Increased risk of idiopathic chronic pancreatitis in cystic fibrosis carriers.
Hum Mutat. 2005 Oct;26(4):303-7., [PMID:16134171]

Abstract [show]
Cystic fibrosis (CF) is a recessive disease caused by mutations of the CF transmembrane conductance regulator (CFTR) gene. The risk of idiopathic chronic pancreatitis (ICP) is increased in individuals who have CFTR genotypes containing a CF-causing mutation plus a second pathogenic allele. It is unknown whether the risk of ICP is increased in CF carriers who have one CF-causing mutation plus one normal allele. In this study, 52 sporadic cases of ICP were ascertained through the European Registry of Hereditary Pancreatitis and Familial Pancreatic Cancer. Individuals with pathogenic cationic trypsinogen mutations were excluded. DNA was comprehensively tested for CFTR mutations using a robotically enhanced, multiplexed, and highly redundant form of single-strand conformation polymorphism (SSCP) analysis followed by DNA sequencing. Fifteen subjects had a total of 18 pathogenic CFTR alleles. Eight subjects had common CF-causing mutations. This group included seven CF carriers in whom the second CFTR allele was normal (4.3 times the expected frequency, P=0.0002). Three subjects had compound heterozygotes genotypes containing two pathogenic alleles (31 times the expected frequency, P<0.0001). A variant allele of uncertain significance (p.R75Q) was detected in eight of the 52 ICP subjects and at a similar frequency (13/96) in random donors. ICP differs from other established CFTR-related conditions in that ICP risk is increased in CF carriers who have one documented normal CFTR allele. Having two CFTR mutations imparts a higher relative risk, while having only one mutation imparts a higher attributable risk.

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93 Abnormal CFTR Genotypes Detected in 52 Patients with ICPa Genotype categorya ] Patients Genotypes detectedb Compound heterozygotes and homozygotes 3 p.F508del / p.L967S p.D1152H / p.D1152H p.V920M / p.L967S Heterozygotes, common mutation causing classic CFa 7 p.F508del /^ ('ve subjects)c p.R560T/^ p.G542X /^ Heterozygotes, uncommon mutation causing variable phenotype 3 p.S1235R /^ p.A209S /^ p.L997F/^ Heterozygotes, common CBAVD-associated mutation 2 IVS8(5T) /^ (two subjects) a Common CF-mutations consistently cause classic CF in compound heterozygotes and homozygotes [Rosenstein and Cutting, 1998]. Login to comment

[hide] A frequent large rearrangement in the CFTR gene in... Genet Test. 2006 Fall;10(3):208-14. Nectoux J, Audrezet MP, Viel M, Leroy C, Raguenes O, Ferec C, Lesure JF, Davy N, Renouil M, Cartault F, Bienvenu T
A frequent large rearrangement in the CFTR gene in cystic fibrosis patients from Reunion Island.
Genet Test. 2006 Fall;10(3):208-14., [PMID:17020473]

Abstract [show]
Reunion Island is a French province, 800 km east of Madagascar and 200 km west of Mauritius. On Reunion Island, the birth prevalence of cystic fibrosis (CF) is particularly high in the population of European origin, approximately 1:1000. In a previous study, we demonstrated that the screening of the 27 exons of the CF transmembrane conductance regulator (CFTR) gene by denaturing high-pressure liquid chromatography (DHPLC) in 114 CF families allowed the detection of about 93% of the molecular defects present on Reunion Island. Unidentified CF mutations may lie in introns or in regulatory regions that are not routinely investigated, or may correspond to gene rearrangements such as large, heterozygous deletions that escape detection using current PCR-based techniques. Using a combination of different methods (such as multiplex ligation-dependent probe amplification), 6 of the 13 unidentified CF alleles (46%) were found to harbor a deletion of 5288 bp, spanning from exon 17a to 18. Identification and examination of the breakpoint sequences showed that this deletion is different from the 3120+1kbdel8.6Kb previously found in the Palestinian Arabs. The chromosomes bearing IVS16+3316_IVS18+644del5288 did not have a common extragenic haplotype. Clinical evaluation of homozygotes (2 unrelated patients) and compound heterozygotes indicated that this deletion represents a severe mutation associated with positive sweat chloride test, pancreatic insufficiency, and early age at diagnosis.

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41 p.F508del and p.G542X were previously associated with the haplotype B (rarely A, D) (Sereth et al. 1993; Cuppens et al. 1994). Login to comment
52 GENOTYPE OF CF PATIENTS FROM REUNION ISLAND WHO WERE FOUND TO CARRY AT LEAST ONE CFTR MUTATION-NEGATIVE ALLELE Present Patient Sex age (years) Cl-test Genotype Phenotype MUC971 F 6 87-91 p.V520I : unidentified PS-CF (bronchitis, mild disease) MUC696 M 10 37-70 c.3849 ϩ 45G l A : unidentified PS-CF (bronchitis) R105C F 82 56-106 p.A309G : unidentified PS-CF (H. influenzae colonization, bronchitis) MUC900 M 7 71-147 p.F508del : unidentified PI-CF R131C F 16 39-70 p.F508del : unidentified PS-CF (B. cepacia colonization, bronchitis) R89C F 20 23-107 p.F508del : unidentified PS-CF R71C F 17 58-90 p.G542X : unidentified PI-CF (S. aureus and P. aeruginosa colonization) R48C F 19 81 Unidentified : unidentified PI-CF (meconium ileus) R44C F 11 70 Unidentified : unidentified PI-CF (died at the age of 11 years) MUC74 M 6 125 Unidentified : unidentified PI-CF (bronchitis, mild disease) PS, pancreatic sufficiency; PI, pancreatic insufficiency; H, Haemophilus; B, Burkholderia; P, Pseudomonas; S, Staphylococcus. Login to comment

[hide] Spectrum of mutations in the CFTR gene in cystic f... Ann Hum Genet. 2007 Mar;71(Pt 2):194-201. Alonso MJ, Heine-Suner D, Calvo M, Rosell J, Gimenez J, Ramos MD, Telleria JJ, Palacio A, Estivill X, Casals T
Spectrum of mutations in the CFTR gene in cystic fibrosis patients of Spanish ancestry.
Ann Hum Genet. 2007 Mar;71(Pt 2):194-201., [PMID:17331079]

Abstract [show]
We analyzed 1,954 Spanish cystic fibrosis (CF) alleles in order to define the molecular spectrum of mutations in the CFTR gene in Spanish CF patients. Commercial panels showed a limited detection power, leading to the identification of only 76% of alleles. Two scanning techniques, denaturing gradient gel electrophoresis (DGGE) and single strand conformation polymorphism/hetroduplex (SSCP/HD), were carried out to detect CFTR sequence changes. In addition, intragenic markers IVS8CA, IVS8-6(T)n and IVS17bTA were also analyzed. Twelve mutations showed frequencies above 1%, p.F508del being the most frequent mutation (51%). We found that eighteen mutations need to be studied to achieve a detection level of 80%. Fifty-one mutations (42%) were observed once. In total, 121 disease-causing mutations were identified, accounting for 96% (1,877 out of 1,954) of CF alleles. Specific geographic distributions for the most common mutations, p.F508del, p.G542X, c.1811 + 1.6kbA > G and c.1609delCA, were confirmed. Furthermore, two other relatively common mutations (p.V232D and c.2789 + 5G > A) showed uneven geographic distributions. This updated information on the spectrum of CF mutations in Spain will be useful for improving genetic testing, as well as to facilitate counselling in people of Spanish ancestry. In addition, this study contributes to defining the molecular spectrum of CF in Europe, and corroborates the high molecular mutation heterogeneity of Mediterranean populations.

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8 Specific geographic distributions for the most common mutations, p.F508del, p.G542X, c.1811 + 1.6kbA > G and c.1609delCA, were confirmed. Login to comment
45 (%) p.F508del # E.10 1009 (51.74) p.G542X # E.11 150 (7.69) p.N1303K # E.21 57 (2.92) c.1811 + 1.6kbA > G I.11 36 (1.84) p.R334W # E.7 35 (1.79) p.L206W E.6a 32 (1.64) c.711 + 1G > T # I.5 31 (1.58) p.Q890X E.15 28 (1.43) p.R1162X # E.19 25 (1.28) c.2789 + 5G > A # I.14b 24 (1.23) p.R1066C E.17b 23 (1.18) p.I507del # E.10 21 (1.07) c.1609delCA E.10 18 (0.92) c.712-1G > T I.5 18 (0.92) c.3272-26A > G I.17a 18 (0.92) c.2183AA > G # E.13 16 (0.82) p.G85E # E.3 15 (0.77) c.2869insG E.15 15 (0.77) p.W1282X # E.20 15 (0.77) p.V232D E.6a 14 (0.71) p.A1006E * E.17a 12 (0.61) c.2184insA E.13 11 (0.56) p.K710X E.13 11 (0.56) TOTAL (n = 23) 1,634 (83.72) * , the complex allele [p.A1006E; p.V562I; IVS8-6(5T)] #, CF mutations identified with the Celera Diagnosis Cystic Fibrosis v2 genotyping assay and the Inno-Lipa CFTR12, CFTR17 + Tn Samples with microsatellite haplotypes 16/45-46-47 (IVS8CA/IVS17bTA) were submitted to direct analysis of the c.1811 + 1.6kbA > G mutation, which was found mainly associated with the 16-46 haplotype. Login to comment
61 The evaluation data from the specific regions contributing to this study showed that the c.2789 + 5G > A mutation was, along with p.G542X, the second most common mutation (9/86, 10.5%) among the Balearic Islands` alleles, whereas mutation c.1609delCA had its highest prevalence in Aragon (10/109, 9%). Login to comment
90 The p.F508del mutation showed frequencies ranging from 86% (North) to 46% (South) in the Iberian Peninsula (Casals et al. 1992; Coto et al. 1994; Borrego et al. 1994; Telleria et al. 1999) as well as a 14% prevalence of the p.G542X mutation in the Mediterranean Coastal area (Casals et al. 1993). Login to comment
94 In fact, this splicing mutation has also been reported with frequencies ≥ 1% in France (Claustres et al. 2000), Italy (Bonizzato et al. 1995), Greece (Kanavakis et al. 2003) and Turkey (Kilinc et al. 2002), suggesting that, like p.G542X, c.2789 + 5G > A was spread across the Mediterranean Sea. Login to comment
99 Some high frequencies such as those found for the p.G542X mutation have been attributed to the Spanish ancestry of the Mexican (Orozco et al. 2000) and Brazilian (Bernardino et al. 2000) populations. Login to comment
105 Our impression is that Table 3 Common CF mutations identified in this study and in several Latin American populations Mutation This study Hispanic1 Mexico2 Colombia3 Brazil4 Argentina5 Chile6 p.F508del 51.7 51.6 40.7 41.8 48.4 58.6 45.0 p.G542X 7.7 4.0 6.2 3.8 8.8 4.1 7.0 p.N1303K 2.9 0.8 2.0 0.5 2.5 2.7 - c.1811 + 1,6kbA > G 1.8 - - 6.5 - 0.9 - p.R334W 1.8 1.6 - 0.5 2.5 1.1 2.0 p.L206W 1.6 - - - 0.6 - - c.711 + 1G > T 1.6 - - - - - - p.Q890X 1.4 - - - - - - p.R1162X 1.3 0.8 - 1.1 2.5 0.4 2.0 c.2789 + 5G > A 1.2 - - 0.5 0.3 0.7 - p.R1066C 1.2 1.6 - 0.5 - 0.2 - p.I507del 1.0 - 2.6 - - 0.7 - c.2183AA > G 0.8 - 1.0 - 0.2 - p.G85E 0.7 0.8 0.5 - 1.3 0.7 - p.W1282X 0.7 0.8 - 1.1 1.3 2.7 5.0 c.3849 + 10kbC > T 0.4 4.0 0.5 - - 0.9 3.0 p.S549N - 2.4 2.6 - - - - c.3120 + 1G > A - 1.6 - 0.5 - - - c.3876delA - 5.6 - - - - - c.406-1G > A - 1.6 1.5 - - - - c.935delA - 1.6 1.0 - - - - p.R75X - 0.8 1.5 - - - - c.2055del9 - - 1.0 - - - - p.I506T - - 1.0 - - - - c.3199del6 - - 1.0 - - - - p.S549R 0.4 - - 2.2 - 0.2 - c.1717-1G > A 0.2 - - - 0.3 1.1 - p.G551D 0.2 0.8 0.5 - - - 1.0 p.R553X 0.4 - 0.5 - 0.6 0.2 1.0 No. Login to comment

[hide] Clinical outcome of preimplantation genetic diagno... Eur J Hum Genet. 2007 Jul;15(7):752-8. Epub 2007 Apr 18. Keymolen K, Goossens V, De Rycke M, Sermon K, Boelaert K, Bonduelle M, Van Steirteghem A, Liebaers I
Clinical outcome of preimplantation genetic diagnosis for cystic fibrosis: the Brussels' experience.
Eur J Hum Genet. 2007 Jul;15(7):752-8. Epub 2007 Apr 18., [PMID:17440499]

Abstract [show]
Preimplantation genetic diagnosis is an alternative for prenatal diagnosis that makes it possible to perform the diagnosis of a chromosomal or monogenic disorder at the preimplantation embryo level. Cystic fibrosis is one of the monogenic diseases for which PGD can be performed. In this study, we looked at the requests and PGD cycles for this particular disorder over an 11-year period. Sixty-eight percent of the requests eventually led to at least one complete PGD cycle. In 80% of the cycles, an embryo transfer was performed and an ongoing pregnancy was obtained in 22.2% of the cycles with oocyte retrieval. After embryo transfer, a couple had 27.8% chance of giving birth to a liveborn child. No misdiagnosis was recorded. The rate of perinatal deaths/stillborn children was relatively high, but no excess of major congenital anomalies was observed in the surviving children.

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66 Table 1 Assessment of CF risk Couples with PGD (n ¼ 47) Couples without PGD (n ¼ 22) All couples (n ¼ 69)(%) CF risk assessment Affected child or foetus 23 14 37 (53.6) CBAVD (without other CF complaints) 7 3 10 (14.5) During fertility work-up (not CBAVD) 10 10 (14.5) Positive family history 3 2 5 (7.2) CF patient (with CBAVD in males) 4 4 (5.8) Unknown 2 2 (2.9) Preconceptual screening 1 1 (1.4) Table 2 Reasons for choosing PGD Couples with PGD (n ¼ 47) Couples without PGD (n ¼ 22) All couples (n ¼ 69) Reason for choosing/informing about PGD Fertility problems 24 7 31 (44.9%) Objection to abortion 15 2 17 (24.6%) History of termination of pregnancy 8 1 9 (13%) Unknown 11 11 (15.9%) Other 1 1 (1.4%) Table 3 Genotypes of the couples with PGD cycles Female partner Male partner Number of couples with this genotype p.F508del/- p.F508del/- 17 p.F508del/- p.R117H/- (7T/9T) 1 p.2789+5G4A/- p.D110H/p.D110H 1 p.G542X/- p.F508del/- 1 p.R334Q/- p.F508del/- 1 p.R553X/- p.F508del/- 2 p.1717-1G4A p.2183AA4G/5T 1 p.F508del/- p.F508del/? Login to comment
67 2 p.1303K/- p.G542X/p.R117H 1 p.F508del/- 5T/? Login to comment
69 2 p.F508del/- p.N1303K/- 1 p.Q493X/- p.F508del/- 1 p.F508del/- p.R1162X/- 1 p.4218insT/- p.N1303K/- 1 p.G673X/- p.F508del/- 1 p.W1282X/- p.G542X/- 1 p.F508del/- p.W1282X/- 1 p.W1282X/- p.F508del/- 2 p.F508del/- p.G551D/- 1 p.D1168G/- p.L206W/- 1 If we express these results per cycle with oocyte retrieval, this means that in each cycle there was an average of 12.5 COCs, giving 5.1 embryos to be biopsied with an 80% chance of having an embryo transfer and a 22.2% chance of having an ongoing pregnancy with the delivery of a child. Login to comment

[hide] Mutations in the amiloride-sensitive epithelial so... Hum Mutat. 2009 Jul;30(7):1093-103. Azad AK, Rauh R, Vermeulen F, Jaspers M, Korbmacher J, Boissier B, Bassinet L, Fichou Y, des Georges M, Stanke F, De Boeck K, Dupont L, Balascakova M, Hjelte L, Lebecque P, Radojkovic D, Castellani C, Schwartz M, Stuhrmann M, Schwarz M, Skalicka V, de Monestrol I, Girodon E, Ferec C, Claustres M, Tummler B, Cassiman JJ, Korbmacher C, Cuppens H
Mutations in the amiloride-sensitive epithelial sodium channel in patients with cystic fibrosis-like disease.
Hum Mutat. 2009 Jul;30(7):1093-103., [PMID:19462466]

Abstract [show]
We investigated whether mutations in the genes that code for the different subunits of the amiloride-sensitive epithelial sodium channel (ENaC) might result in cystic fibrosis (CF)-like disease. In a small fraction of the patients, the disease could be potentially explained by an ENaC mutation by a Mendelian mechanism, such as p.V114I and p.F61L in SCNN1A. More importantly, a more than three-fold significant increase in incidence of several rare ENaC polymorphisms was found in the patient group (30% vs. 9% in controls), indicating an involvement of ENaC in some patients by a polygenetic mechanism. Specifically, a significantly higher number of patients carried c.-55+5G>C or p.W493R in SCNN1A in the heterozygous state, with odds ratios (ORs) of 13.5 and 2.7, respectively.The p.W493R-SCNN1A polymorphism was even found to result in a four-fold more active ENaC channel when heterologously expressed in Xenopus laevis oocytes. About 1 in 975 individuals in the general population will be heterozygous for the hyperactive p.W493R-SCNN1A mutation and a cystic fibrosis transmembrane conductance regulator (CFTR) gene that results in very low amounts (0-10%) functional CFTR. These ENaC/CFTR genotypes may play a hitherto unrecognized role in lung diseases.

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238 The NPD was measured between a Ringer`s solution-filled exploring bridge on the nasal mucosa of the floor of the nasal cavity, and a reference bridge on the abraded skin of the forearm in a CF patient compound heterozygous for p.F508del and p.G542X in CFTR, a patient heterozygous for p.W493R-SCNN1A and p.F508del-CFTR, and a normal subject. Login to comment

[hide] Outcomes of a cystic fibrosis carrier testing clin... Med J Aust. 2009 Nov 2;191(9):499-501. Christie LM, Ingrey AJ, Turner GM, Proos AL, Watts GE
Outcomes of a cystic fibrosis carrier testing clinic for couples.
Med J Aust. 2009 Nov 2;191(9):499-501., 2009-11-02 [PMID:19883345]

Abstract [show]
OBJECTIVE: To review the outcomes of offering carrier testing for cystic fibrosis (CF) to couples considering pregnancy, and to women in early pregnancy and their partners. METHODS: An after-hours clinic was established in Newcastle for discussion of issues related to prenatal testing. Couples were offered CF carrier testing by extracting DNA from a mouthwash sample. An expanded one-step model was used with both partners being tested initially for the p.F508del cystic fibrosis transmembrane conductance regulator gene (CFTR) mutation. If one partner was a p.F508del carrier, the other partner was tested for an additional 28 CFTR mutations. RESULTS: Of 1000 individuals who were offered CF carrier testing, none declined. No re-collections of mouthwash samples were required, and results were available within 14 days. There were 730 individuals who had no family history of CF (73%); 27 were carriers (4%; 95% CI, 2.4%-5.3%), and there were two high-risk couples where both partners were carriers of p.F508del. There were 270 individuals who had an affected family member with CF or a child identified as a CF carrier through newborn screening; 126 were carriers (46%; 95% CI, 40.6%-52.8%), and there were two high-risk couples - one couple where both partners were carriers of p.F508del, and another couple where the woman was homozygous for p.F508del and the man was a p.F508del carrier. The information on carrier status led the four high-risk couples to change their reproductive decisions to avoid having a child with CF. CONCLUSION: CF carrier testing for couples using an expanded one-step model will detect about 80% of high-risk couples and enables various reproductive choices. We believe that all couples considering pregnancy, and women in early pregnancy and their partners, should be offered CF carrier testing.

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72 This provides each individual with information on their carrier status, and accurate residual risks of 1 CFTR mutations tested for in individuals whose partner was a carrier of p.F508del* p.F508del p.F316leufsX p.I507del p.R347P p.G542X p.S1251N p.G551D p.E60X p.N1303K p.W1282X c.1585-1G>A p.D1152H p.R553X c.2988+1G>A c.489+G>T c.2657+5G>A p.R117H c.1766+1G>A p.R1162X c.579+1G>A c.3717+10kbC>T p.G85E p.R334W p.K684fs p.A455E p.I148T p.K684fs p.R560T p.T1176fs CFTR = gene encoding cystic fibrosis transmembrane conductance regulator. Login to comment

[hide] Genetic, epidemiological, and clinical aspects of ... Am J Gastroenterol. 2010 Aug;105(8):1876-83. Epub 2010 May 25. Joergensen MT, Brusgaard K, Cruger DG, Gerdes AM, Schaffalitzky de Muckadell OB
Genetic, epidemiological, and clinical aspects of hereditary pancreatitis: a population-based cohort study in Denmark.
Am J Gastroenterol. 2010 Aug;105(8):1876-83. Epub 2010 May 25., [PMID:20502448]

Abstract [show]
OBJECTIVES: In a population-based, well-defined group of patients first regarded as having pancreatitis of unknown origin (PUO), we identified, described, and compared the clinical and genetic aspects of patients with hereditary pancreatitis (HP) and with cystic fibrosis transmembrane conductance regulator gene (CFTR) and serine protease inhibitor Kazal type 1 gene (SPINK1) mutations with patients who retained the diagnosis of true idiopathic pancreatitis (tIP) after genetic testing for HP, SPINK1, and CFTR mutations. METHODS: Patients with PUO were identified in the Danish National Registry of Patients or were referred by clinicians. DNA from blood was analyzed for cationic trypsinogen (PRSS1), SPINK1, and CFTR mutations. Considering the diagnosis of HP, a pedigree was drawn for each patient. RESULTS: A genetic mutation was found in 40% of 122 patients with PUO. After testing first-degree relatives of the 18 initially identified HP patients, 38 HP patients in total were identified, and 28 patients had SPINK1-CFTR mutations. Among HP patients, no p.N29I mutations were found and the p.A16V mutation was more frequent than previously reported, 45 and 32% had exocrine and endocrine insufficiency, respectively, and among tIP patients 9 and 12%, respectively. Pancreatic cancer was diagnosed in 5% of the HP families. CONCLUSIONS: The genotype of the Danish population with HP differs from that of previously described cohorts. The occurrence of exocrine and endocrine insufficiency is higher among patients with HP than in patients with SPINK1-CFTR mutations and tIP, and more HP families develop pancreatic cancer. Genetic testing thus helps to predict the prognosis of the pancreatitis.

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57 The samples were also tested for 33 CFTR mutations, and all 6 classeswererepresented:394delTT,p.R553X,621+1G>T,p.R1162X, 1717-1G>A,3659delC,p.G542X,2183AA>G,p.W1282X,1078delT, 711+1G>T, F508del, p.S549N, I507del, p.S549R, 2184delA, p.G551D, p.G85E, p.N1303K, p.R560T, p.R117H, p.R347H, p.R347P, p.R334W, 2789+5G>A, 3849+10kbC>T, p.A445E, 3120+1G>A, p.V520F,1898+1G>A,3876delA,3905insT,andIVS8-5T.DNAwas amplified by multiplex PCR (Hybaid 4 A62, Middlesex, UK). Login to comment

[hide] Cystic fibrosis in Chilean patients: Analysis of 3... J Cyst Fibros. 2011 Jan;10(1):66-70. Epub 2010 Oct 30. Lay-Son G, Puga A, Astudillo P, Repetto GM
Cystic fibrosis in Chilean patients: Analysis of 36 common CFTR gene mutations.
J Cyst Fibros. 2011 Jan;10(1):66-70. Epub 2010 Oct 30., [PMID:21036675]

Abstract [show]
BACKGROUND: CFTR gene mutations have worldwide differences in prevalence and data on Chilean patients is scarce. METHODS: We studied 36 of the most common CFTR mutations in Chilean patients from the CF National Program [Programa Nacional de Fibrosis Quistica (PNFQ)] of the Ministry of Health of Chile. RESULTS: Two hundred and eighty-nine patients were studied. Fourteen different mutations were identified with an overall allele detection rate of 42.0%. Mutations with frequencies greater than 1% were p.F508del (30.3% of alleles), p.R334W (3.3%), p.G542X (2.4%), c.3849+10Kb C>T (1.7%), and p.R553X (1.2%). A north to south geographical gradient was observed in the overall rate of detection. CONCLUSIONS: Southern European CFTR mutations predominate in the Chilean population, but a high percentage of alleles remain unknown. Geographical heterogeneity could be explained in part by admixture. Complementary analyses are necessary to allow for effective genetic counselling and improve cost-effectiveness of screening and diagnostic tests.

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5 Mutations with frequencies greater than 1% were p.F508del (30.3% of alleles), p.R334W (3.3%), p.G542X (2.4%), c.3849+10Kb CNT (1.7%), and p.R553X (1.2%). Login to comment
52 In addition, another 4 mutations had a frequency greater than 1% (p.R334W, p.G542X, c.3849+10Kb CNT, and p.R553X), encompassing 8.5% of the total alleles or 20.2% of detected alleles, while 6 mutations were found in only one family. Login to comment
70 Several other prevalent mutations in our Chilean cohort are common in Southern European countries (i.e: p.R334W, p.G542X, and p.R1162X), and even more prevalent in the Canary Islands (4%; 14.3%-25% and 6.1%, respectively) [14,15], a point of halting for the Spanish expeditions to America, including Columbus' first journey [16]. Login to comment
81 Mutation This study Rios et al. [4] Molina et al. [5] Repetto et al. [6] Perez et al. [13] CFGAC [2] (n=578) (%) (n=72) (%) (n=36) (%) (n=100) (%) (n=4102) (%) (n=43,849) (%) Chile Chile Chile Chile Latin-Americaa Worldwide Unknown 58.0 66.6 61.1 34.0 36.7 22.7 p.F508del 30.6 29.2 30.6 45.0 47.1 66.0 p.R334W 3.1 - - 2.0 0.8 0.1 p.G542X 2.4 0 8.3 7.0 5.0 2.4 c.3849+10Kb CNT 1.7 - - 3.0 0.3 0.2 p.R553X 1.2 4.2 0 1.0 0.4 0.7 p.R1162X 0.9 - - 2.0 1.0 0.3 p.1078delT 0.5 - - 0 b0.1 0.1 p.G85E 0.5 - - - 0.8 0.2 p.W1282X 0.2 - - 5.0 1.0 1.2 c.3120+1 GNA 0.2 - - - 0.3 - c.711+1 GNT 0.2 - - - 0.1 0.1 p.R117H 0.2 - - 0 b0.1 0.3 p.A455E 0.2 - - 0 0 0.1 p.I148T 0.2 - - - - - p.G551D 0 0 0 1.0 0.1 1.6 p.N1303K 0 0 0 0 1.8 1.3 c.621+1 GNT 0 - - 0 0.2 0.7 c.1717-1 GNA 0 - - 0 0.3 0.6 p.I507del 0 - - 0 0.2 0.2 p.R347P 0 - - 0 0 0.2 c.2789+5 GNA 0 - - - 0.2 0.1 c.1898+1 GNA 0 - - - 0.1 0.1 c.2184delA 0 - - - b0.1 0.1 p.S549N 0 - 0 - 0.1 0.1 c.3659delC 0 - - 0 0.1 0.1 p.R560T 0 - - - 0 0.1 c.1811+1.6Kb ANG 0 - - - 0.4 - c.2183AANG 0 - - 0 0.1 - p.S549R 0 - - - 0.1 - c.3272-26 ANG 0 - - - 0.1 - c.3199del6 0 - - - b0.1 - p.E60X 0 - - 0 0 - c.3905insT 0 - - - 0 - p.S1251N 0 - - 0 - - CFTRdele2,3 0 - - - - - p.R347H 0 - - - - - p.V520F 0 - - - - - p.Q552X 0 - - - - - c.394delTT 0 - - - - - c.711+1 GNA 0 - - - - - c.2143delT 0 - - - - - c.3876delA 0 - - - - - a Data from Chilean patients published in Rios et al., Molina et al., and Repetto et al. [4-6] included in this publication were excluded in this table to avoid repetition. Login to comment

[hide] An MBL2 haplotype and ABCB4 variants modulate the ... Dig Liver Dis. 2009 Nov;41(11):817-22. Epub 2009 May 20. Tomaiuolo R, Degiorgio D, Coviello DA, Baccarelli A, Elce A, Raia V, Motta V, Seia M, Castaldo G, Colombo C
An MBL2 haplotype and ABCB4 variants modulate the risk of liver disease in cystic fibrosis patients: a multicentre study.
Dig Liver Dis. 2009 Nov;41(11):817-22. Epub 2009 May 20., [PMID:19467940]

Abstract [show]
BACKGROUND: Cystic fibrosis is the most common lethal recessive disorder among Caucasians. Over 1500 mutations have been identified in cystic fibrosis transmembrane conductance regulator disease-gene so far. A large variability of the clinical phenotype has been observed both in cystic fibrosis patients bearing the same genotype, and in affected sibpairs. Thus, genes inherited independently from cystic fibrosis transmembrane conductance regulator could modulate the clinical expression of cystic fibrosis. METHODS: We analysed some putative modifier genes of liver cystic fibrosis phenotype (serpin 1, hemochromatosis, transferrin receptor 2, ferroportin 1, mannose binding lectin and adenosine triphospate-binding cassette subfamily B member 4) in 108 unrelated cystic fibrosis patients with and without liver involvement. RESULTS: HYPD mannose binding lectin haplotype was significantly (p<0.05) more frequent in cystic fibrosis patients with liver disease versus those without liver disease. This haplotype already related to a more severe pulmonary cystic fibrosis phenotype, is associated to a reduced MBL immunological activity. The c.834-66G>T variant of adenosine triphospate-binding cassette subfamily B member 4 gene was significantly (p<0.05) less frequent in cystic fibrosis patients with liver disease as compared to those with no liver disease. CONCLUSIONS: The HYPD mannose binding lectin haplotype may predispose a subgroup of cystic fibrosis patients to a more severe liver involvement impairing the local defence mechanisms whereas the c.834-66G>T adenosine triphospate-binding cassette subfamily B member 4 variant may enhance the activity of the protein and thus exert a protective effect toward liver disease.

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30 Later, a CF patient homozygous for the G542X mutation was described, who had a severe liver phenotype, unlike the six previously reported cases bearing the same CFTR genotype, who were free of any liver involvement [9]. Login to comment
41 To reduce the possible influence of the CFTR genotype on the liver, we selected only patients who were homozygotes for the F508del mutation (about 80%) or compound heterozygotes for the F508del and another severe (class 1, 2 or 3) CFTR mutation (i.e., c.1717-1G>A; p.G542X; p.NI303K; c.1782delT; c.182delT; c.3659delC; c.4016insT; dele17a-18; p.E585X; p.R553X). Login to comment

[hide] Itraconazole treatment of allergic bronchopulmonar... Allergy. 2002 Aug;57(8):723-8. Skov M, Hoiby N, Koch C
Itraconazole treatment of allergic bronchopulmonary aspergillosis in patients with cystic fibrosis.
Allergy. 2002 Aug;57(8):723-8., [PMID:12121192]

Abstract [show]
BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) in cystic fibrosis (CF) patients is a potentially fatal inflammatory disease due to the dual-type immune response provoked by the fungal antigens. Despite serious side effects long-term treatment with corticosteroids is often required. Itraconazole has been reported to be a useful steroid-sparing agent. METHODS: In a retrospective follow-up of 21 CF patients from a total of 250 treated once or twice within a five-year study period (1994-98), 9 patients were treated with systemic glucocorticosteroids in combination with itraconazole and 12 patients were treated with itraconazole (200-600 mg/day) as monotherapy. RESULTS: During treatment the percentage of Aspergillus fumigatus (AF)-positive sputum cultures significantly reduced (P < 0.05); precipitating antibodies to AF decreased significantly in all patients (P < 0.05); forced expiratory volume (FEV1) increased to pre-exacerbation level; total IgE levels decreased in 42% of patients on monotherapy and in 56% on combination therapy. Specific IgE (radioallergosorbant; RAST) level decreased in 6 of 21 patients. Eleven patients had transient increased levels of alanine transaminase (ALAT). One patient had isolated increase in alkaline phosphatase and another in aspartate transaminase (ASAT). CONCLUSIONS: High dose itraconazole as monotherapy or in combination with systemic glucocorticosteroids seems effective in CF patients with ABPA. No hepatotoxicity was observed during long-term therapy.

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62 Af.+others Others Negative Not done 1 M 30 F508/W1282X x x x 2 M 21 x x x x 3 F 27 F508/unknown x x x 4 M 21 F508/3659delC x x x 5 F 15 F508/394delTT x x x 6 M 13 x F508/G542X x x x 7 F 14 x x x x 8 M 13 F508/1571delG x x x 9 M 13 x x x x 10 F 15 x x x x 11 M 13 x x x x 12 F 13 x x x x 13 F 13 x x x x 14 F 12 x x x x 15 M 17 F508/3905insT x x x 16 M 11 x x x x 17 M 9 x x x x 18 F 7 x x x x 19 M 8 x x x x 20 F 16 x x x x 21 F 8 x x x x Totals 11M/10F median 13 n=14 n=7 n=9 n=12 n=14 n=7 n=3 n=6 n=1 n=2 n=9 * Age at end of the study. Login to comment

[hide] CFTR! Am J Physiol. 1992 Aug;263(2 Pt 1):C267-86. Fuller CM, Benos DJ
CFTR!
Am J Physiol. 1992 Aug;263(2 Pt 1):C267-86., [PMID:1381146]

Abstract [show]
Cystic fibrosis (CF) is a fatal genetic disease primarily affecting Caucasians, although cases have been reported from other ethnic groups. CF has a complex etiology, but it is chiefly a disease of electrolyte transport and is characterized by defects in fluid secretion by several epithelia, including the sweat duct, exocrine pancreas, and the pulmonary airways. The link between CF and a defect in cAMP-mediated Cl- transport in secretory epithelia was established in the early 1980s. Since then, numerous electrophysiological studies have focused on the characterization and regulation of individual Cl- channels underlying the macroscopic Cl- currents of secretory epithelia in the airways, sweat ducts, and gut. In this review the results of these studies in the light of current knowledge of the function of the CF gene product, the CF transmembrane conductance regulator (CFTR) protein, will be analyzed. The CFTR protein is a member of a family of ATP-binding proteins that act as unidirectional solute pumps. These proteins are membrane spanning, are found in both prokaryotic and eukaryotic cells, and have two ATP-binding domains. The family includes the p-glycoproteins that are involved with the expression of multidrug resistance in certain tumor cells. The majority of CF chromosomes (70%) have a single codon deletion that translates to a missing phenylalanine residue at position 508 (delta F508) of the protein. Unique for this family of proteins, the CFTR protein possesses an additional highly charged domain (the R domain) containing several consensus polypeptide sequences for kinase phosphorylation. Although CFTR bears structural resemblance to this family of ATP-dependent pumps, overexpression of the protein in a variety of different cell types is associated with the appearence of a cAMP-sensitive Cl- channel. We critically examine current information concerning the structure-function relationships of the CFTR protein obtained from both electrophysiological and biochemical approaches. We also summarize recent evidence suggesting that the CFTR protein may act as a pump and a channel, a hypothesis in keeping with the multifaceted nature of the disease.

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366 AF508/AF508 G551D/G551D G542X/G458V G542X/G542X R553X/W1316X N369X/unknown R553X/R553X G551S/G551S G368Xlunknown AF508/R117H PI PI PI PI PI PI PI PS PS PS Severe 116 Severe 181 Severe 49 Mild 49 Mild 50 Mild 102 Moderate-Severe 13 Mild 181 Mild 102 Mild 55 Comparison of genotype with phenotype for some CF-associated mutations. Login to comment
416 Cutting et al. (50) have investigated two individuals heterozygous for two different nonsense mutations (S 1255X, G542X and Wl316X, R553X). Login to comment
418 Interestingly, an individual homozygous for the G542X mutation had milder clinical symptoms than a relative with severe CF who was heterozygous [G542X, G458V (49)]. Login to comment
419 The G542X mutation falls near the end of the first NBF region; presumably the protein would be truncated after this point. Login to comment

[hide] Quantitative expression patterns of multidrug-resi... Eur J Biochem. 1992 May 15;206(1):137-49. Bremer S, Hoof T, Wilke M, Busche R, Scholte B, Riordan JR, Maass G, Tummler B
Quantitative expression patterns of multidrug-resistance P-glycoprotein (MDR1) and differentially spliced cystic-fibrosis transmembrane-conductance regulator mRNA transcripts in human epithelia.
Eur J Biochem. 1992 May 15;206(1):137-49., [PMID:1375156]

Abstract [show]
P-glycoprotein (MDR1), that confers multidrug resistance in cancer, and the cystic-fibrosis transmembrane-conductance regulator (CFTR), that is causative defective in cystic fibrosis, belong to the family of ATP-binding transport proteins. The expression of MDR1 and CFTR in human epithelial tissues and the cell lines T84 and HT29 was estimated by primer-directed reverse transcription (RT) and subsequent monitoring of the kinetics of cDNA product formation during the polymerase chain reaction (PCR). MDR1 mRNA was found in high levels, 15-50 amol mRNA/microgram RNA, in the intestine, kidney, liver and placenta, and in low levels, 0.2 amol/microgram RNA, in respiratory epithelium. Large amounts of CFTR mRNA were measured in the gastrointestinal tract, whereas the kidney, as the phenotypically normal organ, and the lung, as the most severely affected organ in cystic fibrosis, both contained low amounts, 3 amol CFTR/microgram RNA. CFTR transcript levels of 1-5 amol/microgram RNA were determined in lymphocytes and lymphoblast cell lines, suggesting that lymphoblasts are an accessible source for the study of the molecular pathogenesis of cystic fibrosis. When transcripts were scanned by overlapping RT/PCR analyses, only transcript of expected size was detected for MDR1 mRNA, where variable in-frame deletions of either exon 4, 9 or 12 were observed in CFTR mRNA. The complete loss of single exons was seen at proportions of 1-40% in all investigated tissues and cell lines with large donor-to-donor variation. Exons 9 and 12 of the CFTR gene encode parts of the evolutionarily well-conserved first nucleotide-binding fold including the two Walker motifs. Alternative splicing may give rise to various CFTR forms of different function and localization.

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139 A few other mutations were analyzed either by allele-specific oligonucleotide hybridization (R117H, 1717-1G --f A) (Dean et al., 1990; Kerem et al., 1990) or by allele-specific PCR (G542X, N1303K) (Kerem et al., 1990; Osborne et al., 1991). Login to comment

[hide] Regulation of male fertility by CFTR and implicati... Hum Reprod Update. 2012 Nov;18(6):703-13. doi: 10.1093/humupd/dms027. Epub 2012 Jun 17. Chen H, Ruan YC, Xu WM, Chen J, Chan HC
Regulation of male fertility by CFTR and implications in male infertility.
Hum Reprod Update. 2012 Nov;18(6):703-13. doi: 10.1093/humupd/dms027. Epub 2012 Jun 17., [PMID:22709980]

Abstract [show]
BACKGROUND The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl(-) and HCO(3)(-) conducting channel, mutations of which are known to be associated with male infertility. However, the underlying mechanisms remain elusive. METHODS Literature databases were searched for papers on the topics related to CFTR and male fertility and infertility with relevant keywords. Unpublished data from authors' laboratory were also included for analysis. RESULTS Clinical evidence shows increased mutation frequency or reduced CFTR expression in men with congenital bilateral absence of vas deferens (CBAVD) or sperm abnormalities, such as azoospermia teratospermia and oligoasthenospermia. Studies on primary rodent Sertoli cells and germ cells, as well as testes from CFTR knockout mice or a cryptorchidism model, yield findings indicating the involvement of CFTR in spermatogensis through the HCO(3)(-)/sAC/cAMP/CREB(CREM) pathway and the NF-kappaB/COX-2/PGE(2) pathway. Evidence also reveals a critical role of CFTR in sperm capacitation by directly or indirectly mediating HCO(3)(-) entry that is essential for capacitation. CFTR is emerging as a versatile player with roles in mediating different signaling pathways pertinent to various reproductive processes, in addition to its long-recognized role in electrolyte and fluid transport that regulates the luminal microenvironment of the male reproductive tract. CONCLUSIONS CFTR is a key regulator of male fertility, a defect of which may result in different forms of male infertility other than CBAVD. It would be worthwhile to further investigate the potential of developing novel diagnostic and contraceptive methods targeting CFTR.

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73 Mutations/ variants Phenotypes Reference F508del Non-obstructive azoospermia, oligozoospermia, oligoasthenozoospermia, oligoasthenoteratozoospermia Stuppia et al. (2005), Schulz et al. (2006) R117H Non-obstructive azoospermia, oligozoospermia, oligoasthenozoospermia, oligoasthenoteratozoospermia Schulz et al. (2006) W1282X Non-obstructive azoospermia, Stuppia et al. (2005) G542X Non-obstructive azoospermia, Stuppia et al. (2005) 5T/7T/9T Non-obstructive azoospermia, oligozoospermia Kanavakis et al. (1998), Stuppia et al. (2005), Schulz et al. (2006), Tamburino et al. (2008), Tomaiuolo et al. (2011) Conti, 2003; Wu et al., 2006; Geng et al., 2009; Schmid et al., 2010; Tresguerres et al., 2011). Login to comment
72 Mutations/ variants Phenotypes Reference F508del Non-obstructive azoospermia, oligozoospermia, oligoasthenozoospermia, oligoasthenoteratozoospermia Stuppia et al. (2005), Schulz et al. (2006) R117H Non-obstructive azoospermia, oligozoospermia, oligoasthenozoospermia, oligoasthenoteratozoospermia Schulz et al. (2006) W1282X Non-obstructive azoospermia, Stuppia et al. (2005) G542X Non-obstructive azoospermia, Stuppia et al. (2005) 5T/7T/9T Non-obstructive azoospermia, oligozoospermia Kanavakis et al. (1998), Stuppia et al. (2005), Schulz et al. (2006), Tamburino et al. (2008), Tomaiuolo et al. (2011) Conti, 2003; Wu et al., 2006; Geng et al., 2009; Schmid et al., 2010; Tresguerres et al., 2011). Login to comment

[hide] Fixing cystic fibrosis by correcting CFTR domain a... J Cell Biol. 2012 Oct 15;199(2):199-204. doi: 10.1083/jcb.201208083. Okiyoneda T, Lukacs GL
Fixing cystic fibrosis by correcting CFTR domain assembly.
J Cell Biol. 2012 Oct 15;199(2):199-204. doi: 10.1083/jcb.201208083., [PMID:23071149]

Abstract [show]
For cystic fibrosis (CF) patients most therapies focus on alleviating the disease symptoms. Yet the cellular basis of the disease has been well studied; mutations in the CF gene can impair folding, secretion, cell surface stability, and/or function of the CFTR chloride channel. Correction of these basic defects has been a challenge, but indicates that a deeper understanding of the molecular and cellular mechanism of mutations is a prerequisite for developing more efficient therapies.

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14 Class I mutations include nonsense mutations (G542X and R553X), generating premature termination codons and frame-shift mutations that lead to truncated and/or and nonfunctional protein (Fig. 1). Login to comment
45 (A) Class I mutations (e.g., G542X) impair production of CFTR full-length protein by induction of premature termination codons (PTC). Login to comment

[hide] Cystic fibrosis: insight into CFTR pathophysiology... Clin Biochem. 2012 Oct;45(15):1132-44. doi: 10.1016/j.clinbiochem.2012.05.034. Epub 2012 Jun 12. Lubamba B, Dhooghe B, Noel S, Leal T
Cystic fibrosis: insight into CFTR pathophysiology and pharmacotherapy.
Clin Biochem. 2012 Oct;45(15):1132-44. doi: 10.1016/j.clinbiochem.2012.05.034. Epub 2012 Jun 12., [PMID:22698459]

Abstract [show]
Cystic fibrosis is the most common life-threatening recessively inherited disease in Caucasians. Due to early provision of care in specialized reference centers and more comprehensive care, survival has improved over time. Despite great advances in supportive care and in our understanding of its pathophysiology, there is still no cure for the disease. Therapeutic strategies aimed at rescuing the abnormal protein are either being sought after or under investigation. This review highlights salient insights into pathophysiology and candidate molecules suitable for CFTR pharmacotherapy. Clinical trials using Ataluren, VX-809 and ivacaftor have provided encouraging data. Preclinical data with inhibitors of phosphodiesterase type 5, such as sildenafil and analogs, have highlighted their potential for CFTR pharmacotherapy. Because sildenafil and analogs are in clinical use for other clinical applications, research on this class of drugs might speed up the development of new therapies for CF.

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982 Class Mutation prototypes Consequences Severe CF phenotype I G542X, W1282X, R553X, 3950delT CFTR is not synthesized because of stop codons or splicing defects II F508del, N1303K CFTR is synthesized but in an immature form (only partly glycosylated, misfolded, not released from the endoplasmic reticulum) and is mostly degraded by the ubiquitin-proteasomal pathway III G551D CFTR is synthesized and transported to the plasma membrane, but its activation and regulation by ATP or cAMP are disrupted Milder CF phenotype IV R334W, G314E, R347P, D1152H CFTR is synthesized and expressed at the plasma membrane, but chloride conductance is reduced V 3849+10 kb C>T, 3272-26 A>G CFTR synthesis or processing is partly defective Severe CF phenotype VI 1811+1.6 kb A>G CFTR is synthesized, but membrane stability or conductance of ions other than chloride is reduced Fig. 2. Login to comment

[hide] Hispanic Infants with cystic fibrosis show low CFT... J Genet Couns. 2012 Oct;21(5):671-5. doi: 10.1007/s10897-012-9481-2. Epub 2012 Feb 4. Watts KD, Layne B, Harris A, McColley SA
Hispanic Infants with cystic fibrosis show low CFTR mutation detection rates in the Illinois newborn screening program.
J Genet Couns. 2012 Oct;21(5):671-5. doi: 10.1007/s10897-012-9481-2. Epub 2012 Feb 4., [PMID:22311127]

Abstract [show]
States develop specific protocols for cystic fibrosis (CF) newborn screening to reflect the population served. We hypothesized that mutation distribution and detection rates would differ between Hispanic and non-Hispanic CF patients diagnosed by IL newborn screen with more Hispanic infants carrying mutations not detected by the state panel. Data from CF cases diagnosed via newborn screen in IL between 3/1/2008 and 10/31/2010 were reviewed. More Hispanic infants with CF had one or more undefined mutations after screening, in comparison to non-Hispanic Caucasian patients (40% vs. 9.5%; p < 0.002). The risk of having a positive diagnosis of CF with only one mutation noted by positive newborn screen increases 2-fold in Hispanic Caucasian versus non-Hispanic Caucasian infants (5% vs. 2.4%). Health care providers must be aware of the limitations of CF newborn screening to ensure appropriate counseling and prompt referral for a positive newborn screen, even when zero or one mutations are identified.

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39 Mutation Frequency Table 1 shows the mutations found in Illinois patients diagnosed with CF after a positive NBS and compares these to mutations documented in Hispanic Caucasian Table 1 CFTR mutation frequency detected by Illinois newborn screen Mutation IL Newborn Screen CFF Patient Registry Total alleles Non-Hispanic Caucasian Hispanic Caucasian African American Ethnicity/Race Missing Hispanic Caucasian ΔF508 63.9% 71.6% 36.7% 33.3% 58.3% 44.7% R117H 7.7% 10.1% 3.3% - - 0.3% G542X 1.9% 2.0% - - 4.2% 4.1% 3120+1G>A 1.9% 0.7% 3.3% 33.3% - 0.7% ΔI507 1.4% 0.7% - - 8.3% 1.3% G551D 1.4% 2.0% - - - 0.5% 3659delC 1.4% 1.3% 3.3% - - 0.1% 3849+10 kbC>T 1.4% - 6.7% 16.7% - 1.0% ΔF311 1.4% - 6.7% - 4.2% 0.03% 1288insT 0.5% - 3.3% - - 0% 621+1G>T 0.5% - 3.3% - - 0.4% G85E 1.0% - 3.3% - 4.2% 0.3% 2184delA 0.5% - 3.3% - - 0.2% S549N 0.5% - 3.3% - - 0.7% R334W 1.0% 0.7% - 16.7% - 1.0% N1303K 1.0% - - - 8.3% 1.6% Other 4.4% 6.2%a 0% 0% 0% 12.8%b Unknown 8.2% 4.7% 23.5% 0% 12.5% 15.7% a R347P, 1898+1G>A, 2789+5G>A, 3272-26A>G, 3876delA, CFTRdel2,3, W1282X occurred in non-Hispanic Caucasian patients only with an allele frequency of 0.5% of the entire IL NBS population b In the 2004 CFF Patient Registry 12.8% of alleles are not included in the above table because they occur in less than 1% of the population. Login to comment
42 The most common were ΔF508, R117H, G542X, G551D, 3120 +1G>A, ΔI507, 3659delC, 3849 +10kb C>T, and ΔF311, showing overlap but not concordance with the most common mutations reported by the CF Foundation (CFF) Annual Data Report 2009 (ΔF508, G542X, G551D, R117H, W1282X, N1303K and R553X). Login to comment

[hide] Polymorphisms in ADRB2 gene can modulate the respo... BMC Pulm Med. 2012 Sep 5;12(1):50. Marson FA, Bertuzzo CS, Ribeiro AF, Ribeiro JD
Polymorphisms in ADRB2 gene can modulate the response to bronchodilators and the severity of cystic fibrosis.
BMC Pulm Med. 2012 Sep 5;12(1):50., [PMID:22950544]

Abstract [show]
ABSTRACT: BACKGROUND: The most common cystic fibrosis (CF) manifestation is the progressive chronic obstructive pulmonary disease caused by deficiency, dysfunction, or absence of the CFTR (Cystic Fibrosis Transmembrane Regulator) protein on the apical surface of the cells in the respiratory tract. The use of bronchodilators (BD), and inhaled corticosteroids (IC) have been suggested for the management of airway inflammation in CF. The effectiveness of BD and IC have been verified, proven in laboratory and in the clinical treatment for asthma patients. However, in CF, the effectiveness of these drugs is controversial. The extent of asthma's response to BD depends on the presence of polymorphisms in the ADRB2 gene. In contrast, in CF, little is known about the response to the BD and the association of CF's severity with the different polymorphisms in ADRB2 gene. In this context, our objective was to verify whether the Arg16Gly and Glu27Gln polymorphisms in ADRB2 gene are associated with severity and with the bronchodilator response in CF patients. METHOD: Cross-sectional study of 122 CF patients subjected to analysis of mutations in the CFTR gene, polymorphisms in ADRB2 gene, along with clinical and laboratorial characteristics of severity. Result The Arg16Gly polymorphism in ADRB2 gene was associated with pancreatic insufficiency(p:0.009), Bhalla score(p:0.039), forced expiratory volume in the first second[FEV1(%)](p:0.003), forced expiratory flow between 25 and 75% of the forced vital capacity-FVC[FEF25-75(%)](p:0.008) and lower age at the first isolation of the Pseudomonas aeruginosa(p:0.012). The response to the BD spirometry was associated with clinical severity markers, FEV1(%)(p:0.011) and FEF25-75(%)(p:0.019), for the Arg16Gly polymorphism in the ADRB2 gene. The haplotype analysis showed association with the FEV1/FVC marker from the spirometry test, before and after using the BD, with higher values in the group with Gly/Gly and Glu/Glu, respectively, for the Arg16Gly and Gln27Glu polymorphisms. The analysis by MDR2.0 software, showed association with FEF25-75%; the response to Arg16Gly was respondent by 17.35% and Gln27Glu by 6.8% in variation found. CONCLUSION: There was an association between the Arg16Gly and Gln27Glu polymorphisms in ADRB2 gene with CF's severity and bronchodilator response.

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47 The patients underwent two perspiration tests of chlorine and sodium with chlorine levels equal to or greater than 60 mEq/L, and/or identification of two mutations in CFTR gene [F508del, G542X, G551D, R553X, R1162X, I618T and N1303K]. Login to comment
73 Table 1 Characteristics of patients included in the study (N = 122)1 Male 48.8 % Age 246.68 ± 168,73 months (87 - 932 months) Caucasoid 93.4% BMI - Thinness and Thinness accentuated 22.3% SaO2 94.87 ± 4.53 (66 - 99) Bhalla 9.41 ± 5.57 (0 - 25) Kanga 19.37 ± 5.01 (11 - 40) Shwachman-Kulczycki 65.41 ± 16.02 (20 - 95) FVC (%) 78.27 ± 22.86 (19 - 135) FEV1 (%) 70.28 ± 26.17 (17 - 125) FEV1/FVC (%) 83.83 ± 15.79 (37 - 137) FEF25-75% 58.50 ± 34.83 (7 - 150) FVC (%) reversibility 0.92 ± 10.48 (-27 - 32) FEV1 (%) reversibility 2.15 ± 9.45 (-12 - 31) FEV1/FVC (%) reversibility 2.84 ± 9.69 (-19 - 47) FEF25-75% reversibility 7.24 ± 9.43(-12 - 30) Nasal Polyps 21.7% Diabetes mellitus 20.8% Osteoporosis 20.8% Pancreatic insufficiency 76% Meconium ileus 9.1% P. aeruginosa status 2 53.7% P. aeruginosa mucoid status 2 45.5% B. cepacia status 2 9.1% A. xylosoxidans status 2 9.9% S. aureus status 2 78.5% CFTR mutation F508del/F508del 29 (24%) F508del/G542X 10 (8.3%) F508del/N1303K 3 (2.5%) F508del/R1162X 3 (2.5%) F508del/R553X 1 (0.8%) G542X/I618T 1 (0.8%) G542X/R1162X 1 (0.8%) F508del/No identified mutation 26 (21.5%) G542X/No identified mutation 4 (3.3%) No identified mutation 43 (35.3%) N - Sample size; BMI - body mass index; % - percentage; FVC - forced vital capacity; FEV1 - forced expiratory volume in the first second; FEF25-75% - forced expiratory flow between 25 and 75% of CVF. 1. Continuous variables expressed as mean ± SD (range). Login to comment
42 The patients underwent two perspiration tests of chlorine and sodium with chlorine levels equal to or greater than 60 mEq/L, and/or identification of two mutations in CFTR gene [F508del, G542X, G551D, R553X, R1162X, I618T and N1303K]. Login to comment
93 Buscher et al. (2002) [17] used the following markers: Table 1 Characteristics of patients included in the study (N = 122)1 Male 48.8 % Age 246.68 &#b1; 168,73 months (87 - 932 months) Caucasoid 93.4% BMI - Thinness and Thinness accentuated 22.3% SaO2 94.87 &#b1; 4.53 (66 - 99) Bhalla 9.41 &#b1; 5.57 (0 - 25) Kanga 19.37 &#b1; 5.01 (11 - 40) Shwachman-Kulczycki 65.41 &#b1; 16.02 (20 - 95) FVC (%) 78.27 &#b1; 22.86 (19 - 135) FEV1 (%) 70.28 &#b1; 26.17 (17 - 125) FEV1/FVC (%) 83.83 &#b1; 15.79 (37 - 137) FEF25-75% 58.50 &#b1; 34.83 (7 - 150) FVC (%) reversibility 0.92 &#b1; 10.48 (-27 - 32) FEV1 (%) reversibility 2.15 &#b1; 9.45 (-12 - 31) FEV1/FVC (%) reversibility 2.84 &#b1; 9.69 (-19 - 47) FEF25-75% reversibility 7.24 &#b1; 9.43(-12 - 30) Nasal Polyps 21.7% Diabetes mellitus 20.8% Osteoporosis 20.8% Pancreatic insufficiency 76% Meconium ileus 9.1% P. aeruginosa status 2 53.7% P. aeruginosa mucoid status 2 45.5% B. cepacia status 2 9.1% A. xylosoxidans status 2 9.9% S. aureus status 2 78.5% CFTR mutation F508del/F508del 29 (24%) F508del/G542X 10 (8.3%) F508del/N1303K 3 (2.5%) F508del/R1162X 3 (2.5%) F508del/R553X 1 (0.8%) G542X/I618T 1 (0.8%) G542X/R1162X 1 (0.8%) F508del/No identified mutation 26 (21.5%) G542X/No identified mutation 4 (3.3%) No identified mutation 43 (35.3%) N - Sample size; BMI - body mass index; % - percentage; FVC - forced vital capacity; FEV1 - forced expiratory volume in the first second; FEF25-75% - forced expiratory flow between 25 and 75% of CVF. 1. Continuous variables expressed as mean &#b1; SD (range). Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... J Cyst Fibros. 2012 Sep;11(5):355-62. doi: 10.1016/j.jcf.2012.05.001. Epub 2012 Jun 2. Ooi CY, Durie PR
Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in pancreatitis.
J Cyst Fibros. 2012 Sep;11(5):355-62. doi: 10.1016/j.jcf.2012.05.001. Epub 2012 Jun 2., [PMID:22658665]

Abstract [show]
BACKGROUND: The pancreas is one of the primary organs affected by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. While exocrine pancreatic insufficiency is a well-recognized complication of cystic fibrosis (CF), symptomatic pancreatitis is often under-recognized. RESULTS: The aim of this review is to provide a general overview of CFTR mutation-associated pancreatitis, which affects patients with pancreatic sufficient CF, CFTR-related pancreatitis, and idiopathic pancreatitis. The current hypothesis regarding the role of CFTR dysfunction in the pathogenesis of pancreatitis, and concepts on genotype-phenotype correlations between CFTR and symptomatic pancreatitis will be reviewed. Symptomatic pancreatitis occurs in 20% of pancreatic sufficient CF patients. In order to evaluate genotype-phenotype correlations, the Pancreatic Insufficiency Prevalence (PIP) score was developed and validated to determine severity in a large number of CFTR mutations. Specific CFTR genotypes are significantly associated with pancreatitis. Patients who carry genotypes with mild phenotypic effects have a greater risk of developing pancreatitis than patients carrying genotypes with moderate-severe phenotypic consequences at any given time. CONCLUSIONS: The genotype-phenotype correlation in pancreatitis is unique compared to other organ manifestations but still consistent with the complex monogenic nature of CF. Paradoxically, genotypes associated with otherwise mild phenotypic effects have a greater risk for causing pancreatitis; compared with genotypes associated with moderate to severe disease phenotypes. Greater understanding into the underlying mechanisms of disease is much needed. The emergence of CFTR-assist therapies may potentially play a future role in the treatment of CFTR-mutation associated pancreatitis.

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847 Total PI Total PI+PS PIP score 621+1G>T 96 96 1.00 Classes I - III 711+1G>T 36 36 1.00 Classes I - III R553X 24 24 1.00 Classes I - III I507del 34 34 1.00 Classes I - III G542X 74 75 0.99 Classes I - III F508del 1276 1324 0.96 Classes I - III 1717-1G>A 20 21 0.95 Classes I - III W1282X 19 20 0.95 Classes I - III N1303K 45 48 0.94 Classes I - III R1162X 12 13 0.92 Classes I - III G551D 59 67 0.88 Classes I - III G85E 16 22 0.73 Classes I - III A455E 18 37 0.49 Classes IV - V 2789+5G>A 6 16 0.38 Classes IV - V R334W 1 10 0.10 Classes IV - V 3849+10kbC>T 2 22 0.09 Classes IV - V R117H 1 25 0.04 Classes IV - V Mutation Canadian Consortium for CF Genetic Studies Mutation class The PIP score for a specific mutation is the ratio between the pancreatic insufficient patients carrying the mutation (Total PI) and all pancreatic insufficient and sufficient patients (Total PI+PS) carrying the same mutation in a homozygous state or heterozygous in a combination with a severe mutation such as F508del, G551D or a Class I mutation. Login to comment
855 CFTR mutation Total PI Total PI + PS PIP score CFTR mutation Total PI Total PI + PS PIP score 621+1G>T 96 96 1.00 G542X 74 75 0.99 711+1G>T 36 36 1.00 F508del 1276 1324 0.96 I507del 34 34 1.00 1717-1G>A 20 21 0.95 R553X 24 24 1.00 W1282X 19 20 0.95 Q493X 11 11 1.00 N1303K 45 48 0.94 S489X 11 11 1.00 R1162X 12 13 0.92 1154insTC 10 10 1.00 Y1092X 12 13 0.92 3659delC 9 9 1.00 I148T 10 11 0.91 CFTRdele2 7 7 1.00 V520F 9 10 0.90 4016insT 7 7 1.00 G551D 59 67 0.88 E60X 7 7 1.00 L1077P 5 6 0.83 R560T 7 7 1.00 R1066C 5 6 0.83 R1158X 7 7 1.00 2184insA 9 12 0.75 3905insT 6 6 1.00 2143delT 3 4 0.75 I148T;3199del6 5 5 1.00 1161delC 3 4 0.75 2183AA>G 5 5 1.00 3120+1G>A 3 4 0.75 1898+1G>A 5 5 1.00 S549N 3 4 0.75 2347delG 4 4 1.00 G85E 16 22 0.73 Q1313X 3 3 1.00 R117C 2 3 0.67 Q220X 3 3 1.00 M1101K 19 30 0.63 2184delA 3 3 1.00 P574H 3 5 0.60 1078delT 3 3 1.00 474del13BP 1 2 0.50 L1254X 3 3 1.00 R352Q 1 2 0.50 E585X 3 3 1.00 Q1291H 1 2 0.50 3876delA 2 2 1.00 A455E 18 37 0.49 S4X 2 2 1.00 R347P 6 15 0.40 R1070Q 2 2 1.00 2789+5G>A 6 16 0.38 F508C 2 2 1.00 L206W 6 18 0.33 DELI507 2 2 1.00 IVS8-5T 4 16 0.25 Q1411X 2 2 1.00 3272-26A>G 1 4 0.25 365-366insT 2 2 1.00 R334W 1 10 0.10 R709X 2 2 1.00 3849+10kbC>T 2 22 0.09 1138insG 2 2 1.00 P67L 1 14 0.07 CFTRdele2-4 2 2 1.00 R117H 1 25 0.04 3007delG 2 2 1.00 R347H 0 5 0.00 Q814X 2 2 1.00 G178R 0 3 0.00 394delTT 2 2 1.00 E116K 0 2 0.00 406-1G>A 2 2 1.00 875+1G>C 0 2 0.00 R75X 2 2 1.00 V232D 0 2 0.00 CFTRdel2-3 2 2 1.00 D579G 0 2 0.00 E193X 2 2 1.00 L1335P 0 2 0.00 185+1G>T 2 2 1.00 Mild mutations (based on PIP scores) are shaded in gray. Login to comment

[hide] Newborn screening for cystic fibrosis: Polish 4 ye... Eur J Hum Genet. 2012 Aug 15. doi: 10.1038/ejhg.2012.180. Sobczynska-Tomaszewska A, Oltarzewski M, Czerska K, Wertheim-Tysarowska K, Sands D, Walkowiak J, Bal J, Mazurczak T
Newborn screening for cystic fibrosis: Polish 4 years' experience with CFTR sequencing strategy.
Eur J Hum Genet. 2012 Aug 15. doi: 10.1038/ejhg.2012.180., [PMID:22892530]

Abstract [show]
Newborn screening for cystic fibrosis (NBS CF) in Poland was started in September 2006. Summary from 4 years' experience is presented in this study. The immunoreactive trypsin/DNA sequencing strategy was implemented. The group of 1 212 487 newborns were screened for cystic fibrosis during the programme. We identified a total of 221 CF cases during this period, including, 4 CF cases were reported to be omitted by NBS CF. Disease incidence in Poland based on the programme results was estimated as 1/4394 and carrier frequency as 1/33. The frequency of the F508del was similar (62%) to population data previously reported. This strategy allowed us to identify 29 affected infants with rare genotypes. The frequency of some mutations (eg, 2184insA, K710X) was assessed in Poland for the first time. Thus, sequencing assay seems to be accurate method for screening programme using blood spots in the Polish population.European Journal of Human Genetics advance online publication, 15 August 2012; doi:10.1038/ejhg.2012.180.

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57 Mutations D537N and P731L have not been Period of NBS CF Method The most frequent mutations in Polish population under analysis September 2006 - December 2007 Estonia Asper Biotech assay E60X, G85E, 394delTT, R117H, R117P, R117L, I148T, 621G>A, 711+1G>T, 711+5G>A, 1078delT, R334W, R347H, R347P, R347L, IVS8-T, A455E, I507del, F508del, 1717-1G>A, G542X, p.G551D, Q552X, R553X, R553G, R560T, R560K, 1898+1G>A, 1898+1G>T, 1898+1G>C, 2143delT, 2184delA, 2183AA>G, 2789+5G>A, 3120+1G>A, 3199del6, 3272-26A>G, R1162X, 3659delC, 3849+10kbC>T, 3905insT, S1235R, S1251N, W1282X, W1282C, N1303K, CFTRdele2,3 January 2007 - June 2009 Sanger sequencing of exons: 4, 7, 10, 11, 13, 21, fragment of intron 19 F508del, CFTRdele2,3, 3849+10kbC>T, R117H+IVS8-T*, R334W, R347P, 1717-1G>A, G542X, R553X, K710X, 2184insA, 2143delT, 2183AA>G, N1303K July 2009 - currently Sanger sequencing of exons: 7, 10, 11, 13, 17b, 20, 21, fragment of intron 19 F508del, CFTRdele2,3, 3849+10kbC>T, R334W, R347P, 1717-1G>A, G542X, R553X, K710X, 2184insA, 2143delT, 2183AA>G, N1303K, 3272-26A>G**, W1282X** * removed from DNA analysis since July 2009 , **added into DNA analysis since July 2009 Figure 1 NBS CF in Poland. Login to comment
72 Table 2 Genotypes of CF newborns with mutations not included into common commercial kits applied in Poland and European countries* Genotype Number of cases [F508del]; [1767-8T4A*] 1 [F508del];[2184insA*] 6 [F508del];[E33X*] 1 [F508del];[F1286C*] 1 [F508del];[G314R*] 1 [F508del];[K710X*] 1 [F508del];[W1282R*] 1 [F508del];[1898 þ 1G4C*] 1 [F508del];[3600 þ 2insT*] 1 [F508del];[F1052V*] 1 [F508del];[V1240G*] 1 [F508del];[T582I*] 1 [2143delT];[R1102X*] 1 [2143delT];[2721del11*] 1 [3272-26A4G];[K967S*] 1 [CFTRdele2,3];[Y1092X*] 1 [K710X*];[K710X*] 1 [L732X*];[3600 þ 2insT*] 1 [N1303K];[2184insA*] 1 [N1303L];[T1036I*] 1 [R553X];[3182ins8*] 1 [2143delT];[V1240G*] 1 [R553X];[Trp356X*] 1 [L997F*];[1210-12T[5];1210-13G4T] 1 Total 29 Table 3 Frequency of CFTR mutations in Polish CF patients from newborns screening programme CFTR mutations Frequency according to Bobadilla et al15 Frequency according to NBS CF results (all ¼ 442 CF alleles) Name Position % % F508del Exon11 57.1 62.4 3849 þ 10kbC4T Intron 22 2.7 3.0 G542X Exon 12 2.6 1.6 1717-1G4A Intron 11 2.4 1.4 R553X Exon 12 1.9 2.5 CFTRdele2,3 Exons 2 and 3 1.8 6.2 N1303K Exon 24 1.8 2.1 2143delT Exon 14 No data 2.8 2184insA Exon 14 No data 1.8 2183AA4G Exon 14 No data 1.6 W1282X Exon 23 0.7 1.5 R334W Exon 8 No data 0.7 R347P Exon 8 No data 0.5 G551D Exon 12 0.5 0.0 K710X Exon 14 No data 0.7 3272-26A4G Intron 19 No data 0.7 3600 þ 2insT Intron 21 No data 0.5 1898 þ 1G4C Intron 13 No data 0.5 V1240G Exon 23 No data 0.5 Othersa - No data 10.0 Abbreviations: CF, cystic fibrosis; NBS CF, newborn screening for CF. Login to comment

[hide] The ACE gene D/I polymorphism as a modulator of se... BMC Pulm Med. 2012 Aug 8;12:41. Marson FA, Bertuzzo CS, Hortencio TD, Ribeiro JD, Bonadia LC, Ribeiro AF
The ACE gene D/I polymorphism as a modulator of severity of cystic fibrosis.
BMC Pulm Med. 2012 Aug 8;12:41., [PMID:22874010]

Abstract [show]
ABSTRACT: BACKGROUND: Cystic Fibrosis (CF) is a monogenic disease with complex expression because of the action of genetic and environmental factors. We investigated whether the ACE gene D/I polymorphism is associated with severity of CF. METHODS: A cross-sectional study was performed, from 2009 to 2011, at University of Campinas - UNICAMP. We analyzed 180 patients for the most frequent mutations in the CFTR gene, presence of the ACE gene D/I polymorphism and clinical characteristics of CF. RESULTS: There was an association of the D/D genotype with early initiation of clinical manifestations (OR: 1.519, CI: 1.074 to 2.146), bacterium Burkholderia cepacia colonization (OR: 3.309, CI: 1.476 to 6.256) and Bhalla score (BS) (p = 0.015). The association was observed in subgroups of patients which were defined by their CFTR mutation genotype (all patients; subgroup I: no mutation detected; subgroup II: one CFTR allele identified to mutation class I, II or III; subgroup III: both CFTR alleles identified to mutation class I, II and/or III). CONCLUSION: An association between the D allele in the ACE gene and the severity of CF was found in our study.

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28 Determination of mutations in the CFTR gene Determination of mutations in the CFTR gene was performed in the Laboratory of Molecular Genetics for mutations by polymerase chain reaction (F508del) and restriction fragment length polymorphism method (G542X, R1162X, R553X, G551D and N1303K). Login to comment
70 The patients` CFTR genotypes were: 44 patients (24.44%) without identified mutation, 51 (28.33%) with one identified mutation (25% F508del/-, 2.78% G542X/-, 0.56% R1162X/-) and 85 (47.22%) patients with two identified mutations (31.67% F508del/F508del, 6.67% F508del/G542X, 2.78% F508del/R1162X, 2.22% F508del/N1303K, 0.56% F508del/ R553X, 0.56% F508del/S4X, 0.56% F508del/1717-1 G > A, 0.56% G542X/R1162X, 0.56% G542X/I618T, 0.56% G542X/2183A > G and 0.56% R1162X/R1162X). Login to comment
27 Determination of mutations in the CFTR gene Determination of mutations in the CFTR gene was performed in the Laboratory of Molecular Genetics for mutations by polymerase chain reaction (F508del) and restriction fragment length polymorphism method (G542X, R1162X, R553X, G551D and N1303K). Login to comment
69 The patients` CFTR genotypes were: 44 patients (24.44%) without identified mutation, 51 (28.33%) with one identified mutation (25% F508del/-, 2.78% G542X/-, 0.56% R1162X/-) and 85 (47.22%) patients with two identified mutations (31.67% F508del/F508del, 6.67% F508del/G542X, 2.78% F508del/R1162X, 2.22% F508del/N1303K, 0.56% F508del/ R553X, 0.56% F508del/S4X, 0.56% F508del/1717-1 G > A, 0.56% G542X/R1162X, 0.56% G542X/I618T, 0.56% G542X/2183A > G and 0.56% R1162X/R1162X). Login to comment

[hide] Prospective and parallel assessments of cystic fib... Eur J Pediatr. 2012 Aug;171(8):1223-9. Epub 2012 May 12. Krulisova V, Balascakova M, Skalicka V, Piskackova T, Holubova A, Paderova J, Krenkova P, Dvorakova L, Zemkova D, Kracmar P, Chovancova B, Vavrova V, Stambergova A, Votava F, Macek M Jr
Prospective and parallel assessments of cystic fibrosis newborn screening protocols in the Czech Republic: IRT/DNA/IRT versus IRT/PAP and IRT/PAP/DNA.
Eur J Pediatr. 2012 Aug;171(8):1223-9. Epub 2012 May 12., [PMID:22581207]

Abstract [show]
Cystic fibrosis (CF) is a life-threatening disease for which early diagnosis following newborn screening (NBS) improves the prognosis. We performed a prospective assessment of the immunoreactive trypsinogen (IRT)/DNA/IRT protocol currently in use nationwide, versus the IRT/pancreatitis-associated protein (PAP) and IRT/PAP/DNA CF NBS protocols. Dried blood spots (DBS) from 106,522 Czech newborns were examined for IRT concentrations. In the IRT/DNA/IRT protocol, DNA-testing was performed for IRT >/= 65 ng/mL. Newborns with IRT >/= 200 ng/mL and no detected cystic fibrosis transmembrane conductance regulator gene (CFTR) mutations were recalled for a repeat IRT. In the same group of newborns, for both parallel protocols, PAP was measured in DBS with IRT >/= 50 ng/mL. In PAP-positive newborns (i.e., >/=1.8 if IRT 50-99.9 or >/=1.0 if IRT >/= 100, all in ng/mL), DNA-testing followed as part of the IRT/PAP/DNA protocol. Newborns with at least one CFTR mutation in the IRT/DNA/IRT and IRT/PAP/DNA protocols; a positive PAP in IRT/PAP; or a high repeat IRT in IRT/DNA/IRT were referred for sweat testing. CONCLUSION: the combined results of the utilized protocols led to the detection of 21 CF patients, 19 of which were identified using the IRT/DNA/IRT protocol, 16 using IRT/PAP, and 15 using IRT/PAP/DNA. Decreased cut-offs for PAP within the IRT/PAP protocol would lead to higher sensitivity but would increase false positives. Within the IRT/PAP/DNA protocol, decreased PAP cut-offs would result in high sensitivity, an acceptable number of false positives, and would reduce the number of DNA analyses. Thus, we concluded that the IRT/PAP/DNA protocol would represent the most suitable protocol in our conditions.

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81 According to the protocol, this result indicated the sequencing of the Table 1 Parallel comparison of CF NBS protocols IRT/DNAa /IRT IRT/PAP IRT/PAP/DNAa Newborns screened (N) 106,522 106,522 106,522 IRT positives (N; %) 1,158 (1.09) 3,155 (2.96) 3,155 (2.96) PAP positives (N; %) - 260 (0.24) 260 (0.24) Median age (range) at the availability of DNA-testinga results (days) 36 (9-222b ) - 36 (9-222b ) 1 and/or 2 CF mutations detected (N; %) 76 (0.07) - 27 (0.03) Recalled newborns for repeated IRT examination (N; %) 47 (0.04) - - Positive CF NBS (N; %) 123 (0.12) 260 (0.24) 27 (0.03) Positive IRT in newborns recalled for repeated examination (N) 1 - - ST indicated (N; %) 77 (0.07) 260 (0.24) 27 (0.03) ST carried out (N; % of indicated ST) 72c (93.51) 204c (78.46) 24c (88.89) CF carriers (N) 55 - 12 Prevalence of CF carriers 1 in 21 - 1 in 22 Diagnosed CF patients (N) 19 16 15 False positives based on performed ST (N; % of all cases screened) 99d (0.09) 188 (0.18) 9 (0.01) Newborns with equivocal diagnosis [F508del/R117H-IVS-8 T(7) and ST<30 mmol/L; N] 2 - 0 False negatives (N) 2 5 6 Total of CF patients detected (N) 21e Median age (range) at diagnosis (days) 36 (9-57)e CF prevalence 1 in 5,072e Sensitivity (TP/TP+FN) 0.9048 0.7619 0.7142 Specificity (TN/TN+FP) 0.9991 0.9982 0.9999 PPV (TP/TP+FP) 0.1610 0.0784 0.625 N number, % of all cases screened, TP true positives, FN false negatives, TN true negatives, FP false positives, PPV positive predictive value, ST sweat test a CF-causing mutations covered by Elucigene assays ("legacy" nomenclature) with the CF-EU1Tm accounting for: p.Arg347Pro (R347P), c.2657+ 5G>A (2789+5G>A), c.2988+1G>A (3120+1G>A), c.579+1G>T (711+1G>T), p.Arg334Trp (R334W), p.Ile507del (I507del), p.Phe508del (F508del), c.3718-2477C>T (3849+10kbC>T), p.Phe316LeufsX12 (1078delT), p.Trp1282X (W1282X), p.Arg560Thr (R560T), p.Arg553X (R553X), p.Gly551Asp (G551D), p.Met1101Lys (M1101K), p.Gly542X (G542X), p.Leu1258PhefsX7 (3905insT), p.Ser1251Asn (S1251N), c.1585-1G>A (1717-1G>A), p.Arg117His (R117H), p.Asn1303Lys (N1303K), p.Gly85Glu (G85E), c.1766+1G>A (1898+1G>A), p.Lys684AsnfsX38 (2184delA), p.Asp1152His (D1152H), c.54-5940_273+10250del (CFTRdele2,3), p.Pro67Leu (P67L), p.Glu60X (E60X), p.Lys1177SerfsX15 (3659delC), c.489+1G>T (621+1G>T), p.Ala455Glu (A455E), p.Arg1162X (R1162X), p.Leu671X (2143delT), c.1210-12T[n] (IVS8-T(n) variant), including additional mutations in the CF-EU2Tm : p.Gln890X (Q890X), p.Tyr515X (1677delTA), p.Val520Phe (V520F), c.3140-26A>G (3272-26A>G), p.Leu88IlefsX22 (394delTT), p.Arg1066Cys (R1066C), p.Ile105SerfsX2 (444delA), p.Tyr1092X (C>A) (Y1092X(C>A)), p.Arg117Cys (R117C), p.Ser549Asn (S549N), p.Ser549ArgT>G (S549R T>G), p.Tyr122X (Y122X), p.Arg1158X (R1158X), p.Leu206Trp (L206W), c.1680-886A>G (1811+1.6kbA>G), p.Arg347His (R347H), p.Val739TyrfsX16 (2347delG) and p.Trp846X (W846X) b failed DNA isolation from DBS, including repetition of DNA-testing c deceased patient or non-compliance with referrals (five CF carriers in IRT/DNA/IRT, 56 newborns in IRT/PAP, three CF carriers in IRT/PAP/DNA) d comprising newborns with repeated IRT (47 newborns) e aggregate data from all protocols entire CFTR coding region in both newborns, and led to the identification of p.Ile336Lys (I336K) and p.Glu1104Lys (E1104K) mutations. Login to comment
109 Table 2 False negatives due to mean PAP concentrations below the cut-off IRT (ng/mL) PAP (ng/mL) CFTR Genotypea Sweat chloride concentration (mmol/L) Patient 1 174 0.93 F508del/ F508del 109.6 Patient 2 337 0.49 F508del/ F508del 98.7 Patient 3 203 0.42 F508del/ F508del 103.7 Patient 4 115 0.67 F508del/ I507del 93.2 Patient 5b 87.8 1.43 G542X/ E1104K 74.5 a legacy nomenclature b pancreatic sufficient patient (fecal Elastase-1 level was 507 μg/g) IRT/PAP and IRT/PAP/DNA protocols Five CF patients were false negative within the IRT/PAP protocol due to low PAP concentrations (Table 2). Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Ann Pharmacother. 2012 Jul-Aug;46(7-8):1065-75. Epub 2012 Jun 26. Pettit RS
Cystic fibrosis transmembrane conductance regulator-modifying medications: the future of cystic fibrosis treatment.
Ann Pharmacother. 2012 Jul-Aug;46(7-8):1065-75. Epub 2012 Jun 26., [PMID:22739718]

Abstract [show]
OBJECTIVE: To review and evaluate cystic fibrosis transmembrane conductance regulator (CFTR) modulators for the treatment of cystic fibrosis (CF). DATA SOURCES: Literature was accessed through MEDLINE (1977-January 2012), the Cochrane Library, and International Pharmaceutical Abstracts (1977-March 2012). Search terms included ivacaftor, VX-770, VX-809, ataluren, PTC 124, CFTR modulator, and cystic fibrosis. STUDY SELECTION AND DATA EXTRACTION: All English-language articles identified from the data sources were evaluated for inclusion. Clinical trials and relevant review articles were evaluated for each CFTR modulator. DATA SYNTHESIS: CF is caused by a mutation in the gene that encodes for the CFTR protein; mutations can be separated into 5 different classes. Ivacaftor is a new CFTR potentiator that helps the CFTR channel open properly in patients with the CFTR mutation, G551D. Patients in one study had significant decreases in sweat chloride values and increases in pulmonary function tests. Ivacaftor was approved by the Food and Drug Administration (FDA) to be taken orally at a dose of 150 mg twice a day in G551D CF patients older than 6 years. Additional studies are investigating the use of ivacaftor in other gating mutations and in younger patients. VX-809 is a CFTR corrector that modulates the folding and trafficking of CFTR. VX-809 was originally studied alone in patients with F508del mutation but is now being used in combination with ivacaftor in Phase 2 studies. Ataluren allows the read through of premature stop codons, and studies in patients with CF with nonsense mutations show an increase in chloride transportation. Ataluren requires 3 times a day dosing and is currently in a Phase 3 placebo-controlled study. CONCLUSIONS: Three new agents, ivacaftor, VX-809, and ataluren, target the basic defects in CFTR production. Ivacaftor was recently FDA approved, while the other 2 agents are still in clinical trials. Patients with CF will benefit from personalized medicine based on their specific genotype.

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140 Ataluren not only has therapeutic applicability in patients with CF and class I CFTR mutations, but may be effective in other diseases, such as Duchenne muscular dystrophy.27 Ataluren was originally studied in a mouse model, where it restored chloride to 24-29% of normal levels in mice with CF and a G542X mutation.28 Following positive animal studies, ataluren was administered to 62 healthy adults in increasing doses, from 3 mg/kg to 200 mg/kg per dose.29 The doses were well tolerated until more than 150 mg/kg was administered; these doses caused headache, dizziness, and gastrointestinal adverse effects. Login to comment
246 Du M, Liu X, Welch EM, et al. PTC124 is an orally bioavailable compound that promotes suppression of the human CFTR-G542X nonsense allele in a CF mouse model. Login to comment

[hide] Retrospective analysis of stored dried blood spots... J Cyst Fibros. 2012 Jul;11(4):332-6. doi: 10.1016/j.jcf.2012.01.001. Epub 2012 Feb 1. Barben J, Gallati S, Fingerhut R, Schoeni MH, Baumgartner MR, Torresani T
Retrospective analysis of stored dried blood spots from children with cystic fibrosis and matched controls to assess the performance of a proposed newborn screening protocol in Switzerland.
J Cyst Fibros. 2012 Jul;11(4):332-6. doi: 10.1016/j.jcf.2012.01.001. Epub 2012 Feb 1., [PMID:22300503]

Abstract [show]
BACKGROUND: Newborn screening (NBS) for Cystic Fibrosis (CF) has been introduced in many countries, but there is no ideal protocol suitable for all countries. This retrospective study was conducted to evaluate whether the planned two step CF NBS with immunoreactive trypsinogen (IRT) and 7 CFTR mutations would have detected all clinically diagnosed children with CF in Switzerland. METHODS: IRT was measured using AutoDELFIA Neonatal IRT-Kit in stored NBS cards. RESULTS: Between 2006 and 2009, 66 children with CF were reported, 4 of which were excluded for various reasons (born in another country, NBS at 6 months, no informed consent). 98% (61/62) had significantly higher IRT compared to matched control group. There was one false negative IRT result in an asymptomatic child with atypical CF (normal pancreatic function and sweat test). CONCLUSIONS: All children but one with atypical CF would have been detected with the planned two step protocol.

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28 If IRT is elevated (N99th percentile) a screening test with the seven most common CFTR mutations in Switzerland (F508del, 3905insT, G542X, R553X, W1282X, 1717-1 GNA, N1303K) [12] will be used to confirm the suspicion. Login to comment
46 In brief, this assay is based on DNA amplification of four fragments containing the mutations (F508del, 3905insT, G542X, R553X, W1282X, 1717-1 GNA, and N1303K) by PCR, followed by hybridization with short, allele-specific oligonucleotide probes labeled with europium, terbium, or samarium chelates. Login to comment
80 CFTR mutations Alleles found Percentage of total Homozygous (n) F508del a 86 68.2 30 3905insT a 4 3.2 1 G542X a 3 2.4 - R553X a 3 2.4 1 W1282X a 2 1.6 - 1717-1 GNA a 2 1.6 - N1303K a 0 0.0 - S549R 3 2.4 1 Q525X 3 2.4 - Y1092X 2 1.6 - 3120+1 GNA b 2 1.6 1 2347delG 2 1.6 - 2176insC 1 0.8 - 3659delC 1 0.8 - 3359delCTCTG 1 0.8 - W1089X 1 0.8 - 711+1 GNT 1 0.8 - D1152H 1 0.8 - G1244E 1 0.8 - R1066C 1 0.8 - R31C 1 0.8 - R347P 1 0.8 - R74W 1 0.8 - S945L 1 0.8 - T501I 1 0.8 - K68X 1 0.8 - Total 126 100.0% 34 a Seven most common CF-gene mutations in Switzerland ("Swiss panel")=79.4% (100/126) of alleles. Login to comment

[hide] Improving test properties for neonatal cystic fibr... J Inherit Metab Dis. 2012 Jul;35(4):635-40. Cornel MC, Gille JJ, Loeber JG, Vernooij-van Langen AM, Dankert-Roelse J, Bolhuis PA
Improving test properties for neonatal cystic fibrosis screening in the Netherlands before the nationwide start by May 1st 2011.
J Inherit Metab Dis. 2012 Jul;35(4):635-40., [PMID:22302635]

Abstract [show]
When new technical possibilities arise in health care, often attunement is needed between different actors from the perspectives of research, health care providers, patients, ethics and policy. For cystic fibrosis (CF) such a process of attunement in the Netherlands started in a committee of the Health Council on neonatal screening in 2005. In the balancing of pros and cons according to Wilson and Jungner criteria, the advantages for the CF patient were considered clear, even though CF remains a severe health problem with treatment. Nevertheless, screening was not started then, mainly since the specificity of the tests available at that time was considered too low. Many healthy infants would have been referred for sweat testing and much uncertainty would arise in their parents. Also the limited sensitivity for immigrants and the detection of less severe phenotypes and carriers were considered problematic. The Health Council recommended a pilot screening project which was subsequently performed in some provinces, leading to a 4-step protocol: IRT, PAP, screening for a CFTR mutation panel, and sequencing of the CFTR gene. This would lead to the identification of 23 cases of classical CF, two infants with less severe forms and 12 carriers per year in the Netherlands. Thus many CF patients can be diagnosed early, while limiting the number of referrals, the number of infants with less severe forms diagnosed and the number of carriers identified. Technical solutions were found to limit the ethical problems. A nationwide program using this four step protocol started by 1 May 2011.

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69 This protocol was expected to identify 25 CF patients on an annual basis, additional to four infants already diagnosed because of meconium ileus (Health Council of 1 Using the LiPA test (INNO-LiPA CFTR 19 en INNO-LiPA CFTR 17+Tn; Innogenetics, Gent, Belgium) the following CFTR mutations can be detected: exon 2-3del (21 kb), 394delTT, E60X, G85E, R117H, 621+1G>T, 711+1G>T, 711+5G>A, 1078delT, R334W, R347P, A455E, I507del, F508del, 1717-1G>A, G542X, G551D, Q552X, R553X, R560T, 1898+1G>A, 2143delT, 2183AA>G, 2184delA, 2789+5G>A, 3120+1G>A, 3199del6, 3272-26A>G, 3659delC, R1162X, 3849+10kbC>T, 3905insT, S1251N, W1282X en N1303K. Login to comment
70 This test also identifies the CFTR polymorphism Tn in intron 8 which is important in cases where the mutation R117H is detected. Login to comment

[hide] Link between CFTR mutations and ABPA: a systematic... Mycoses. 2012 Jul;55(4):357-65. doi: 10.1111/j.1439-0507.2011.02130.x. Epub 2011 Oct 17. Agarwal R, Khan A, Aggarwal AN, Gupta D
Link between CFTR mutations and ABPA: a systematic review and meta-analysis.
Mycoses. 2012 Jul;55(4):357-65. doi: 10.1111/j.1439-0507.2011.02130.x. Epub 2011 Oct 17., [PMID:21999194]

Abstract [show]
Summary There is a biological plausibility on the link between cystic fibrosis transmembrane conductance regulator (CFTR) mutations and allergic bronchopulmonary aspergillosis (ABPA). The aim of the systematic review was to investigate this link by determining the frequency of CFTR mutations in ABPA. We searched the PubMed and EmBase databases for studies reporting CFTR mutations in ABPA. We pooled the odds ratio (OR) and 95% confidence intervals (CI) from individual studies using both fixed and random effects model. Statistical heterogeneity was evaluated using the I(2) test and the Cochran-Q statistic. Publication bias was assessed using both graphical and statistical methods. Our search yielded four studies (79 ABPA, 268 controls). The odds of encountering CFTR mutation was higher in ABPA compared with the control group (OR 10.39; 95% CI, 4.35-24.79) or the asthma population (OR 5.53; 95% CI 1.62-18.82). There was no evidence of statistical heterogeneity or publication bias. There is a possible pathogenetic link between CFTR mutations and ABPA. However, because of the small numbers of patients, further studies are required to confirm this finding. Future studies should adopt a uniform methodology and should screen for the entire genetic sequence of the CFTR gene.

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56 (1996)[30] 11ABPA53chronic bronchitis Asthma,pulmonaryinfiltrates,CB, immediateAfskintestpositivity,totalIgE >1000ngml)1 ,positiveAfprecipitins, elevatedAfIgG/IgE,bloodeosinophilia, sweatchloride<40mmoll)1 /(United States) BothgroupssixmutationsF508del, G542X,GS51D,R553X,W1282X andN1303K;ninemoremutations inABPA:R117H,R347P,R347H, R334W,A455E,G551S, 2789+5G>A,D1152H,and 3849+10kbC>T ReverseASOanalysis andDGGEwithDNA sequencing 1patientcarried2CF (F508del;R347H)and5 carried1CF(4F508del; 1R117H).Mutationsseenin 6/11ABPAvs.1/53 controls Aronetal. Login to comment
58 (2001)[32] 21ABPA43allergic asthma; 142healthy controls Asthma,pulmonaryinfiltrates,CB, immediateAfskintestpositivity,totalIgE >450IUml)1 ,positiveAfprecipitins, elevatedAfIgG/IgE,bloodeosinophilia >500ll)1 .Sweatchloride <60mmoll)1 /(Belgium) R117H,621-1G>T,R334W, F508del,I507del10,1717-1G>A, G542X,R553X,G551D,R1162X, 3849+10kbC>T,W1282X, N1303K Heteroduplexand acrylamidegel electrophoresis, ARMS,nestedPCR followedby electrophoresisand DNAsequencing OneCFTRmutationin6/21 patients(F508del[n=2], G542X[n=1],R1162X [n=1],1717-1G>A [n=1],andR117H[n=1]) vs.2/43asthmatics(1CFTR mutation;(F508del, 1717-1G>Aand6/142 controls Eatonetal. Login to comment
59 (2002)[33] 31ABPAHealthycontrols (n=34) Asthma(n=51) Asthma,positiveSPTtoAf,totalIgE >1000ngml)1 ,elevatedAf-IgE,positive precipitinstoAf,bloodeosinophilia >350ll)1 ,pulmonaryinfiltratesonCXR orCBonCT/(NewZealand) 16CFmutations-F508del,I507del, R117H,W1282X,621+1G>T, R334W,R347P,A455E, 1717-1G>A,G542X,5549N, G551D,R553X,R560T,N1303Kand 3849+10kbC>T ASOhybridisationand DGGEwithDNA sequencing 4/31(F508del[n=3], R117H[n=1])vs.2/51 asthma(F508del[n=1], R117H[n=1])vs.1/34 healthycontrols ABPA,allergicbronchopulmonaryaspergillosis;ARMS,amplificationrefractorymutationsystem;ASO,allele-specificoligonucleotide;CB,centralbronchiectasis;CFTR,cysticfibrosis transmembraneconductanceregulator;DGGE,denaturinggradientgelelectrophoresis;OR,oddsratio CFTRmutationclass(classI--1717-1G>A,R1162X,G542X;classII--F508del,N1303K;classIV--R347H,R117H). Login to comment

[hide] The F508del mutation in cystic fibrosis transmembr... Am J Pathol. 2012 May;180(5):2068-75. Epub 2012 Mar 23. Le Henaff C, Gimenez A, Hay E, Marty C, Marie P, Jacquot J
The F508del mutation in cystic fibrosis transmembrane conductance regulator gene impacts bone formation.
Am J Pathol. 2012 May;180(5):2068-75. Epub 2012 Mar 23., [PMID:22449949]

Abstract [show]
The F508del mutation in the cystic fibrosis transmembrane conductance regulator (Cftr) gene is believed to be an independent risk factor for cystic fibrosis-related bone disease. In this study, we evaluated the bone mineral density as well as the histomorphometric parameters of bone formation and bone mass in both F508del-Cftr homozygous mice (F508del Cftr(tm1Eur)) and Cftr(+/+) littermate controls at 6 (prepubertal), 10 (pubertal), and 14 (young adult) weeks of age in both sexes. The bone architecture of F508del Cftr(tm1Eur) and wild-type (WT) littermate mice was evaluated by bone densitometry, microcomputed tomography, and analysis of the dynamic parameters of bone formation. Serum levels of both insulin-like growth factor 1 and osteocalcin also were determined. Reduced bone mineral density, lower femoral bone mass, and altered trabecular bone architecture were observed in F508del Cftr(tm1Eur) mice compared with controls at 6, 10, and 14 weeks of age. A decrease in the bone formation rate in F508del Cftr(tm1Eur) mice was shown compared with control mice, independently of age and sex. In addition, we found lower insulin-like growth factor 1 levels in F508del Cftr(tm1Eur) mice compared with age-matched controls, whereas osteocalcin levels were normal. Severe osteopenia and altered bone architecture were found in young and mature adult F508del Cftr(tm1Eur) mice. Our findings show that the F508del mutation in CFTR impacts trabecular bone mass by reducing bone formation.

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20 The expression of CFTR protein has been identified by immunohistochemistry in human bone cells.19 We previously reported the expression of CFTR mRNA and protein in primary human osteoblasts (cells that form bone) and showed that inhibition of CFTR-mediated Cl- channel activity affects the release of osteoprotegerin and prostaglandin E2, two key regulators of bone formation.20 We recently discovered a defective CFTR Cl- channel activity and a deficit of osteoprotegerin production by primary osteoblasts from a 25-year-old CF patient with the F508del/G542X mutation in CFTR.21 One study in patients with CF with at least one F508del allele showed a direct association between the F508del mutation and low BMD in both sexes.22 However, the impact of the F508del allele mutation in CFTR on bone formation and bone mass remains unknown. Login to comment
19 The expression of CFTR protein has been identified by immunohistochemistry in human bone cells.19 We previously reported the expression of CFTR mRNA and protein in primary human osteoblasts (cells that form bone) and showed that inhibition of CFTR-mediated Clafa; channel activity affects the release of osteoprotegerin and prostaglandin E2, two key regulators of bone formation.20 We recently discovered a defective CFTR Clafa; channel activity and a deficit of osteoprotegerin production by primary osteoblasts from a 25-year-old CF patient with the F508del/G542X mutation in CFTR.21 One study in patients with CF with at least one F508del allele showed a direct association between the F508del mutation and low BMD in both sexes.22 However, the impact of the F508del allele mutation in CFTR on bone formation and bone mass remains unknown. Login to comment

[hide] A large deletion causes apparent homozygosity for ... Gene. 2012 Apr 10;497(1):90-2. Epub 2012 Jan 31. Diana A, Tesse R, Polizzi AM, Santostasi T, Manca A, Leonetti G, Seia M, Porcaro L, Cavallo L
A large deletion causes apparent homozygosity for the D1152H mutation in the cystic fibrosis transmembrane regulator (CFTR) gene.
Gene. 2012 Apr 10;497(1):90-2. Epub 2012 Jan 31., [PMID:22310382]

Abstract [show]
We report the case of a patient with an apparent homozygosity for the D1152H mutation located in exon 18 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The parents had no personal history of cystic fibrosis (CF) and referred to our laboratory after the diagnosis of fetal bowel hyperechogenicity. The proband presented with meconium ileus and normal sweat chloride test. Sequencing of the CFTR exon 18 together with quantitative genomic assays, such as real-time PCR and the multiplex ligation probe amplification (MLPA) techniques, were performed and revealed that the father was heterozygous for the D1152H mutation and the mother carried a large deletion of the CFTR gene encompassing the genomic sequence including the same mutation. The child inherited D1152H from his father and the large deletion of the CFTR gene from his mother. We suggest that D1152H likely acts as a mild mutation with a dominant effect on the severe deletion of exon 18, considering that after 3 years of clinical examinations the child shows no classical signs and symptoms of CF. Not testing for large deletions in subjects with apparent homozygosity for a mutated CFTR allele could lead to the misidentification of CFTR mutation carrier status.

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63 We speculate that the D1152H, acting as a mild mutation, has a dominant effect on the severe dele17a-18. Login to comment
64 Orgad et al. (2002) reported a case of hyperechogenic bowel loops and MI in a fetus carrying the combination of D1152H and G542X mutations, but they did not describe the clinical follow-up of the newborn. Login to comment

[hide] Rapid detection of the ACMG/ACOG-recommended 23 CF... J Biomol Tech. 2012 Apr;23(1):24-30. Elliott AM, Radecki J, Moghis B, Li X, Kammesheidt A
Rapid detection of the ACMG/ACOG-recommended 23 CFTR disease-causing mutations using ion torrent semiconductor sequencing.
J Biomol Tech. 2012 Apr;23(1):24-30., [PMID:22468138]

Abstract [show]
Cystic fibrosis (CF) is one of the most frequently diagnosed autosomal-recessive diseases in the Caucasian population. For general-population CF carrier screening, the American College of Medical Genetics (ACMG)/American College of Obstetricians and Gynecologists (ACOG) have recommended a core panel of 23 mutations that will identify 49-98% of carriers, depending on ethnic background. Using a genotyping technology that can rapidly identify disease-causing mutations is important for high-throughput general-population carrier screening, confirming clinical diagnosis, determining treatment options, and prenatal diagnosis. Here, we describe a proof-of-concept study to determine whether the Ion Torrent Personal Genome Machine (PGM) sequencer platform can reliably identify all ACMG/ACOG 23 CF transmembrane conductance regulator (CFTR) mutations. A WT CF specimen along with mutant DNA specimens representing all 23 CFTR mutations were sequenced bidirectionally on the Ion Torrent 314 chip to determine the accuracy of the PGM for CFTR variant detection. We were able to reliably identify all of the targeted mutations except for 2184delA, which lies in a difficult, 7-mer homopolymer tract. Based on our study, we believe PGM sequencing may be a suitable technology for identifying CFTR mutations in the future. However, as a result of the elevated rate of base-calling errors within homopolymer stretches, mutations within such regions currently need to be evaluated carefully using an alternative method.

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26 Amplicons were then pooled together in equimolar concentrations and purified using the T A B L E 1 Data Generation from Three PGM Runs Run Total number of reads Total bases (Mbp) AQ17 total bases (Mbp) AQ17 avg. read length CF WT 101,211 8.5 6.5 68 CF 23 pooled mutants 222,247 18.6 12.52 64 CF mutant 135,000 11.7 8.8 72 T A B L E 2 CFTR Variant Coverage, Mutant Read Percentage, and Base-Call Accuracy from a WT Library Using PGM Sequencing Variant cDNA position Coverage Mutant read % Accuracy/base G85E c.254G Ͼ A 408 0 99.5 R117H c.350G Ͼ A 3627 0 99.9 621 ϩ 1G Ͼ T c.489 ϩ 1G Ͼ T 245 0 99.6 711 ϩ 1G Ͼ T c.579 ϩ 1G Ͼ T 2660 0 99.9 R334W c.1000C Ͼ T 5419 0 99.7 R347P c.1040G Ͼ C 3562 0 99.4 A455E c.1364C Ͼ A 10,340 0 99.9 ⌬I507 c.1519_1521delATC 6507 0 98.6 ⌬F508 c.1521_1523delCTT 6507 0 99.4 1717-1G Ͼ A c.1585-1G Ͼ A 2086 0 99.2 G542X c.1624G Ͼ T 854 0 97.8 G551D c.1652G Ͼ A 3901 0 99 R553X c.1657C Ͼ T 3915 0 99.9 R560T c.1679G Ͼ C 3924 0 99.6 1898 ϩ 1G Ͼ A c.1766 ϩ 1G Ͼ A 1793 0 97.6 2184delAa c.2052delA 2001 35% 63.6 2789 ϩ 5G Ͼ A c.2657 ϩ 5G Ͼ A 293 0 100 3120 ϩ 1G Ͼ A c.2988 ϩ 1G Ͼ A 2408 0 100 R1162X c.3484C Ͼ T 9610 0 98.1 3659delC c.3528delC 9271 0 100 3849 ϩ 10kbC Ͼ T c.3717 ϩ 12191C Ͼ T 10,157 0 99.9 W1282X c.3846G Ͼ A 4789 0 95.6 N1303K c.3909C Ͼ G 3236 0 99.5 a The 2184delA variant lies in a homopolymer stretch of seven adenines and is not detected accurately as a result of homopolymer-length sequencing errors. Login to comment
67 For this data set, the PGM 314 chip output was 18.6 Mbp, with ϳ67% aligning to the CFTR T A B L E 3 PGM CFTR Variant Coverage and Mutant Read Percentage from a Pooled Mutant Library Representing All 23 ACMG/ACOG Mutations Variant cDNA position Coverage Mutant read % Predicted read % Genotype G85E c.254G Ͼ A 93 33 50 Het R117H c.350G Ͼ A 6228 39 50 Het 621 ϩ 1G Ͼ T c.489 ϩ 1G Ͼ T 1243 46 50 Het 711 ϩ 1G Ͼ T c.579 ϩ 1G Ͼ T 1352 29 50 Het R334W c.1000C Ͼ T 13,284 8 25 Het R347P c.1040G Ͼ C 9454 27 25 Het A455E c.1364C Ͼ A 19,527 43 50 Het ⌬I507 c.1519_1521delATC 15,587 14 25 Het ⌬F508 c.1521_1523delCTT 15,587 68 50 Homo 1717-1G Ͼ A c.1585-1G Ͼ A 3584 36 50 Het G542X c.1624G Ͼ T 610 41 50 Het G551D c.1652G Ͼ A 6714 16 17 Het R553X c.1657C Ͼ T 6670 15 17 Het R560T c.1679G Ͼ C 6395 22 17 Het 1898 ϩ 1G Ͼ A c.1766 ϩ 1G Ͼ A 3293 49 50 Het 2184delAa c.2052delA 2256 63 50 Het 2789 ϩ 5G Ͼ A c.2657 ϩ 5G Ͼ A 1765 54 50 Het 3120 ϩ 1G Ͼ A c.2988 ϩ 1G Ͼ A 7447 40 50 Het R1162X c.3484C Ͼ T 19,060 54 50 Het 3659delC c.3528delC 28,321 30 50 Het 3849 ϩ 10kbC Ͼ T c.3717 ϩ 12191C Ͼ T 27,102 46 50 Het W1282X c.3846G Ͼ A 9219 48 50 Het N1303K c.3909C Ͼ G 4842 49 50 Het a The 2184delA variant lies in a homopolymer stretch of seven adenines and is not accurately detected as a result of homopolymer-length sequencing errors. Login to comment
83 Analysis of the data correctly identified the two heterozygous mutations ⌬F508 and G542X, with mutant read distributions of 47% and 41%, respectively (Table 4 and Fig. 2). Login to comment
86 Using samples characterized previously, we analyzed the PGM`s data out- T A B L E 4 PGM CFTR Variant Coverage and Mutant Read Percentage from an Individual Harboring Two Disease-Causing CFTR Mutations Variant cDNA position Coverage Mutant read % G85E c.254G Ͼ A 237 0 R117H c.350G Ͼ A 3774 0 621 ϩ 1G Ͼ T c.489 ϩ 1G Ͼ T 936 0 711 ϩ 1G Ͼ T c.579 ϩ 1G Ͼ T 2018 0 R334W c.1000C Ͼ T 10,899 0 R347P c.1040G Ͼ C 7720 0 A455E c.1364C Ͼ A 14,525 0 ⌬I507 c.1519_1521delATC 8855 0 ⌬F508 c.1521_1523delCTT 8855 47 1717-1G Ͼ A c.1585-1G Ͼ A 2216 0 G542X c.1624G Ͼ T 2035 41 G551D c.1652G Ͼ A 4581 0 R553X c.1657C Ͼ T 4545 0 R560T c.1679G Ͼ C 4774 0 1898 ϩ 1G Ͼ A c.1766 ϩ 1G Ͼ A 2702 0 2184delAa c.2052delA 2837 18.5 2789 ϩ 5G Ͼ A c.2657 ϩ 5G Ͼ A 860 0 3120 ϩ 1G Ͼ A c.2988 ϩ 1G Ͼ A 4347 0 R1162X c.3484C Ͼ T 12,039 0 3659delC c.3528delC 7169 0 3849 ϩ 10kbC Ͼ T c.3717 ϩ 12191C Ͼ T 11,588 0 W1282X c.3846G Ͼ A 6187 0 N1303K c.3909C Ͼ G 4479 0 a The 2184delA variant lies in a homopolymer stretch of seven adenines and is not accurately detected as a result of homopolymer-length sequencing errors. Login to comment
96 In agreement, using the GAIIx, we were FIGURE 2 SoftGenetics PGM sequence analysis illustrating the CFTR mutations ⌬F508 (A) and G542X (B) in a single CF sample. Login to comment
66 For this data set, the PGM 314 chip output was 18.6 Mbp, with b03;67% aligning to the CFTR T A B L E 3 PGM CFTR Variant Coverage and Mutant Read Percentage from a Pooled Mutant Library Representing All 23 ACMG/ACOG Mutations Variant cDNA position Coverage Mutant read % Predicted read % Genotype G85E c.254G b0e; A 93 33 50 Het R117H c.350G b0e; A 6228 39 50 Het 621 af9; 1G b0e; T c.489 af9; 1G b0e; T 1243 46 50 Het 711 af9; 1G b0e; T c.579 af9; 1G b0e; T 1352 29 50 Het R334W c.1000C b0e; T 13,284 8 25 Het R347P c.1040G b0e; C 9454 27 25 Het A455E c.1364C b0e; A 19,527 43 50 Het èc;I507 c.1519_1521delATC 15,587 14 25 Het èc;F508 c.1521_1523delCTT 15,587 68 50 Homo 1717-1G b0e; A c.1585-1G b0e; A 3584 36 50 Het G542X c.1624G b0e; T 610 41 50 Het G551D c.1652G b0e; A 6714 16 17 Het R553X c.1657C b0e; T 6670 15 17 Het R560T c.1679G b0e; C 6395 22 17 Het 1898 af9; 1G b0e; A c.1766 af9; 1G b0e; A 3293 49 50 Het 2184delAa c.2052delA 2256 63 50 Het 2789 af9; 5G b0e; A c.2657 af9; 5G b0e; A 1765 54 50 Het 3120 af9; 1G b0e; A c.2988 af9; 1G b0e; A 7447 40 50 Het R1162X c.3484C b0e; T 19,060 54 50 Het 3659delC c.3528delC 28,321 30 50 Het 3849 af9; 10kbC b0e; T c.3717 af9; 12191C b0e; T 27,102 46 50 Het W1282X c.3846G b0e; A 9219 48 50 Het N1303K c.3909C b0e; G 4842 49 50 Het a The 2184delA variant lies in a homopolymer stretch of seven adenines and is not accurately detected as a result of homopolymer-length sequencing errors. Login to comment
82 Analysis of the data correctly identified the two heterozygous mutations èc;F508 and G542X, with mutant read distributions of 47% and 41%, respectively (Table 4 and Fig. 2). Login to comment
85 Using samples characterized previously, we analyzed the PGM`s data out- T A B L E 4 PGM CFTR Variant Coverage and Mutant Read Percentage from an Individual Harboring Two Disease-Causing CFTR Mutations Variant cDNA position Coverage Mutant read % G85E c.254G b0e; A 237 0 R117H c.350G b0e; A 3774 0 621 af9; 1G b0e; T c.489 af9; 1G b0e; T 936 0 711 af9; 1G b0e; T c.579 af9; 1G b0e; T 2018 0 R334W c.1000C b0e; T 10,899 0 R347P c.1040G b0e; C 7720 0 A455E c.1364C b0e; A 14,525 0 èc;I507 c.1519_1521delATC 8855 0 èc;F508 c.1521_1523delCTT 8855 47 1717-1G b0e; A c.1585-1G b0e; A 2216 0 G542X c.1624G b0e; T 2035 41 G551D c.1652G b0e; A 4581 0 R553X c.1657C b0e; T 4545 0 R560T c.1679G b0e; C 4774 0 1898 af9; 1G b0e; A c.1766 af9; 1G b0e; A 2702 0 2184delAa c.2052delA 2837 18.5 2789 af9; 5G b0e; A c.2657 af9; 5G b0e; A 860 0 3120 af9; 1G b0e; A c.2988 af9; 1G b0e; A 4347 0 R1162X c.3484C b0e; T 12,039 0 3659delC c.3528delC 7169 0 3849 af9; 10kbC b0e; T c.3717 af9; 12191C b0e; T 11,588 0 W1282X c.3846G b0e; A 6187 0 N1303K c.3909C b0e; G 4479 0 a The 2184delA variant lies in a homopolymer stretch of seven adenines and is not accurately detected as a result of homopolymer-length sequencing errors. Login to comment
95 In agreement, using the GAIIx, we were FIGURE 2 SoftGenetics PGM sequence analysis illustrating the CFTR mutations èc;F508 (A) and G542X (B) in a single CF sample. Login to comment

[hide] Novel strategies in newborn screening for cystic f... Thorax. 2012 Apr;67(4):289-95. Epub 2012 Jan 23. Vernooij-van Langen AM, Loeber JG, Elvers B, Triepels RH, Gille JJ, Van der Ploeg CP, Reijntjens S, Dompeling E, Dankert-Roelse JE
Novel strategies in newborn screening for cystic fibrosis: a prospective controlled study.
Thorax. 2012 Apr;67(4):289-95. Epub 2012 Jan 23., [PMID:22271776]

Abstract [show]
CONTEXT: Newborn screening for cystic fibrosis (CF) is included in many routine programmes but current strategies have considerable drawbacks, such as false-positive tests, equivocal diagnosis and detection of carriers. OBJECTIVE: To assess the test performance of two newborn screening strategies for CF. DESIGN, SETTING AND PARTICIPANTS: In 2008 and 2009, CF screening was added to the routine screening programme as a prospective study in part of The Netherlands. INTERVENTIONS: Two strategies were performed in all newborns. In the first strategy, concentrations of immunoreactive trypsinogen (IRT) and pancreatitis-associated protein (PAP) were measured. In the second method, samples with IRT >/=60 mug/litre were analysed for 36 CFTR mutations, followed by sequencing when a single mutation was detected. Tests were positive only with two identified CFTR mutations. MAIN OUTCOME: Sensitivity, specificity and positive predictive value (PPV) of both screening strategies. RESULTS: 145,499 infants were screened. The IRT/PAP approach showed a sensitivity of 95.0%, a specificity of 99.897% and a PPV of 12.3%. Test properties for the IRT/DNA/sequencing strategy were respectively 100%, 100% and 64.9%. Combining both strategies (IRT/PAP/DNA/sequencing) led to a sensitivity of 95.0%, a specificity of 100% and a PPV of 87.5%. CONCLUSION: In conclusion, all strategies performed well. Although there was no statistically significant difference in test performance, the IRT/DNA/sequencing strategy detected one infant that was missed by IRT/PAP (/DNA/sequencing). IRT/PAP may be the optimal choice if the use of DNA technology must be avoided. If identification of carriers and equivocal diagnosis is considered an important disadvantage, IRT/PAP/DNA/sequencing may be the best choice.

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110 Table 2 Immunoreactive trypsinogen and pancreatitis-associated protein concentrations, CFTR gene mutation analysis and sweat tests in infants with cystic fibrosis detected by newborn screening IRT (mg/litre) PAP (mg/litre) Mutation 1 Mutation 2 Sweat test chloride (mmol/litre) 1 438 5.3 F508del F508del 74 2 284 1.8 F508del F508del 88 3 266 9.8 F508del F508del 97 4 237 1.8 F508del F508del 11 and 74 5 197 4.3 F508del F508del 69 6* 191 12.6 F508del C.3889dupT 94 7 164 14.4 F508del G542X 102 8 129 4.3 F508del F508del Failed 9 110 2.2 F508del F508del 94 10 109 2.0 F508del F508del 51 11 105 4.4 F508del F508del 149 12 155 2.6 F508del F508del 111 13 191 12.6 F508del F508del 4 14 116 15.8 F508del F508del Failed 3 times 15 293 5.7 F508del 2184A 120 16* 228 15.8 F508del 1294_1300del 99 17 218 4.5 F508del G85E 99 18 153 4.0 F508del S1251N 77 19* 141 15.8 F508del E730X 82 20z 78 0.8 F508del A455E 65 21y 114 11.2 F508del F508del Failed 22y 109 0.8 F508del F508del 78 23y 93 1.3 F508del F508del e 24y 75 6.7 F508del F508del 78 *Second mutation detected by sequencing. Login to comment
109 Table 2 Immunoreactive trypsinogen and pancreatitis-associated protein concentrations, CFTR gene mutation analysis and sweat tests in infants with cystic fibrosis detected by newborn screening IRT (mg/litre) PAP (mg/litre) Mutation 1 Mutation 2 Sweat test chloride (mmol/litre) 1 438 5.3 F508del F508del 74 2 284 1.8 F508del F508del 88 3 266 9.8 F508del F508del 97 4 237 1.8 F508del F508del 11 and 74 5 197 4.3 F508del F508del 69 6* 191 12.6 F508del C.3889dupT 94 7 164 14.4 F508del G542X 102 8 129 4.3 F508del F508del Failed 9 110 2.2 F508del F508del 94 10 109 2.0 F508del F508del 51 11 105 4.4 F508del F508del 149 12 155 2.6 F508del F508del 111 13 191 12.6 F508del F508del 4 14 116 15.8 F508del F508del Failed 3 times 15 293 5.7 F508del 2184A 120 16* 228 15.8 F508del 1294_1300del 99 17 218 4.5 F508del G85E 99 18 153 4.0 F508del S1251N 77 19* 141 15.8 F508del E730X 82 20z 78 0.8 F508del A455E 65 21y 114 11.2 F508del F508del Failed 22y 109 0.8 F508del F508del 78 23y 93 1.3 F508del F508del e 24y 75 6.7 F508del F508del 78 *Second mutation detected by sequencing. Login to comment

[hide] CFTR, SPINK1, CTRC and PRSS1 variants in chronic p... Gut. 2012 Mar 17. Rosendahl J, Landt O, Bernadova J, Kovacs P, Teich N, Bodeker H, Keim V, Ruffert C, Mossner J, Kage A, Stumvoll M, Groneberg D, Kruger R, Luck W, Treiber M, Becker M, Witt H
CFTR, SPINK1, CTRC and PRSS1 variants in chronic pancreatitis: is the role of mutated CFTR overestimated?
Gut. 2012 Mar 17., [PMID:22427236]

Abstract [show]
OBJECTIVE: In chronic pancreatitis (CP), alterations in several genes have so far been described, but only small cohorts have been extensively investigated for all predisposing genes. DESIGN: 660 patients with idiopathic or hereditary CP and up to 1758 controls were enrolled. PRSS1, SPINK1 and CTRC were analysed by DNA sequencing, and cystic fibrosis transmembrane conductance regulator (CFTR) by melting curve analysis. RESULTS: Frequencies of CFTR variants p.R75Q, p.I148T, 5T-allele and p.E528E were comparable in patients and controls. We identified 103 CFTR variants, which represents a 2.7-fold risk increase (p<0.0001). Severe cystic fibrosis (CF)-causing variants increased the risk of developing CP 2.9-fold, and mild CF-causing variants 4.5-fold (p<0.0001 for both). Combined CF-causing variants increased CP risk 3.4-fold (p<0.0001), while non-CF-causing variants displayed a 1.5-fold over-representation in patients (p=0.14). CFTR compound heterozygous status with variant classes CF-causing severe and mild represented an OR of 16.1 (p<0.0001). Notably, only 9/660 (1.4%) patients were compound heterozygotes in this category. Trans-heterozygosity increased CP risk, with an OR of 38.7, with 43/660 (6.5%) patients and 3/1667 (0.2%) controls being trans-heterozygous (p<0.0001). CONCLUSIONS: Accumulation of CFTR variants in CP is less pronounced than reported previously, with ORs between 2.7 and 4.5. Only CF-causing variants reached statistical significance. Compound and trans-heterozygosity is an overt risk factor for the development of CP, but the number of CFTR compound heterozygotes in particular is rather low. In summary, the study demonstrates the complexity of genetic interactions in CP and a minor influence of CFTR alterations in CP development.

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72 The following CFTR variants were analysed with specific FRET probes: p.E60X, p.R75Q, p.G85E, p.R117H, p.I148T, c.621 +1G>T (IVS4+1G>T), c.711+1G>T (IVS5+1G>T), c.1078delT, p.R334W, p.R347P, 9-13TG, 5-9T, p.A455E, p.M470V, p.F508del, c.1716G>A (p.E528E), c.1717-1G>A (IVS10-1G>A), p.G542X, p.S549N, p.R553X, p.R560T, c.1898+1G>A (IVS12 +1G>A), c.2143delT, c.2183AA>G, c.2562T>G, c.2657+5G>A (IVS14B+5G>A), p.L997F, p.I1005R, p.Y1092X, p.D1152H, p.R1162X, c.3659delC, p.S1235R, p.S1251N, p.W1282X, p.N1303K, and c.4389G>A. Login to comment
140 Variant distribution in patients aged >20 and <20 years In younger patients, overall PRSS1 variants were 2.9-fold more common (>20 years: 9/239, 3.8%; <20 years: 46/421, 10.9%; p¼0.001, OR 3.1, 95% CI 1.5 to 6.5), whereas overall SPINK1 variants were similarly distributed (56/239, 23.4%; 73/421, Table 2 CFTR variants detected by melting curve analysis Gene Variant Patients Controls p Value OR (95% CI) CFTR (CF-causing, severe) p.F508del 44/660 (6.7%) 48/1758 (2.7%) <0.0001 2.5 (1.7 to 3.9) p.R117H (5T/7T) 2/660 (0.3%) 1/1758 (0.06%) NS e p.G542X 1/660 (0.2%) 1/1758 (0.06%) NS e c.1717-1G>A 3/660 (0.5%) 1/1758 (0.06%) NS e p.E585X 0/660 1/1758 (0.06%) NS e c.2183AA>G 0/660 1/1758 (0.06%) NS e p.R1158X 1/660 (0.2%) 0/1758 NS e p.R1162X 1/660 (0.3%) 0/1758 NS e p.N1303K 3/660 (0.5%) 0/1758 NS e Total 55/660 (8.3%) 53/1758 (3%) <0.0001 2.9 (2 to 4.3) CFTR (CF-causing mild) p.R117H (7T/7T) 13/660 (2%) 8/1758 (0.5%) 0.0009 4.4 (1.8 to 10.7) p.R117H (7T/9T) 3/660 (0.5%) 1/1758 (0.06%) NS e p.R347H 1/660 (0.2%) 0/1758 NS e p.R347P 1/660 (0.2%) 0/1758 NS e p.A455E 1/660 (0.2%) 0/1758 NS e c.2657+5G>A 1/660 (0.2%) 0/1758 NS e p.D1152H 3/660 (0.5%) 5/1758 (0.3%) NS e Total 23/660 (3.5%) 14/1758 (0.8%) <0.0001 4.5 (2.3 to 8.8) CFTR (non CF-causing) p.R74Q 2/660 (0.3%) 0/1758 NS e p.R75Q (het)* 29/660 (4.4%) 59/1758 (3.4%) NS e p.R75Q (hom)* 2/660 (0.3%) 1/1758 (0.06%) NS e p.Y84H 0/660 1/1758 (0.06%) NS e p.A120T 0/660 1/1758 (0.06%) NS e p.I148T* 4/660 (0.6%) 11/1758 (0.6%) NS e p.I507V 1/660 (0.2%) 2/1758 (0.1%) NS e p.F508C 1/660 (0.2%) 0/1758 NS e c.1716+12T>C 0/660 1/1758 (0.06%) NS e p.E528E (het)* 36/660 (5.5%) 82/1758 (4.7%) NS e p.E528E (hom)* 0/660 2/1758 (0.1%) NS e c.1898+8C>G 0/660 1/1758 (0.06%) NS e p.H667Y 1/660 (0.2%) 0/1758 NS e p.R668C 5/660 (0.8%) 3/1758 (0.2%) NS e p.G691R 0/660 1/1758 (0.06%) NS e p.L997F 5/660 (0.8%) 6/1758 (0.3%) NS e p.S1235R 10/660 (1.5%) 18/1758 (1.0%) NS e Total (excluded)* 25/660 (3.8%) 45/1758 (2.6%) NS e CFTR (CF-causing) Total (all) 78/660 (11.8%) 67/1758 (3.8%) <0.0001 3.4 (2.4 to 4.8) CFTR (all) Total (excluded)* 103/660 (15.6%) 112/1758 (6.4%) <0.0001 2.7 (2 to 3.6) The table is divided into three parts. Login to comment
69 The following CFTR variants were analysed with specific FRET probes: p.E60X, p.R75Q, p.G85E, p.R117H, p.I148T, c.621 +1G>T (IVS4+1G>T), c.711+1G>T (IVS5+1G>T), c.1078delT, p.R334W, p.R347P, 9-13TG, 5-9T, p.A455E, p.M470V, p.F508del, c.1716G>A (p.E528E), c.1717-1G>A (IVS10-1G>A), p.G542X, p.S549N, p.R553X, p.R560T, c.1898+1G>A (IVS12 +1G>A), c.2143delT, c.2183AA>G, c.2562T>G, c.2657+5G>A (IVS14B+5G>A), p.L997F, p.I1005R, p.Y1092X, p.D1152H, p.R1162X, c.3659delC, p.S1235R, p.S1251N, p.W1282X, p.N1303K, and c.4389G>A. Login to comment
135 Variant distribution in patients aged >20 and <20 years In younger patients, overall PRSS1 variants were 2.9-fold more common (>20 years: 9/239, 3.8%; <20 years: 46/421, 10.9%; p&#bc;0.001, OR 3.1, 95% CI 1.5 to 6.5), whereas overall SPINK1 variants were similarly distributed (56/239, 23.4%; 73/421, Table 2 CFTR variants detected by melting curve analysis Gene Variant Patients Controls p Value OR (95% CI) CFTR (CF-causing, severe) p.F508del 44/660 (6.7%) 48/1758 (2.7%) <0.0001 2.5 (1.7 to 3.9) p.R117H (5T/7T) 2/660 (0.3%) 1/1758 (0.06%) NS e p.G542X 1/660 (0.2%) 1/1758 (0.06%) NS e c.1717-1G>A 3/660 (0.5%) 1/1758 (0.06%) NS e p.E585X 0/660 1/1758 (0.06%) NS e c.2183AA>G 0/660 1/1758 (0.06%) NS e p.R1158X 1/660 (0.2%) 0/1758 NS e p.R1162X 1/660 (0.3%) 0/1758 NS e p.N1303K 3/660 (0.5%) 0/1758 NS e Total 55/660 (8.3%) 53/1758 (3%) <0.0001 2.9 (2 to 4.3) CFTR (CF-causing mild) p.R117H (7T/7T) 13/660 (2%) 8/1758 (0.5%) 0.0009 4.4 (1.8 to 10.7) p.R117H (7T/9T) 3/660 (0.5%) 1/1758 (0.06%) NS e p.R347H 1/660 (0.2%) 0/1758 NS e p.R347P 1/660 (0.2%) 0/1758 NS e p.A455E 1/660 (0.2%) 0/1758 NS e c.2657+5G>A 1/660 (0.2%) 0/1758 NS e p.D1152H 3/660 (0.5%) 5/1758 (0.3%) NS e Total 23/660 (3.5%) 14/1758 (0.8%) <0.0001 4.5 (2.3 to 8.8) CFTR (non CF-causing) p.R74Q 2/660 (0.3%) 0/1758 NS e p.R75Q (het)* 29/660 (4.4%) 59/1758 (3.4%) NS e p.R75Q (hom)* 2/660 (0.3%) 1/1758 (0.06%) NS e p.Y84H 0/660 1/1758 (0.06%) NS e p.A120T 0/660 1/1758 (0.06%) NS e p.I148T* 4/660 (0.6%) 11/1758 (0.6%) NS e p.I507V 1/660 (0.2%) 2/1758 (0.1%) NS e p.F508C 1/660 (0.2%) 0/1758 NS e c.1716+12T>C 0/660 1/1758 (0.06%) NS e p.E528E (het)* 36/660 (5.5%) 82/1758 (4.7%) NS e p.E528E (hom)* 0/660 2/1758 (0.1%) NS e c.1898+8C>G 0/660 1/1758 (0.06%) NS e p.H667Y 1/660 (0.2%) 0/1758 NS e p.R668C 5/660 (0.8%) 3/1758 (0.2%) NS e p.G691R 0/660 1/1758 (0.06%) NS e p.L997F 5/660 (0.8%) 6/1758 (0.3%) NS e p.S1235R 10/660 (1.5%) 18/1758 (1.0%) NS e Total (excluded)* 25/660 (3.8%) 45/1758 (2.6%) NS e CFTR (CF-causing) Total (all) 78/660 (11.8%) 67/1758 (3.8%) <0.0001 3.4 (2.4 to 4.8) CFTR (all) Total (excluded)* 103/660 (15.6%) 112/1758 (6.4%) <0.0001 2.7 (2 to 3.6) The table is divided into three parts. Login to comment

[hide] Role of Cystic Fibrosis Transmembrane Conductance ... Chest. 2012 Mar 15. Gonska T, Choi P, Stephenson A, Ellis L, Martin S, Solomon M, Dupuis A, Dorfman R, Zielenski J, Ooi CY, Weiser W, Durie PR, Tullis E
Role of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in patients with chronic sinopulmonary disease.
Chest. 2012 Mar 15., [PMID:22423042]

Abstract [show]
ABSTRACT INTRODUCTION:Previous studies report a high frequency of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in patients with idiopathic bronchiectasis. However, most studies have based their findings on pre-selected patient groups or have performed limited testing for CFTR dysfunction. The objective of our study was to evaluate the prevalence of CFTR gene mutations and/or CFTR-related ion channel abnormalities among subjects with idiopathic chronic sinopulmonary disease and the prevalence of CF or a CFTR-related disorder in this population. METHODS:We evaluated 72 prospectively enrolled patients from 1995-2005 at the Hospital for Sick Children and St. Michael's Hospital with idiopathic chronic sinopulmonary disease for evidence of CFTR-mediated abnormalities. We performed CFTR genotyping and assessed CFTR function using sweat testing and nasal potential difference testing. The results were compared with data from healthy controls, CF heterozygotes and CF patients. RESULTS:The CFTR functional tests in idiopathic sinopulmonary patients showed a continuous spectrum, ranging from normal to values typically seen in individuals with CF. Forty eight patients (66%) demonstrated CFTR mutations and/or abnormalities of CFTR function. Twenty two (31%) fulfilled criteria for a CF diagnosis and 26 (36%) for a CFTR-related disorder with a strong female preponderance. Functional tests, more than genotyping, were instrumental in establishing a CF diagnosis. Clinical features failed to distinguish CF subjects from those with CFTR-related or idiopathic disease. CONCLUSION:The high prevalence of CF and CFTR dysfunction among patients with idiopathic chronic sinopulmonary disease underscores the need for extensive diagnostic evaluation for CF.

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66 All P values are two-sided with a Table 2-CFTR Genotypes Identified in Subjects With Idiopathic Sinopulmonary Disease CF Causing/CF Causing CF Causing/CFTR Mutation CFTR Mutation/CFTR Mutation CF Causing/Unknown CFTR Mutation/Unknown F508del/A455E 3x F508del /D1152H 2x D579G/D579G 2x F508del /26x R764X/2 F508del/S1251N R75X/V456A 758delC/2 F508del/L967S 1716G.A/5T 1716G.A/2 F508del/5T R75Q/5T R117H (7T)/23x F508del/3212T.C 5T/23x G542X/D1152H 1717-1G.A/Q1291H Patients are grouped according to the identified CFTR alterations on allele 1/allele 2. Login to comment

[hide] CFTR mutation analysis and haplotype associations ... Mol Genet Metab. 2012 Feb;105(2):249-54. doi: 10.1016/j.ymgme.2011.10.013. Epub 2011 Oct 26. Cordovado SK, Hendrix M, Greene CN, Mochal S, Earley MC, Farrell PM, Kharrazi M, Hannon WH, Mueller PW
CFTR mutation analysis and haplotype associations in CF patients.
Mol Genet Metab. 2012 Feb;105(2):249-54. doi: 10.1016/j.ymgme.2011.10.013. Epub 2011 Oct 26., [PMID:22137130]

Abstract [show]
Most newborn screening (NBS) laboratories use second-tier molecular tests for cystic fibrosis (CF) using dried blood spots (DBS). The Centers for Disease Control and Prevention's NBS Quality Assurance Program offers proficiency testing (PT) in DBS for CF transmembrane conductance regulator (CFTR) gene mutation detection. Extensive molecular characterization on 76 CF patients, family members or screen positive newborns was performed for quality assurance. The coding, regulatory regions and portions of all introns were sequenced and large insertions/deletions were characterized as well as two intronic di-nucleotide microsatellites. For CF patient samples, at least two mutations were identified/verified and four specimens contained three likely CF-associated mutations. Thirty-four sequence variations in 152 chromosomes were identified, five of which were not previously reported. Twenty-seven of these variants were used to predict haplotypes from the major haplotype block defined by HapMap data that spans the promoter through intron 19. Chromosomes containing the F508del (p.Phe508del), G542X (p.Gly542X) and N1303K (p.Asn1303Lys) mutations shared a common haplotype subgroup, consistent with a common ancient European founder. Understanding the haplotype background of CF-associated mutations in the U.S. population provides a framework for future phenotype/genotype studies and will assist in determining a likely cis/trans phase of the mutations without need for parent studies.

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7 Chromosomes containing the F508del (p.Phe508del), G542X (p.Gly542X) and N1303K (p.Asn1303Lys) mutations shared a common haplotype subgroup, consistent with a common ancient European founder. Login to comment
101 CFTR mutation 1 Gene location CFTR mutation 2 Gene location CFTR mutation 3 Gene location S549N Ex12 3120+1G→A Intron 18 -102T→A Promoter F508del Ex11 G542X Ex12 185+4A→T Intron1 F508del Ex11 F508del Ex11 I1027T Ex19 F508del Ex11 W1282X Ex23 I1027T Ex19 only by the number of repeats of the IVS8CA microsatellite; 32 chromosomes contained 17 repeats and 29 chromosomes contained 23 repeats. Login to comment
102 The F508del haplotype that contained 23 repeats of the IVS8CA microsatellite was identical to the predicted haplotypes associated with G542X (N=6 of 6), N1303K (N=6 of 6), del Ex17a, b and 18 (N=1 of 1), and 3849+10 kb C→T (N=1 of 2). Login to comment
104 Mutation N alleles c.966T>G(5'flanking) c.234T>A(5'flanking)a c.-8G>C(5'UTR) c.-4G>C(Exon1) c.274-179G>A(Intron3) c.743+40A>G(Intron6) c.744-31TTGA(5_7)(Intron6) c.869+11C>T(Intron7) c.869+88T>A(Intron7) c.1209+43T>G(Intron9) IVS8CA(15-23)(Intron9) TG(10-13)_T(5-9)(Intron9) c.1393-61A>G(Intron10) M470V(Exon11) F508del(Exon11) c.1766+152T>A(Intron13) c.1767-231T>C(Intron13) c.1767-136T>C(Intron13) c.1767-132A>G(Intron13) c.2562T>G(Exon15) c.2604A>G(Exon15) c.2619+86_2619+87del(Intron15) c.2619+106T>A(Intron15) c.2909-92G>A(Intron17) IVS17bCA(11-17)(Intron20) c.3368-140A>C(Intron20) c.3469-65C>A(Intron21) F508del 32 TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- GA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- A5- 55- 55- 55- 66- 66- 66- 66- 66- 66- 66- 66- 66- 66- 55- 55- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TC- TT- TT- TT- TC- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TG- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- T17- 10_9- G- F508del- TA- 13C F508del 29 G23- 10_9- G- F508del- TA- 13C F508del 1 G21- 10_9- G- GG- G-F508del- TA- 13C F508del 1 G17- 10_9- G- F508del- A- G- delTA- 17- C- A N1303K 6 G542X 6 3849+10kbC→T 1 del Ex17a, b, Ex18 1 GG- GG- GG- 23- 10_9- GG-F508- T- TA- 13- C A455E 1 G22- 10_9- G- F508- T- TA- 13- C 621+1G→T 5 G21- 10_9- G- GG- GG- F508C- TA- 13- C 711+1G→T 3 3272-26A→G 2 3659delC 2 R347P 2 G16- 11_7- A- A-F508- TA- 13C del Ex 2, 3 2 del Ex 17a,17b 2 Normal 1 R334W 2 G17- 11_7- A- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA-AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- A-AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- F508- TA- 13C 2183AA→G 2 G16- 10_7- F508- TATA- TATA- TATA- TATA- TATA- TATA- 13C del Ex 2 1 G16- 11_7- F508- 14C 1288insTA 1 G16- 12_7- F508- 13C Normal 1 G16- 12_7- F508- 13C R1162X 1 G17- 10_7- F508- 13C del Ex 2,3 1 G16- 11_7- F508- A17- C del Ex 17a,17b 1 GA- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT-16- 11_7- F508- 14- C G85E 1 G16- 11_7- F508- 15C 1898+1G→A 1 G16- 11_7- F508- G13- C no mut detected 1 GT- TT- T16- 10_7- F508- 13C no mut detected 1 G16- 10_7- F508- 17A W1282X 2 G17- 10_7- F508- 17A W1282X 4 GC- CC- C17- 10_7- F508- delTA- 17- A Q39X 1 I507del 1 3849+10kbC→T 1 R560T 2 1717-1G→A 2 G551D 3 G16- 10_7- F508- delTA- 17- A G551D 2 1154insTC 1 G16- 10_7- F508- delTA- 17- 1717- 17A 1717-1G→A 1 2789+5G→A 1 GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- G17- 10_7- F508- AdelTA- A R1066C 1 GG- 17- 10_7- F508- delTA- A R1066H 1 GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- G17- 9_7- F508- delTAC R553X 3 GG- GG- CA- AA- AA- AA- A17- 12_7- F508- delTA- 11- C 3121-1G→A 1 C17- 12_7- F508- delTA- 11- C R334W 1 G17- 12_7- F508- TA- 13- C (TG)13T5b 1 G17- 13_5- F508- delTA- 13- C CC- CC- CC- CC- CC- CC- CC- CC- CC- CC- CC- CC- CC- CC- CC- CC- CC- R117H 1 CA- 6C- TT- 15- 12_5- AG- F508- T- TT- AT- ATA- TG- 13A- C R117H1 1 CA- 6C- TT- 16- 12_5- AG- F508- T- TT- AT- ATA- TG- 13A- C 1717-1G→A 1 R117Hb 1 GA- 6C- TT- 16- 10_7- AA- F508- A- TC- AG- AdelTA- TG- 13A- C 144c a Variation found in a sample where the haplotype could not be predicted. Login to comment
118 Half (N=77) of the 152 chromosomes examined shared a common haplotype subgroup which was associated with three of the most prevalent CF-causing mutations, F508del, G542X, and N1303K. Login to comment
119 To understand CFTR mutations, a previous study assessing the origin of 27,177 CF chromosomes from 29 European countries and three North African countries described the five most common CF-causing mutations: F508del (66.8%), G542X (2.6%), N1303K (1.6%), G551D (1.5%) and W1282X (1.0%) [22]. Login to comment
120 Similarly, Bobadilla et al. described the five most common CF-causing mutations in the U.S., which included F508del (68.6%), G542X (2.4%), G551D (2.1%), W1282X (1.4%) and N1303K (1.3%) [23]. Login to comment
121 Hence, F508del, G542X, and N1303K are the more common mutations in Caucasians from Europe and the United States. Login to comment
123 The haplotypes identified in the present study were consistent with the Spanish patient findings, showing that F508del, G542X, and N1303K again share a common haplotype subgroup [24]. Login to comment
124 Of our 62 F508del containing chromosomes (excluding the one probable recombinant), 29 predicted haplotypes are identical across 27 polymorphisms from the promoter to intron 21 to the G542X containing haplotypes (N=6) and the N1303K containing haplotypes (N=6). Login to comment
140 For example, when a newborn specimen is positive for R117H and either F508del, G542X or N1303K, and also carries both an 5T and a 9T variant, a clinician could use haplotype information to proceed with a strong probability that the 9T variant is in cis with F508del, G542X or N1310K and not R117H (Table 3). Login to comment

[hide] Implications for health and disease in the genetic... Genome Biol. 2012 Jan 25;13(1):R2. Guha S, Rosenfeld JA, Malhotra AK, Lee AT, Gregersen PK, Kane JM, Pe'er I, Darvasi A, Lencz T
Implications for health and disease in the genetic signature of the Ashkenazi Jewish population.
Genome Biol. 2012 Jan 25;13(1):R2., [PMID:22277159]

Abstract [show]
BACKGROUND: Relatively small, reproductively isolated populations with reduced genetic diversity may have advantages for genomewide association mapping in disease genetics. The Ashkenazi Jewish population represents a unique population for study based on its recent (< 1,000 year) history of a limited number of founders, population bottlenecks and tradition of marriage within the community. We genotyped more than 1,300 Ashkenazi Jewish healthy volunteers from the Hebrew University Genetic Resource with the Illumina HumanOmni1-Quad platform. Comparison of the genotyping data with that of neighboring European and Asian populations enabled the Ashkenazi Jewish-specific component of the variance to be characterized with respect to disease-relevant alleles and pathways. RESULTS: Using clustering, principal components, and pairwise genetic distance as converging approaches, we identified an Ashkenazi Jewish-specific genetic signature that differentiated these subjects from both European and Middle Eastern samples. Most notably, gene ontology analysis of the Ashkenazi Jewish genetic signature revealed an enrichment of genes functioning in transepithelial chloride transport, such as CFTR, and in equilibrioception, potentially shedding light on cystic fibrosis, Usher syndrome and other diseases over-represented in the Ashkenazi Jewish population. Results also impact risk profiles for autoimmune and metabolic disorders in this population. Finally, residual intra-Ashkenazi population structure was minimal, primarily determined by class 1 MHC alleles, and not related to host country of origin. CONCLUSIONS: The Ashkenazi Jewish population is of potential utility in disease-mapping studies due to its relative homogeneity and distinct genomic signature. Results suggest that Ashkenazi-associated disease genes may be components of population-specific genomic differences in key functional pathways.

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344 Lerer I, Sagi M, Cutting GR, Abeliovich D: Cystic fibrosis mutations delta F508 and G542X in Jewish patients. Login to comment
343 Lerer I, Sagi M, Cutting GR, Abeliovich D: Cystic fibrosis mutations delta F508 and G542X in Jewish patients. Login to comment

[hide] Lessons learned from 20 years of newborn screening... Med J Aust. 2012 Jan 16;196(1):67-70. Massie RJ, Curnow L, Glazner J, Armstrong DS, Francis I
Lessons learned from 20 years of newborn screening for cystic fibrosis.
Med J Aust. 2012 Jan 16;196(1):67-70., [PMID:22256939]

Abstract [show]
OBJECTIVE: To compare three cystic fibrosis (CF) newborn screening strategies used in Victoria since 1989. DESIGN, SETTING AND PARTICIPANTS: Retrospective review of newborn screening and clinical records for people with CF born in Victoria between 1989 and 2008 to compare screening strategies: repeat immunoreactive trypsinogen (IRT) testing (IRT/IRT, 1989-1990), IRT and p.F508del mutation analysis (IRT/p.F508del, 1991-2006) and IRT with analysis of 12 CFTR mutations (IRT/12 mutations, 2007-2008). MAIN OUTCOME MEASURES: Total number of infants screened, people identified with CF (by screening or clinical diagnosis), number of CF-affected terminations of pregnancy, and number of carriers detected. RESULTS: There were 420 people born with CF (live-birth prevalence, 1/3139; 95% CI, 1/2853-1/3462) and 78 CF-affected pregnancy terminations (overall prevalence, 1/2647; 95% CI, 1/2425-1/2896). Of the babies born with CF, 283 (67.4%) were detected by newborn screening alone, 61 (14.5%) had meconium ileus, 33 (7.9%) had a family history of CF, nine (2.1%) were diagnosed antenatally, and 34 (8.1%) were missed by screening (17 missed because IRT level was < 99th percentile, two with repeat IRT level not elevated, 14 without a screened CFTR mutation, and one with missing data). The sensitivities of the protocols were 86.6% for IRT/IRT, 89.9% for IRT/p.F508del, and 95.8% for IRT/12 mutations. Including 12 mutations in the analysis detected one patient who would otherwise have been missed and, had this protocol been implemented from 1989, it would have detected four others. CONCLUSION: Most babies with CF without meconium ileus, a family history or antenatal diagnosis are detected by newborn screening. Despite improved sensitivity with the 12-mutation analysis, most infants detected would have been diagnosed using the IRT/p.F508del protocol.

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14 From 1991 to 2006, babies with an IRT level > 99th percentile had CFTR gene mutation analysis for p.F508del and, from 2007, for 12 CFTR mutations (p.F508del, p.G551D, p.G542X, p.N1303K, c.1585- 1G>A, p.I507del, p.R560T, p.W1282X, p.V520F, c.489+1G>T, p.R553X, c.3718-2477C>T). Login to comment

[hide] Extensive molecular analysis of patients bearing C... J Mol Diagn. 2012 Jan;14(1):81-9. Epub 2011 Oct 20. Amato F, Bellia C, Cardillo G, Castaldo G, Ciaccio M, Elce A, Lembo F, Tomaiuolo R
Extensive molecular analysis of patients bearing CFTR-related disorders.
J Mol Diagn. 2012 Jan;14(1):81-9. Epub 2011 Oct 20., [PMID:22020151]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR)-related disorders (CFTR-RDs) may present with pancreatic sufficiency, normal sweat test results, and better outcome. The detection rate of mutations is lower in CFTR-RD than in classic CF: mutations may be located in genes encoding proteins that interact with CFTR or support channel activity. We tested the whole CFTR coding regions in 99 CFTR-RD patients, looking for gene mutations in solute carrier (SLC) 26A and in epithelial Na channel (ENaC) in 33 patients who had unidentified mutations. CFTR analysis revealed 28 mutations, some of which are rare. Of these mutations, RT-PCR demonstrated that the novel 1525-1delG impairs exon 10 splicing; by using minigene analysis, we excluded the splicing effect of three other novel intronic variants. Analysis of SLC26A genes revealed several variants, some of which are novel, that did not affect mRNA expression. Other mutations occurred in the ENaC genes encoding the ENaC subunits, but their frequency did not significantly differ between patients and controls. Our data, although obtained on a preliminary cohort of CFTR-RD patients, exclude a role of mutations in SLC26A and in SCNN genes in the pathogenesis of such disease; we confirm that CFTR analysis has a relevant role in CFTR-RD patients; and it appears mandatory to use CFTR scanning techniques and approaches to reveal the effect of novel mutations.

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69 Allele Frequency and CFTR Mutations in Patients Bearing CFTR-RDs Mutation (traditional name) HGVS nomenclature15 CBAVD (118 alleles)* RP (42 alleles)* DB (38 alleles)* Total (198 alleles)* TG12-T5-470V 34 (28.8) 2 (4.8) 10 (26.3) 46 (23.2) F508del c.1521_1523del 19 (16.1) 7 (16.7) 4 (10.5) 30 (15.2) 3195del6 c.3063_3069del 9 (7.6) 0 0 9 (4.5) N1303K c.3909CϾG 3 (2.5) 1 (2.4) 4 (10.5) 8 (4.0) G542X c.1624GϾT 4 (3.4) 1 (2.4) 1 (2.6) 6 (3.0) D1152H c.3454GϾC 1 (0.8) 2 (4.8) 2 (5.3) 5 (2.5) G85E c.254GϾA 2 (1.7) 3 (7.1) 0 5 (2.5) 1525-1delG c.1394de 3 (2.5) 1 (2.4) 0 4 (3.0) 4016insT c.3885insT 2 (1.7) 1 (2.4) 0 3 (1.5) 2789ϩ5GϾA c.2657ϩ5GϾA 0 3 (7.1) 0 3 (1.5) Q1476X c.4426CϾT 3 (2.5) 0 0 3 (1.5) 2183AAϾG c.2051_2052delinsG 1 (0.8) 1 (2.4) 0 2 (1.0) R553X c.1657CϾT 1 (0.8) 1 (2.4) 0 2 (1.0) L568F c.1704GϾT 2 (1.7) 0 0 2 (1.0) R1158X c.3472CϾT 2 (1.7) 0 0 2 (1.0) V920M c.2758GϾA 1 (0.8) 0 1 (2.6) 2 (1.0) 711ϩ1GϾT c.579ϩ1GϾT 0 1 (2.4) 0 1 (0.5) D614G c.1841AϾG 1 (0.8) 0 0 1 (0.5) 2184insA c.2052del 0 1 (2.4) 0 1 (0.5) 621ϩ1GϾT c.489ϩ1GϾT 1 (0.8) 0 0 1 (0.5) R1438W c.4312CϾT 0 1 (2.4) 0 1 (0.5) E193X c.577GϾT 0 1 (2.4) 0 1 (0.5) G1244E c.3731GϾA 1 (0.8) 0 0 1 (0.5) K68E c.202AϾG 1 (0.8) 0 0 1 (0.5) R347P c.1040GϾC 1 (0.8) 0 0 1 (0.5) 621ϩ3AϾG c.489ϩ3AϾG 1 (0.8) 0 0 1 (0.5) L997F c.2991GϾC 0 1 (2.4) 0 1 (0.5) F508C c.1523TϾG 1 (0.8) 0 0 1 (0.5) Total 94 (79.7) 28 (66.7) 22 (57.9) 144 (72.7) Undetected 24 (20.3) 14 (33.3) 16 (42.1) 54 (27.3) *Data are given as number (percentage). Login to comment
86 Six mutations were present in Ͼ2.0% of chromosomes (namely, 3195del6, N1303K, G542X, D1152H, 1525-1delG, and G85E). Login to comment

[hide] Measurements of CFTR-Mediated Cl(-) Secretion in H... PLoS One. 2012;7(10):e47708. doi: 10.1371/journal.pone.0047708. Epub 2012 Oct 17. Sousa M, Servidoni MF, Vinagre AM, Ramalho AS, Bonadia LC, Felicio V, Ribeiro MA, Uliyakina I, Marson FA, Kmit A, Cardoso SR, Ribeiro JD, Bertuzzo CS, Sousa L, Kunzelmann K, Ribeiro AF, Amaral MD
Measurements of CFTR-Mediated Cl(-) Secretion in Human Rectal Biopsies Constitute a Robust Biomarker for Cystic Fibrosis Diagnosis and Prognosis.
PLoS One. 2012;7(10):e47708. doi: 10.1371/journal.pone.0047708. Epub 2012 Oct 17., [PMID:23082198]

Abstract [show]
BACKGROUND: Cystic Fibrosis (CF) is caused by approximately 1,900 mutations in the CF transmembrane conductance regulator (CFTR) gene encoding for a cAMP-regulated chloride (Cl(-)) channel expressed in several epithelia. Clinical features are dominated by respiratory symptoms, but there is variable organ involvement thus causing diagnostic dilemmas, especially for non-classic cases. METHODOLOGY/PRINCIPAL FINDINGS: To further establish measurement of CFTR function as a sensitive and robust biomarker for diagnosis and prognosis of CF, we herein assessed cholinergic and cAMP-CFTR-mediated Cl(-) secretion in 524 freshly excised rectal biopsies from 118 individuals, including patients with confirmed CF clinical diagnosis (n = 51), individuals with clinical CF suspicion (n = 49) and age-matched non-CF controls (n = 18). Conclusive measurements were obtained for 96% of cases. Patients with "Classic CF", presenting earlier onset of symptoms, pancreatic insufficiency, severe lung disease and low Shwachman-Kulczycki scores were found to lack CFTR-mediated Cl(-) secretion (<5%). Individuals with milder CF disease presented residual CFTR-mediated Cl(-) secretion (10-57%) and non-CF controls show CFTR-mediated Cl(-) secretion >/=30-35% and data evidenced good correlations with various clinical parameters. Finally, comparison of these values with those in "CF suspicion" individuals allowed to confirm CF in 16/49 individuals (33%) and exclude it in 28/49 (57%). Statistical discriminant analyses showed that colonic measurements of CFTR-mediated Cl(-) secretion are the best discriminator among Classic/Non-Classic CF and non-CF groups. CONCLUSIONS/SIGNIFICANCE: Determination of CFTR-mediated Cl(-) secretion in rectal biopsies is demonstrated here to be a sensitive, reproducible and robust predictive biomarker for the diagnosis and prognosis of CF. The method also has very high potential for (pre-)clinical trials of CFTR-modulator therapies.

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64 CFTR Genotyping Following screening of the 6 most common CFTR-disease causing mutations in the region of Campinas (Brazil) [27-29]: F508del, G551D, G542X, R1162X, N1303K, R553X, an extended CFTR mutation search (see Methods S1) was performed when only one/none mutation was found (Table S2, with both traditional and standard nomenclatures [30]). Login to comment
103 As to the 5 individuals showing inconclusive Ussing chamber measurements, one individual had one CF-disease causing mutation (G542X) and two individuals had RD- related mutations (V562I and G576A). Login to comment
105 Functional classification of rarer mutations also results from these analyses, namely (Table S1): 3120+1G.A as class I (2 siblings with 3120+1G.A/R1066C, absence of CFTR-function and severe phenotypes); 1716+18672A.G as class V (2 other siblings with F508del/1716+18672A.G, residual CFTR function 228-34%- and mild CF); I618T as class IV (in a patient with G542X/I618T, 37% CFTR function and mild disease); and L206W as class IV or CFTR-RD mutation (in a patient with F508del/L206W and the highest CFTR function 257%- and very mild disease). Login to comment

[hide] Nonvisualization of fetal gallbladder increases th... Prenat Diagn. 2012 Jan;32(1):21-8. doi: 10.1002/pd.2866. Epub 2011 Nov 2. Dugueperoux I, Scotet V, Audrezet MP, Saliou AH, Collet M, Blayau M, Schmitt S, Kitzis A, Fresquet F, Muller F, Ferec C
Nonvisualization of fetal gallbladder increases the risk of cystic fibrosis.
Prenat Diagn. 2012 Jan;32(1):21-8. doi: 10.1002/pd.2866. Epub 2011 Nov 2., [PMID:22052729]

Abstract [show]
OBJECTIVE: The aim of our study is to evaluate the prevalence of cystic fibrosis (CF) in fetuses referred for genetic testing because of ultrasonographic sign (nonvisualized fetal gallbladder--NVFGB). METHOD: We reviewed the results of CFTR gene analysis over the period 2002 to 2009 in all consecutive cases referred because of NVFGB in Western France. We correlated these data with the presence of a more classical ultrasonographic finding (fetal echogenic bowel - FEB). RESULTS: Cystic fibrosis was diagnosed in 5 of the 37 fetuses with NVFGB (13.5%, 95% confidence interval (CI): [2.5%; 24.5%]) and in only 9 of the 229 other cases referred because of FEB (3.9%, 95% CI: [3.2%; 14.7%]). In our series, all CF-affected fetuses with NVFGB also had FEB. The risk of CF was 11.6-fold higher in fetuses with both indications (NVFGB + FEB) than in fetuses with isolated FEB (45.5% vs 3.9%, RR = 11.6, 95% CI: [4.7%; 28.8%], p = 0.0001). We also estimated that the residual risk of CF was less than 1 in 68 (1.5%) when a single mutation was identified in the fetus by our molecular protocol. CONCLUSION: Ultrasonographic evidence of NVFGB is an additional risk factor for CF in cases with FEB.

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107 The gene analysis indicated that the couple carried the F508del (c.1521_1523delCTT) and the G542X (c.1624 G > T) mutations and that the fetus was CF-affected. Login to comment

[hide] CFTR mutation screening in an assisted reproductiv... Aust N Z J Obstet Gynaecol. 2011 Dec;51(6):536-9. doi: 10.1111/j.1479-828X.2011.01348.x. Epub 2011 Aug 22. Field PD, Martin NJ
CFTR mutation screening in an assisted reproductive clinic.
Aust N Z J Obstet Gynaecol. 2011 Dec;51(6):536-9. doi: 10.1111/j.1479-828X.2011.01348.x. Epub 2011 Aug 22., [PMID:21875427]

Abstract [show]
INTRODUCTION: Cystic fibrosis (CF) is the most common deleterious single-gene recessive disorder in non-Hispanic Caucasians. Mutations within the CF transmembrane receptor (CFTR) gene produce a variable phenotype, including pulmonary disease, pancreatic insufficiency, meconium ileus and infertility. Screening of antenatal/preconception patients to identify CFTR mutation carriers has been shown to reduce the incidence of CF-affected babies at birth. The application of preconception screening to assisted reproductive technology (ART) patients enables carrier couples a choice between prenatal screening and preimplantation genetic diagnosis (PGD). AIM: To screen patients entering an infertility clinic, for 30 common CFTR mutations, and to detect carrier patients prior to initiating assisted reproductive treatment. METHOD: DNA from 5600 infertility patients was screened using a PCR/OLA kit for 30 CFTR mutations. All identified carriers and carrier couples were offered genetic counselling. Prenatal testing and PGD for CFTR mutations were offered to carrier couples where appropriate. RESULTS: A total of 5600 patients were screened for 30 CFTR mutations with 261 carriers being identified and at a significantly increased carrier rate of one in 21.5 (4.66% +/- 0.55%). R117H/c.350G>A was significantly increased in this infertile population and accounted for 13.8% of all mutations identified. Twelve carrier couples were identified, and nine carrier couples had at least one cycle of PGD for CFTR mutations. CONCLUSION: The carrier rate of CFTR mutations is elevated in patients presenting for infertility treatment, and preconception screening should be encouraged in all patients entering ART clinics.

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36 In total, 17 different mutations were identified in this cohort of patients presenting for infertility care, with G551D/c.1652G>A, G542X/c.1624G>T, N1303K/c.3909C>G and 621+ 1G>T/c.489+1G>T being identified at a higher rate (but not significant) than the antenatal population.4 Conversely, F508delCTT/c.1521_1523delCTT, 3849+10kbC>T/c. Login to comment
37 Table 1 A breakdown of the CFTR mutations identified in the infertile patient population, the percentage of those mutations identified, the percentage of the infertile population screened, the percentage of the same mutations identified in the antenatal population by Massie et al. and figures published by Bobadilla et al. for the corresponding CFTR mutations in a global population study 'Legacy Mutation Name` and HGVS convention nomenclature* Number of mutations identified in infertile population Percentage of mutations identified in infertile population (%) Percentage of mutations identified in antenatal population4 (%) Percentage of mutations identified in a global population5 (%) F508delCTT / c.1521_1523delCTT 185 70.9 88.89 75.48 R117H / c.350G>A 36 13.8 0.63 G551D / c.1652G>A 12 4.6 2.78 3.82 G542X / c.1624G>T 6 2.3 0.93 1.83 N1303K / c.3909C>G 4 1.5 0.93 0.95 621+1G>T / c.489+1G>T 5 1.9 0.93 0.96 I507del / c.1519-1521delATC 2 0.8 0.53 3659delC / c.3528delC 2 0.8 R1162X / c.3484C>T 1 0.4 0.20 3120+1G>A / c.2988+1G>A 1 0.4 2184-delA / c.2052delA 1 0.4 3849+10kbC>T / c.3717-2477C>T 1 0.4 4.63 2789+5G>A / c.2657+5G>A 1 0.4 0.93 R347P / c.1040G>A 1 0.4 0.16 1717-1G>A / c.1585-1G>A 1 0.4 0.81 R553X / c.1657C>T 1 0.4 S549R / c.1647T>G 1 0.4 Total CFTR mutations identified 261 Total patients screened 5600 Incidence of CF carriers at QFG 1 in 21.5 (4.66%) CF, cystic fibrosis; CFTR, CF transmembrane receptor. Login to comment

[hide] Cystic fibrosis mutations for p.F508del compound h... Clin Genet. 2012 Dec;82(6):546-551. doi: 10.1111/j.1399-0004.2011.01804.x. Epub 2011 Nov 29. Sebro R, Levy H, Schneck K, Dimmock D, Raby B, Cannon C, Broeckel U, Risch N
Cystic fibrosis mutations for p.F508del compound heterozygotes predict sweat chloride levels and pancreatic sufficiency.
Clin Genet. 2012 Dec;82(6):546-551. doi: 10.1111/j.1399-0004.2011.01804.x. Epub 2011 Nov 29., [PMID:22035343]

Abstract [show]
Sebro R, Levy H, Schneck K, Dimmock D, Raby BA, Cannon CL, Broeckel U, Risch NJ. Cystic fibrosis mutations for p.F508del compound heterozygotes predict sweat chloride levels and pancreatic sufficiency. Cystic fibrosis (CF) is a monogenetic disease with a complex phenotype. Over 1500 mutations in the CFTR gene have been identified; however, the p.F508del mutation is most common. There has been limited correlation between the CFTR mutation genotype and the disease phenotypes. We evaluated the non-p.F508del mutation of 108 p.F508del compound heterozygotes using the biological classification method, Grantham and Sorting Intolerant from Tolerant (SIFT) scores to assess whether these scoring systems correlated with sweat chloride levels, pancreatic sufficiency, predicted FEV(1) , and risk of infection with Pseudomonas aeruginosa in the last year. Mutations predicted to be 'mild' by the biological classification method are associated with more normal sweat chloride levels (p < 0.001), pancreatic sufficiency (p < 0.001) and decreased risk of infection with Pseudomonas in the last year (p = 0.014). Lower Grantham scores are associated with more normal sweat chloride levels (p < 0.001), and pancreatic sufficiency (p = 0.014). Higher SIFT scores are associated with more normal sweat chloride levels (p < 0.001) and pancreatic sufficiency (p = 0.011). There was no association between pulmonary function measured by predicted FEV(1) and the biological classification (p = 0.98), Grantham (p = 0.28) or SIFT scores (p = 0.62), which suggests the pulmonary disease related to CF may involve other modifier genes and environmental factors.

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64 CFTR mutation classification for compound heterozygotesa Mutations n (%) Biological classification Grantham score SIFT Q493X 3 (3) Ib - - G542X 21 (20) Ib,c,e - - R553X 4 (4) Ib,e - - Y1092X 2 (2) Ib - - R1158X 1 (1) NA - - W1282X 9 (9) Ib,e - - G85E 4 (4) IIIb 98 0.01 R117H 4 (4) IVb,c 29 0.60 R334W 1 (1) IVb 101 0.02 R347P 1 (1) IVb 103 0.05 R352Q 1 (1) NA 43 0.35 G551D 20 (19) IIIb,c 94 0.00 R560T 3 (3) IIIb 71 0.00 D1270N 1 (1) NA 23 0.01 N1303K 6 (6) IIg 94 0.00 I507del 3 (3) IId - - 394delTT 1 (1) NAc - - 621+1G>T 7 (7) Ib,f - - 711+1G>T 2 (2) Ib - - 1717-1G>A 5 (5) Ib,c,e,f - - 1898+1G>A 2 (2) NA - - 2789+5G>A 3 (3) Vb - - 3659delC 1 (1) Ib - - 3849+10kbC>T 2 (2) Vb,c,f - - 3905insT 1 (1) Ib - - NA, not applicable; SIFT, Sorting Intolerant from Tolerant. a The following mutations biological classification scores could not be verified: 1898+G-A, 394delTT, D1270N, R352Q, and R1158X. Login to comment

[hide] The prevalence of common CFTR mutations in Iranian... J Assist Reprod Genet. 2011 Oct 6. Safinejad K, Darbouy M, Kalantar SM, Zeinali S, Mirfakhraie R, Yadegar L, Houshmand M
The prevalence of common CFTR mutations in Iranian infertile men with non-CAVD obstructive azoospermia by using ARMS PCR techniques.
J Assist Reprod Genet. 2011 Oct 6., [PMID:21976147]

Abstract [show]
PURPOSE: To evaluate five common cystic fibrosis trans-membrane conductance regulator (CFTR) mutations (DeltaF508, G542X, R117H, W1282X and N1303K) in the Iranian infertile men with noncongenital absence of vas deferens (CAVD) obstructive azoospermia. METHODS: The common CFTR gene mutations were tested on blood samples from 53 infertile men with non-CAVD obstructive azoospermia and 50 normal men as control individuals. Genomic DNA is extracted from the whole blood and the common CFTR mutations have been detected by the amplification refractory mutation system (ARMS) techniques. RESULTS: The common CFTR mutations were found positive in 5/53)9.43%(for DeltaF508 and 4/53)7.55%(for G542X mutation of all patients tested. Also, no CFTR mutations were detected in the normal men. CONCLUSION: The common CFTR mutations were detected in 9/53(17%) infertile men with non-CAVD obstructive azoospermia. Pre-treatment CFTR mutation analysis remains critical to distinguish cystic fibrosis (CF) genotypes for men with non CAVD obstructive azoospermia.

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0 GENETICS The prevalence of common CFTR mutations in Iranian infertile men with non-CAVD obstructive azoospermia by using ARMS PCR techniques Kyumars Safinejad & Mojtaba Darbouy & Sayed Mahdi Kalantar & Sirus Zeinali & Reza Mirfakhraie & Leila Yadegar & Masoud Houshmand Received: 9 May 2011 /Accepted: 24 August 2011 /Published online: 6 October 2011 # Springer Science+Business Media, LLC 2011 Abstract Purpose To evaluate five common cystic fibrosis transmembrane conductance regulator (CFTR) mutations (ΔF508, G542X, R117H, W1282X and N1303K) in the Iranian infertile men with noncongenital absence of vas deferens (CAVD) obstructive azoospermia. Login to comment
3 Results The common CFTR mutations were found positive in 5/53)9.43%(for ΔF508 and 4/53)7.55%(for G542X mutation of all patients tested. Login to comment
34 The aim of this study was to evaluate five common CF mutations (ΔF508, G542X, R117H,W1282X, N1303K)by use of the multiplex and single ARMS system among Iranian men with non-CAVD obstructive azoospermia (including those with idiopathic epididymal or ejaculatory duct obstruction) as the first descriptive study. Login to comment
38 The diagnosis of non-CAVD obstructive azoospermia is based on the following examinations: normal semen volume; normal testicular size; presence of the vas deferens by clinical examination; normal levels of serum follicle-stimulating hormone (FSH);azoospermia; absence or low levels of fructose and presence of spermatozoa in sample extracted by percutaneous sperm aspiration(PESA).No other symptoms of CF such as chronic lung inflammation/infection, pancreatic Table 1 Allelic and Genotypic Frequencies in Iranian infertile men with non-CAVD obstructive azoospermia Mutation No. of chromosomes carry CF allele %(Allelic frequencies) Genotype No. of patients %(Genotypic frequencies) ΔF508 5/106 4.7 ΔF508/+ 5 9.43 G542X 4/106 3.77 G542X/+ 4 7.55 R117H 0/106 0 R117H/+ 0 0 W1282X 0/106 0 W1282X/+ 0 0 N1303K 0/106 0 N1303K/+ 0 0 Normal 97/106 91.5 +/+ 44 83 Total 106/106 100.00 Total 53 100.00 insufficiency and intestinal obstruction have been reported in clinical file of our patients. Login to comment
40 All DNA samples were analyzed, using the primer sequence and single and multiplex ARMS-PCR technique as described by Ferrie et al. [21], for the following mutations: ΔF508, N1303K, G542X,W1282X,R117H mutations.W1282X and R117H mutations were analyzed by single ARMS-PCR technique and ΔF508, N1303K and G542X mutations were analyzed simultaneously by multiplex ARMS-PCR technique. Login to comment
41 ARMS PCR program for ΔF508, N1303K, G542X,R117H began with a 5 min incubation at 94°C,andProceeded with 28 cycles, each containing 15 s of denaturation at 94°C,30 s of annealing at appropriate temperature and 30 s of extension at 72°C;with a 10 min incubation at 72°C completing the amplification. Login to comment
43 Results Heterozygote frequency for ΔF508 mutation has been 5/53 (%9.43) and for G542X mutation, it has been 4/53(%7.55) in all patients tested where as other common mutations (R117H, W1282X, N1303K) were not detected in our samples. Login to comment
45 In other words, frequency for ΔF508 mutation was 5/106 (%4.7) and for G542X mutation it proved 4/106(3.77) in all chromosomes tested. Login to comment
52 Among 53 patients with non-CAVD obstructive azoospermia, five were heterozygotes for ΔF508 mutation (9.43%), and four patients carried G542X mutation (7.55%) whereas other mutations (N1303K, W1282X andR117H) were not detected in our samples. Login to comment

[hide] First study of the F508del mutation in Malaysian c... J Paediatr Child Health. 2011 Aug;47(8):573-5. doi: 10.1111/j.1440-1754.2011.02149.x. Nathan AM, Thong MK, deBruyne J, Ariffin H
First study of the F508del mutation in Malaysian children diagnosed with cystic fibrosis.
J Paediatr Child Health. 2011 Aug;47(8):573-5. doi: 10.1111/j.1440-1754.2011.02149.x., [PMID:21843195]

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48 Letters to the Editor Journal of Paediatrics and Child Health 47 (2011) 572-575 (c) 2011 The Authors Journal of Paediatrics and Child Health (c) 2011 Paediatrics and Child Health Division (Royal Australasian College of Physicians) Table1Summaryoftheclinicalcharacteristics,sweattestresultsandcysticfibrosistransmembraneconductanceregulatormutationstudiesofthepatientsdiagnosedwithcysticfibrosisinUniversity MalayaMedicalCenterfrom2000to2009 PatientAgeat presentation PresentingsymptomsOtherfindingsConsanguinityRaceSweatconductivity (mmol/l) KS score Mutations Rin3monthsRecurrentpneumoniaandFTTPseudo-Bartter`ssyndromeYesIndian13440†Nonedetected Nes8yearsSeverepersistentasthmaFTTNoIndian12450F508del/unknown Abd4monthsSeverepneumoniaandventilator dependent FTTYesYemeni11730F508del/F508del Ben7yearsCirrhosisoftheliverwithportal hypertension FTTUnknown(adopted)Unknown14080†Nonedetected(7T polymorphism) Sak3monthsRecurrentpneumoniaandFTTNDYesIndian11350F508del/F508del Ngan3yearsPseudo-Bartter`ssyndromeNDNoChinese13790Notdone(parentsrefused) LJH5monthsPseudo-Bartter`ssyndromeRecurrentpneumoniaNoChinese/Indonesian9465F508delnegative Josh5monthsPseudo-Bartter`ssyndromeandFTTNDNoIndian8585†Nonedetected Nur3monthsChronicdiarrhoeaandFTTPseudo-Bartter`ssyndromeNoMalay/Chinese13085‡†R553X/nonedetected Vin4monthsRecurrentpneumoniaandFTTNDNoChinese12260F508delnegative Muh5yearsPoorlycontrolledasthmaNDNoMalay10765F508delnegative Naz3monthsFTTandsteatorrhoeaRecurrentlunginfectionsand pseudo-Bartter`s NoMalay14675F508delnegative Additionalmutationsscreenedinthefourpatients:†F508del,I506/7del,G551D,G542X,R553X,R117C,R117H,621+1G>T,V520F,A455E,N1303K,3849+10kbC>T.‡R334W,R347P,A455E,S549N,R560T, 3659delC,W1282X.FTT,failuretothrive;KS,Schwachman-Kulczycki(KS)score;ND,nodata. Login to comment

[hide] Genotype-phenotype correlation in cystic fibrosis ... Genet Mol Biol. 2011 Jul;34(3):416-20. Epub 2011 Jul 1. Polizzi A, Tesse R, Santostasi T, Diana A, Manca A, Logrillo VP, Cazzato MD, Pantaleo MG, Armenio L
Genotype-phenotype correlation in cystic fibrosis patients bearing [H939R;H949L] allele.
Genet Mol Biol. 2011 Jul;34(3):416-20. Epub 2011 Jul 1., [PMID:21931512]

Abstract [show]
Cystic fibrosis (CF) is caused by CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations. We ascertained five patients with a novel complex CFTR allele, with two mutations, H939R and H949L, inherited in cis in the same exon of CFTR gene, and one different mutation per patient inherited in trans in a wide population of 289 Caucasian CF subjects from South Italy. The genotype-phenotype relationship in patients bearing this complex allele was investigated. The two associated mutations were related to classical severe CF phenotypes.

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60 The other four patients were compound heterozygotes respectively for G542X, 1259insA, G1349D, F508del and the two associated mutation in exon 15 [H939R;H949L]. Login to comment
61 The mutations G542X, 1259insA, G1349D, F508del have already been described as severe CF-asssociated mutation (Casals et al., 1993; Morral et al., 1993; Morral et al., 1994; Kerem et al., 1995; Estivill et al., 1997; Shrimpton et al., 1997; Rowntree and Harris 2003; Bompadre et al., 2007; Castellani et al., 2008). Login to comment
64 Also the G542X prevents the synthesis of full-length, normal CFTR protein due to the creation of a premature termination codon (Rich et al., 1993; Rowntree and Harris, 2003). Login to comment
66 Patients characteris* Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Mutation in trans with [H939R;H949L] R248T G542X 1259insA G1349D F508del Sex male male male male male Present age (years) 15 15 17 20 25 Age at diagnosis (years) 14 3 0 10 10 Airways colonization No SA SA SA PA, BC Age of first colonization (years) / 9 6 12 14 BMI (kg/m2 ) 21.9 17.0 15.1 17.6 17.5 FEV1 as % predicted 84.4 114.8 80.9 93.2 53.7 Sweat chloride concentration (mEq/L) 78 100 108 92 95 S-K score 100 70 60 75 40 Brasfield scorez N/A 5 11 7 21 Pancreas status PS PI PI PI PI Diagnosis CFTR-RD CF CF CF CF SA = Staphilococcus aureus, PA = Pseudomonas aeruginosa, BC = Burkholderia cepacia; N/A = not applicable; S-K = Shwachman-Kulczycki: the system is based on four parameters (general activity, physical examination, growth and nutrition and chest radiograph x-ray), and is rated as a) excellent: 86-100 b) good: 71-85, c) mild: 56-70, d) moderate: 41-55, and e) severe: < 40 (Shwachman and Kulczyzki, 1958); z scoring system from 3 "mild" to 25 "most severe" (Brett et al., 1992) after x-ray; PS/PI = Pancreatic sufficiency/insufficiency. Login to comment
71 In our study, the four patients carrying the complex allele [H939R;H949L] associated in trans with the severe mutations G542X, 1259insA, G1349D and F508del presented the classic CF phenotype. Login to comment

[hide] Twenty years after cystic fibrosis gene identifica... Pathol Biol (Paris). 2011 Jun;59(3):131-3. Epub 2009 Nov 5. Edelman A, Fritsch J, Ollero M
Twenty years after cystic fibrosis gene identification: Where are we and what are we up to?
Pathol Biol (Paris). 2011 Jun;59(3):131-3. Epub 2009 Nov 5., [PMID:19896304]

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97 Our initial pilot study showed that systemic administration of gentamycin, an antibiotic known to suppress two PTCs found in CFTR (G542X and R553X) when expressed in HeLa cells, improves the clinical status of patients bearing the Y122X mutation. Login to comment

[hide] Hypertonic saline solutions do not influence the s... Arch Med Sci. 2011 Apr;7(2):326-31. Epub 2011 May 17. Barboza MA, Brandao de Mattos CC, Ferreira AI, Barja PR, Santos de Faria Junior N, de Oliveira LV, de Mattos LC
Hypertonic saline solutions do not influence the solubility of sputum from secretor and non-secretor cystic fibrosis patients.
Arch Med Sci. 2011 Apr;7(2):326-31. Epub 2011 May 17., [PMID:22291775]

Abstract [show]
INTRODUCTION: Functional alterations of the cystic fibrosis transmembrane conductance regulator gene (CFTR) increase the viscoelasticity of pulmonary secretions of cystic fibrosis (CF) patients and require the use of therapeutic aerosols. The biochemical properties of exocrine secretions are influenced by the expression of the FUT2 gene which determine the secretor and non-secretor phenotypes of the ABH glycoconjugates. The aim of this study was to determine the influence of secretor and non-secretor phenotypes by means of photoacoustic analysis, both the typical interaction time (t(0)) and the solubilization interval (Deltat) of the sputum of secretor and non-secretor CF patients nebulized by hypertonic saline solutions at different concentrations. MATERIAL AND METHODS: Sputum samples were obtained by spontaneous expectoration from 6 secretor and 4 non-secretor patients with CF. Each sample was nebulized with 3%, 6%, and 7% hypertonic saline solutions in a photoacoustic cell. The values of t(0) and Deltat were determined using the Origin 7.5((R)) computer program (Microcal Software Inc.). The t-test was employed using the GraphPad Instat 3.0((R)) computer program to calculate the mean and standard deviation for each parameter. RESULTS: For all hypertonic saline solutions tested, the mean values of t(0) and Deltat do not show statistically significant differences between secretor and non-secretor patients. CONCLUSIONS: The secretor and non-secretor phenotypes do not influence the in vitro solubilization of the sputum nebulized by hypertonic saline solutions at different concentrations when analysed by photoacoustic technique.

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27 Seven patients had the ΔF508 mutation and one had the G542X mutation in the CFTR gene. Login to comment

[hide] Detecting Common CFTR Mutations by Reverse Dot Blo... Iran J Pediatr. 2011 Mar;21(1):51-7. Dooki MR, Akhavan-Niaki H, Juibary AG
Detecting Common CFTR Mutations by Reverse Dot Blot Hybridization Method in Cystic Fibrosis First Report from Northern Iran.
Iran J Pediatr. 2011 Mar;21(1):51-7., [PMID:23056764]

Abstract [show]
OBJECTIVE: Cystic fibrosis and its distribution vary widely in different countries and/or ethnic groups. Common cystic fibrosis transmembrane conductance regulator (CFTR) mutations were reported from Iran, but the northern population was not or underrepresented in those studies. The aim of this study was to determine the frequency of common CFTR mutations in children from northern Iran. METHODS: Thirty unrelated Iranian cystic fibrosis patients aged less than 11 years and living in Mazandaran province (in Iran) were screened for 5 common CFTR gene mutations. deltaF508, N1303K, G542X, R347H and W1282X using Reverse Dot Blot method. FINDINGS: Only one mutation, DeltaF508, was found in 7 patients accounting for 21.7% (13/60) of alleles. CONCLUSION: These findings can be used for planning future screening and appropriate genetic counseling programs in Iranian CF families.

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8 deltaF508, N1303K, G542X, R347H and W1282X using Reverse Dot Blot method. Login to comment
22 Only four (p.G542X, p.N1303K, p.G551D and p.W1282X) have overall frequencies higher than 1%[5]. Login to comment
23 Intriguingly, p.G542X and p.N1303K are found on the same haplotype background as ∆F508, suggesting that they arose in the same population[6]. Login to comment
27 We selected five mutations, deltaF508, N1303K, G542X, R347H and W1282X based on previous reports in Iran and neighboring countries. Login to comment
67 Table 1: Human cystic fibrosis transconductance regulator probes CFTR probe sequenceLocation in the CFTR geneProbe name 5'-NH2-GAAACACCAAAGATGATA-3'Exon10∆F508-N 5'-NH2-GGAAACACCAATGATATT-3'Exon10∆F508-MUT 5'-NH2-TATAGTTCTTGGAGAAGGTG3'Exon11G542X-N 5'-NH2-TATAGTTCTTTGAGAAGGTG-3'Exon11G542X-MUT 5'-NH2-GCTTTCCTCCACTGTTG-3'Exon20W1282X-N 5'-NH2-CAACAGTGAAGGAAAGC-3'Exon20W1282X-MUT 5'-NH2-AGAAAAAACTTGGATCC-3'Exon21N1303K-N 5'-NH2-GGGATCCAACTTTTTTCT-3'Exon21N1303K-MUT 5'-NH2-AATTGTTCTGCGCATGG-3'Exon7R347H-N 5'-NH2-CATTGTTCTGCCCATGGC-3'Exon7R347H-MUT Table 2: Human cystic fibrosis transmembrane conductance regulator primers Cystic fibrosis primer sequence Exon amplified Cystic fibrosis primer name Cystic fibrosis mutation tested 5'-Biotin-AGACCATGCTCAGATCTTCCAT-3' 5'-Biotin-GCAAAGTTCATTAGAACTGATC-3' 7 CF7-F CF7-R R347P 5'-Biotin-GCAGAGTACCTGAAACAGGA-3' 5'-Biotin-CATTCACAGTAGCTTACCCA-3' 10 CF10-F CF10-R ∆F508 5'-Biotin-CAACTGTGGTTAAAGCAATAGTGT-3' 5'-Biotin-GCACAGATTCTGAGTAACCATAAT-3' 11 CF11-F CF11-R G542X 5'-Biotin-TGGGCCTCTTGGGAAGAACT-3' 5'-Biotin-CTCACCTGTGGTATCACTCC-3' 20 CF20-F CF20-R W1282X 5'-Biotin-GGTAAGTACATGGGTGTTTC-3' 5'-Biotin-CAAAAGTACCCTGTTGCTCCA-3' 21 CF21-F CF21-R N1303K Genotype Analysis: Mutation screening of the CFTR gene in 60 alleles by reverse dot blot hybridization for five common mutations showed that 13 (21.6%) alleles were ∆F508. Login to comment
69 The other four mutations tested (N1303K, G542X, R 347H and W1282X) were not encountered in these patients. Login to comment
80 The other four mutations tested: N1303K, G542X, R347H and W1282X, were not found in these patients. Login to comment
88 Specifically, the four most common Turkish mutations were found in Iran, including ∆F508, c.1677delTA, p.G542X, and c.2183AA>G. Login to comment
89 The "Mediterranean mutation", p.G542X, is reported to be of Phoenician origin[7-9,29,31]. Login to comment
98 Consequently, it is believed that the incidence of cystic fibrosis is also similarly high, and that the low incidence commonly believed to be associated cystic fibrosis is also similarly high, and that the Table 5: Comparison of the frequency of common CFTR mutations ∆F508, N1303K, G542X, R347H , W1282X in Europe and North Africa with Iran and some neighboring countries Region or Country Mutation Type Reference ∆F508 W1282X N1303K G542X R347H Europe and N Africa 66.8 1 1.6 2.6 0.8-3.6 6 Turkey 24.5-27 ND 2.9-3.7 2.6-4.9 3-3.6 6, 23, 25 Saudi Arabia 13 ND 2 ND ND 27, 28 India 19-27 ND ND ND ND 24, 26 Iran 16-17.8 0-4 4.3-5.5 1.6-3.6 1.6-3.6 7, 8, 9 Mazandaran 21.6 0 0 0 0 Present study ND: Not detected low incidence commonly believed to be associated with this non-European population is likely to be due to under-diagnosis. Login to comment
139 Jalalirad M, Houshmand M, Mirfakhraie R, et al. First study of CF mutations in the CFTR gene of Iranian patients: detection of DeltaF508, G542X, W1282X, A120T, R117H, and R347H mutations. Login to comment
219 Loirat F, Hazout S, Lucotte G. G542X as a probable Phoenician cystic fibrosis mutation. Login to comment
140 Jalalirad M, Houshmand M, Mirfakhraie R, et al. First study of CF mutations in the CFTR gene of Iranian patients: detection of DeltaF508, G542X, W1282X, A120T, R117H, and R347H mutations. Login to comment
222 Loirat F, Hazout S, Lucotte G. G542X as a probable Phoenician cystic fibrosis mutation. Login to comment

[hide] The D1152H cystic fibrosis mutation in prenatal ca... J Med Screen. 2011;18(4):169-72. Epub 2011 Dec 7. Peleg L, Karpati M, Bronstein S, Berkenstadt M, Frydman M, Yonath H, Pras E
The D1152H cystic fibrosis mutation in prenatal carrier screening, patients and prenatal diagnosis.
J Med Screen. 2011;18(4):169-72. Epub 2011 Dec 7., [PMID:22156145]

Abstract [show]
OBJECTIVE: To assess the frequency of the D1152H mutation in the CFTR gene in normal individuals, in cystic fibrosis (CF) patients and in the setting of prenatal diagnosis. SETTING: A database analysis of sequential screening results seen at the Sheba Medical Center, Israel, between 2001 and 2010. METHODS: We retrospectively analyzed the frequency of D1152H in a large cohort of healthy individuals who were screened as part of a routine prenatal care programme, in individuals referred due to CF-related symptoms and in the setting of prenatal diagnosis. RESULTS: We found one asymptomatic homozygous female and 195 D1152H carriers among 49,940 healthy individuals screened, establishing a carrier rate of 1:255 for this mutation. We detected D1152H in nine of 103 individuals referred due to CF-related symptoms. Four suffered from respiratory symptoms and five from congenital bilateral absence of the vas deferens (CBAVD). During this period D1152H was detected in three pregnancies, two of which were aborted. CONCLUSION: The increased frequency of D1152H in individuals referred due to CF-related symptoms compared with healthy individuals included in the CF carrier screening programme (P < 0.001) clearly indicates that it is a disease-causing mutation.

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180 of mutations Group of mutations 2001 Ashkenazi Jews 7 Group A Non-Ashkenazi Jews 11 Group A þ B Georgian Jews 12 Group A þ B þ T360K/Q359K 9.2004-7.2005 Yemenite Jews 12 Groups A þ B þ I1234V Iraqi Jews 12 Groups A þ B þY1092X 8.2005-12.2007 Iraqi Jews 14 Groups A þ B þY1092X þ 3121-1G-A 1.2008-2010 14 mutations for all 14 Groups A þ B þ C Georgian Jews 15 Groups A þ B þ C þ T360K/Q359K Arabic population 19 Groups A þ B þ C þ D Group A: G542X, W1282X, N1303K, F508del, 3849 þ 10KbC-T, 1717-1G-A, D1152H Group B: W1089X, G85E, 405 þ 1G-A, S549R(T-G) Group C: Y1092X, 3121-1G-A, I1234V Group D: 4010delTATT, S549I, 3120 þ 1Kbdel18.6Kb, 2183AA-G, R75X Between 2005-2008 the Iraqi population was screened for an additional mutation 2751 þ 1insT. Login to comment

[hide] The K+ channel opener 1-EBIO potentiates residual ... PLoS One. 2011;6(8):e24445. Epub 2011 Aug 31. Roth EK, Hirtz S, Duerr J, Wenning D, Eichler I, Seydewitz HH, Amaral MD, Mall MA
The K+ channel opener 1-EBIO potentiates residual function of mutant CFTR in rectal biopsies from cystic fibrosis patients.
PLoS One. 2011;6(8):e24445. Epub 2011 Aug 31., [PMID:21909392]

Abstract [show]
BACKGROUND: The identification of strategies to improve mutant CFTR function remains a key priority in the development of new treatments for cystic fibrosis (CF). Previous studies demonstrated that the K(+) channel opener 1-ethyl-2-benzimidazolone (1-EBIO) potentiates CFTR-mediated Cl(-) secretion in cultured cells and mouse colon. However, the effects of 1-EBIO on wild-type and mutant CFTR function in native human colonic tissues remain unknown. METHODS: We studied the effects of 1-EBIO on CFTR-mediated Cl(-) secretion in rectal biopsies from 47 CF patients carrying a wide spectrum of CFTR mutations and 57 age-matched controls. Rectal tissues were mounted in perfused micro-Ussing chambers and the effects of 1-EBIO were compared in control tissues, CF tissues expressing residual CFTR function and CF tissues with no detectable Cl(-) secretion. RESULTS: Studies in control tissues demonstrate that 1-EBIO activated CFTR-mediated Cl(-) secretion in the absence of cAMP-mediated stimulation and potentiated cAMP-induced Cl(-) secretion by 39.2+/-6.7% (P<0.001) via activation of basolateral Ca(2)(+)-activated and clotrimazole-sensitive KCNN4 K(+) channels. In CF specimens, 1-EBIO potentiated cAMP-induced Cl(-) secretion in tissues with residual CFTR function by 44.4+/-11.5% (P<0.001), but had no effect on tissues lacking CFTR-mediated Cl(-) conductance. CONCLUSIONS: We conclude that 1-EBIO potentiates Cl(-)secretion in native CF tissues expressing CFTR mutants with residual Cl(-) channel function by activation of basolateral KCNN4 K(+) channels that increase the driving force for luminal Cl(-) exit. This mechanism may augment effects of CFTR correctors and potentiators that increase the number and/or activity of mutant CFTR channels at the cell surface and suggests KCNN4 as a therapeutic target for CF.

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46 CFabsent CFresidual CFTR genotype Number of individuals CFTR genotype Number of individuals F508del/F508del 10 F508del/Y161C 1 F508del/W57X 1 F508del/V232D 1 F508del/G85E 3 F508del/R334W 2 F508del/120del23 1 F508del/T338I 1 F508del/182delT 1 F508del/I1234V 1 F508del/G542X 1 F508del/3272-26 A.G 1 F508del/A561E 1 F508del/3849+10 kb C.T 1 F508del/Y1092X 1 F508del/4005 +5727 A.G 1 F508del/N1303K 1 F508del/G576A 1 F508del/1525-1 G.A 2 N1303K/R334W 1 F508del/Q39X 1 F1052V/M1137R 1 F508del/Q552X 1 1898+3 A.G/ 1898+3 A.G 1 G85E/G85E 1 R334W/3199del6 1 Q552X/R1162X 1 R334W/X 1 A561E/A561E 2 dele2,3/X 1 R764X/1717-1 G.A 1 R1158X/2183AA.G 1 R1158X/R560T 1 doi:10.1371/journal.pone.0024445.t001 luminal and basolateral surfaces of the epithelium were perfused continuously with a solution of the following composition (mmol/ L): NaCl 145, KH2PO4 0.4, K2HPO4 1.6, D-glucose 5, MgCl2 1, Ca-gluconate 1.3, pH 7.4, at 37uC. Login to comment
203 Du M, Liu X, Welch EM, Hirawat S, Peltz SW, et al. (2008) PTC124 is an orally bioavailable compound that promotes suppression of the human CFTR- G542X nonsense allele in a CF mouse model. Login to comment

[hide] Molecular basis of cystic fibrosis disease: an Ind... Indian J Clin Biochem. 2010 Oct;25(4):335-41. Epub 2010 Nov 19. Prasad R, Sharma H, Kaur G
Molecular basis of cystic fibrosis disease: an Indian perspective.
Indian J Clin Biochem. 2010 Oct;25(4):335-41. Epub 2010 Nov 19., [PMID:21966101]

Abstract [show]
Cystic fibrosis is a common autosomal recessive disorder usually found in population of white Caucasian descent. Now it is well documented the presence of CF disease in India with the advancement of laboratory testing. As once it was thought non existence of this disease in our population. Most of the phenotype of CF disease was in accordance of western population. Genetic analysis of CFTR gene in Indian CF patients revealed that most common mutation was delta F508 mutation. However, it was less than Caucasian population. CFTR mutations are also a causative factor in the pathogenesis of male infertility due to obstructive azoospermia. There are two most common mutation viz. IVS8-T5 and delta F508 which are responsible for congenital absence of vas deferens in male infertility patients. Elevated levels of sweat chloride at two occasions along with the presence of two mutations in CFTR gene was gold standard method for diagnosis of CF disease. It is noteworthy here that due to magnitude of Indian population, the total CF disease load would be more than many European countries. Clinical data demonstrate the prevalence of both classical and genetic form of CF in India.

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106 G542X, second most common mutation (5%) in Hispanic Caucasians [42], was not found in our population. Login to comment

[hide] The use of DHPLC (Denaturing High Performance Liqu... J Prenat Med. 2010 Jul;4(3):45-8. Mesoraca A, Di Natale M, Cima A, Di Giacomo G, Sarti M, Barone MA, Bizzoco D, Cignini P, Mobili L, D'emidio L, Giorlandino C
The use of DHPLC (Denaturing High Performance Liquid Chromatography) in II level screening of the CFTR gene in Prenatal Diagnosis.
J Prenat Med. 2010 Jul;4(3):45-8., [PMID:22439061]

Abstract [show]
OBJECTIVE: The aim of the study is to evaluate the role of Denaturing High Performance Liquid Chromatography (DHPLC) in the second level screening of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. METHODS: A 9-month prospective study, between June 2008 and March 2009 at Artemisia Fetal Medical Centre, included 3829 samples of amniotic fluid collected from women undergoing mid-trimester amniocentesis.The genetic diagnosis of CF was based on research of the main mutations of the CFTR gene on fetal DNA extracted from the amniocytes, (first level screening) using different commercial diagnostic systems. A second level screening using DHPLC, on the amniotic fluid and on a blood sample from the couple, was offered in case of fetuses heterozygous at first level screening. RESULTS: Of 3829 fetuses, 134 were found to be positive, 129 heterozygous and 5 affected. Of the 129 couples, following appropriate genetic counselling, 53 requested a second level screening. Through the use of DHPLC, 44 couples were found to be negative, and in nine couples, nine rare mutations were identified. CONCLUSIONS: The first level screening can be useful to evidence up to 75% of the CF mutations. The second level screening can identify a further 10% of mutant alleles. DHPLC was found to be a reliable and specific method for the rapid identification of the rare CFTR mutations which were not revealed in initial first level screening.

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100 48 Journal of Prenatal Medicine 2010; 4 (3): 45-50 Table III Mutations found with II level screening through DHPLC Mutations of mutated alleles DF508 29 W1282X 3 N1303K 8 1717-1G®A 2 3659delC 1 G85E 1 2789 +5G®A 2 R553X 2 R1162X 1 R117H 1 G542X 3 Total 53Table I Mutations found through I level screeningMutations analysed with I level screening through OLA CFTR Mutations Position on the CFTR gene DF508 Exon 10 3849+10KbC®T Intron 19 R334W Exon 7 W1282X Exon 10 V520F Exon 10 3905insT Exon 20 N1303K Exon 21 3876delA Exon 20 1717-1G®A Exon 11 3659delC Exon 19 DI507 Exon 10 A455E Exon 9 G85E Exon 3 2789 +5G®A Exon 14 / Intron 14 2183AA®G Exon 13 1898+1G®A Exon 12 / Intron 12 R347P Exon 7 R347H Exon 7 R560T Exon 11 1078delT Exon 7 R553X Exon 11 711+1G®T Exon 5 / Intron 5 G551D Exon 11 R1162X Exon 19 S549R Exon 11 R117H Exon 4 S549N Exon 11 621+1G®T Exon 4 G542X Exon 11 394delTT Exon 3 3120+1G®ðA Exon 16/ Intron 16 2184delA Exon 13 Table II Mutations found through I level screening Mutations Positions on CFTR gene R1066C Exon 17 b L1065P Exon 17 b A1006E Exon 19 R75Q Exon 3 D537E Exon 11 W1134X Exon 18 W1145X Exon 18 L1077P Exon 17b C524X Exon 11 Total 9 The use of DHPLC (Denaturing High Performance Liquid Chromatography) in II level screening of the CFTR gene in Prenatal Diagnosis Journal of Prenatal Medicine 2010; 4 (3): 45-50 49 tion was to provide the couple with adequate counselling in order to better understand the genotype-phenotype correlation in the various associations of mutations. Login to comment

[hide] An immunocytochemical assay to detect human CFTR e... Mol Cell Probes. 2009 Dec;23(6):272-80. Epub 2009 Jul 15. Davidson H, Wilson A, Gray RD, Horsley A, Pringle IA, McLachlan G, Nairn AC, Stearns C, Gibson J, Holder E, Jones L, Doherty A, Coles R, Sumner-Jones SG, Wasowicz M, Manvell M, Griesenbach U, Hyde SC, Gill DR, Davies J, Collie DD, Alton EW, Porteous DJ, Boyd AC
An immunocytochemical assay to detect human CFTR expression following gene transfer.
Mol Cell Probes. 2009 Dec;23(6):272-80. Epub 2009 Jul 15., [PMID:19615439]

Abstract [show]
BACKGROUND: To assess gene therapy treatment for cystic fibrosis (CF) in clinical trials it is essential to develop robust assays that can accurately detect transgene expression in human airway epithelial cells. Our aim was to develop a reproducible immunocytochemical assay for human CFTR protein which can measure both endogenous CFTR levels and augmented CFTR expression after gene delivery. METHODS: We characterised an antibody (G449) which satisfied the criteria for use in clinical trials. We optimised our immunocytochemistry method and identified G449 dilutions at which endogenous CFTR levels were negligible in CF samples, thus enhancing detection of transgenic CFTR protein. After developing a transfection technique for brushed human nasal epithelial cells, we transfected non-CF and CF cells with a clinically relevant CpG-free plasmid encoding human CFTR. RESULTS: The optimised immunocytochemistry method gave improved discrimination between CF and non-CF samples. Transfection of a CFTR expression vector into primary nasal epithelial cells resulted in detectable RNA and protein expression. CFTR protein was present in 0.05-10% of non-CF cells and 0.02-0.8% of CF cells. CONCLUSION: We have developed a sensitive, clinically relevant immunocytochemical assay for CFTR protein and have used it to detect transgene-expressed CFTR in transfected human primary airway epithelial cells.

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162 TwotransfectedCF samples (1DF508/G542X,11585-1G>A/P67L) and three transfected non-CF samples were negative for plasmid-expressed CFTR. Login to comment
165 TwotransfectedCF samples (1DF508/G542X,11585-1G>A/P67L) and three transfected non-CF samples were negative for plasmid-expressed CFTR. Login to comment

[hide] Long-term gas exchange characteristics as markers ... Respir Res. 2009 Nov 12;10:106. Kraemer R, Latzin P, Pramana I, Ballinari P, Gallati S, Frey U
Long-term gas exchange characteristics as markers of deterioration in patients with cystic fibrosis.
Respir Res. 2009 Nov 12;10:106., [PMID:19909502]

Abstract [show]
BACKGROUND AND AIM: In patients with cystic fibrosis (CF) the architecture of the developing lungs and the ventilation of lung units are progressively affected, influencing intrapulmonary gas mixing and gas exchange. We examined the long-term course of blood gas measurements in relation to characteristics of lung function and the influence of different CFTR genotype upon this process. METHODS: Serial annual measurements of PaO2 and PaCO2 assessed in relation to lung function, providing functional residual capacity (FRCpleth), lung clearance index (LCI), trapped gas (VTG), airway resistance (sReff), and forced expiratory indices (FEV1, FEF50), were collected in 178 children (88 males; 90 females) with CF, over an age range of 5 to 18 years. Linear mixed model analysis and binary logistic regression analysis were used to define predominant lung function parameters influencing oxygenation and carbon dioxide elimination. RESULTS: PaO2 decreased linearly from age 5 to 18 years, and was mainly associated with FRCpleth, (p < 0.0001), FEV1 (p < 0.001), FEF50 (p < 0.002), and LCI (p < 0.002), indicating that oxygenation was associated with the degree of pulmonary hyperinflation, ventilation inhomogeneities and impeded airway function. PaCO2 showed a transitory phase of low PaCO2 values, mainly during the age range of 5 to 12 years. Both PaO2 and PaCO2 presented with different progression slopes within specific CFTR genotypes. CONCLUSION: In the long-term evaluation of gas exchange characteristics, an association with different lung function patterns was found and was closely related to specific genotypes. Early examination of blood gases may reveal hypocarbia, presumably reflecting compensatory mechanisms to improve oxygenation.

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84 According to the frequencies in our Table 1: Patient cohort (A), data base characteristics (B), and distribution of CFTR mutations (C) taken from the Bernese CF Registry (n = 178, 87.3% of a total number of 204 CF patients) A Patient cohort follow-up statistics Gender distribution of patients Blood gas tests within age periods n % - males 88 49.4 5 to 8 y 427/1457 29,3% - females 90 50.6 9 to 14 y 527/1457 36.2% 178 100 15 to 18 y 503/1457 34.5% From entire database, 26 patients (12.7%) excluded because of insufficient number of tests, (6) or age < 6 years (20) B Blood gas test and lung function measurement follow-up statistics Number of blood gas tests median (range) Blood gas tests per year of observation Total of tests 1457 1987 to 1993 326/1457 22.4% per child 8.1 (3-15) 1994 to 2000 539/1457 37.0% per year of observation 68.2 (37-90) 2001 to 2008 592/1457 40.6% C Distribution of CFTR mutations n % Inframe/inframe (F508del[2]) a 103 57.9 Inframe/nonsense b 22 12.4 Frameshift/F508del c 19 10.7 Frameshift/non-F508del d 12 6.7 Inframe/splicesite e 7 3.9 Miscellaneous f 15 8.4 Total 178 100.0 Equal distribution of CFTR genotypes over age range and over years of observation CFTR: cystic fibrosis transmembrane regulator population-specific CFTR genotype distribution, the patients were stratified into 6 groups consisting of (a) F508del homozygotes F508del[2| (inframe/inframe): n = 103 (57.9%), (b) R553X, G542X, Q525X and E585X compound heterozygotes with F508del (inframe/nonsense mutations): n = 22, (12.4%), (c) 3905insT compound heterozygotes 3905insT/F508del (frameshift/F508del): n = 19, (10.7%), (d) 3905insT compound heterozygotes with other than F508del (frameshift/non-F508del): n = 12, (6.7%), (e) 1717-1G>A, 621+1G<T and 4005+1G>A compound heterozygotes with F508del (inframe/splicesite): n = 7 (3.9%), and (f) miscellaneous genotypes n = 15, (8.4%). Login to comment
103 frequent CFTR genotypes inframe/inframe (F508del[2]), inframe/nonsense mutations (F508del/R553X, F508del/ G542X, F508del/Q524, F508del/E553), inframe/ frameshift (mainly F508del/3905insT), non-F508del/ frameshift, (mainly non-F508del/3905insT) and inframe/ splicesite genotypes were incorporated as fixed effects with "age at time of annual test" as covariate, and the patient-specific intercept as a random effect. Login to comment

[hide] CFTR H609R mutation in Ecuadorian patients with cy... J Cyst Fibros. 2009 Jul;8(4):280-1. Epub 2009 May 19. Moya-Quiles MR, Glover G, Mondejar-Lopez P, Pastor-Vivero MD, Fernandez-Sanchez A, Sanchez-Solis M
CFTR H609R mutation in Ecuadorian patients with cystic fibrosis.
J Cyst Fibros. 2009 Jul;8(4):280-1. Epub 2009 May 19., [PMID:19457724]

Abstract [show]
Mutation epidemiology in each ethnic group is important for cystic fibrosis diagnosis and genetic counselling. To date, little has been reported on the prevalence of cystic fibrosis in the Ecuadorian population where the mutation distribution appears to differ from that of Europe. We present a series of four Ecuadorian patients homozygous for the H609R mutation in the CFTR gene. This is the first report of detection of this mutation in the Ecuadorian population. Taking advantage of the homozygous status of the patients, an evaluation of the most important clinical parameters is presented. From the diagnostic point of view, the information provided by our study is of relevance in designing an appropriate strategy for genetic testing of patients in Ecuador and in European countries where immigration from Ecuador is common.

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14 There are two other reports of mutations on the CFTR gene in CF patients from Ecuador; in the first, the estimated Ecuadorian CF incidence was 1:11,252 and mutations were, in order of frequency, F508del (37.1%), G85E (8.9%), G542X (2.4%), N1303K (2.4%), G551D (1.6%) and R334W (0.8%), with a detection rate of 53.22% of the total CF chromosomes studied [7]. Login to comment
15 In the second report, a compilation of data from CFTR gene analysis in Latin American CF patients, four mutations were found: F508del (31.37%), G542X (1.96%), G85E (1.96%) and N1303K (1.96%), with 63.7% of Ecuadorian CF mutations remaining unidentified [6]. Login to comment
13 There are two other reports of mutations on the CFTR gene in CF patients from Ecuador; in the first, the estimated Ecuadorian CF incidence was 1:11,252 and mutations were, in order of frequency, F508del (37.1%), G85E (8.9%), G542X (2.4%), N1303K (2.4%), G551D (1.6%) and R334W (0.8%), with a detection rate of 53.22% of the total CF chromosomes studied [7]. Login to comment

[hide] Borderline sweat test: Utility and limits of genet... Clin Biochem. 2009 May;42(7-8):611-6. Epub 2009 Jan 24. Seia M, Costantino L, Paracchini V, Porcaro L, Capasso P, Coviello D, Corbetta C, Torresani E, Magazzu D, Consalvo V, Monti A, Costantini D, Colombo C
Borderline sweat test: Utility and limits of genetic analysis for the diagnosis of cystic fibrosis.
Clin Biochem. 2009 May;42(7-8):611-6. Epub 2009 Jan 24., [PMID:19318035]

Abstract [show]
OBJECTIVE: The sweat test remains the gold standard for the diagnosis of Cystic Fibrosis (CF) even despite the availability of molecular analysis of Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR). We investigated the relationship between CFTR mutation analysis and sweat chloride concentration in a cohort of subjects with borderline sweat test values, in order to identify misdiagnosis of CF. DESIGN AND METHODS: In the period between March 2006 and February 2008 we performed 773 sweat tests in individuals referred for suspect CF. Ninety-one subjects had chloride values in the border-line range. Clinicians required CFTR gene complete scanning on 66 of them. RESULTS: The mean value of sweat chloride in the DNA negative subjects was lower than in those with at least one CFTR mutation. Our data indicate that 39 mEq/l is the best sensitivity trade off for the sweat test with respect to genotype. CONCLUSIONS: To optimise diagnostic accuracy of reference intervals, it may be useful to modify from 30 to 39 mEq/l the threshold for sweat chloride electrolytes.

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59 In order to evaluate the relationship between the presence of CFTR mutation and sweat chloride concentration, we focused our attention on the 91 individuals (11.8%) in whom borderline sweat chloride values (31-59 mEq/l) were recorded (mean sweat electrolyte value was 40.0 mEq/l): 25 refused to be referred to the local Table 2 Demographic and clinical features of subjects with positive DNA analysis Patient Initials Gender Age at test years/ months Sweat chloride mEq/l Clinical indication DNA results IRT Right arm Left arm 1 CA M 49y5m 34 34 CBAVD G542X/5T-TG12 ND 2 SA M 45y2m 45 43 Pancreatitis F508del/R117H-7T ND 3 PD F 43y7m 33 38 Recurrent bronchitis F508del/5T-TG12 ND 4 CA M 36y1m 31 29 CBAVD R117H-7T/R117C-7T ND 5 SC M 36y1m 33 40 Pneumonia F508del/D1152H ND 6 MG M 25Y5m 41 45 CBAVD Q552X/D1152H NEG 7 SG M 18y5m 49 54 Pancreatitis 4016insT/dupl.prom.-3 ND 8 LS F 10y4m 41 38 Pancreatitis D1152H/L997F NEG 9 CM M 8y3m 30 31 Pneumonia F1052V/A120T NEG 10 PT M 7y3m 41 39 Positive screening F508del/Y1032C POS 11 ME F 7y1m 44 44 Positive screening 2789+5GNA/5T-TG12 POS 12 PM F 6y4m 35 36 Positive screening 2183AANG/5T-TG12 POS 13 BM F 6y3m 36 39 Positive screening F508del/5T-TG12 POS 14 CD M 5y8m 40 41 Chronic bronchitis 5T-TG12/5T-TG12 NEG 15 CG F 4y5m 33 37 Recurrent bronchitis R553X/L997F POS 16 CS F 3y8m 53 58 Family history G542X/D614G POS 17 VA M 4y2m 49 43 Pneumonia E831X/5T-TG12 ND 18 SC M 3y4m 39 39 Positive screening R352Q/G213E POS 19 CC F 2y3m 31 31 Positive screening F508del/5T-TG12 POS 20 CA F 2y5m 51 52 Recurrent bronchitis E831X/5T-TG12 ND 21 MR F 3y+7m 29 31 Family history G542X/5T-TG12 POS 22 CM F 2y3m 60 58 Pneumonia T338I/L997F POS 23 LM F 2y1m 50 52 Positive screening F508del/E1473X POS 24 CGE F 0y8m 46 47 Positive screening E92K/5T-TG13 POS 25 NF M 0y7m 32 30 Positive screening F508del/P5L POS 26 RG M 0y7m 45 40 Positive screening N1303K/P5L POS 27 PE M 47y4m 60 58 Nasal polyposis R1066H/UN ND 28 LS M 39y9m 39 38 Azoospermy N1303K/UN ND 29 TM M 38y4m 40 45 Azoospermy N1303K/UN ND 30 DF M 34y2m 52 58 Bronchiectasis 3849+10 kbCNT/UN ND 31 TV F 30y5m 35 34 Recurrent bronchitis L997F/UN ND 32 FA F 18y7m 53 49 Family history Del es.2/UN NEG 33 DG M 17y8m 43 47 Recurrent bronchitis 5T-TG12/UN NEG 34 LN F 13y7m 54 53 Nasal poliposis, malnutrition R74W-V855I/UN NEG 35 FKT M 15y4m 54 53 Chronic bronchitis R352Q/UN NEG 36 BM M 10y9m 48 51 Chronic bronchitis T1263I/UN NEG 37 SV F 11y1m 60 58 Chronic bronchitis R347H/UN NEG 38 CV F 10y10m 38 39 Recurrent bronchitis 5T-TG12/UN NEG 39 BF F 9y10m 37 38 Chronic bronchitis L997F/UN NEG 40 CA M 8y2m 33 32 Pneumonia F508del/UN NEG 41 RX F 8y7m 29 31 Chronic bronchitis V920L/UN NEG 42 MG F 4y3m 51 51 Positive screening F508del/UN POS Sweat chloride concentration and mutations/variants detected are also reported. Login to comment
98 This represents the most frequent variant in borderline subjects according to literature [4], followed by F508del (7.69%), G542X (2.31%) and N1303K (2.31%) that are the most common mutations in Caucasian population. Login to comment
57 In order to evaluate the relationship between the presence of CFTR mutation and sweat chloride concentration, we focused our attention on the 91 individuals (11.8%) in whom borderline sweat chloride values (31-59 mEq/l) were recorded (mean sweat electrolyte value was 40.0 mEq/l): 25 refused to be referred to the local Table 2 Demographic and clinical features of subjects with positive DNA analysis Patient Initials Gender Age at test years/ months Sweat chloride mEq/l Clinical indication DNA results IRT Right arm Left arm 1 CA M 49y5m 34 34 CBAVD G542X/5T-TG12 ND 2 SA M 45y2m 45 43 Pancreatitis F508del/R117H-7T ND 3 PD F 43y7m 33 38 Recurrent bronchitis F508del/5T-TG12 ND 4 CA M 36y1m 31 29 CBAVD R117H-7T/R117C-7T ND 5 SC M 36y1m 33 40 Pneumonia F508del/D1152H ND 6 MG M 25Y5m 41 45 CBAVD Q552X/D1152H NEG 7 SG M 18y5m 49 54 Pancreatitis 4016insT/dupl.prom.-3 ND 8 LS F 10y4m 41 38 Pancreatitis D1152H/L997F NEG 9 CM M 8y3m 30 31 Pneumonia F1052V/A120T NEG 10 PT M 7y3m 41 39 Positive screening F508del/Y1032C POS 11 ME F 7y1m 44 44 Positive screening 2789+5GNA/5T-TG12 POS 12 PM F 6y4m 35 36 Positive screening 2183AANG/5T-TG12 POS 13 BM F 6y3m 36 39 Positive screening F508del/5T-TG12 POS 14 CD M 5y8m 40 41 Chronic bronchitis 5T-TG12/5T-TG12 NEG 15 CG F 4y5m 33 37 Recurrent bronchitis R553X/L997F POS 16 CS F 3y8m 53 58 Family history G542X/D614G POS 17 VA M 4y2m 49 43 Pneumonia E831X/5T-TG12 ND 18 SC M 3y4m 39 39 Positive screening R352Q/G213E POS 19 CC F 2y3m 31 31 Positive screening F508del/5T-TG12 POS 20 CA F 2y5m 51 52 Recurrent bronchitis E831X/5T-TG12 ND 21 MR F 3y+7m 29 31 Family history G542X/5T-TG12 POS 22 CM F 2y3m 60 58 Pneumonia T338I/L997F POS 23 LM F 2y1m 50 52 Positive screening F508del/E1473X POS 24 CGE F 0y8m 46 47 Positive screening E92K/5T-TG13 POS 25 NF M 0y7m 32 30 Positive screening F508del/P5L POS 26 RG M 0y7m 45 40 Positive screening N1303K/P5L POS 27 PE M 47y4m 60 58 Nasal polyposis R1066H/UN ND 28 LS M 39y9m 39 38 Azoospermy N1303K/UN ND 29 TM M 38y4m 40 45 Azoospermy N1303K/UN ND 30 DF M 34y2m 52 58 Bronchiectasis 3849+10 kbCNT/UN ND 31 TV F 30y5m 35 34 Recurrent bronchitis L997F/UN ND 32 FA F 18y7m 53 49 Family history Del es.2/UN NEG 33 DG M 17y8m 43 47 Recurrent bronchitis 5T-TG12/UN NEG 34 LN F 13y7m 54 53 Nasal poliposis, malnutrition R74W-V855I/UN NEG 35 FKT M 15y4m 54 53 Chronic bronchitis R352Q/UN NEG 36 BM M 10y9m 48 51 Chronic bronchitis T1263I/UN NEG 37 SV F 11y1m 60 58 Chronic bronchitis R347H/UN NEG 38 CV F 10y10m 38 39 Recurrent bronchitis 5T-TG12/UN NEG 39 BF F 9y10m 37 38 Chronic bronchitis L997F/UN NEG 40 CA M 8y2m 33 32 Pneumonia F508del/UN NEG 41 RX F 8y7m 29 31 Chronic bronchitis V920L/UN NEG 42 MG F 4y3m 51 51 Positive screening F508del/UN POS Sweat chloride concentration and mutations/variants detected are also reported. Login to comment
96 This represents the most frequent variant in borderline subjects according to literature [4], followed by F508del (7.69%), G542X (2.31%) and N1303K (2.31%) that are the most common mutations in Caucasian population. Login to comment

[hide] Common mutations in Cuban cystic fibrosis patients... J Cyst Fibros. 2009 Jan;8(1):47-9. Epub 2008 Oct 19. Collazo T, Bofill AM, Clark Y, Hernandez Y, Gomez M, Rodriguez F, Ramos MD, Gimenez J, Casals T, Rojo M
Common mutations in Cuban cystic fibrosis patients.
J Cyst Fibros. 2009 Jan;8(1):47-9. Epub 2008 Oct 19., [PMID:18938114]

Abstract [show]
So far, more than 1500 mutations have been reported in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Mutational spectrum varies in accordance with geographic and/or ethnic origin. In this study, we have analyzed seven common CF mutations (p.F508del, p.G542X, p.R1162X, p.N1303K, p.R334W, p.R553X and c.3120+1G>A) taking into account the ethnic origin of the Cuban population which is mainly influenced by Spanish and sub-Sahara African contribution. All but p.N1303K have been detected in our patients, the p.F508del being the most prevalent (37.9%). Overall, six mutations showed frequencies above 1% accounting for 55.5% of the Cuban CF alleles.

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2 In this study, we have analyzed seven common CF mutations (p.F508del, p.G542X, p.R1162X, p.N1303K, p.R334W, p.R553X and c.3120+1G>A) taking into account the ethnic origin of the Cuban population which is mainly influenced by Spanish and sub-Sahara African contribution. Login to comment
25 Seven CF mutations were analyzed: p.F508del, p.G542X, p.R1162X, p.N1303K, p.R334W, p.R553X, c.3120+ 1G>A in all patients. Login to comment
26 Amplification Refractory of Mutations Specific (ARMS) [6] was carried out to detect four mutations: p.F508del, p.G542X, p.R1162X, p.N1303K. Login to comment
36 Concerning the frequency of p.G542X mutation (6.8%), a high prevalence of this mutation has also been reported in Mexico (6.2%) [14] and Costa Rica (25%) [18], strongly suggesting the Spanish influence in all cases. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... J Hepatol. 2009 Jan;50(1):150-7. Epub 2008 Oct 7. Henckaerts L, Jaspers M, Van Steenbergen W, Vliegen L, Fevery J, Nuytten H, Roskams T, Rutgeerts P, Cassiman JJ, Vermeire S, Cuppens H
Cystic fibrosis transmembrane conductance regulator gene polymorphisms in patients with primary sclerosing cholangitis.
J Hepatol. 2009 Jan;50(1):150-7. Epub 2008 Oct 7., [PMID:18992954]

Abstract [show]
BACKGROUND/AIMS: Primary sclerosing cholangitis (PSC) is a progressive cholestatic disease commonly associated with inflammatory bowel disease (IBD) and characterized by fibrosing inflammatory destruction of bile ducts. The histological features in the liver of PSC patients are similar to those observed in cystic fibrosis (CF). Our aim was to study whether variants in the CFTR gene are associated with the occurrence and/or evolution of PSC. METHODS: PSC patients (n=140) were genotyped for F508del, the TGmTn variants, and four additional polymorphic loci (1001+11 C>T, M470V, T854T and Q1463Q), and compared to 136 matched healthy controls. RESULTS: The 1540G-allele, encoding V470, was less frequent in PSC (52%) than in controls (64%, p=0.003), and was associated with protection against PSC in individuals without IBD (OR 0.25, 95% CI 0.12-0.52, p=0.0002). Also TG11-T7 was less frequent in PSC (53%) than in controls (61%, p=0.04), this haplotype was associated with reduced risk for PSC (OR 0.34, 95% CI 0.17-0.70, p=0.003) in individuals without IBD. CONCLUSIONS: In this cohort of PSC patients, several CFTR-variants affecting the functional properties of the CFTR protein seem to offer protection against the development of PSC, confirming our hypothesis that CFTR might be implicated in the pathogenesis of PSC.

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91 There was Table 4 Summary of the 37 CFTR variants studied in the exploratory phase INNO-LiPA CFTR 19 INNO-LiPA CFTR17+Tn Update F508del 621+1GfiT G542X 3849+10kbCfiT N1303K 2183AAfiG W1282X 394delTT G551D 2789+5GfiA 1717-1GfiA R1162X R553X 3659delC CFTRdele2,3(21kb) R117H I507del R334W 711+1GfiT R347P 3272-26AfiG G85E 3905insT 1078delT R560T A455E 1898+1GfiA 2143delT S1251N E60X I148T 2184delA 3199del6 711+5GfiA 3120+1GfiA Tn Q552X Fig. 1. Login to comment

[hide] Increased SULT1E1 activity in HepG2 hepatocytes de... Steroids. 2009 Jan;74(1):20-9. Epub 2008 Sep 11. Li L, He D, Wilborn TW, Falany JL, Falany CN
Increased SULT1E1 activity in HepG2 hepatocytes decreases growth hormone stimulation of STAT5b phosphorylation.
Steroids. 2009 Jan;74(1):20-9. Epub 2008 Sep 11., [PMID:18831980]

Abstract [show]
Mouse models of cystic fibrosis (CF) display increased sulfotransferase 1E1 (SULT1E1) activity in hepatocytes of cystic fibrosis transmembrane receptor (CFTR)-deficient animals. SULT1E1 is responsible for the sulfation and inactivation of beta-estradiol (E2) at physiological concentrations. IGF-1 message levels in CFTR(-/-) mouse livers were positively correlated with body weight and negatively correlated with SULT1E1 activity. Growth hormone (GH) is important in the regulation of hepatic IGF-1 expression indicating that E2 levels are involved with GH signaling in hepatocytes. To investigate the effects of E2 and SULT1E1 activity on GH signal transduction in human hepatocytes, SULT1E1 was stably expressed in HepG2 cells. Effects of increased E2 sulfation on the GH signaling pathway and E2-regulated gene expression were examined. Pretreatment of HepG2 cells with 10nM E2 prior to GH stimulation increased STAT5b phosphorylation and IGF-1 expression. In SULT1E1-transfected HepG2 cells, GH-stimulated STAT5b phosphorylation was significantly decreased. E2 treatment had no effect on STAT5b phosphorylation in the absence of GH stimulation. E2 also had no effect on Jak-2 phosphorylation. E2 has an apparent rapid action on increasing GH-stimulated STAT5b phosphorylation that was not attenuated by the estrogen receptor antagonist, ICI 182,780. Physiological levels of E2 in HepG2 cells increase GH stimulation of IGF-1 production apparently through increased phosphorylated STAT5b levels and transcriptional activation of the IGF-1 gene. The enhanced SULT1E1 activity may have a role in inhibiting GH-stimulated STAT5b phosphorylation and IGF-1 synthesis via the sulfation and inactivation of E2.

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349 [24] Du M, Jones JR, Lanier J, Keeling KM, Lindsey JR, Tousson A, et al. Aminoglycoside suppression of a premature stop mutation in a Cftr-/- mouse carrying a human CFTR-G542X transgene. Login to comment
354 Aminoglycoside suppression of a premature stop mutation in a Cftr-/- mouse carrying a human CFTR-G542X transgene. Login to comment

[hide] Improvement in clinical markers in CF patients usi... J Cyst Fibros. 2008 Sep;7(5):433-6. Epub 2008 May 21. Visca A, Bishop CT, Hilton SC, Hudson VM
Improvement in clinical markers in CF patients using a reduced glutathione regimen: an uncontrolled, observational study.
J Cyst Fibros. 2008 Sep;7(5):433-6. Epub 2008 May 21., [PMID:18499536]

Abstract [show]
CFTR mutation, which causes cystic fibrosis (CF), has also recently been identified as causing glutathione system dysfunction and systemic deficiency of reduced glutathione (GSH). Such dysfunction and deficiency regarding GSH may contribute to the pathophysiology of CF. We followed 13 patients (age range 1-27 years) with cystic fibrosis who were using a regimen of reduced glutathione (GSH), including oral glutathione and inhaled buffered glutathione in an uncontrolled, observational study. Dosage ranged from 66-148 mg/kg/day in divided doses, and the term examined was the initial 5.5 months of GSH use (45 days of incrementally adjusted dose, plus 4 months of use at full dosage). Baseline and post-measurements of FEV1 percent predicted, BMI percentile, and weight percentile were noted, in addition to bacterial status and pulmonary exacerbations. Significant improvement in the following clinical parameters was observed: average improvement in FEV1 percent predicted (N=10) was 5.8 percentage points (p<0.0001), average weight percentile (N=13) increased 8.6 points (p<0.001), BMI percentile (N=11) improved on average 1.22 points (p<0.001). All patients improved in FEV1 and BMI, if measured in their case; 12 of 13 patients improved in weight percentile. Positive sputum cultures of bacteria in 11 patients declined from 13 to 5 (p<0.03) with sputum cultures of Pseudomonas aeruginosa becoming negative in 4 of 5 patients previously culturing PA, including two of three patients chronically infected with PA as determined by antibody status. Use of a daily GSH regimen appears to be associated in CF patients with significant improvement in lung function and weight, and a significant decline in bacteria cultured in this uncontrolled study. These findings bear further clinical investigation in larger, randomized, controlled studies.

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43 65 64 71 28.5 28 30 9 5 6 PA PA none 3, F, 19 DF508/G1244E 47 48 55 43 43 48 3 3 9 PA b PAb PA 4, M, 5 DF508/R347P NA NA NA 17 17.5 19 33 33 40 SA SA none 5, M, 24 W1282G/G542X 60 58 70 51 51.5 57 3 3 7 BC BC BC 6, F, 14 3659delC/? Login to comment
64 15.1 15.3 1 0 36 30 3, F, 19 DF508/G1244E 16.3 18.3 0 0 9 18 4, M, 5 DF508/R347P 14.7 15.4 0 1 11 11 5, M, 24 W1282G/G542X 17.8 19.7 1 0 16 20 6, F, 14 3659delC/? Login to comment

[hide] The study of cystic fibrosis transmembrane conduct... J Cyst Fibros. 2008 Sep;7(5):423-8. Epub 2008 May 7. Frentescu L, Brownsell E, Hinks J, Malone G, Shaw H, Budisan L, Bulman M, Schwarz M, Pop L, Filip M, Tomescu E, Mosescu S, Popa I, Benga G
The study of cystic fibrosis transmembrane conductance regulator gene mutations in a group of patients from Romania.
J Cyst Fibros. 2008 Sep;7(5):423-8. Epub 2008 May 7., [PMID:18467194]

Abstract [show]
BACKGROUND: Cystic fibrosis (CF) is produced by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator Gene (CFTR) gene. METHODS: One hundred twenty eight patients with CF were analysed for mutations in the CFTR gene in order to establish the frequency of CF mutations in the Romanian population. The chief methods of analysis were polymerase chain reaction (PCR) of DNA extracted from blood and electrophoresis of PCR products. RESULTS: The frequency of F508del in CF chromosomes from Romania is approximately 56.3%. Other frequent mutations noted are: G542X (3.9%), W1282X (2.3%), and CFTRdele2,3(21 kb)(1.6%); the remaining mutations have frequencies below 1%. CONCLUSIONS: We consider that the frequency of F508del in CF patients from Romania is higher than in previous reports, reaching 56.3%, probably owing to more rigorous selection of patients for genetic testing, allowing improved calculation of mutation frequencies.

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5 Other frequent mutations noted are: G542X (3.9%), W1282X (2.3%), and CFTRdele2,3(21 kb)(1.6%); the remaining mutations have frequencies below 1%. Login to comment
35 Nine patients were tested for 13 mutations [F508del, 1677delTA, I507del, R117H, R553X, 621+ 1GNT, R334W, R347P, G55D, G542X, W1282X, N1303K, CFTR dele2,3(21 kb)] in the Department of Human Genomics, Institute for Molecular Biology and Genetics, National Academy of Science, Kiev, Ukraine (Table 1). Login to comment
39 The following 117 samples were analyzed in DCMB, for a variable number of common mutations between 3 and 26, starting with F508del, I507del and 1677delTA, and continuing with the commercial kits CF-3 (G542X, W1282X and N1303K), CF-8 [F508del, I507del, 1677delTA, CFTRdele2,3 (21 kb), 2143delT, 2184insA, 394delTT, 3821delT], both produced by the Research Center for Medical Genetics, Moscow, Russia, and Elucigene CF20. Login to comment
47 For other Table 1 PCR primers and references for the analysis of 13 common mutations in the CFTR gene Mutation Name of primers Restriction enzyme Reference R334W 7F MspI [10] R347P 7R Hin6I R117H 4A Hin6I [11] 621+1GNT 4B HincII N1303K N1303F DdeI [12] N1303R W1282X W1182F MnlI [13] W1282R [14] G551D 11i5 HincII [15] R553X 11i3 Sau3A G542X 11ex3` MvaI [11] G542X F508del CF2 [3] I507del CF3 [16] 1677delTA C16B [17] C16D [18] [19] CFTRdele2,3(21 kb) CFTRdel2,3F [20] CFTRdel2,3R [13] Control primers for exon 3: 3i-5 3i-3 common mutations, the CF-3 kit was used, and/or restriction enzyme digestions of PCR products were performed, followed by the analysis of restriction products by agarose gel electrophoresis (Table 1); alternatively, the kits from Belgium and UK mentioned above, were used for selected samples, especially for heterozygous patients with F508del and an unknown mutation. Login to comment
60 From the total number of 128 patients with CF we detected both mutations in the majority of them (77), one mutation in 30 Table 2 Distribution of CFTR gene mutations in the group of 128 patients with CF Mutation Number of chromosomes Percent of chromosomes (128 patients, 256 chromosomes) Cumulative frequency F508del 144 56.3% 56.3% G542X 10 3.9% 60.2% W1282X 6 2.3% 62.5% CFTRdele2,3(21 kb) 4 1.6% 64.1% 621+1GNT 2 0.8% 64.8% N1303K 2 0.8% 65.6% 2183AANG 2 0.8% 66.4% R1070Q 2 0.8% 67.2% 457TATNG 1 0.4% 67.6% R117H 1 0.4% 68.0% R334W 1 0.4% 68.4% R735K 1 0.4% 68.8% R785X 1 0.4% 69.1% E831X 1 0.4% 69.5% 3849+10 kb(CNT) 1 0.4% 69.9% R1162X 1 0.4% 70.3% 3272-26ANG 1 0.4% 70.7% 1677delTA 1 0.4% 71.1% 1717-2ANG 1 0.4% 71.5% E585X 1 0.4% 71.9% 2789+5GNA 1 0.4% 72.3% Unknown 71 27.7% 100.0% Total 256 100.0% Fig. 1. Login to comment
66 In the cohort of 142 relatives tested, we found 61 chromosomes with F508del, 6 with W1282X, 4 with G542X, and one of each with N1303K, CFTRdele2,3(21 kb), and 1717-2ANG. Login to comment
77 The most frequent five mutations in Europe are: F508del 66.8%; G542X 2.6%; N1303K 1.6%; G551D 1.5% and W1282X 1% [5]. Login to comment
92 Regarding the mutations detected, we noted a moderate heterogeneity with 21 mutations detected, the Table 3 Distribution of genotypes in CF patients from Romania (n=128; 256 chromosomes) Genotype Number Ethnicity F508del/F508del 46 Romanian 42 Hungarian 3 Gypsy 1 F508del/x 25 Romanian 23 Hungarian 1 Turkish-Romanian 1 F508del/G542X 8 Romanian F508del/CFTRdele2,3(21 kb) 4 Romanian 3 Hungarian 1 F508del/W1282X 3 Romanian F508del/F508del/R117H 1 Romanian F508del/R334W 1 Romanian F508del/621+1GNT 1 Romanian F508del/N1303K 1 Romanian F508del/2183AANG 1 Romanian F508del/3849+10 kb(CNT) 1 Romanian F508del/3272-26ANG 1 Romanian F508del/R1162X 1 Romanian F508del/R785X 1 Romanian F508del/1717-2ANG 1 Romanian F508del/2789+5GNA 1 Romanian G542X/G542X 1 Romanian W1282X/W1282X 1 Romanian N1303K/457TATNG 1 Romanian 621+1GNT/2183AANG 1 Romanian W1282X/x 1 Romanian R1070Q/E585X 1 Romanian R1070Q/x 1 Romanian E831X/x 1 Gypsy R735K/x 1 Romanian 1677delTA/x 1 Romanian x/x 21 Romanian 18 Hungarian 2 Gypsy 1 presence of common mutations (excepting the Celtic mutation G551D), and a similarity with the mutations detected in Italy, France and Spain [5]. Login to comment

[hide] Cystic fibrosis carrier frequency and estimated pr... J Cyst Fibros. 2008 Sep;7(5):440-3. Epub 2008 Feb 1. Ratbi I, Genin E, Legendre M, Le Floch A, Costa C, Cherkaoui-Deqqaqi S, Goossens M, Sefiani A, Girodon E
Cystic fibrosis carrier frequency and estimated prevalence of the disease in Morocco.
J Cyst Fibros. 2008 Sep;7(5):440-3. Epub 2008 Feb 1., [PMID:18243066]

Abstract [show]
BACKGROUND: The epidemiology of cystic fibrosis (CF) is poorly known in North African populations, in particular in Morocco and the CF carrier frequency in the general Moroccan population has never been evaluated. METHODS: To estimate the prevalence of CF mutations in Morocco, blood samples from 150 healthy Moroccans were tested for frequent CFTR mutations and the intron 8 polyT variant. RESULTS: Two subjects were heterozygous for F508del and eight others for the (T)5 variant. CONCLUSION: These findings indicate that the Moroccan population is at risk for CF and CFTR-related disorders. CF prevalence could be in the range of that found in European populations. Wider studies are necessary to identify the clinical pattern and accurately determine the prevalence and molecular basis of CF in Morocco.

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27 We screened for 32 CFTR gene mutations (G85E, 394delTT, R117H, 621+1GNT, 711+1GNT, R334W, R347P, R347H, 1078delT, A455E, I507del, F508del, V520F, 1717-1GNA, G542X, G551D, R553X, R560T, S549R(TNG), S549N, 1898+1GNA, 2183AANG, 2184delA, 2789+5GNA, 3120 + 1G NA, R1162X, 3659delC, 3849 + 10kbC NT, W1282X, 3905insT, 3876delA, N1303K) and the (T)5 splicing variant of intron 8, using a commercial kit (CF v3 Genotyping Assay, Abbott, Rungis, France). Login to comment
64 Beside F508del, other frequent mutations were found among North African populations, in particular 711+1GNT, W1282X, N1303K, G542X and R1162X [1,4,6]. Login to comment

[hide] Validation of high-resolution DNA melting analysis... J Mol Diagn. 2008 Sep;10(5):424-34. Epub 2008 Aug 7. Audrezet MP, Dabricot A, Le Marechal C, Ferec C
Validation of high-resolution DNA melting analysis for mutation scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.
J Mol Diagn. 2008 Sep;10(5):424-34. Epub 2008 Aug 7., [PMID:18687795]

Abstract [show]
High-resolution melting analysis of polymerase chain reaction products for mutation scanning, which began in the early 2000s, is based on monitoring of the fluorescence released during the melting of double-stranded DNA labeled with specifically developed saturation dye, such as LC-Green. We report here the validation of this method to scan 98% of the coding sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We designed 32 pairs of primers to amplify and analyze the 27 exons of the gene. Thanks to the addition of a small GC-clamp at the 5' ends of the primers, one single melting domain and one identical annealing temperature were obtained to co-amplify all of the fragments. A total of 307 DNA samples, extracted by the salt precipitation method, carrying 221 mutations and 21 polymorphisms, plus 20 control samples free from variations (confirmed by denaturing high-performance liquid chromatography analysis), was used. With the conditions described in this study, 100% of samples that carry heterozygous mutations and 60% of those with homozygous mutations were identified. The study of a cohort of 136 idiopathic chronic pancreatitis patients enabled us to prospectively evaluate this technique. Thus, high-resolution melting analysis is a robust and sensitive single-tube technique for screening mutations in a gene and promises to become the gold standard over denaturing high-performance liquid chromatography, particularly for highly mutated genes such as CFTR, and appears suitable for use in reference diagnostic laboratories.

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51 Sequences of the Primers Used for CFTR Analysis by HRM, GC Size, Amplicon Length, Number of Positive Controls Validated for Each Exon, and Positive Controls for Routine Analysis Exon Primer Sequences GC length Amplicon length (bp) Introns Number of heterozygous- positive controls Number of homozygous- positive controls Recommended control 1 LSCFE1Fmod 5Ј-CCGCCGCCGTTGAGCGGCAGGCACC-3Ј 8 200 bp 74 4 125GϾC LSCFE1Rmod 5Ј-CCGCCGCCGGCACGTGTCTTT CCGAAGCT-3Ј 8 19 M1I 2 2i5b 5Ј-CAAATCTGTATGGAGACC-3Ј 0 194 bp 39 5 R31C 2i3Љ 5Ј-CAACTAAACAATGTACATGAAC-3Ј 0 4 296ϩ1GϾT 3 LSCFe3Fmod LSCFe3Rmod 5Ј-CGCCGTTAAGGGAAATAGGACAA CTAAAATA-3Ј 5 276 bp 44 10 2 R75Q 5Ј-CCGCCGATTCACCAGATTTCGTAGTC-3Ј 6 66 G85V 4 LSCFe4FmodC 5Ј-CCGCCGCCGCCCGTGTTGAAATT CTCAGGGT-3Ј 12 361 bp 52 14 1 R117H LSCFe4RmodC 5Ј-CCGCCGCCCACATGTACGATAC AGAATATATGTGCC-3Ј 9 26 574delA 5 LSCFE5Fmod 5Ј-CCGCCGGTTGAAATTATCTAACTTTCC-3Ј 6 201 bp 13 8 624delT LSCFE5Rmod 5Ј-CCGAACTCCGCCTTTCCAGTTGT-3Ј 3 48 711ϩ1GϾT 6a LSCF6aFmod2 5Ј-CCGCCGGGGTGGAAGAT ACAATGACACCTG-3Ј 5 317 bp 25 8 C225X LSCF6aRmod2 5Ј-CCGCCGCCGCGATGCATAGAG CAGTCCTGGTT-3Ј 11 66 L206W 6b LSCFE6bFmod 5Ј-CGCGCCGCCGGATTTAC AGAGATCAGAGAG-3Ј 10 239 bp 0 2 1 R258G LSCFE6Brmod 5Ј-CCGCCGCCGAGGTGGA GTCTACCATGA-3Ј 8 66 1001ϩ11CϾT 7 LSCFE7Fmod2 5Ј-CCGCCGCCCTCTCCCTGAATTT TATTGTTATTGTTT-3Ј 13 326 bp 7 11 1078delT LSCFE7Rmod2 5Ј-CCCGCCGCCCTATAATGCAG CATTATGGT-3Ј 10 7 1248ϩ1GϾT 8 LSCFE8Fmod 5Ј-CCGGAATGCATTAATGCTAT TCTGATTC-3Ј 4 199 bp 32 7 W401X LSCFE8Rmod 5Ј-CCCGCAGTTAGGTGTTTAG AGCAAACAA-3Ј 4 18 1249-5AϾG 9 LSCFe9Fmod2 5Ј-CCGCCGCCGGGAATTATTTGAGAA AGCAAAACA-3Ј 8 279 bp 0 3 D443Y LSCFe9Rmod2 5Ј-CCGCCGCGAAAATACCTTCCAG CACTACAAACTAGAAA-3Ј 8 57 A455E 10 LSCF10FmodD 5Ј-CGCCGTTATGGGAGAACTGG AGCCTTCAGAG-3Ј 5 275 bp 0 15 1 F508del LSCF10RmodD 5Ј-CCGCAGACTAACCGATTGAAT ATGGAGCC-3Ј 4 68 E528E 11 h11i5 5Ј-TGCCTTTCAAATTCAGATTGAGC-3Ј 0 197 bp 42 13 2 G542X 11i3ter 5Ј-ACAGCAAATGCTTGCTAGACC-3Ј 0 17 G551D 12 LSCFE12Fmod 5Ј-CGCGTCATCTACACTAGATGACCAG-3Ј 4 244 bp 43 15 G576A 1898 ϩ 1GϾALSCFE12Rmod 5Ј-CCGGAGGTAAAATGCAATCTATGATG-3Ј 3 63 13 LSCF13AFmod 5Ј-CCGCCGCCGGAGACATATTG CAATAAAGTAT-3Ј 9 38 20 I601F LSCF13ARmod 5Ј-GCCTGTCCAGGAGACAGGA GCATCTC-3Ј 2 R668C LSCF13BFmod 5Ј-CCGCCGCAATCCTAACTGAG ACCTTACACCG-3Ј 2 R668C LSCF13BRmod 5Ј-CCGCCGATCAGGTTCAGGA CAGACTGC-3Ј 3 346 bp 2184insA LSCF13CFmod 5Ј-CCGCGGTGATCAGCACTGGCCC-3Ј 6 301 bp 77 L749L LSCF13CRmod 5Ј-CCGCGCGCGCGGCCAGTTTCTTG AGATAACCTTCT-3Ј 13 259 bp V754M LSCF13DFmod 5Ј-CGTGTCACTGGCCCCTCAGGC-3Ј 1 221 bp I807M LSCF13DRmof 5Ј-CCGCCGCCGCTAATCCTATGA TTTTAGTAAAT-3Ј 9 220 bp 2622ϩ1GϾA LSCf13FFmod 5Ј-CGCGGTGCAGAAAGAAGAAAT TCAATCCTAACTG-3Ј 4 R668C LSCF13FRmod 5Ј-CCGCCGTGCCATTCATTTGT AAGGGAGTCT-3Ј 6 2184insA 14a LSCF14aFmodB 5Ј-CCGACCACAATGGTGGCAT GAAACTG-3Ј 3 239 bp 35 7 1 T854T LSCF14aRmodB 5Ј-CCGCCGACTTTAAATCCAGTAAT ACTTTACAATAGAACA-3Ј 6 7 W846X 14b LSCF14bFmod 5Ј-CCGGAGGAATAGGTGAAGAT-3Ј 2 179 bp 38 4 2752-5GϾT LSCF14bRmodb 5Ј-CCGTACATACAAACATAGTGGATT-3Ј 3 59 2789ϩ5GϾT 15 LSCFE15Fmod 5Ј-CGCGCCGTGTATTGGAAA TTCAGTAAGTAACTTTGG-3Ј 7 412 bp 33 16 T908S LSCFE15Rmod 5Ј-CCGCAGCCAGCACTGCCAT TAGAAA-3Ј 4 68 S945L (table continues) phisms that we have chosen to exclude. Login to comment
75 In addition to heterozygous variants, 10 homozygote samples carrying F508del, 394delT, R117H, G542X, S549R, 4016inT homozygous mutations, and R75Q, T854T, 1001ϩ11 CϾT, and Q1463Q homozygous variants, were also tested. Login to comment
76 Only mutations 394delT, R117H, G542X, 4016insT, and variants R75Q and 1001ϩ11 CϾ T provided positive results (Figure 5). Login to comment
115 Some homozygous variants detected: 394delT in exon 3, 1001ϩ11CϾT in exon 6b, G542X in exon 11, and 4016insT in exon 21. already been successfully applied to the analysis of TP53,22 phenylalanine hydroxylase gene,23 factor VIII,24 factor II, factor V, HFE,11,25,26 C-kit,27 EGFR HER2,28 RET,29 and CFTR.30 In this last study, the authors report the CFTR scanning by HRM after PCR amplification of 37 exon/intron fragments in 2 panels of 96 random white UK blood donors and 30 blinded DNA samples enriched for CF-causing variants. Login to comment
171 Results of CFTR Analysis by HRM on 136 Samples of Patients with Idiopathic Chronic Pancreatitis (ICP) Exon Number of positive samples Mutations identified Variants identified New positive controls 1 14 14 125GϾC 2 1 1 R31C 3 9 1 G85E 7 R75Q 1 R74W 4 4 1 R117G 1 I148T R117G 1 R117H 1 A120T 5 1 1 L188P L188P 6a 5 1 V201M 1 A221A A221A 3 875ϩ40 AϾG 6b 27 1 M284T 26 1001ϩ11CϾT M284T 7 1 1 L320V L320V 8 0 0 9 1 1 D443Y 10 16 8 F508del 8 E528E 11 1 1 G542X 12 6 4 G576A 1 Y577Y L568F 1 L568F 13 7 1 S737F 4 R668C S737F 1 V754M L644L 1 L644L 14a 53 52 T854T T854TϩI853I 1 T854TϩI853I 14b 0 0 15 3 1 L967S T908S 1 T908S 1 S945L 16 0 0 17a 10 7 L997F 1 3271ϩ18CϾT 3271 ϩ 3AϾG 1 3271 ϩ 3 AϾG 1 Y1014C 17b 3 1 L1096L L1096L 1 H1054DϩG1069R 1 3272-33AϾG H1054DϩG1069R 3272-33AϾG 18 2 1 D1152H E1124del 1 E1124del 19 5 5 S1235R poly 20 7 1 W1282X 5 P1290P 1 D1270N 21 2 1 N1303K 1 T1299T 22 0 0 23 1 0 4374ϩ13 AϾG 24 43 40 Q1463Q 2 Y1424Y 1 Q1463QϩY1024Y ing domain of a gene brings an excellent sensitivity for heterozygote detection that is very close to 100%. Login to comment

[hide] Effectiveness of PTC124 treatment of cystic fibros... Lancet. 2008 Aug 30;372(9640):719-27. Epub 2008 Aug 20. Kerem E, Hirawat S, Armoni S, Yaakov Y, Shoseyov D, Cohen M, Nissim-Rafinia M, Blau H, Rivlin J, Aviram M, Elfring GL, Northcutt VJ, Miller LL, Kerem B, Wilschanski M
Effectiveness of PTC124 treatment of cystic fibrosis caused by nonsense mutations: a prospective phase II trial.
Lancet. 2008 Aug 30;372(9640):719-27. Epub 2008 Aug 20., [PMID:18722008]

Abstract [show]
BACKGROUND: In about 10% of patients worldwide and more than 50% of patients in Israel, cystic fibrosis results from nonsense mutations (premature stop codons) in the messenger RNA (mRNA) for the cystic fibrosis transmembrane conductance regulator (CFTR). PTC124 is an orally bioavailable small molecule that is designed to induce ribosomes to selectively read through premature stop codons during mRNA translation, to produce functional CFTR. METHODS: This phase II prospective trial recruited adults with cystic fibrosis who had at least one nonsense mutation in the CFTR gene. Patients were assessed in two 28-day cycles. During the first cycle, patients received PTC124 at 16 mg/kg per day in three doses every day for 14 days, followed by 14 days without treatment; in the second cycle, patients received 40 mg/kg of PTC124 in three doses every day for 14 days, followed by 14 days without treatment. The primary outcome had three components: change in CFTR-mediated total chloride transport; proportion of patients who responded to treatment; and normalisation of chloride transport, as assessed by transepithelial nasal potential difference (PD) at baseline, at the end of each 14-day treatment course, and after 14 days without treatment. The trial was registered with who.int/ictrp, and with clinicaltrials.gov, number NCT00237380. FINDINGS: Transepithelial nasal PD was evaluated in 23 patients in the first cycle and in 21 patients in the second cycle. Mean total chloride transport increased in the first treatment phase, with a change of -7.1 (SD 7.0) mV (p<0.0001), and in the second, with a change of -3.7 (SD 7.3) mV (p=0.032). We recorded a response in total chloride transport (defined as a change in nasal PD of -5 mV or more) in 16 of the 23 patients in the first cycle's treatment phase (p<0.0001) and in eight of the 21 patients in the second cycle (p<0.0001). Total chloride transport entered the normal range for 13 of 23 patients in the first cycle's treatment phase (p=0.0003) and for nine of 21 in the second cycle (p=0.02). Two patients given PTC124 had constipation without intestinal obstruction, and four had mild dysuria. No drug-related serious adverse events were recorded. INTERPRETATION: In patients with cystic fibrosis who have a premature stop codon in the CFTR gene, oral administration of PTC124 to suppress nonsense mutations reduces the epithelial electrophysiological abnormalities caused by CFTR dysfunction.

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38 §Based on normative data for age, sex, and height.15 Table 1: Baseline characteristics Allele 1 Allele 2 Stop codon Total Treatment response* Change to normal range†; G542X ΔF508 UGA 3 3 (100%) 3 (100%) G542X W1282X UGA/UGA 1 0 1 (100%) G542X N1303K UGA 1 1 (100%) 1 (100%) W1282X ΔF508 UGA 13 10 (77%) 9 (69%) W1282X W1282X UGA/UGA 3 1 (33%) 1 (33%) W1282X 3849+10kB C→T‡ UGA/UAA 1 1 (100%) 1 (100%) 3849+10kB C→T‡ ΔF508 UAA 1 1 (100%) 1 (100%) Data are n or n (%). Login to comment
86 The predominant premature stop mutations in these 23 patients were W1282X and G542X (table 2). Login to comment
94 Table 3 also shows that the proportion of patients who had normal chloride transport (predefined as nasal PD at least as electrically negative as -5 mV) increased during PTC124 treatment (first cycle p=0·0003, second cycle p=0·020).14 Results showed that participants who had all three genotypes for premature stop mutations (G542X, W1282X, and 3849+10 kB C→T), including patients who had a nonsense mutation in a single CFTR allele or in both CFTR alleles, had a total chloride transport response or normalisation during at least one cycle of PTC124 treatment (table 2). Login to comment
104 In the two patients whose only nonsense mutation was G542X, total chloride transport became more electrically negative than -5 mV (ie, within the normal range) although in both patients the proportion of CFTR mRNA that contained a nonsense mutation was less than 10% of wild-type CFTR mRNA. Login to comment
141 We did not include cystic fibrosis patients who did not have a CFTR nonsense mutation (eg, ΔF508 homozygous) on the basis that safety data for PTC124 in these patients were not sufficient at the beginning of the G542X only W1282X only 3849+10KB 3849+10KB+W1282X G542x+W1282X r=0·57 R2 =0·32 p=0·046 0 -20·0 -17·5 -15·0 -12·5 -10·0 -7·5 -5·0 -2·5 0·0 10 20 30 40 50 Proportion of CFTR mRNA with a nonsense mutation relative to wild-type mRNA (%) Typical range for patients with cystic fibrosis* Normal range* Nonsense mutation NasalPD(mV) Figure 4: Correlation of most normal nasal potential difference (PD) during treatment in either cycle with proportion of CFTR mRNA that contained a nonsense mutation relative to wild-type MRNA CFTR=cystic fibrosis transmembrane conductance regulator. mRNA=messenger RNA. Login to comment
156 In two patients who had only the G542X mutation, total chloride transport changed to within the normal range although the proportion of their CFTR mRNA that contained a nonsense mutation was less than 10% of 65 60 55 50 45 40 35 30 25 20 15 10 5 0 60 50 55 45 40 35 30 25 20 15 10 5 0 80 85 90 95 100 75 70 65 80 85 90 95 100 75 70 65 60 50 55 45 p=0·037 p=0·350 p=0·027 p=0·212 p=0·448 p=0·015p<0·0001 p=0·625 40 35 30 25 20 15 10 5 0 0 1000 Bodyweight Time (days) Treatment phase 1* Treatment phase 2* Treatment phase 1* Treatment phase 2* Weight(kg) AbsoluteneutrophilcountpermLProportionofpredictednormalvalue(%) Proportionofpredictednormalvalue(%) 0 14 28 42 Time (days) 0 14 28 42 FEV1 FVC Absolute neutrophil count 2000 3000 4000 5000 6000 7000 Figure 5: Mean clinical measurements at baseline and end of each treatment phase FEV1=forced expiratory volume in 1 second. FVC=forced vital capacity. Login to comment
163 In this regard, preclinical work with PTC124 shows that ribosomal read-through of UGA-G (the sequence of G542X) is more efficient than that for UGA-A (the sequence of W1282X).8 Treatment with PTC124 was associated with small increases in FEV₁, FVC, and bodyweight in most patients, and with a reduction in neutrophil counts. Login to comment

[hide] Consensus on the use and interpretation of cystic ... J Cyst Fibros. 2008 May;7(3):179-96. Castellani C, Cuppens H, Macek M Jr, Cassiman JJ, Kerem E, Durie P, Tullis E, Assael BM, Bombieri C, Brown A, Casals T, Claustres M, Cutting GR, Dequeker E, Dodge J, Doull I, Farrell P, Ferec C, Girodon E, Johannesson M, Kerem B, Knowles M, Munck A, Pignatti PF, Radojkovic D, Rizzotti P, Schwarz M, Stuhrmann M, Tzetis M, Zielenski J, Elborn JS
Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice.
J Cyst Fibros. 2008 May;7(3):179-96., [PMID:18456578]

Abstract [show]
It is often challenging for the clinician interested in cystic fibrosis (CF) to interpret molecular genetic results, and to integrate them in the diagnostic process. The limitations of genotyping technology, the choice of mutations to be tested, and the clinical context in which the test is administered can all influence how genetic information is interpreted. This paper describes the conclusions of a consensus conference to address the use and interpretation of CF mutation analysis in clinical settings. Although the diagnosis of CF is usually straightforward, care needs to be exercised in the use and interpretation of genetic tests: genotype information is not the final arbiter of a clinical diagnosis of CF or CF transmembrane conductance regulator (CFTR) protein related disorders. The diagnosis of these conditions is primarily based on the clinical presentation, and is supported by evaluation of CFTR function (sweat testing, nasal potential difference) and genetic analysis. None of these features are sufficient on their own to make a diagnosis of CF or CFTR-related disorders. Broad genotype/phenotype associations are useful in epidemiological studies, but CFTR genotype does not accurately predict individual outcome. The use of CFTR genotype for prediction of prognosis in people with CF at the time of their diagnosis is not recommended. The importance of communication between clinicians and medical genetic laboratories is emphasized. The results of testing and their implications should be reported in a manner understandable to the clinicians caring for CF patients.

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1236 Table 1 Geographical distribution of the most common mutations E60X Southern European S549N Indian CFTR Slavic - Eastern European G551D United Kingdom, Central Europe R75X Southern European, US-Hispanic Q552X Southern European, Italian 394delTT Nordic - Baltic sea region R553X Central European G85E Southern Europe A559T African-American 406-1GNA US-Hispanic R560T Northern Irish R117H European-derived populations 1811+1.6kbANG Spanish, US-Hispanic R117C Northern European 1898+1GNA United Kingdom, Central Europe 621+1GNT Southern European 1898+5GNT East Asian populations 711+1GNT French, French Canadian 2143delT Slavic - Eastern European 711+5GNA US-Hispanic 2183delAANG Southern Europe, Middle Eastern, Iranian, Latin American L206W Spanish and US-Hispanic 2184delA European-derived populations V232D Spanish and US-Hispanic 2789+5GNA European-derived populations 1078delT French Brittany Q890X Southern European R334W Southern European, Latin American 3120+1GNA African, Arabian, African-American, Southern Europe 1161delC Indian 3272-26ANG European-derived populations R347P European-derived, Latin America 3659delC Scandinavian R347H Turkish 3849+10kbCNT Ashkenazi-Jewish, Southern European, Middle Eastern, Iranian, Indian A455E Dutch R1066C Southern European 1609delCA Spanish, US-Hispanic Y1092X (CNA) Southern European I506T Southern European, Spanish M1101K US-Hutterite I507del European-derived populations 3905insT Swiss F508del European-derived populations D1152H European-derived populations 1677delTA Southern European, Middle Eastern R1158X Southern European 1717-GNA European-derived populations R1162X Italian, Amerindian, Latin America V520F Irish S1251N European-derived populations G542X Southern European, Mediterranean W1282X Ashkenazi-Jewish, Middle Eastern S549R(TNG) Middle Eastern N1303K Southern European, Middle Eastern Legend: these alleles occur with a frequency superior to 0.1% in selected populations. Login to comment
1239 Table 1 Geographical distribution of the most common mutations E60X Southern European S549N Indian CFTR Slavic - Eastern European G551D United Kingdom, Central Europe R75X Southern European, US-Hispanic Q552X Southern European, Italian 394delTT Nordic - Baltic sea region R553X Central European G85E Southern Europe A559T African-American 406-1GNA US-Hispanic R560T Northern Irish R117H European-derived populations 1811+1.6kbANG Spanish, US-Hispanic R117C Northern European 1898+1GNA United Kingdom, Central Europe 621+1GNT Southern European 1898+5GNT East Asian populations 711+1GNT French, French Canadian 2143delT Slavic - Eastern European 711+5GNA US-Hispanic 2183delAANG Southern Europe, Middle Eastern, Iranian, Latin American L206W Spanish and US-Hispanic 2184delA European-derived populations V232D Spanish and US-Hispanic 2789+5GNA European-derived populations 1078delT French Brittany Q890X Southern European R334W Southern European, Latin American 3120+1GNA African, Arabian, African-American, Southern Europe 1161delC Indian 3272-26ANG European-derived populations R347P European-derived, Latin America 3659delC Scandinavian R347H Turkish 3849+10kbCNT Ashkenazi-Jewish, Southern European, Middle Eastern, Iranian, Indian A455E Dutch R1066C Southern European 1609delCA Spanish, US-Hispanic Y1092X (CNA) Southern European I506T Southern European, Spanish M1101K US-Hutterite I507del European-derived populations 3905insT Swiss F508del European-derived populations D1152H European-derived populations 1677delTA Southern European, Middle Eastern R1158X Southern European 1717-GNA European-derived populations R1162X Italian, Amerindian, Latin America V520F Irish S1251N European-derived populations G542X Southern European, Mediterranean W1282X Ashkenazi-Jewish, Middle Eastern S549R(TNG) Middle Eastern N1303K Southern European, Middle Eastern Legend: these alleles occur with a frequency superior to 0.1% in selected populations. Login to comment

[hide] FIT probes: peptide nucleic acid probes with a flu... Anal Biochem. 2008 Apr 15;375(2):318-30. Epub 2008 Jan 12. Socher E, Jarikote DV, Knoll A, Roglin L, Burmeister J, Seitz O
FIT probes: peptide nucleic acid probes with a fluorescent base surrogate enable real-time DNA quantification and single nucleotide polymorphism discovery.
Anal Biochem. 2008 Apr 15;375(2):318-30. Epub 2008 Jan 12., [PMID:18249184]

Abstract [show]
The ability to accurately quantify specific nucleic acid molecules in complex biomolecule solutions in real time is important in diagnostic and basic research. Here we describe a DNA-PNA (peptide nucleic acid) hybridization assay that allows sensitive quantification of specific nucleic acids in solution and concomitant detection of select single base mutations in resulting DNA-PNA duplexes. The technique employs so-called FIT (forced intercalation) probes in which one base is replaced by a thiazole orange (TO) dye molecule. If a DNA molecule that is complementary to the FIT-PNA molecule (except at the site of the dye) hybridizes to the probe, the TO dye exhibits intense fluorescence because stacking in the duplexes enforces a coplanar arrangement even in the excited state. However, a base mismatch at either position immediately adjacent to the TO dye dramatically decreases fluorescence, presumably because the TO dye has room to undergo torsional motions that lead to rapid depletion of the excited state. Of note, we found that the use of d-ornithine rather than aminoethylglycine as the PNA backbone increases the intensity of fluorescence emitted by matched probe-target duplexes while specificity of fluorescence signaling under nonstringent conditions is also increased. The usefulness of the ornithine-containing FIT probes was demonstrated in the real-time PCR analysis providing a linear measurement range over at least seven orders of magnitude. The analysis of two important single nucleotide polymorphisms (SNPs) in the CFTR gene confirmed the ability of FIT probes to facilitate unambiguous SNP calls for genomic DNA by quantitative PCR.

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51 Human genomic DNA samples containing the G542X and the G551D mutations were obtained from the Coriell Institute for Medical Research and stored in H2O at a concentration of 100 ng/ll at À80 °C. Login to comment
177 In a paradigm study, we explored the usefulness of FIT probes by addressing the two most common cystic fibrosis single base mutations: G542X and G551D. Login to comment
192 For reasons of comparison, we chose to analyze the G542X mutation site by applying the well-established dual-labeled probe technology [20]. Login to comment
291 The analysis of two important single nucleotide polymorphisms in the CFTR gene, the G542X and G551D mutations, confirmed the ability of Orn(TO) FIT probes to make unambiguous SNP calls for genomic DNA by qPCR. Login to comment
52 Human genomic DNA samples containing the G542X and the G551D mutations were obtained from the Coriell Institute for Medical Research and stored in H2O at a concentration of 100 ng/ll at 80 &#b0;C. Login to comment
178 In a paradigm study, we explored the usefulness of FIT probes by addressing the two most common cystic fibrosis single base mutations: G542X and G551D. Login to comment
193 For reasons of comparison, we chose to analyze the G542X mutation site by applying the well-established dual-labeled probe technology [20]. Login to comment
292 The analysis of two important single nucleotide polymorphisms in the CFTR gene, the G542X and G551D mutations, confirmed the ability of Orn(TO) FIT probes to make unambiguous SNP calls for genomic DNA by qPCR. Login to comment

[hide] Estimating the age of CFTR mutations predominantly... J Cyst Fibros. 2008 Mar;7(2):168-73. Epub 2007 Sep 6. Fichou Y, Genin E, Le Marechal C, Audrezet MP, Scotet V, Ferec C
Estimating the age of CFTR mutations predominantly found in Brittany (Western France).
J Cyst Fibros. 2008 Mar;7(2):168-73. Epub 2007 Sep 6., [PMID:17825628]

Abstract [show]
BACKGROUND: Disparities in the spectrum of mutations within the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene are commonly observed in populations from different ethnical and/or geographical origins. The occurrence of CF in Brittany (western France) is one of the highest in populations from Caucasian origin (<1/2000 in specific areas). The W846X(2), 1078delT and G551D mutations, as well as the I1027T polymorphism in cis with the DeltaF508 mutation (currently referred to as p.F508del) are particularly frequent in this area. We investigated the age of the respective variants in the region of interest. METHODS: Several polymorphic markers surrounding the CFTR gene were genotyped. Allele frequencies as well as mutation rates and other parameters were used to calculate the respective age of the most recent common ancestors in the region of interest by a previously employed, simple likelihood-based method. RESULTS: Following haplotype reconstruction and simulation, the ages were estimated to be approximately 600, 1000, 1200 and 600 years, respectively (with a 95% confidence interval). CONCLUSIONS: These datings thus provide historical insights in the context of understanding population migrations. They also underline the usefulness of this method for estimating the age of rare mutations with a limited number of carriers.

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16 Only four additional mutations (i.e., G542X, G551D, Journal of Cystic Fibrosis 7 (2008) 168-173 www.elsevier.com/locate/jcf ☆ Data were presented at The American Society of Human Genetics 56th Annual Meeting, New Orleans, Louisiana, USA, October 9-13, 2006. Login to comment
30 Likewise, the G542X and N1303K mutations were estimated to be N34000 years old [14]. Login to comment
51 Primers amplifying the regions of interest were designed with PrimerQuestSM from Table 1 Genotypes of CF patients W846X2 1078delT G551D Mutation in trans Number Mutation in trans Number Mutation in trans Number ΔF508 6 ΔF508 21 ΔF508 18 R117C 1 1078delTa 2 E60K 1 ΔI507 1 4005+1GNA 2 W79X 1 Y563N 1 L610S 1 C225X 1 1078delTb 1 W846X2 b 1 F311L 1 621+1GNT 1 R1066H 1 R347H 1 2789+5GNA 1 1221delCT 1 G542X 1 3849+4ANG 1 1717-1GNA 1 G551D 1 3659delC 1 R553G 1 S942F 1 Y1092X 1 621+1GNT 1 2789+5GNA 1 4006-1GNA 1 Unidentified 1 Total 13 Total 31 Total 32 a One particular case: in this individual, the two chromosomes 7 are identical by descent. Login to comment

[hide] Analysis of the CFTR gene in Iranian cystic fibros... J Cyst Fibros. 2008 Mar;7(2):102-9. Epub 2007 Jul 27. Alibakhshi R, Kianishirazi R, Cassiman JJ, Zamani M, Cuppens H
Analysis of the CFTR gene in Iranian cystic fibrosis patients: identification of eight novel mutations.
J Cyst Fibros. 2008 Mar;7(2):102-9. Epub 2007 Jul 27., [PMID:17662673]

Abstract [show]
BACKGROUND: Cystic fibrosis (CF) is the most common inherited disorder in Caucasian populations, with over 1400 mutations identified in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. Mutations in the CFTR gene may be also causative for CBAVD (Congenital Bilateral Absence of the Vas Deferens). The type and distribution of mutations varies widely between different countries and/or ethnic groups, and is relatively unknown in Iran. We therefore performed a comprehensive analysis of the CFTR gene in Iranian CF patients. METHODS: 69 Iranian CF patients, and 1 CBAVD patient, were analysed for mutations in the complete coding region, and its exon/intron junctions, of their CFTR genes, using different methods, such as ARMS (amplification refractory mutation system)-PCR, SSCP (single stranded conformation polymorphism) analysis, restriction enzyme digestion analysis, direct sequencing, and MLPA (Multiplex Ligation-mediated Probe Amplification). RESULTS: CFTR mutation analysis revealed the identification of 37 mutations in 69 Iranian CF patients. Overall, 81.9% (113/138) CFTR genes derived from Iranian CF patients could be characterized for a disease-causing mutation. The CBAVD patient was found to be homozygous for the p.W1145R mutation. The most common mutations were p.F508del (DeltaF508) (18.1%), c.2183_2184delAAinsG (2183AA>G) (6.5%), p.S466X (5.8%), p.N1303K (4.3%), c.2789+5G>A (4.3%), p.G542X (3.6%), c.3120+1G>A (3.6%), p.R334W (2.9%) and c.3130delA (2.9%). These 9 types of mutant CFTR genes totaled for 52% of all CFTR genes derived from the 69 Iranian CF patients. Eight mutations, c.406-8T>C, p.A566D, c.2576delA, c.2752-1_2756delGGTGGCinsTTG, p.T1036I, p.W1145R, c.3850-24G>A, c.1342-?_1524+?del, were found for the first time in this study. CONCLUSIONS: We identified 37 CFTR mutations in 69 well characterized Iranian CF patients, obtaining a CFTR mutation detection rate of 81.9%, the highest detection rate obtained in the Iranian population so far. These findings will assist in genetic counseling, prenatal diagnosis and future screening of CF in Iran.

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8 The most common mutations were p.F508del (ΔF508) (18.1%), c.2183_2184delAAinsG (2183AANG) (6.5%), p.S466X (5.8%), p.N1303K (4.3%), c.2789+5GNA (4.3%), p.G542X (3.6%), c.3120+1GNA (3.6%), p.R334W (2.9%) and c.3130delA (2.9%). Login to comment
27 A few mutations, such as p.F508del, p.N1303K and p.G542X, are frequent worldwide. Login to comment
37 1 c.406-3TNC I3 T to C at 406-3 mRNA splicing defect 1 p.R170H E5 G to A at 641 Arg to His at 170 1 p.D192G E5 A to G at 707 Asp to Gly at 192 2 p.R334W E7 C to T at 1132 Arg to Trp at 334 4 c.1525-1GNA I9 G to A at 1525-1 mRNA splicing defect 2 p.F508del E10 Deletion of CTT from 1653 Deletion of Phe at 508 25 p.S466X E10 C to G at 1529 Ser to stop at 466 8 c.1677delTA E10 Deletion of TA from 1677 Frame shift 2 p.G542X E11 G to T at 1756 Gly to stop at 542 5 p.S549R E11 T to G at 1779 Ser to Arg at 549 2 p.A566D E12 C to A at 1829 Ala to Asp at 566 2 c.1898+1GNT I12 G→T at 1898+1 mRNA splicing defect 2 c.2183_2184delAAinsG E13 A to G at 2183 and deletion of A at 2184 Frame shift 9 c.2576delA E13 Deletion of A at 2576 Frame shift 1 c.2043delG E13 Deletion of A at 2043 Frame shift 1 c.2184insA E13 Insertion of A after 2184 Frame shift 1 p.R785X E13 C to T at 2485 Arg to stop at 785 2 c.2752-1_2756delGGTGGCinsTTG I14a/ Deletion of GGTGGC mRNA splicing defect 2 E14b From 2752-1 to 2756 and insertion TTG c.2789+5GNA I14b G to A at 2789+5 mRNA splicing defect 6 p.S945L E15 C to Tat 2966 Ser to Leu at 945 2 c.3120+1GNA I16 G to A at 3120+1 mRNA splicing defect 5 c.3121-1GNA I16 G to A at 3121-1 mRNA splicing defect 2 c.3130delA E17a Deletion of A at 3130 Frame shift 4 p.T1036I E17a C to T at 3239 Thr to Ile at 1036 1 p.R1066C E17b C to T at 3328 Arg to Cys at 1066 1 p.L1077P E17b T to C at 3362 Leu to Pro at 1077 1 p.T1086I E17b C to T at 3389 Thr to Ile at 1086 1 p.R1162X E19 C to T at 3616 Arg to stop at 1162 2 p.K1177X E19 A to T at 3361 Lys to stop at 1177 2 c.3850-24GNA I19 G to A at 3850-24 mRNA splicing defect? Login to comment
50 Mutations were detected as follows: In a first phase, all subjects were analyzed with an amplification refractory mutation system assay (ARMS-PCR), as described by Ferrie et al. [20], detecting the following mutations: p.F508del, p.N1303K, p.G542X, c.1717-1GNA, p.R553X, p.W1282X, p.G551D, c.621+1GNT, c.I507del and p.R560T. Login to comment
66 Results A total of 69 unrelated CF patients (38 male and 31 female; aged between 2 months and 15 years) of Iranian Table 2 Genotype of CFTR genes in 53 Iranian patients Genotype Exon/intron Number of patients p.F508del/p.F508del E10/E10 10 p.F508del/p.R1162X E10/E19 2 p.F508del/p.T1036I E10/E17a 1 p.F508del/p.R1066C E10/E17b 1 p.F508del/c.1342-?_1524+?del E10/E9 1 p.S466X/p.S466X E10/E10 4 c.2183_2184delAAinsG/ c.2183_2184delAAinsG E13/E13 4 c.2183_2184delAAinsG/c.186- ?_296+?del E13/E2 1 p.N1303K/p.N1303K E21/E21 2 p.N1303K/p.S945L E21/E15 1 p.N1303K/c.1677delTA E21/E10 1 p.G542X/p.G542X E11/E11 2 p.G542X/c.2789+5GNA E11/I14b 1 c.3120+1GNA/c.3120+1GNA I16/I16 2 c.3120+1GNA/c.3121-1GNA I16 1 c.3121-1GNA/p.T1086I I16/E17b 1 c.3130delA/c.3130delA E17a/E17a 2 p.D192G/p.D192G E5/E5 1 p.R334W/p.R334W E7/E7 1 p.R334W/p.S945L E7/E15 1 p.R334W/p.L1077P E7/E17b 1 c.1525-1GNA/c.1525-1GNA I9/I9 1 p.S549R/p.S549R E11/E11 1 p.A566D/p.A566D E12/E12 1 c.1898+1GNT/c.1898+1GNT I12/I12 1 c.2576delA/p.S1455X/ E13/E24 1 c.2184insA/c.1677delTA E10/E13 1 p.R785X/p.R785X E13/E13 1 c.2752-1_2756delGGTGGCinsTTG/ c.2752-1_2756delGGTGGCinsTTG I14a/E14b 1 c.2789+5GNA/c.2789+5GNA I14b/I14b 1 p.K1177X/p.K1177X E19/E19 1 c.406-?_1716+?del/c.406-?_1716+?del E4-E10/E4-E10 1 Total 53 origin were extensively studied for the presence of mutations in the CFTR gene, for the presence of the deep intronic 3849+10 kbC→T mutation, and large deletions/ duplications. Login to comment
67 Screening of the samples for ten mutations with an ARMS-PCR assay only revealed the identification of three mutations: p.F508del was found in 25 (18.1%) alleles, p.N1303K in six (4.3%) alleles, and p.G542X in five (3.6%) alleles (Table 1), the remainder mutations in the CFTR coding region, and its exon/intron junctions, were found by sequencing and the MLPA assay, which are given in Table 1. Login to comment
90 Eight other mutations were found with a frequency greater than 2%: c.2183_2184delAAinsG (6.5%), p.S466X (5.8%), p.N1303K (4.3%), c.2789+5GNA (4.3%), p.G542X (3.6%), c.3120+ 1GNA (3.6%), p.R334W (2.9%), and c.3130delA (2.9%). Login to comment
142 The p.G542X mutation accounts for 2.4% of the CFTR mutations worldwide [13,21]. Login to comment
144 Two patients carried p.G542X in homozygous state, of which one was consanguineous, and one was compound heterozygous with c.2789+5GNA. Login to comment
155 Possible explanations for failure to detect all mutations are: the mutations that are in intron sequences far from coding Table 3 CFTR mutation panel recommended for screening in Iranian CF patients Mutation Number of chromosomes Frequency p.F508del 25 18.1% c.2183_2184delAAinsG 9 6.5% p.S466X 8 5.8% p.N1303K 6 4.3% c.2789+5GNA 6 4.3% p.G542X 5 3.6% c.3120+1GNA 5 3.6% p.R334W 4 2.9% c.3130delA 4 2.9% Total 72 52.0% Table 4 Clinical features and some polymorphisms in 7 Iranian patients; in these patients a mutation could only be found on one CFTR gene Genotype PI/PS Sweat (Cl- ) TGm Tn (In8) GATT (In6a) 1001+11 (In6b) M470V p.K68E/U⁎ PI 80 TG10-T7_TG10-T7 GATT 7/7 C A c.406-8TNC/U PI 50 TG12-T7_TG11-T7 GATT 6/7 C A/G c.406-3TNC/U PI 90 TG11-T7_TG11-T7 GATT 7/7 C G p.R170H/U PS 80 TG11-T7_TG10-T7 GATT 7/7 C A/G c.3850-24GNA/U PI 55 TG11-T7_TG11-T7 GATT 7/7 C G c.2789+5GNA/U PI 50 TG11-T7_TG10-T7 GATT 7/7 C A/G c.2043delG/U PS 70 TG12-T7_TG10-T7 GATT 6/7 C A ⁎Unknown mutations; PS, indicates pancreatic sufficient; PI, pancreatic sufficient. Login to comment
65 Results A total of 69 unrelated CF patients (38 male and 31 female; aged between 2 months and 15 years) of Iranian Table 2 Genotype of CFTR genes in 53 Iranian patients Genotype Exon/intron Number of patients p.F508del/p.F508del E10/E10 10 p.F508del/p.R1162X E10/E19 2 p.F508del/p.T1036I E10/E17a 1 p.F508del/p.R1066C E10/E17b 1 p.F508del/c.1342-?_1524+?del E10/E9 1 p.S466X/p.S466X E10/E10 4 c.2183_2184delAAinsG/ c.2183_2184delAAinsG E13/E13 4 c.2183_2184delAAinsG/c.186- ?_296+?del E13/E2 1 p.N1303K/p.N1303K E21/E21 2 p.N1303K/p.S945L E21/E15 1 p.N1303K/c.1677delTA E21/E10 1 p.G542X/p.G542X E11/E11 2 p.G542X/c.2789+5GNA E11/I14b 1 c.3120+1GNA/c.3120+1GNA I16/I16 2 c.3120+1GNA/c.3121-1GNA I16 1 c.3121-1GNA/p.T1086I I16/E17b 1 c.3130delA/c.3130delA E17a/E17a 2 p.D192G/p.D192G E5/E5 1 p.R334W/p.R334W E7/E7 1 p.R334W/p.S945L E7/E15 1 p.R334W/p.L1077P E7/E17b 1 c.1525-1GNA/c.1525-1GNA I9/I9 1 p.S549R/p.S549R E11/E11 1 p.A566D/p.A566D E12/E12 1 c.1898+1GNT/c.1898+1GNT I12/I12 1 c.2576delA/p.S1455X/ E13/E24 1 c.2184insA/c.1677delTA E10/E13 1 p.R785X/p.R785X E13/E13 1 c.2752-1_2756delGGTGGCinsTTG/ c.2752-1_2756delGGTGGCinsTTG I14a/E14b 1 c.2789+5GNA/c.2789+5GNA I14b/I14b 1 p.K1177X/p.K1177X E19/E19 1 c.406-?_1716+?del/c.406-?_1716+?del E4-E10/E4-E10 1 Total 53 origin were extensively studied for the presence of mutations in the CFTR gene, for the presence of the deep intronic 3849+10 kbC࢐T mutation, and large deletions/ duplications. Login to comment
89 Eight other mutations were found with a frequency greater than 2%: c.2183_2184delAAinsG (6.5%), p.S466X (5.8%), p.N1303K (4.3%), c.2789+5GNA (4.3%), p.G542X (3.6%), c.3120+ 1GNA (3.6%), p.R334W (2.9%), and c.3130delA (2.9%). Login to comment
141 The p.G542X mutation accounts for 2.4% of the CFTR mutations worldwide [13,21]. Login to comment
143 Two patients carried p.G542X in homozygous state, of which one was consanguineous, and one was compound heterozygous with c.2789+5GNA. Login to comment
154 Possible explanations for failure to detect all mutations are: the mutations that are in intron sequences far from coding Table 3 CFTR mutation panel recommended for screening in Iranian CF patients Mutation Number of chromosomes Frequency p.F508del 25 18.1% c.2183_2184delAAinsG 9 6.5% p.S466X 8 5.8% p.N1303K 6 4.3% c.2789+5GNA 6 4.3% p.G542X 5 3.6% c.3120+1GNA 5 3.6% p.R334W 4 2.9% c.3130delA 4 2.9% Total 72 52.0% Table 4 Clinical features and some polymorphisms in 7 Iranian patients; in these patients a mutation could only be found on one CFTR gene Genotype PI/PS Sweat (Cl- ) TGm Tn (In8) GATT (In6a) 1001+11 (In6b) M470V p.K68E/UÌe; PI 80 TG10-T7_TG10-T7 GATT 7/7 C A c.406-8TNC/U PI 50 TG12-T7_TG11-T7 GATT 6/7 C A/G c.406-3TNC/U PI 90 TG11-T7_TG11-T7 GATT 7/7 C G p.R170H/U PS 80 TG11-T7_TG10-T7 GATT 7/7 C A/G c.3850-24GNA/U PI 55 TG11-T7_TG11-T7 GATT 7/7 C G c.2789+5GNA/U PI 50 TG11-T7_TG10-T7 GATT 7/7 C A/G c.2043delG/U PS 70 TG12-T7_TG10-T7 GATT 6/7 C A Ìe;Unknown mutations; PS, indicates pancreatic sufficient; PI, pancreatic sufficient. Login to comment

[hide] CFTR mutations in the Algerian population. J Cyst Fibros. 2008 Jan;7(1):54-9. Epub 2007 Jun 14. Loumi O, Ferec C, Mercier B, Creff J, Fercot B, Denine R, Grangaud JP
CFTR mutations in the Algerian population.
J Cyst Fibros. 2008 Jan;7(1):54-9. Epub 2007 Jun 14., [PMID:17572159]

Abstract [show]
The nature and frequency of the major CFTR mutations in the North African population remain unclear, although a small number of CFTR mutation detection studies have been done in Algeria and Tunisia, showing largely European mutations such as F508del, G542X and N1303K, albeit at different frequencies, which presumably emerged via population admixture with Caucasians. Some unique mutations were identified in these populations. This is the first study that includes a genetic and clinical evaluation of CF patients living in Algeria. In order to offer an effective diagnostic service and to make accurate risk estimates, we decided to identify the CFTR mutations in 81 Algerian patients. We carried out D-HPLC, chemical-clamp denaturing gradient gel electrophoresis, multiplex amplification analysis of the CFTR gene and automated direct DNA sequencing. We identified 15 different mutations which account for 58.5% of the CF chromosomes. We used a quantitative PCR technique (quantitative multiplex PCR short fragment fluorescence analysis) to screen for deletion/duplication in the 27 exons of the gene. Taking advantage of the homogeneity of the sample, we report clinical features of homozygous CF patients. As CFTR mutations have been detected in males with infertility, 46 unrelated Algerian individuals with obstructive azoospermia were also investigated.

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0 CFTR mutations in the Algerian population O. Loumia,⁎, C. Ferecb,⁎, B. Mercier b , J. Creff b , B. Fercot b , R. Denine c , J.P. Grangaudd a Faculté des Sciences Biologiques, Université des Sciences et de la Technologie Houari Boumediene, Bab-Ezzouar Alger, Algérie b INSERM U613, Laboratoire de Génétique Moléculaire, 46 rue Félix Le Dantec - 29200 Brest, France c Hôpital ISSAD HASANI Beni-messous, Laboratoire de Biochimie, Algérie d Faculté de Médecine, Université d`Alger, Algérie Received 22 March 2006; received in revised form 19 April 2007; accepted 24 April 2007 Available online 14 June 2007 Abstract The nature and frequency of the major CFTR mutations in the North African population remain unclear, although a small number of CFTR mutation detection studies have been done in Algeria and Tunisia, showing largely European mutations such as F508del, G542X and N1303K, albeit at different frequencies, which presumably emerged via population admixture with Caucasians. Login to comment
58 The V754M (G to A at position 2392) mutation has previously been reported to the Cystic Fibrosis Genetic Analysis Consortium by Roger Mountford and seems to confer moderate disease when it is associated either with 1812-1G→A or G542X. Login to comment
90 Table 1 CFTR mutations detected in 36 Algerian patients (N=72 CF chromosomes) Mutations Substitution nucleotide Substitution amino acid Localisation N % Cum. fr. hF508del del CTT Del phe 507/508 Exon 10 12 16.7 16.7 N1303K C→G 4041 Asn→Lys 1303 Exon 21 6 8.3 25.0 711+1G→T G→T711+1 MRNA splicing defect Intron 5 6 8.3 33.3 2183AA/G del A2184 Frameshift Exon 13 3 4.2 37.5 A→G 2183 1609delCA delCA Frameshift Exon 10 2 2.8 40.3 1812-1G→A G→A 1812-1 mRNA splicing defect Intron 11 2 2.8 43.1 V562I G→A 1816 Val→Ile 562 Exon 12 2 2.8 45.9 V754M G→A 2392 Val→Met 754 Exon 13 1 1.4 47.3 W1282X G→A 3978 Trp→Stop 1282 Exon 20 3 4.2 51.5 621+3A/Ga A→G 621+3 mRNA splicing defect Intron 4 1 1.4 52.9 4332delTGa delTG4332 Frameshift Exon 23 G542X G→T 1756 Gly→Stop 542 Exon 11 1 1.4 54.3 4271delC del A 4271 Frameshift Exon 23 1 1.4 55.7 S977F C→T 3062 Ser→Phe 97 Exon 16 1 1.4 57.1 21Kb del 21-kb del Del AA E2-E3 1 1.4 58.5 R74W C→T 352 Arg→Trp 74 Exon 3 0 0 D1270N G→A 3940 Asp→Asn 1270 Exon 20 0 0 Total 43 58.5 N=number of chromosomes; Cum. fr.=cumulative frequency. Login to comment
135 Surprisingly, none of the defined mutations (R553X, G551D, 1717-1G→A, G542X), which occur relatively frequently in exon 11 in Caucasian populations, was identified in our Algerian population except the G542X that has been identified once. Login to comment
140 The 21-kb deletion seems to confer severe disease when it is associated with the G542X mutation. Login to comment

[hide] ENaCbeta and gamma genes as modifier genes in cyst... J Cyst Fibros. 2008 Jan;7(1):23-9. Epub 2007 Jun 7. Viel M, Leroy C, Hubert D, Fajac I, Bienvenu T
ENaCbeta and gamma genes as modifier genes in cystic fibrosis.
J Cyst Fibros. 2008 Jan;7(1):23-9. Epub 2007 Jun 7., [PMID:17560176]

Abstract [show]
BACKGROUND: Clinical phenotype varies among cystic fibrosis (CF) patients with identical CF transmembrane conductance regulator (CFTR)genotype, suggesting that genetic modifiers exist. Transgenic mice that overexpress SCNN1beta present CF-like lung disease symptoms. Mutations or variants in SCNN1beta may therefore potentially modulate the clinical phenotype in CF patients. METHODS: We analysed by DHPLC SCNN1beta and SCNN1gamma genes in 56 patients with classical CF. Patients were classified into two groups according to their CFTR genotype and their severity: 38 patients with severe genotype and an unexpectedly mild lung phenotype, and 18 patients with mild genotype and a severe lung phenotype. RESULTS: We found 3 patients out of 56 carrying at least one missense mutation. Two were novel (p.Thr313Met in SCNN1beta, p.Leu481Gln in SCNN1gamma) and two were previously described (p.Gly589Ser in SCNN1beta and p.Val546Ileu in SCNNgamma). p.Thr313Met has been identified in a CF patient with mild genotype and severe lung phenotype suggesting that it could act in increasing ENaC activity. The three other variants have been identified in CF patients with severe genotype and mild lung phenotype suggesting that they might decrease ENaC activity. However, the function of ENaC in the nasal epithelia of these patients, evaluated by nasal potential difference measurements, did not support the fact that these variants were functional, at least in nasal epithelium. CONCLUSION: Our results suggest that genetic variants in ENaCbeta and gamma genes do not modulate disease severity in the majority of CF patients.

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72 Twenty-one were homozygous for the Phe508del mutation and 17 were compound heterozygous or homozygous for two severe mutations (R553X:1717-1GNA, Phe508del:W1282X, Phe508del:1717-1GNA, 2 Phe508del:3659delC, Phe508del:N1303K, Phe508del:W57X, Phe508del:Q1411X, G542X:1380insT, Phe508del:R553X, Table 1 Parameters for amplification of the ENaCβ and ENaCγ gene fragments (GenBank accession number NM_000336 and NM_001039, respectively) Fragment Sequence of primers Annealing temperature (°C) ENaCβ Exon 2 2F 5' gtgtcccagctgatgtgcgt 3' 55 2R 5' tgaggccagctgtgcactcc 3' Exon 3 3F 5' acagactactatggagtggg 3' 55 3R 5' aagaaacacccatcagcctc 3' Exon 4 4F 5' gtcctgctagcagctcccac 3' 59 4R 5' caaccgtaacatgccactgt 3' Exon 5 5F 5' ctgccctgcagctgatgctg 3' 55 5R 5' ccctgcaacagctgatggtc 3' Exon 6 6F gtctcctttctgcctcagga 3' 59 6R 5' tcagaccctctaggactgcc 3' Exon 7 7F 5' aggtgcagaaagggcttcct 3' 63 7R 5' catgaggcgtgcaccaccttcccac 3' Exon 8 8F 5' ctgaccatgcctgtgttctc 3' 59 8R 5' ctctatggtcagagcctctg 3' Exon 9-10 9F 5' cagaggctcagcagggaaca 3' 63 10R 5' catcttatgcccagacttgt 3' Exon 11 11F 5' gatgctgcagatggcaactt 3' 55 11R 5' gagctgtcctgtgtccaaac 3' Exon 12 12F 5' acattagtcccggcccttct 3' 55 12R 5' ggtattgggagactcctaaa 3' Exon 13 13F 5' fgaggcaagaatgtgtggcct 3' 59 13R 5' tcttggctgctcagtgagtt 3' ENaCγ Exon 2 2F 5' agcacgcccgtcctcagagt 3' 57 2R 5' ccagtgtgtcactttcggga 3' Exon 3 3F 5' tgaggctgacacgtgttgat 3' 55 3R 5' tgcccctaagcagtgaaaga 3' Exon 4 4F 5' agtagcgataggaccgatgg 3' 55 4R 5' tcagagctgccagtccttag 3' Exon 5 5F 5' cccaacttcagctaagatgc 3' 55 5R 5' agatctccttggcacaggtt 3' Exon 6 6F 5' ttggatcacagcaggttgtc 3' 55 6R 5' gatctgttctctccaagcct 3' Exon 7 7F 5' ctgtctggtgctccttgcaa 3' 55 7R 5' ccagcttagatataactttg 3' Exon 8 8F 5' tgagcaaagacatgaatggc 3' 57 8R 5' agtgcctattgccaggacta 3' Exon 9-10-11 9F 5' tccaaagctcatgctgccct 3' 57 11R 5' acagaggaacagggtagagg 3' Exon 12 12F 5' ggatgccaaggctcttgatt 3' 52 12R 5' gccaggaagatgctcacatt 3' Exon 13 13F 5' aggttcctcttgatggtgt 3' 55 13R 5' ggtcctgactagatctgtct 3' Table 2 Parameters for dHPLC conditions Fragment Temperature (°C) ENaCβ Exon 2 62.3/63.3 Exon 3 59.5/60.7 Exon 4 62.2/63.4 Exon 5 59.5/61 Exon 6 63 Exon 7 61.6/62.6/63.6 Exon 8 62.8/64.8 Exon 9-10 61.5/62.5/65 Exon 11 61/62/63.5 Exon 12 69 Exon 13 61/63.3/64.8 ENaCγ Exon 2 60.8/63.2/66 Exon 3 61/61.4 Exon 4 60.6 Exon 5 59.5/60.5 Exon 6 56.5/59/60.5 Exon 7 63/63.6 Exon 8 59.5/63 Exon 9-10-11 60.7/61.5/62.7/64.7 Exon 12 59.5/61.7 Exon 13 61/62.2 Phe508del:I507del, Phe508del:4382delA, S549R:3120+ 1GNA, Phe508del:3120+1GNA, Y122X:Y122X; Phe508del:W846X; Phe508del:E60X). Login to comment
74 Their genotypes were: 2 G542X:3849+10kbCNT, Phe508del:3276-26ANG, Phe508del:A455E, 297-3CNT:W361R, S1251N:3849+10kbCNT, Phe508del:G178R, Phe508del:3849+10kbCNT, Phe508del:A561E, Phe508del: G1061R, 1717-1GNA:3272-26ANG, 2 Phe508del:R347P, Phe508del:G85E, L227R:L227R, R300G:3007delG, Phe508del:R347H, Phe508del:G1244E. Login to comment

[hide] High incidence of the CFTR mutations 3272-26A-->G ... J Cyst Fibros. 2007 Nov 30;6(6):371-5. Epub 2007 May 3. Storm K, Moens E, Vits L, De Vlieger H, Delaere G, D'Hollander M, Wuyts W, Biervliet M, Van Schil L, Desager K, Nothen MM
High incidence of the CFTR mutations 3272-26A-->G and L927P in Belgian cystic fibrosis patients, and identification of three new CFTR mutations (186-2A-->G, E588V, and 1671insTATCA).
J Cyst Fibros. 2007 Nov 30;6(6):371-5. Epub 2007 May 3., [PMID:17481968]

Abstract [show]
We have analyzed 143 unrelated Belgian patients with a positive diagnosis of cystic fibrosis (CF) for mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. An initial screening for 29 CFTR mutations led to mutation identification in 89.9% of the tested chromosomes. Subsequently an extensive analysis of the CFTR gene was performed by denaturating gradient gel electrophoresis (DGGE) in those patients with at least one unknown mutation after preliminary screening. In addition to 10 previously reported mutations we identified 2 new mutations 186-2A-->G and E588V. A third new mutation 1671insTATCA was identified during routine screening for DeltaF508. Two mutations were detected with a higher frequency than expected: 3272-26A-->G, which is the second most common mutation after DeltaF508 in our CF population with a frequency of 3.8%, and L927P (2.4%). The clinical data is presented for the mutations 186-2A-->G, E588V, 3272-26A-->G and L927P. The mutation data are useful for the Belgian population to supplement the initial screening set of mutations.

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31 The Inno Lipa™ CFTR12 assay contains normal and mutant probes for 12 different CFTR mutations (ΔF508, G542X, N1303K, 1717-1G→A, W1282X, G551D, R553X, S1251N, R560T, 3905insT, Q552X, ΔI507). Login to comment
60 L927P (together with G542X and S1251N) is the fourth most common CFTR mutation, with a frequency of 2.4%. Login to comment

[hide] Highly preferential association of NonF508del CF m... J Cyst Fibros. 2007 Jan;6(1):15-22. Epub 2006 Jun 19. Ciminelli BM, Bonizzato A, Bombieri C, Pompei F, Gabaldo M, Ciccacci C, Begnini A, Holubova A, Zorzi P, Piskackova T, Macek M Jr, Castellani C, Modiano G, Pignatti PF
Highly preferential association of NonF508del CF mutations with the M470 allele.
J Cyst Fibros. 2007 Jan;6(1):15-22. Epub 2006 Jun 19., [PMID:16784904]

Abstract [show]
BACKGROUND: On the basis of previous findings on random individuals, we hypothesized a preferential association of CF causing mutations with the M allele of the M470V polymorphic site of the CFTR gene. METHODS: We have determined the M/V-CF mutation haplotype in a series of 201 North East Italian and 73 Czech CF patients who were not F508del homozygotes, as F508del was already known to be fully associated with the M allele. RESULTS: Out of 358 not F508del CF genes, 84 carried the V allele and 274 the less common M allele. In the N-E Italian population, MM subjects have a risk of carrying a CF causing mutation 6.9x greater than VV subjects when F508del is excluded and 15.4x when F508del is included. In the Czech population a similar, although less pronounced, association is observed. CONCLUSIONS: Besides the possible biological significance of this association, the possibility of exploiting it for a pilot screening program has been explored in a local North East Italian population for which CF patients were characterized for their CF mutation. General M470V genotyping followed by common CF mutation screening limited to couples in which each partner carries at least one M allele would need testing only 39% of the couples, which contribute 89% of the total risk, with a cost benefit.

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62 In particular, 12 Italian and the 3 Czech CF patients carried the F508del (that has been unambiguously assigned to the M allele 118/118 and 57/57 times in the present two samples, respectively, in agreement with previous reports); 1 carried the 2183AAYG (21/21); 1 carried the I507del (7/7), 1 carried the 1717- 1GYA (20/20) and 1 carried the G542X (19/19). Login to comment
121 Table 5 shows the estimated residual Table 4 CF mutations found in the 53 CF patients of the Bolzano province CF mutation Absolute and relative (%) frequencies Associated with(1) F508del 56 (52.8) M 711+5 G>A 10 (9.4) M R347P 3 (2.8) V S466X 1 (0.9) M 1717-1 G>A 1 (0.9) M G542X 1 (0.9) M G551D 2 (1.9) V 1874insT 1 (0.9) V 2183AA>G 3 (2.8) M 2789+5G>A 1 (0.9) M R1162X 24 (22.6) M N1303K 2 (1.8) M (1) Based on data of Table 1. Login to comment

[hide] Mutational spectrum of cystic fibrosis patients fr... Mol Genet Metab. 2006 Apr;87(4):370-5. Epub 2006 Jan 19. Ramirez AM, Ramos MD, Jimenez J, Ghio A, de Botelli MM, Rezzonico CA, Marques I, Pereyro S, Casals T, de Kremer RD
Mutational spectrum of cystic fibrosis patients from Cordoba province and its zone of influence: implications of molecular diagnosis in Argentina.
Mol Genet Metab. 2006 Apr;87(4):370-5. Epub 2006 Jan 19., [PMID:16423550]

Abstract [show]
Cystic Fibrosis (CF) is an autosomal recessive disorder affecting 1/2000-4000 newborns in Caucasian populations. This lethal disease mainly affects respiratory and digestive organs as well as fertility in man. So far, the CF prevalence and mutational spectrum have showed specificity among populations and regions, making it necessary to establish them in each one. In this study, we present the spectrum and frequency of CFTR gene mutations in CF patients from Cordoba (a province with 3.1 millions inhabitants in the middle of Argentina) and its zone of influence, to offer an accurate genetic testing. The study includes 78 families in which 98 patients fulfilled clinical criteria to CF diagnosis. The strategy for the molecular diagnosis comprised analysis of 21 common mutations, microsatellite haplotypes and the complete CFTR gene analysis using scanning techniques followed by sequencing of the abnormal migration patterns. Our first step led us to the identification of 10 mutations that represented 76% of alleles. Another four mutations (p.R1066C, c.1811 + 1.6 kbA > G, c.711 + 1G > T, and p.G85E) were found based on the microsatellite haplotype-mutation association. Finally, 14 mutations were characterized after the CFTR gene scanning, three of them are not previously described (p.G27R, c.622-2A > G, and p.W277R). In summary, we have identified 27 mutations accounting for 94.23% of CF alleles. This characteristic mutational spectrum highlights the 14 most frequent mutations (>1%) in the Cordoba region.

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16 The most frequent mutations worldwide (p.F508del, p.G542X, p.G551D, p.N1303K, and p.W1282X) have shown considerable diVerences in their frequencies depending on ethnic origin and geographic areas. Login to comment
44 Mutations (p.F508del, p.N1303K, p.G542X, p.R334W, c.2789 + 5G > A, c.3659delC, p.R553X, c.3849 + 10kbC > T, p.R1162X, c.621 + 1G > T, p.W1282X, p.R117H, c.1078delT, p.E60X, p.R347P, p.A455E, p.I507del, c.1717-1G > A, p.G551D, [c.2183A > G; c.2184delA] and p.S1251N) were analyzed by heteroduplex analysis on polyacrylamide gel electrophoresis [11,12] and by ampliWcation refractory mutation system [13] in all 78 patients. Login to comment
85 Haplotype (n D 20) No. of chromosomes (n D 64)a Mutations associated (No. of chromosomes) 23-31 14 p.F508del 17-31 7 p.F508del 17-7 7 p.R1066C (3), p.W277R, c.2789 + 5G > A, c.3120 + 1G > A, c.3849 + 10KbC > T 16-7 6 c.3272-26A > G (2), p.G27R, c.622-2A > G, unknown (2) 16-32 5 p.S589I (2), unknown (3) 16-30 3 IVS8-5T (2), unknown 23-33 2 p.G542X, p.R1283M 23-32 2 p.G542X 23-30 2 p.F508del, p.N1303K 24-31 2 p.N1303K 16-24 2 p.G85E 16-31 3 c.1898 + 1G > A, p.W1089X, unknown 16-46 2 c.1811 + 1.6KbA > G 16-25 1 c.711 + 1G > T 16-33 1 Unknown 16-44 1 c.1898 + 1G > A 16-45 1 p.Y913C 16-47 1 c.4005 + 1G > A 17-30 1 Unknown 23-7 1 [c.3199_3204delATAGTG; p.I148T] Table 2 Frequency of the mutations in the 78 CF Argentinean patients of Córdoba region a IdentiWed novel mutations. Login to comment
86 Mutation Exon/Intron CF alleles % p.F508del Exon 10 94 60.26 p.N1303K Exon 21 8 5.13 p.G542X Exon 11 7 4.49 p.R334W Exon 7 3 1.93 p.R1066C Exon 17b 3 1.93 c.2789 + 5G > A Intron 14b 3 1.93 p.G85E Exon 3 2 1.28 c.3659del C Exon 19 2 1.28 c.1811 + 1.6kbA > G Intron 11 2 1.28 c.1898 + 1G > A Intron 12 2 1.28 c.3272-26A > G Intron 17a 2 1.28 p.S589I Exon 12 2 1.28 p.R553X Exon 11 2 1.28 IVS8-5T Intron 8 2 1.28 c.3849 + 10kb C > T Intron 19 1 0.64 c.621 + 1G > T Intron 4 1 0.64 p.R1162X Exon 19 1 0.64 c.711 + 1G > T Intron 5 1 0.64 c.3120 + 1G > A Intron 16 1 0.64 p.Y913C Exon 15 1 0.64 c.4005 + 1G > A Intron 20 1 0.64 p.W1089X Exon 17b 1 0.64 p.R1283M Exon 20 1 0.64 [p.I148T;c.3199_3204del ATAGTG] Exon 4, Exon 17a 1 0.64 p.G27Ra Exon 2 1 0.64 p.W277Ra Exon 6b 1 0.64 c.622-2A > Ga Intron4 1 0.64 Unknown allele - 9 5.77 Wrst year of life he required several internments, for hydroelectric desequilibrium and persistent pulmonary infections causing failure to thrive. Login to comment
89 Fourteen mutations have a frequency higher than 1%, p.F508del (60.26%), p.N1303K (5.13%), p.G542X (4.49%), and three mutations, p.R334W, p.R1066C, c.2789 + 5G> A (1.93%), and another eight, p.G85E, c.3659delC, c.1811 + 1.6kbA > G, c.1898 + 1G > A, c.3272-26A > G, p.S589I, p.R553X, and 5T (1.28%). Login to comment
119 Other most signiWcant diVerences were that in Buenos Aires, besides the p.F508del, only Wve mutations showed frequencies higher than 1%, being their percentages the following ones: p.F508del 58.64%, p.G542X 4.1%, p.W1282X 2.73%, p.N1303K 2.73%, and the last two ones p.R334W and c.1717-1G > A with 1.14%. Login to comment
123 In addition, it is important to denote that in our series the most frequent mutations found were p.F508del, p.N1303K, p.G542X, p.R334W, p.R1066C, and c.2789+5G>A, however, the last two ones were rare in Buenos Aires series (p.R1066C, 0.23%) and others were not found (p.S589I, c.3272-26A>G, c.1898+1G>A, c.711+1G>T, c.3199_ 3204delATAGTG, p.W1089X, p.R1283M, p.Y913C, c.4005+1G>A, c.3120 +1G >A, p.G27R, p.W277R, and c.622-2A>G). Login to comment
117 Other most signiWcant diVerences were that in Buenos Aires, besides the p.F508del, only Wve mutations showed frequencies higher than 1%, being their percentages the following ones: p.F508del 58.64%, p.G542X 4.1%, p.W1282X 2.73%, p.N1303K 2.73%, and the last two ones p.R334W and c.1717-1G > A with 1.14%. Login to comment
121 In addition, it is important to denote that in our series the most frequent mutations found were p.F508del, p.N1303K, p.G542X, p.R334W, p.R1066C, and c.2789+5G>A, however, the last two ones were rare in Buenos Aires series (p.R1066C, 0.23%) and others were not found (p.S589I, c.3272-26A>G, c.1898+1G>A, c.711+1G>T, c.3199_ 3204delATAGTG, p.W1089X, p.R1283M, p.Y913C, c.4005+1G>A, c.3120 +1G >A, p.G27R, p.W277R, and c.622-2A>G). Login to comment

[hide] Molecular screening of CFTR gene in Brazilian men ... Hum Fertil (Camb). 2006 Mar;9(1):53-6. Bertuzzo CS, Pinto W
Molecular screening of CFTR gene in Brazilian men with bilateral agenesis of the vas deferens.
Hum Fertil (Camb). 2006 Mar;9(1):53-6., [PMID:16581722]

Abstract [show]
Infertility is a common symptom of cystic fibrosis, especially in men (95% become sterile). It is caused by blockage of the vas deferens and the epididymis, which result in degeneration of the tubules. The purpose of this study was to verify the frequency of CFTR gene mutation in patients with bilateral agenesis of the vas deferens using SSCP and sequencing. The study population consisted of 40 white individuals with agenesis of the vas deferens as well as their 12 siblings without agenesis of the vas deferens. CTFR gene mutation was found in 22 of the 40 patients (55%) and it was possible to detect both mutating alleles in these 22 patients. The most frequent genotype found was ?F508/IVS8-5T. There was no genotype concordance in siblings. Our results show the importance of the investigation of CFTR mutation in patients with vas deferens agenesis.

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57 When we compared the mutations detected by the present study with those found in fibrocystic patients in our region, we verify that among fibrocystics the most frequent mutations are: DF508, G542X, G551D, R553X and N1303K (Bernardino et al., 2000; Martins et al., 1993; Raskin et al., 1999), while in the present study they were the DF508, IVS8-5T, R117H and N1303K. Login to comment

[hide] A haplotype framework for cystic fibrosis mutation... J Mol Diagn. 2006 Feb;8(1):119-27. Elahi E, Khodadad A, Kupershmidt I, Ghasemi F, Alinasab B, Naghizadeh R, Eason RG, Amini M, Esmaili M, Esmaeili Dooki MR, Sanati MH, Davis RW, Ronaghi M, Thorstenson YR
A haplotype framework for cystic fibrosis mutations in Iran.
J Mol Diagn. 2006 Feb;8(1):119-27., [PMID:16436643]

Abstract [show]
This is the first comprehensive profile of cystic fibrosis transmembrane conductance regulator (CFTR) mutations and their corresponding haplotypes in the Iranian population. All of the 27 CFTR exons of 60 unrelated Iranian CF patients were sequenced to identify disease-causing mutations. Eleven core haplotypes of CFTR were identified by genotyping six high-frequency simple nucleotide polymorphisms. The carrier frequency of 2.5 in 100 (1 in 40) was estimated from the frequency of heterozygous patients and suggests that contrary to popular belief, cystic fibrosis may be a common, under-diagnosed disease in Iran. A heterogeneous mutation spectrum was observed at the CFTR locus in 60 cystic fibrosis (CF) patients from Iran. Twenty putative disease-causing mutations were identified on 64 (53%) of the 120 chromosomes. The five most common Iranian mutations together represented 37% of the expected mutated alleles. The most frequent mutation, DeltaF508 (p.F508del), represented only 16% of the expected mutated alleles. The next most frequent mutations were c.1677del2 (p.515fs) at 7.5%, c.4041C>G (p.N1303K) at 5.6%, c.2183AA>G (p.684fs) at 5%, and c.3661A>T (p.K1177X) at 2.5%. Three of the five most frequent Iranian mutations are not included in a commonly used panel of CF mutations, underscoring the importance of identifying geographic-specific mutations in this population.

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32 Only four (p.G542X, p.N1303K, p.G551D, and p.W1282X) have overall frequencies greater than 1%.12 Intriguingly, p.G542X and p.N1303K are found on the same haplotype background as ⌬F508, suggesting that they arose in the same population.13 Two previous reports of CFTR mutations in Iran have been published. Login to comment
111 of Patients Total alleles* Associated haplotype Global distributionHom Het Exon 1 c.134TϾC M1T 1 1 Rare Exon 3 c.386GϾA G85E 1 1 Global Exon 4 c.460GϾC D110H 1 1 H2 Europe Exon 7 c.1132CϾT R334W 1 1 H2 Global Exon 7 c.1145CϾT T338I 1 1 Europe Intron 9 c.1525-1GϾA Mis-splicing 1 1 H8 Pakistan Exon 10 c.1529CϾG S466X 1 2 H4 Germany Exon 10 c.1531CϾT L467F 1 1 Rare Exon 10 c.1649TϾC I506T 1 2 H8 Lebanon Exon 10 c.1652del3† ⌬F508 6 7 19 H5 Global Exon 10 c.1677delTA 515fs 4 1 9 H1 Europe Exon 11 c.1756GϾT G542X 1 1 H5 Global Exon 12 c.1821CϾA Y563X 2 2 Europe Exon 13 c.2183AAϾG 684fs 3 6 H3 Europe Exon 17a c.3170CϾT P1013L 1 1 Turkey Exon 19 c.3616CϾT R1162X 2 2 H2 Germany Exon 19 c.3661AϾT K1177X 1 1 3 H2 Bahrain Intron 20 c.4005ϩ1GϾA Mis-splicing 1 2 H2 Europe Exon 21 c.4041CϾG N1303K 3 1 7 H5 Global Exon 23 c.4363CϾT Q1412X 1 1 Rare *A total of 64 (53%) of the 120 expected alleles were observed. Login to comment
175 Specifically, the four most common Turkish mutations were found in Iran, including ⌬F508, c.1677delTA, p.G542X, and c.2183AAϾG.9,29 The p.G542X "Mediterranean mutation," purported to be of Phoenician origin, was found on only one Iranian chromosome, whereas it was relatively frequent (3.6%) among the Turkish CF chromosomes.6,51 Another common mutations in Iran, p.K1177X, was not found in Turkey but was reported in Bahrain.39 In contrast, three mutations commonly found in Europe, including p.W1282X, p.G551D, and c.1717-1GϾA,12 were not found in Iran. Login to comment

[hide] Increased frequency of cystic fibrosis transmembra... Fertil Steril. 2006 Jan;85(1):135-8. Schulz S, Jakubiczka S, Kropf S, Nickel I, Muschke P, Kleinstein J
Increased frequency of cystic fibrosis transmembrane conductance regulator gene mutations in infertile males.
Fertil Steril. 2006 Jan;85(1):135-8., [PMID:16412743]

Abstract [show]
OBJECTIVE: To investigate the frequency of mutations of the cystic fibrosis transmembrane regulator (CFTR) gene in males with reduced sperm quality before intracytoplasmic sperm injection (ICSI). DESIGN: The nine most frequent cystic-fibrosis-causing mutations in the German population and IVS8T alleles were analyzed. SETTING: University-based centers for reproductive medicine and clinical genetics. PATIENT(S): An unselected group of 597 males with oligo-, astheno-, terato-, crypto-, oligoasthenoteratozoospermia, or azoospermia, which underwent pre-ICSI genetic counseling over a 5-year period. INTERVENTION(S): Blood samples were collected from the patients during genetic counseling. MAIN OUTCOME MEASURE(S): Frequency of mutations of CFTR gene in infertile males. RESULT(S): A heterozygous CFTR mutation was observed in 34 of 597 patients (5.70%). None of the patients had two CFTR mutations. Given that our mutation panel recognizes about 82% of heterozygotes, it can be assumed that the frequency of CFTR heterozygotes in our cohort is about 6.94%. The frequency of CFTR mutations in our cohort did not correlate with a reduced sperm count. CONCLUSION(S): The frequency of cystic fibrosis in the German population is 1:3300. Thus, a CFTR heterozygosity of 3.42% can be estimated. This indicates that in our cohort of infertile males, the frequency of CFTR heterozygosity is twofold higher than in the general population (P<.0001).

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45 Mutations R117H, R347P, G542X, G551D, R553X, 3849ϩ10kbCϾT, and N1303K were analyzed by PCR and restriction enzyme cleavage. Login to comment
47 Among CFTR mutations detected in the German population, F508del, R117H, R347P, G542X, G551D, R553X, 3849ϩ10kbCϾT, N1303K, and CFTR2,3dele(21kb) occur with a frequency of 72%, 1%, 1.2%, 1.2%, 0.9%, 2%, 1%, 1.8%, and 1.2%, respectively (9-11). Login to comment

[hide] Cystic fibrosis mutations and genotype-pulmonary p... J Cyst Fibros. 2006 Jan;5(1):33-41. Epub 2005 Nov 4. Braun AT, Farrell PM, Ferec C, Audrezet MP, Laxova A, Li Z, Kosorok MR, Rosenberg MA, Gershan WM
Cystic fibrosis mutations and genotype-pulmonary phenotype analysis.
J Cyst Fibros. 2006 Jan;5(1):33-41. Epub 2005 Nov 4., [PMID:16275171]

Abstract [show]
BACKGROUND: Although there are more than 1000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, most of them are uncommon and only limited information exists regarding genotype-pulmonary phenotype relationships. METHODS: We determined and classified the CFTR mutations using denaturing high-performance liquid chromatography and developed new, quantitative methods to categorize pulmonary phenotypes. RESULTS: Two novel alleles were discovered, namely G1047R and 1525-2A-->G, which were accompanied by F508del and G551D mutations, respectively. Assessment of numerous options revealed that CF pulmonary phenotype categorization in children cannot be accomplished with clinical or pulmonary function data but is facilitated by longitudinal quantitative chest radiology. It was most useful to categorize pulmonary disease status by evaluating the typical pattern of abnormalities in patients homozygous for the F508del mutation, and then compare patients with minor mutations to this typical CF pulmonary phenotype. By this method, both patients with novel mutations have pulmonary phenotypes typical of F508del homozygotes. However, patients with class IV mutations (e.g., R347P) or with pancreatic sufficiency showed serial chest radiographs that were atypically mild. CONCLUSIONS: Longitudinal quantitative chest radiography provides a new strategy for CF pulmonary phenotype categorization that should be useful for genotype-phenotype delineation in individual patients and in both epidemiologic studies and clinical trials involving groups of children with CF.

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80 Thereafter, the longitudinal patterns of WCXR and BCXR for the two patients with novel mutations (i.e., G1047R and 1525-2AYG) were superimposed on the Table 1 Summary of patient characteristics Characteristics F508del homozygote group (n =38) Pancreatic sufficiency groupa (n =19) Sex Male 25 8 Female 13 11 Center Madison 21 12 Milwaukee 17 7 Group Screened 38 3 Control 0 14 Other 0 2 Meconium ileus Yes 6 0 No 32 19 Mean age at diagnosis (weeks)TS.D. 7.15T2.4 193.1T192 Mean sweat Cl mEq/lTS.D. Login to comment
81 101.0T9.5 83.5T21.2 CXR scores at diagnosis WCXR 2.48T32b 4.68T71 BCXR 21.9T0.3 21.1T.48 Pulmonary function at 8 years FEV1 (%)c 97T4 104T2 FVC (%)c 103T3 103T2 FEV1/FVC% 0.92T0.03 0.98T.01 FEF25 - 75% 99T11 104T5 a Mild pancreatic phenotype mutations include: R117H occurring with F508del (n =5) and G542X (n =1); R117C with F508del (n =2); R347P with F508del (n =1), R1066H (n =1) and 2184insA (n À1), 2789+5G>A with F508del (n =3); 3272À26A>G with F508del (n =1); 3849+10kbC>T with F508del (n =1); L138ins with 3272À26A>G (n =1); R352Q with F508del (n =1); and 1336K with F508del (n =1). Login to comment

[hide] Detection of CFTR mutations using ARMS and low-den... Biosens Bioelectron. 2005 Dec 15;21(6):933-9. Eaker S, Johnson M, Jenkins J, Bauer M, Little S
Detection of CFTR mutations using ARMS and low-density microarrays.
Biosens Bioelectron. 2005 Dec 15;21(6):933-9., [PMID:15890513]

Abstract [show]
The amplification refractory mutation system (ARMS) is routinely used for the identification of specific mutations within genomes. This PCR-based assay, although simple, is performed at a low-throughput scale, usually requiring gel-electrophoresis for the identification of specific mutations. We have applied the ARMS technology to a low-density microarray system to facilitate the needs of the medical clinic; high-throughput capabilities and ease-of-use. Mutations within the cystic fibrosis transmembrane regulator (CFTR) gene (DeltaF508, 1717-1G>A, G542X, 621+1G>T, and N1303K) were detected by multiplex-ARMS-PCR, and fragments were post-PCR labeled with Cy5. Amine-modified probes specific for both the wild-type and mutant forms of each mutation site were attached to glass substrates. Following hybridization of the PCR fragments to the attached probes (in a low-density microarray format), confirmation of the presence of specific sequences was achieved using a commercial scanner, as well as a fabricated low-cost fluorescent detector and applicable software. The novel combination of the ARMS and low-density microarray technologies allows for a high-throughput, simple means to rapidly identify multiple known mutations for many genetic diseases including cystic fibrosis.

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3 Mutations within the cystic fibrosis transmembrane regulator (CFTR) gene ( F508, 1717-1G>A, G542X, 621+1G>T, and N1303K) were detected by multiplex-ARMS-PCR, and fragments were post-PCR labeled with Cy5. Login to comment
88 Each mutation on the panel ( F508, 1717-1G>A, G542X, 621+1G>T, and N1303K) was tested individually (Fig. 1), and in a multiplex reaction containing all five primer pairs (Fig. 1B, lanes 8 and 9). Login to comment
97 PCR reactions containing various DNA templates to evaluate the performance of the ARMS reaction to detect the specific mutations are listed as follows: (A2) wild-type (wt); (A3) N1303K; (A4) wt; (A5) 621+1G>T; (A7) wt; (A8) G542X; (B1) wt; (B2) F508; (B3) wt; (B4) W1282X; (B6) wt; (B7) 1717-1G>A; (B8) wt; (B9) multiplex F508 and W1282X (using F508/W1282X compound heterozygous DNA template). Login to comment
98 Table 1 Probe sequences specific for each mutation, 5 -amine-modified with C6 spacers Mutant probe Sequence 621+1G>T TTT GAT TTA TAA GAA GTT AAT ACT TCC TTG CAC AGG F508 GGC ACC ATT AAA GAA AAT ATC ATT GGT GTT TCC TA 1717-1G>A CTA TTT TTG GTA ATA AGA CAT CTC CAA GTT TGC AG G542X ATA GTT CTT TGA GAA GGT GGA ATC ACA CTG N1303K TAG AAA AAA GTT GGA TCC CTA TGA ACA GTG G W1282X TTT GCA ACA GTG AAG GAA AGC CTT T To each array/glass slide, Cy5-labeled PCR products were hybridized to each array/glass slide, and the fluorescence measured by both a commercial scanner and an inexpensive detection device designed in-house (Fig. 2). Login to comment
101 Each spot is 1 mm in diameter and contains: (1) F508; (2) 1717-1G>A; (3) N1303K; (4) 621+1G>T; (5) W1282X; (6) G542X; and (R) reference spot containing amino-linked 15-mer with a terminal Cy5 label (seen as a smear in B and E due to bleaching of the scanner. Login to comment
102 Each box represents a separate hybridization with the PCR products from ARMS reactions containing known DNA: (A) wt; (B) 1717-1G>A; (C) N1303K; (D) 621+1G>T; (E) F508; (F) G542X. Login to comment
105 After establishing a baseline fluorescence for each slide, ARMS-products from F508, 1717-1G>A, G542X, 621+1G>T, and N1303K DNA templates were hybridized, and detection on each corresponding spot was achieved, leaving the other mutant spots unbound and unlabeled. Login to comment
107 Fig. 2F shows that the ARMS product from G542X DNA bound both to the G542X probe as well as the 1717-1G>A probe. Login to comment
108 This arose due to the fact that the G542X ARMS primers overlap the 1717-1G>A region. Login to comment
109 However, the 1717-1G>A ARMS primers do not overlap the G542X sequence, and does not produce a product which binds to the G542X probe (Fig. 2B). Login to comment
121 (A) and (B) show the readings (screen shots) using slides from Fig. 2(A) N1303K and (B), G542X. Login to comment
143 Multiple spots can be displayed as well, seen in Fig. 4B using the G542X slide from Fig. 2F (reference, blue bar; 1717-1G>A, green bar; G542X, yellow bar). Login to comment
142 Multiple spots can be displayed as well, seen in Fig. 4B using the G542X slide from Fig. 2F (reference, blue bar; 1717-1G>A, green bar; G542X, yellow bar). Login to comment

[hide] Spectrum of mutations in CFTR in Finland: 18 years... J Cyst Fibros. 2005 Dec;4(4):233-7. Epub 2005 Jul 26. Kinnunen S, Bonache S, Casals T, Monto S, Savilahti E, Kere J, Jarvela I
Spectrum of mutations in CFTR in Finland: 18 years follow-up study and identification of two novel mutations.
J Cyst Fibros. 2005 Dec;4(4):233-7. Epub 2005 Jul 26., [PMID:16051530]

Abstract [show]
BACKGROUND: The incidence of cystic fibrosis (CF) is low in the isolated Finnish population and the Finnish CF mutation spectrum has differed from many European countries. METHODS: We have analyzed the mutation spectrum and the geographical distribution of CF mutations in Finland covering the last 18 years (1987-2004). RESULTS: A total of 14 mutations were identified; two of them new, 774insT and S589T (G>C at 1,898). The overall coverage of mutations was 97% (99/102 chromosomes). The most frequent mutations were F508del and 394delTT, found in 36% (37/102) and 35% (36/102) of the CF chromosomes respectively. Of the rare mutations, a mutation of presumable Slavic origin, CFTRdele2.3 (21 kb), was enriched in a rural isolate with a frequency of 5,9% (6/102), and a mutation that possibly indicates Swedish influence, 3659delC, was scattered throughout the country with a similar frequency of 5,9% (6/102). G542X, R1162X, R117H, 3732delA, 1,898 + 3A >C, S1196X, S945L, W57R, 774insT and S589T were each identified in a number of chromosomes from one to three. CONCLUSIONS: Our observations of the Finnish CF mutation spectrum fit well with the characteristics of Finland as a population of multiple local founder effects.

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6 G542X, R1162X, R117H, 3732delA, 1898+3A>C, S1196X, S945L, W57R, 774insT and S589T were each identified in a number of chromosomes from one to three. Login to comment
22 Journal of Cystic Fibrosis 4 (2005) 233 - 237 www.elsevier.com/locate/jcf Only two other mutations, G542X (c.1624G>T, p.Gly542X) and 3732delA (c.3600delA, p.Asp1201fs), were each identified in one CF chromosome out of 40. Login to comment
36 The InnoLipa assay recognizes 36 mutations: E60X (c.178G>T, p.Glu60X), G85E (c.254G>A, p.Gly85- Glu), 394delTT, R117H (c.350G>A, p.Arg117His), I148T (c.443T>C, p.Ile148Thr), 621+1G>T (c.489+1G>T), 711+1G>T (c.579+1G>T), 711+5G>A (c.579+5G>A), 1078delT (c.948delT, p.Phe316fs), R334W (c.1000C>T, p.Arg334Trp), R347P (c.1040G>C, p.Arg347Pro), A455E (c.1364C>A, p.Ala455Glu), I507del (c.1519_1521delATC, p.Ile507del), F508del, 1717À1G>A (c.1585À1G>A), G542X, G551D (c.1652G >A, p.Gly551Asp), Q552X (c.1654C > T, p.Gln552X), R553X (c.1657C > T, p.Arg553X), R560T (1679G>vC, p.Arg560Thr), 1898+ 1G > A (c.1766 + 1G > A), 2143delT (c.2012delT, p.Leu671fs), 2183AA > G (c.2051_2052delAAinsG, p.Lys684fs), 2184delA (c.2052delA, p.Lys684fs), 2789+ 5G>A (c.2657+5G>A), 3120+1G>A (c.2988+1G>A), 3199del6 (c.3067_3072del, p.Ile1023_Val1024del), 3272À 26A > G (c.3140 À26A > G), R1162X (c.3484C > T, p.Arg1162X), 3849+10kbCYT, 3659delC (c.3528delC, p.Lys1177fs), S1251N (c.3752G > A, p.Ser1251Asn), 3905insT (c.3773dupT, p.Leu1258fs), W1282X (c.3846G> A, p.Trp1282X), N1303K (c.3909C>G, p.Asn1303Lys), CFTRdele2,3(21kb) and Tn-polymorphism on intron 8. Login to comment
94 394delTT has been suggested to have a Table 1 Spectrum of CFTR mutations in Finland Mutation Recommended nomenclature/nucleotide Recommended nomenclature/protein Exon/Intron N % F508del c.1520_1522delTCT p.Phe508del E 10 37 36 394delTT c.262_263delTT p.Leu88fs E 3 36 35 CFTRdele2,3(21kb) E2 and E3 6 5.9 3659delC c.3528delC p.Lys1177fs E 19 6 5.9 1898+3A>C c.1766+3A>C I 12 3 2.9 R117H c.350G>A p.Arg117His E 4 2 2 S945L c.2834C>T p.Ser945Leu E 15 2 2 W57R c.169T>C p.Trp57Arg E 3 1 1 774insT c.642_643insT p.Ile215fs E 6a 1 1 G542X c.1624G>T p.Gly542X E 11 1 1 S589T c.1766G>C p.Ser589Thr E 12 1 1 R1162X c.3484C>T p.Arg1162X E 19 1 1 S1196X c.3587C>G p.Ser1196X E 19 1 1 3732delA c.3600delA p.Asp1201fs E 19 1 1 Unknown 2.9 Total 102 100 Reference sequence is Genbank NM_000492.2. Login to comment
109 An exception is G542X, which is one of the five mutations that have relative world frequencies higher than 1%. Login to comment
23 Journal of Cystic Fibrosis 4 (2005) 233 - 237 www.elsevier.com/locate/jcf Only two other mutations, G542X (c.1624G>T, p.Gly542X) and 3732delA (c.3600delA, p.Asp1201fs), were each identified in one CF chromosome out of 40. Login to comment
37 The InnoLipa assay recognizes 36 mutations: E60X (c.178G>T, p.Glu60X), G85E (c.254G>A, p.Gly85Glu), 394delTT, R117H (c.350G>A, p.Arg117His), I148T (c.443T>C, p.Ile148Thr), 621+1G>T (c.489+1G>T), 711+1G>T (c.579+1G>T), 711+5G>A (c.579+5G>A), 1078delT (c.948delT, p.Phe316fs), R334W (c.1000C>T, p.Arg334Trp), R347P (c.1040G>C, p.Arg347Pro), A455E (c.1364C>A, p.Ala455Glu), I507del (c.1519_1521delATC, p.Ile507del), F508del, 1717 1G>A (c.1585 1G>A), G542X, G551D (c.1652G >A, p.Gly551Asp), Q552X (c.1654C > T, p.Gln552X), R553X (c.1657C > T, p.Arg553X), R560T (1679G>vC, p.Arg560Thr), 1898+ 1G > A (c.1766 + 1G > A), 2143delT (c.2012delT, p.Leu671fs), 2183AA > G (c.2051_2052delAAinsG, p.Lys684fs), 2184delA (c.2052delA, p.Lys684fs), 2789+ 5G>A (c.2657+5G>A), 3120+1G>A (c.2988+1G>A), 3199del6 (c.3067_3072del, p.Ile1023_Val1024del), 3272 26A > G (c.3140 26A > G), R1162X (c.3484C > T, p.Arg1162X), 3849+10kbCYT, 3659delC (c.3528delC, p.Lys1177fs), S1251N (c.3752G > A, p.Ser1251Asn), 3905insT (c.3773dupT, p.Leu1258fs), W1282X (c.3846G> A, p.Trp1282X), N1303K (c.3909C>G, p.Asn1303Lys), CFTRdele2,3(21kb) and Tn-polymorphism on intron 8. Login to comment
95 394delTT has been suggested to have a Table 1 Spectrum of CFTR mutations in Finland Mutation Recommended nomenclature/nucleotide Recommended nomenclature/protein Exon/Intron N % F508del c.1520_1522delTCT p.Phe508del E 10 37 36 394delTT c.262_263delTT p.Leu88fs E 3 36 35 CFTRdele2,3(21kb) E2 and E3 6 5.9 3659delC c.3528delC p.Lys1177fs E 19 6 5.9 1898+3A>C c.1766+3A>C I 12 3 2.9 R117H c.350G>A p.Arg117His E 4 2 2 S945L c.2834C>T p.Ser945Leu E 15 2 2 W57R c.169T>C p.Trp57Arg E 3 1 1 774insT c.642_643insT p.Ile215fs E 6a 1 1 G542X c.1624G>T p.Gly542X E 11 1 1 S589T c.1766G>C p.Ser589Thr E 12 1 1 R1162X c.3484C>T p.Arg1162X E 19 1 1 S1196X c.3587C>G p.Ser1196X E 19 1 1 3732delA c.3600delA p.Asp1201fs E 19 1 1 Unknown 3 2.9 Total 102 100 Reference sequence is Genbank NM_000492.2. Login to comment
110 An exception is G542X, which is one of the five mutations that have relative world frequencies higher than 1%. Login to comment

[hide] Reduced exhaled NO is related to impaired nasal po... Vascul Pharmacol. 2005 Dec;43(6):385-9. Epub 2005 Sep 22. Texereau J, Fajac I, Hubert D, Coste J, Dusser DJ, Bienvenu T, Dall'Ava-Santucci J, Dinh-Xuan AT
Reduced exhaled NO is related to impaired nasal potential difference in patients with cystic fibrosis.
Vascul Pharmacol. 2005 Dec;43(6):385-9. Epub 2005 Sep 22., [PMID:16182611]

Abstract [show]
Nitric oxide (NO) plays a central role in many airway physiological functions, and its production appears to be related with progression of lung disease in patients with cystic fibrosis (CF). However, underlying mechanisms which specifically link NO and CF-related lung disease remain unclear. Following in vitro and animal studies suggesting a role for NO in ion transport in various epithelia, this work investigates the relationship between transepithelial baseline potential difference (BPD), an index of airway ion transport, and exhaled NO in the airways of adult patients with CF. Association with other phenotypic traits, lung function tests and CFTR genotype was also assessed. Using simple linear regression, F(E)NO and transepithelial BPD values were significantly inversely correlated (p<0.001, r=-0.53). Polynomial analysis evidenced an asymptotic relationship between F(E)NO and BPD values, yielding a plateau for absolute BPD values above 50 mV. This relation was not altered by adjustment for clinical and genetic characteristics of the patients. The relationship between exhaled NO and transepithelial BPD suggests that low NO concentrations likely worsens airway ion transport impairment resulting from CFTR defect. These results fit with experimental studies that suggest the inhibitory effect of NO on sodium absorption, which is the main determinant of airway basal transepithelial conductance.

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65 Amongst the patients with normal BPD values, four were homozygous for DF508, one was compound heterozygous for DF508 and two were compound heterozygous 1717-1G-A/3272-26G-A and G542X/3849+10KbC-T. Login to comment

[hide] Hyperechogenic fetal bowel: counseling difficultie... Eur J Med Genet. 2005 Oct-Dec;48(4):421-5. Marcus-Soekarman D, Offermans J, Van den Ouweland AM, Mulder AL, Muntjewerff N, Vossen M, Kleijer W, Schrander-Stumpel C, Dooijes D
Hyperechogenic fetal bowel: counseling difficulties.
Eur J Med Genet. 2005 Oct-Dec;48(4):421-5., [PMID:16378926]

Abstract [show]
The detection of echodense fetal bowel on ultrasound examination in the second trimester of pregnancy justifies invasive procedures such as amniocentesis to detect an underlying cause. We present a case in which initial tests identified only one mutation in the cystic fibrosis transmembrane regulator (CFTR)-gene of the fetus, the family history being negative for CF. Strongly reduced intestinal enzyme activities suggested intestinal obstruction and further increased the estimated risk for CF. After the 24th gestational week, a second mutation was found, confirming cystic fibrosis in this child. Problems in counseling in this particular case are discussed.

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36 Routine CFTR-mutation analysis identified the G542X CFTR-mutation in fetal DNA and DNA from the mother (results available in the 21st week of pregnancy). Login to comment
45 Compound heterozygosity for the G542X and 1677delTA mutations was subsequently demonstrated in fetal DNA confirming the diagnosis of cystic fibrosis in the fetus. Login to comment
67 Routine CFTR-mutation analysis, using Table 1 CFTR-mutations screened for in the first step E60X 2143delT G542X G85E 2183AA-G G551D 394delTT 2184delA Q552X 621 + 1G-T 2789 + 5G-A R553X R117H 3849 + 10kbC-T R560T 711 + 5G-A R1162X S1251N 1078delT 3659delC 390insT R334W delta I507 W1282X R347P delta F508 N1303K A455E 1717-1G-A a panel of 29 CFTR-mutations, detects only 41.6% of CFTR-mutations in the Turkish population [1]. Login to comment
70 The G542X mutation, initially detected in this case, is commonly found in Mediterranean populations and is present on 3.6% of the CF chromosomes in the Turkish population [1]. Login to comment

[hide] MBL2 polymorphisms screening in a regional Italian... J Cyst Fibros. 2005 Sep;4(3):189-91. Trevisiol C, Boniotto M, Giglio L, Poli F, Morgutti M, Crovella S
MBL2 polymorphisms screening in a regional Italian CF Center.
J Cyst Fibros. 2005 Sep;4(3):189-91., [PMID:16046196]

Abstract [show]
We performed MBL2 genotyping in 47 CF patients-cared of at the regional CF Centre of Trieste-trying to establish a correlation within allelic variants of MBL2 and modification of patients' clinical outcome. FEV1 values were significantly lowered and a significantly earlier age at onset of Pseudomonas aeruginosa colonisation was found in CF patients with at least one MBL2 variant.

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42 Table 4 CFTR and MBL2 genotypes CFTR genotypes MBL2 genotypes AA A0 00 Severe/Severe CFTR genotype deltaF508/deltaF508 (20) 10 8 2 deltaF508/N1303K (1) 0 1 0 deltaF508/621+1GYT (3) 2 1 0 1717-1GYA/1717-1GYA (1) 1 0 0 deltaF508/1677delTA (1) 1 0 0 G542X/G542X (1) 0 1 0 deltaF508/1717-1GYA (1) 0 1 0 Total 28 14 12 2 Mild; unknown/unknown CFTR genotype R1162X/2789+5GYA (6) 3 3 0 2183 AAYG/4016insT (4) 2 2 0 R1162X/R1162X (3) 1 2 0 DI507/2183 AAYG (4) 2 1 0 S466X/R1070Q; T (2) 2 1 0 Total 19 10 9 0 C. Trevisiol et al. / Journal of Cystic Fibrosis 4 (2005) 189-191190 0/0 CF patients (6.29 years) when compared to A/A patients (11.24; p =0.037). Login to comment

[hide] Genotyping microarray for the detection of more th... J Mol Diagn. 2005 Aug;7(3):375-87. Schrijver I, Oitmaa E, Metspalu A, Gardner P
Genotyping microarray for the detection of more than 200 CFTR mutations in ethnically diverse populations.
J Mol Diagn. 2005 Aug;7(3):375-87., [PMID:16049310]

Abstract [show]
Cystic fibrosis (CF), which is due to mutations in the cystic fibrosis transmembrane conductance regulator gene, is a common life-shortening disease. Although CF occurs with the highest incidence in Caucasians, it also occurs in other ethnicities with variable frequency. Recent national guidelines suggest that all couples contemplating pregnancy should be informed of molecular screening for CF carrier status for purposes of genetic counseling. Commercially available CF carrier screening panels offer a limited panel of mutations, however, making them insufficiently sensitive for certain groups within an ethnically diverse population. This discrepancy is even more pronounced when such carrier screening panels are used for diagnostic purposes. By means of arrayed primer extension technology, we have designed a genotyping microarray with 204 probe sites for CF transmembrane conductance regulator gene mutation detection. The arrayed primer extension array, based on a platform technology for disease detection with multiple applications, is a robust, cost-effective, and easily modifiable assay suitable for CF carrier screening and disease detection.

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51 Complete List of Mutations Detectable with the CF APEX Assay CFTR location Amino acid change Nucleotide change 1 E 1 Frameshift 175delC 2 E 2,3 Frameshift del E2, E3 3 E 2 W19C 189 GϾT 4 E 2 Q39X 247 CϾT 5 IVS 2 Possible splicing defect 296 ϩ 12 TϾC 6 E 3 Frameshift 359insT 7 E 3 Frameshift 394delTT 8 E 3 W57X (TAG) 302GϾA 9 E 3 W57X (TGA) 303GϾA 10 E 3 E60X 310GϾT 11 E 3 P67L 332CϾT 12 E 3 R74Q 353GϾA 13 E 3 R75X 355CϾT 14 E 3 G85E 386GϾA 15 E 3 G91R 403GϾA 16 IVS 3 Splicing defect 405 ϩ 1GϾA 17 IVS 3 Possible splicing defect 405 ϩ 3AϾC 18 IVS 3 Splicing defect 406 - 1GϾA 19 E 4 E92X 406GϾT 20 E 4 E92K 406GϾA 21 E 4 Q98R 425AϾG 22 E 4 Q98P 425AϾC 23 E 4 Frameshift 444delA 24 E 4 Frameshift 457TATϾG 25 E 4 R117C 481CϾT 26 E 4 R117H 482GϾA 27 E 4 R117P 482GϾC 28 E 4 R117L 482GϾT 29 E 4 Y122X 498TϾA 30 E 4 Frameshift 574delA 31 E 4 I148T 575TϾC 32 E 4 Splicing defect 621GϾA 33 IVS 4 Splicing defect 621 ϩ 1GϾT 34 IVS 4 Splicing defect 621 ϩ 3AϾG 35 E 5 Frameshift 624delT 36 E 5 Frameshift 663delT 37 E 5 G178R 664GϾA 38 E 5 Q179K 667CϾA 39 IVS 5 Splicing defect 711 ϩ 1GϾT 40 IVS 5 Splicing defect 711 ϩ 1GϾA 41 IVS 5 Splicing defect 712 - 1GϾT 42 E 6a H199Y 727CϾT 43 E 6a P205S 745CϾT 44 E 6a L206W 749TϾG 45 E 6a Q220X 790CϾT 46 E 6b Frameshift 935delA 47 E 6b Frameshift 936delTA 48 E 6b N287Y 991AϾT 49 IVS 6b Splicing defect 1002 - 3TϾG 50 E 7 ⌬F311 3-bp del between nucleotides 1059 and 1069 51 E 7 Frameshift 1078delT 52 E 7 Frameshift 1119delA 53 E 7 G330X 1120GϾT 54 E 7 R334W 1132CϾT 55 E 7 I336K 1139TϾA 56 E 7 T338I 1145CϾT 57 E 7 Frameshift 1154insTC 58 E 7 Frameshift 1161delC 59 E 7 L346P 1169TϾC 60 E 7 R347H 1172GϾA 61 E 7 R347P 1172GϾC 62 E 7 R347L 1172GϾT 63 E 7 R352Q 1187GϾA 64 E 7 Q359K/T360K 1207CϾA and 1211CϾA 65 E 7 S364P 1222TϾC 66 E 8 Frameshift 1259insA 67 E 8 W401X (TAG) 1334GϾA 68 E 8 W401X (TGA) 1335GϾA 69 IVS 8 Splicing changes 1342 - 6 poly(T) variants 5T/7T/9T 70 IVS 8 Splicing defect 1342 - 2AϾC Table 1. Continued CFTR location Amino acid change Nucleotide change 71 E 9 A455E 1496CϾA 72 E 9 Frameshift 1504delG 73 E 10 G480C 1570GϾT 74 E 10 Q493X 1609CϾT 75 E 10 Frameshift 1609delCA 76 E 10 ⌬I507 3-bp del between nucleotides 1648 and 1653 77 E 10 ⌬F508 3-bp del between nucleotides 1652 and 1655 78 E 10 Frameshift 1677delTA 79 E 10 V520F 1690GϾT 80 E 10 C524X 1704CϾA 81 IVS 10 Possible splicing defect 1717 - 8GϾA 82 IVS 10 Splicing defect 1717 - 1GϾA 83 E 11 G542X 1756GϾT 84 E 11 G551D 1784GϾA 85 E 11 Frameshift 1784delG 86 E 11 S549R (AϾC) 1777AϾC 87 E 11 S549I 1778GϾT 88 E 11 S549N 1778GϾA 89 E 11 S549R (TϾG) 1779TϾG 90 E 11 Q552X 1786CϾT 91 E 11 R553X 1789CϾT 92 E 11 R553G 1789CϾG 93 E 11 R553Q 1790GϾA 94 E 11 L558S 1805TϾC 95 E 11 A559T 1807GϾA 96 E 11 R560T 1811GϾC 97 E 11 R560K 1811GϾA 98 IVS 11 Splicing defect 1811 ϩ 1.6 kb AϾG 99 IVS 11 Splicing defect 1812 - 1GϾA 100 E 12 Y563D 1819TϾG 101 E 12 Y563N 1819TϾA 102 E 12 Frameshift 1833delT 103 E 12 D572N 1846GϾA 104 E 12 P574H 1853CϾA 105 E 12 T582R 1877CϾG 106 E 12 E585X 1885GϾT 107 IVS 12 Splicing defect 1898 ϩ 5GϾT 108 IVS 12 Splicing defect 1898 ϩ 1GϾA 109 IVS 12 Splicing defect 1898 ϩ 1GϾC 110 IVS 12 Splicing defect 1898 ϩ 1GϾT 111 E 13 Frameshift 1924del7 112 E 13 del of 28 amino acids 1949del84 113 E 13 I618T 1985TϾC 114 E 13 Frameshift 2183AAϾG 115 E 13 Frameshift 2043delG 116 E 13 Frameshift 2055del9ϾA 117 E 13 D648V 2075TϾA 118 E 13 Frameshift 2105-2117 del13insAGAA 119 E 13 Frameshift 2108delA 120 E 13 R668C 2134CϾT 121 E 13 Frameshift 2143delT 122 E 13 Frameshift 2176insC 123 E 13 Frameshift 2184delA 124 E 13 Frameshift 2184insA 125 E 13 Q685X 2185CϾT 126 E 13 R709X 2257CϾT 127 E 13 K710X 2260AϾT 128 E 13 Frameshift 2307insA 129 E 13 V754M 2392GϾA 130 E 13 R764X 2422CϾT 131 E 14a W846X 2670GϾA 132 E 14a Frameshift 2734delGinsAT 133 E 14b Frameshift 2766del8 134 IVS 14b Splicing defect 2789 ϩ 5GϾA 135 IVS 14b Splicing defect 2790 - 2AϾG 136 E 15 Q890X 2800CϾT 137 E 15 Frameshift 2869insG 138 E 15 S945L 2966CϾT 139 E 15 Frameshift 2991del32 140 E 16 Splicing defect 3120GϾA interrogation: ACCAACATGTTTTCTTTGATCTTAC 3121-2A3G,T S; 5Ј-ACCAACATGTTTTCTTTGATCTTAC A GTTGTTATTAATTGTGATTGGAGCTATAG-3Ј; CAACAA- TAATTAACACTAACCTCGA 3121-2A3G,T AS. Login to comment
73 Genomic DNA Samples Used for Mutation Evaluation on the APEX Array Mutations validated with native DNA CFTRdel 2,3 (21 kb) 394delTT G85E R75X 574delA Y122X R117C R117H 621 ϩ 1GϾT 621 ϩ 3AϾG 711 ϩ 1GϾT I336K R334W R347P IVS8-5T IVS8-7T IVS8-9T A455E ⌬F508 ⌬I507 1677delTA 1717 - 1GϾA G542X G551D R553X R560T S549N 1898 ϩ 1GϾA 1898 ϩ 1GϾC 2183AAϾG 2043delG R668C 2143delT 2184delA 2184insA 2789 ϩ 5GϾA S945L 3120 ϩ 1GϾA I1005R 3272 - 26AϾG R1066C G1069R Y1092X (CϾA) 3500 - 2AϾT R1158X R1162X 3659delC S1235R 3849 ϩ 10 kb CϾT W1282X primer. Login to comment

[hide] Gene SNPs and mutations in clinical genetic testin... Mutat Res. 2005 Jun 3;573(1-2):195-204. Lee JE, Choi JH, Lee JH, Lee MG
Gene SNPs and mutations in clinical genetic testing: haplotype-based testing and analysis.
Mutat Res. 2005 Jun 3;573(1-2):195-204., [PMID:15829248]

Abstract [show]
Haplotype-based analysis using high-density single nucleotide polymorphism (SNP) markers have gained increasing attention in evaluating candidate genes in various clinical situations. For example, haplotype information is useful for predicting the severity and prognosis of certain genetic disorders. The intragenic cis-interactions between the common polymorphisms and the pathogenic mutations of prion protein (PRNP) and cystic fibrosis transmembrane conductance regulator (CFTR) genes greatly influence the phenotypes and the disease penetrance of hereditary Creutzfeldt-Jakob disease and cystic fibrosis. Merits of haplotype study are more evident in the fine mapping of complex diseases and in identifying genetic variations that influence individual's response to drugs. Knowledge-based approaches and/or linkage analyses using SNP tagged haplotypes are effective tools in detecting genetic associations. For example, haplotype studies in the inflammatory bowel disease susceptibility loci revealed diverse cis and trans gene-gene interactions, which can affect the clinical outcomes. Although currently, we have very limited knowledge on haplotype-phenotypic characterizations of most genes, these examples demonstrate that increased understanding of the clinically relevant haplotypes will provide better results in the diagnosis and possibly in the treatment of both monogenic and polygenic diseases.

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713 Several mutations of the CFTR gene, such as F508del, G542X and N1303K are associated with the severe CF phenotypes and display a high disease penetrance [23]. Login to comment
749 It was observed that the three most common disease-causing mutations, F508del, G542X and N1303K are found in a specific haplotype background (haplotype IIIa by Cuppens et al. [23]). Login to comment
712 Several mutations of the CFTR gene, such as F508del, G542X and N1303K are associated with the severe CF phenotypes and display a high disease penetrance [23]. Login to comment
748 It was observed that the three most common disease-causing mutations, F508del, G542X and N1303K are found in a specific haplotype background (haplotype IIIa by Cuppens et al. [23]). Login to comment

[hide] Diagnostic testing by CFTR gene mutation analysis ... J Mol Diagn. 2005 May;7(2):289-99. Schrijver I, Ramalingam S, Sankaran R, Swanson S, Dunlop CL, Keiles S, Moss RB, Oehlert J, Gardner P, Wassman ER, Kammesheidt A
Diagnostic testing by CFTR gene mutation analysis in a large group of Hispanics: novel mutations and assessment of a population-specific mutation spectrum.
J Mol Diagn. 2005 May;7(2):289-99., [PMID:15858154]

Abstract [show]
Characterization of CFTR mutations in the U.S. Hispanic population is vital to early diagnosis, genetic counseling, patient-specific treatment, and the understanding of cystic fibrosis (CF) pathogenesis. The mutation spectrum in Hispanics, however, remains poorly defined. A group of 257 self-identified Hispanics with clinical manifestations consistent with CF were studied by temporal temperature gradient electrophoresis and/or DNA sequencing. A total of 183 mutations were identified, including 14 different amino acid-changing novel variants. A significant proportion (78/85) of the different mutations identified would not have been detected by the ACMG/ACOG-recommended 25-mutation screening panel. Over one third of the mutations (27/85) occurred with a relative frequency >1%, which illustrates that the identified mutations are not all rare. This is supported by a comparison with other large CFTR studies. These results underscore the disparity in mutation identification between Caucasians and Hispanics and show utility for comprehensive diagnostic CFTR mutation analysis in this population.

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81 G542X, which is a common allele of European origin, occurred a total of 7 times (1%), including once in homozygosity, while R334W and R553X occurred twice each. Login to comment
83 While ⌬F508 was homozygous in six subjects, seven other less common alleles (G542X, W1204X, R75X, V232D, E116K, T501A, 3272-26 AϾG) were also seen in the homozygous state. Login to comment
85 1429del7bp A one-year-old Hispanic female has a novel 1429del7bp in combination with the well-established G542X mutation (exon 11). Login to comment
98 Spectrum of CFTR Sequence Variants in 257 Hispanic Patients Who Underwent Diagnostic DNA Testing for CF Mutations in 257 patients Allele counts of each mutation % of variant alleles (183) % of all alleles tested (514) ACMG/ACOG recommended 25 mutation panel* DeltaF508 53 28.96 10.31 G542X 7 3.83 1.36 R334W 2 1.09 0.39 R553X 2 1.09 0.39 DeltaI507 1 0.55 0.19 1717 - 1 GϾA 1 0.55 0.19 3120 ϩ 1 GϾA 1 0.55 0.19 7 different mutations 67 36.61 13.04 All mutations included ACMG/ACOG 1248 ϩ 1 GϾA 1 0.55 0.19 1249 - 29delAT 1 0.55 0.19 1288insTA1288insTA 1 0.55 0.19 1341 ϩ 80 GϾA1341 ϩ 80 GϾA 1 0.55 0.19 1429del71429del7 1 0.55 0.19 1525 - 42 GϾA1525 - 42 GϾA 1 0.55 0.19 1717 - 1 GϾA 1 0.55 0.19 1717 - 8 GϾA 2 1.09 0.39 1811 ϩ 1 GϾA1811 ϩ 1 GϾA 1 0.55 0.19 2055del9-ϾA 3 1.64 0.58 2105-2117del13insAGAAA 1 0.55 0.19 2215insG 1 0.55 0.19 2585delT2585delT 1 0.55 0.19 2752 - 6 TϾC 1 0.55 0.19 296 ϩ 28 AϾG 1 0.55 0.19 3120 ϩ 1 GϾ A 1 0.55 0.19 3271 ϩ 8 AϾG3271 ϩ 8 AϾG 1 0.55 0.19 3271delGG 1 0.55 0.19 3272 - 26 AϾG 2 1.09 0.39 3876delA 2 1.09 0.39 4016insT 1 0.55 0.19 406 - 1 GϾA 6 3.28 1.17 406 - 6 TϾC 1 0.55 0.19 4374 ϩ 13 A ϾG 1 0.55 0.19 663delT 1 0.55 0.19 874insTACA874insTACA 1 0.55 0.19 A1009T 2 1.09 0.39 A559T 1 0.55 0.19 D1152H 1 0.55 0.19 D1270N 3 1.64 0.58 D1445N 2 1.09 0.39 D836Y 1 0.55 0.19 DeltaF311 1 0.55 0.19 DeltaF508 53 28.96 10.31 DeltaI507 1 0.55 0.19 E116K 2 1.09 0.39 E585X 1 0.55 0.19 E588VE588V 2 1.09 0.39 E831X 1 0.55 0.19 F311L 1 0.55 0.19 F693L 1 0.55 0.19 G1244E 1 0.55 0.19 G542X 7 3.83 1.36 G576A 1 0.55 0.19 H199Y 3 1.64 0.58 I1027T 3 1.64 0.58 I285FI285F 1 0.55 0.19 L206W 3 1.64 0.58 L320V 1 0.55 0.19 L967S 1 0.55 0.19 L997F 3 1.64 0.58 P1372LP1372L 1 0.55 0.19 P205S 1 0.55 0.19 P439SP439S 1 0.55 0.19 Q1313X 1 0.55 0.19 Q890X 2 1.09 0.39 Q98R 1 0.55 0.19 R1066C 1 0.55 0.19 R1066H 1 0.55 0.19 (Table continues) missense variant, I1027T (3212TϾC), in exon 17a.25 Family studies have not been performed to identify which allele carries two mutations. Login to comment
102 Novel Variants Detected in 257 Hispanic Patients Patient Novel variant 1 Other variants Age and symptoms 1 1429del7bp G542X Newborn with intestinal blockage 2 S573C None 9 years old, pancreatitis, limited clinical history 3 Y913X deltaF508/I1027T 1 month old, vomiting, weight loss, diarrhea 4 E588V deltaF508/R1438W Identified one time in a family, family studies revealed deltaF508 and R1438W are in cis 5 E588V G542X Newborn with pneumonia and sweat chloride of 59 mmol/L 6 P439S R668C 10 years old with mild CF symptoms; another patient with CBAVD has P439S/R334W 7 T604S deltaF508 1 month old 8 874insTACA deltaF508 Newborn with meconium ileus and IUGR 9 2585delT deltaF508/I1027T 13 years old with CF 10 1811 ϩ 1 G to A None 44 years old with positive sweat chloride; also seen in 5-year-old CF patient with 3821delT mutation 11 I285F None 1 year old with chronic respiratory problems, also carries a silent mutation at A455 12 P1372L None 1 month old, rule out CF 13 3271 ϩ 8 A to G None 16 years old, borderline sweat test 14 1341 ϩ 80 G to A None Recurrent sinusitis 15 1525 - 42 G to A None Two patients, one 9 years old with FTT, and one 18 months old with chronic lung disease, pulmonary hypotension, hypoxia CBAVD, congenital bilateral absence of the vas deference; IUGR, intrauterine growth retardation. Login to comment
114 This was a newborn with pneumonia and a borderline sweat chloride result of 59 mmol/L, who also carried a G542X mutation. Login to comment
170 Mutations G542X and 406-1GϾA accounted for 3.8% and 2.5% respectively, and 3849 ϩ 10kbCϾT was present at 1.6%. Login to comment
173 Of the 22 mutations present at a relative frequency of 1% or more, only eight are currently included in the standard 25 mutation panel recommended (I148T, R334W, ⌬F508, G542X, R553X, 1717-1GϾA, 3120 ϩ 1GϾA, and 3849 ϩ 10kbCϾT), although a recent ACMG revision will remove variant I148T.13 The California Department of Health Services is also tracking Hispanic mutations.15 However, these may duplicate some of those described in the other reports and therefore are not included in this analysis. Login to comment
176 In comprehensive non-U.S. studies from Brazil, Colombia, and Spain, 420 mutations were identified (231, 117, and 72, respectively).33-35 Only seven occurred with a relative frequency Ͼ1%: ⌬F508 (67.4%), G542X (9%), N1303K (2.4%), R1162X (2.4%), R334W (2.1%), W1282X (1.2%), and S549R (1%). Login to comment
187 CFTR Sequence Variants Identified in Five Comprehensive CFTR Studies in US Hispanics CFTR mutations Alleles Relative mutation frequency (%) (of 317) deltaF508 123 38.80 3876delA 15 4.70 G542X 12 3.80 406 - 1GϾA 8 2.50 3849 ϩ 10kbCϾT 5 1.60 R75X 4 1.30 935delA 4 1.30 S549N 4 1.30 W1204X 4 1.30 R334W 4 1.30 2055del9ϾA 3 1 R74W 3 1 H199Y 3 1 L206W 3 1 663delT 3 1 3120 ϩ 1GϾA 3 1 L997F 3 1 I1027T 3 1 R1066C 3 1 W1089X 3 1 D1270N 3 1 2105del13insAGAAA 3 1 Q98R 2 Ͻ1 E116K 2 Ͻ1 I148T 2 Ͻ1 R668C 2 Ͻ1 P205S 2 Ͻ1 V232D 2 Ͻ1 S492F 2 Ͻ1 T501A 2 Ͻ1 1949del84 2 Ͻ1 Q890X 2 Ͻ1 3271delGG 2 Ͻ1 3272 - 26AϾG 2 Ͻ1 G1244E 2 Ͻ1 D1445N 2 Ͻ1 R553X 2 Ͻ1 E588V 2 Ͻ1 1717 - 8GϾA 2 Ͻ1 A1009T 2 Ͻ1 S1235R 2 Ͻ1 G85E 1 Ͻ1 296 ϩ 28AϾG 1 Ͻ1 406 - 6TϾC 1 Ͻ1 V11I 1 Ͻ1 Q179K 1 Ͻ1 V201 mol/L 1 Ͻ1 874insTACA 1 Ͻ1 I285F 1 Ͻ1 deltaF311 1 Ͻ1 F311L 1 Ͻ1 L320V 1 Ͻ1 T351S 1 Ͻ1 R352W 1 Ͻ1 1248 ϩ 1GϾA 1 Ͻ1 1249 - 29delAT 1 Ͻ1 1288insTA 1 Ͻ1 1341 ϩ 80GϾA 1 Ͻ1 1429del7 1 Ͻ1 1525 - 42GϾA 1 Ͻ1 P439S 1 Ͻ1 1717 - 1GϾA 1 Ͻ1 1811 ϩ 1GϾA 1 Ͻ1 deltaI507 1 Ͻ1 G551D 1 Ͻ1 A559T 1 Ͻ1 Y563N 1 Ͻ1 (Table continues) In this study, we used temporal temperature gradient gel electrophoresis (TTGE) and direct DNA sequencing to increase the sensitivity of mutation detection in U.S. Hispanics, and to determine whether additional mutations are recurrent. Login to comment
199 The pooled data set demonstrates that the most frequently seen mutations are: ⌬F508, G542X, 406-1GϾA, W1204X, R75X, 2055del9ϾA, 3876delA, ⌬I507, S549N, I148T, N1303K, 935delA, and 3849 ϩ 10kbCϾT. Login to comment
201 Comparison of Relative Frequencies of CFTR Sequence Variants in Comprehensive CFTR Studies in US and Mexican Hispanics This study % Orozco 2000 % US/ Mexican % deltaF508 28.96 54.48 43.72 G542X 3.83 8.28 5.19 406 - 1GϾA 3.28 2.07 2.38 W1204X 2.19 Ͻ1 1.08 R74W 1.64 Ͻ1 R75X 1.64 2.07 1.51 H199Y 1.64 Ͻ1 Ͻ1 L206W 1.64 Ͻ1 L997F 1.64 Ͻ1 I1027T 1.64 Ͻ1 2055del9ϾA 1.64 1.38 1.27 D1270N 1.64 Ͻ1 E116K 1.09 Ͻ1 V232D 1.09 Ͻ1 R334W 1.09 Ͻ1 S492F 1.09 Ͻ1 T501A 1.09 Ͻ1 R553X 1.09 Ͻ1 Ͻ1 E588V 1.09 Ͻ1 R668C 1.09 Ͻ1 Q890X 1.09 Ͻ1 W1089X 1.09 Ͻ1 S1235R 1.09 Ͻ1 D1445N 1.09 Ͻ1 3876delA 1.09 3.24 1717 - 8GϾA 1.09 Ͻ1 3272 - 26AϾG 1.09 Ͻ1 A1009T 1.09 Ͻ1 deltaI507 Ͻ1 3.45 1.30 S549N Ͻ1 3.45 1.95 G567A Ͻ1 Ͻ1 I148T 2.07 1.08 I506T 1.38 Ͻ1 N1303K 2.76 1.08 935delA 1.38 1.30 2183AAϾG 1.38 Ͻ1 3199del6 1.38 Ͻ1 3849 ϩ 10kbCϾT Ͻ1 1.30 ACMG/ACOG italicized. Login to comment
202 which occur with a relative frequency above 1% in the pooled data set, only six (⌬F508, G542X, ⌬I507, I148T, N1303K, and 3849 ϩ 10kbCϾT) were included in the ACMG/ACOG 25-mutation screening panel12 and in the recent revision exclusion of I148T has been recommended.13 The most frequently seen mutations in the U.S. and Mexican studies combined (n ϭ 462 identified mutations) include the 10 most frequent mutations observed in the Mexican study.36 They also include all but one mutation (R334W) occurring with a relative frequency above 1% in the five combined studies performed in the U.S. In that group, only ⌬I507, N1303K and I148T were present at a relative frequency below 1%. Login to comment

[hide] Complete gene scanning by temperature gradient cap... J Mol Diagn. 2005 Feb;7(1):111-20. Chou LS, Gedge F, Lyon E
Complete gene scanning by temperature gradient capillary electrophoresis using the cystic fibrosis transmembrane conductance regulator gene as a model.
J Mol Diagn. 2005 Feb;7(1):111-20., [PMID:15681482]

Abstract [show]
Many inherited diseases involve large genes with many different mutations. Identifying a wide spectrum of mutations requires an efficient gene-scanning method. By differentiating thermodynamic stability and mobility of heteroduplexes from heterozygous samples, temperature gradient capillary electrophoresis (TGCE) was used to scan the entire coding region of the cystic fibrosis transmembrane conductance regulator gene. An initial panel (29 different mutations) showed 100% agreement between TGCE scanning and previously genotyped results for heterozygous samples. Different peak patterns were observed for single base substitutions and base insertions/deletions. Subsequently, 12 deidentified clinical samples genotyped as wild type for 32 mutations were scanned for the entire 27 exons. Results were 100% concordance with the bidirectional sequence analysis. Ten samples had nucleotide variations including a reported base insertion in intron 14b (2789 + 2insA) resulting in a possible mRNA splicing defect, and an unreported missense mutation in exon 20 (3991 G/A) with unknown clinical significance. This methodology does not require labeled primers or probes for detection and separation through a temperature gradient eliminates laborious temperature optimization required for other technologies. TGCE automation and high-throughput capability can be implemented in a clinical environment for mutation scanning with high sensitivity, thus reducing sequencing cost and effort.

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75 Mutation Samples with Known Genotypes Scanned by TGCE* Exon Mutation† Amplicon size (bp) Location of mutation from 5Ј end (bp) Base change Detection‡ 3 G85E 234 124 G to A 1/1 3 394delTT 234 132 del TT 1/1 4 R117H 270 83 G to T 2/2 4 I148T 270 176 T to C 3/3 Intron 4 621 ϩ 1 G/T 270 233 G to T 1/1 5 663delT/663delT 186 75 del T 0/1 Intron 5 711 ϩ 1 G/T 186 124 G to T 1/1 7 R334W 345 208 C to T 1/1 7 R347P 345 248 G to C 1/1 9 A455E 263 155 C to A 2/2 10 I506V 292 168 A to G 1/1 10 ⌬I507 292 171 del ATC 2/2 10 ⌬F508 292 174 del TTT 2/2 10 ⌬F508/⌬F508 292 174 del TTT 0/1 10 F508C 292 175 T to G 1/1 10 V520F 292 210 G to T 1/1 Intron 10 1717-1 G/A 175 50 G to A 1/1 11 G542X 175 90 G to T 2/2 11 G542X/G542X 175 90 G to T 0/1 11 G551D 175 118 G to A 3/3 11 R553X 175 123 C to T 3/3 11 R560T 175 145 G to C 2/2 13 2184delA 834 356 del A 1/1 Intron 14b 2789 ϩ 5G/A 192 102 G to A 1/1 Intron 16 3120 ϩ 1G/A 216 111 G to A 1/1 19 R1162X 322 68 C to T 1/1 19 3659delC 322 111 del C 1/1 20 W1282X 206 154 G to A 1/1 21 N1303K 250 175 C to G 2/2 Total exon/intron Overall accuracy 17 93% *Samples were compared with their respective wild-type control (confirmed by sequencing). Login to comment
76 † All genotypes were heterozygous except homozygous sample 663delT/663delT, ⌬F508/ ⌬F508, and G542X/G542X. Login to comment
135 In our study, homozygous mutants 663 delT (exon 5), ⌬F508 (exon 10), and G542X (exon 11) were not initially detected. Login to comment
179 Discussion More than 1000 mutations have been reported since the CFTR gene was cloned and characterized in 1989.23,25 Of these mutations, ⌬F508 (a 3-base deletion) is the most frequent mutation and results in a defective cAMP-regulated chloride transport in epithelial cells.26 Other mutations in the CFTR gene such as G542X, G551D, and N1303K occur in greater than 1% in the CF population and are associated with severe pancreatic insufficiency.23,27 Recently, carriers of the I148T mutation have received more attention because I148T has been found in association with the 3199del6 mutation, which may be necessary for the classic CF phenotype.28 Because of the complexity of both the mutations and the phenotypes, a high-throughput mutation scanning method to screen the entire coding region of the CFTR gene may provide valuable clinical information regarding CF genotypes and respective phenotypes. Login to comment

[hide] Rapid screening for 31 mutations and polymorphisms... Methods Mol Med. 2005;114:147-71. Dunbar SA, Jacobson JW
Rapid screening for 31 mutations and polymorphisms in the cystic fibrosis transmembrane conductance regulator gene by Lminex xMAP suspension array.
Methods Mol Med. 2005;114:147-71., [PMID:16156102]

Abstract [show]
A suspension array hybridization assay is described for the detection of 31 mutations and polymorphisms in the cystic fibrosis transmembrane conductance regulator (CFTR) gene using Luminex xMAP technology. The Luminex xMAP system allows simultaneous detection of up to 100 different targets in a single multiplexed reaction. Included in the method are the procedures for design of oligonucleotide capture probes and PCR amplification primers, coupling oligonucleotide capture probes to carboxylated microspheres, hybridization of coupled microspheres to oligonucleotide targets, production of targets from DNA samples by multiplexed PCR amplification, and detection of PCR-amplified targets by direct hybridization to probe-coupled microspheres. Mutation screening with the system is rapid, requires relatively few sample manipulations, and provides adequate resolution to reliably genotype the 25 CFTR mutations and 6 CFTR polymorphisms contained in the ACMG/ACOG/NIH-recommended core mutation panel for general population CF carrier screening.

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25 A 635-nm 10-mW red diode laser excites the two fluo- 148 Dunbar and Jacobson xMAP™ 149 Table 1 Recommended Core Mutation Panel for General Population Cystic Fibrosis (CF) Carrier Screening Standard mutation panel ΔF508 ΔI507 G542X G551D W1282X N1303K R553X 621+1G→T R117H 1717-1G→A A455E R560T R1162X G85E R334W R347P 711+1G→T 1898+1G→A 2184delA 1078delT 3849+10kbC→T 2789+5G→A 3659delC 1148T 3120+1G→A Reflex tests I506Va I507Va F508Ca 5T/7T/9Tb a Benign variants. Login to comment
94 Methods The methods described below include: (1) design of oligonucleotide capture probes, (2) design of PCR amplification primers and multiplexed PCR reactions, (3) preparation of the probe-conjugated microsphere sets, (4) verification 150 Dunbar and Jacobson xMAPTM Table 2 Oligonucleotide Capture Probesa Target Microsphere Probe sequence Modificationb Sequence 5' → 3' set Standard mutation panel 1c I507 & F508 5'-AmMC12 AACACCAAAGATGATATTTT 006 2B DI507 5'-AmMC12 ACACCAAAGATATTTTCTT 008 3B DF508 5'-AmMC12 AAACACCAATGATATTTTC 015 4B W1282 5'-AmMC12 CAACAGTGGAGGAAAGCC 012 5B W1282X 5'-AmMC12 CAACAGTGAAGGAAAGCC 020 6 1717-1G 5'-AmMC12 TTGGTAATAGGACATCTCCA 017 7 1717-1GÆA 5'-AmMC12 TTGGTAATAAGACATCTCCA 019 8B G542 5'-AmMC12 TATAGTTCTTGGAGAAGGTGGA 026 9B G542X 5'-AmMC12 TATAGTTCTTTGAGAAGGTGGA 028 10C G551 & R553 5'-AmMC12 AGTGGAGGTCAACGAGCAA 038 11B G551D 5'-AmMC12 GTGGAGATCAACGAGCAA 030 12C R553X 5'-AmMC12 GTGGAGGTCAATGAGCAA 032 13 R560 5'-AmMC12 CTTTAGCAAGGTGAATAACT 035 14 R560T 5'-AmMC12 CTTTAGCAACGTGAATAACT 039 15 R117 5'-AmMC12 AGGAGGAACGCTCTATCGCG 042 16 R117H 5'-AmMC12 AGGAGGAACACTCTATCGCG 025 17B I148 5'-AmMC12 CTTCATCACATTGGAATGCAGA 034 18B I148T 5'-AmMC12 CTTCATCACACTGGAATGCAGA 045 19C 621+1G 5'-AmMC12 TTTATAAGAAGGTAATACTTCCT 046 20E 621+1G→T 5'-AmMC12 ATTTATAAGAAGTTAATACTTCCTT 048 21 N1303 5'-AmMC12 GGGATCCAAGTTTTTTCTAA 051 22 N1303K 5'-AmMC12 GGGATCCAACTTTTTTCTAA 052 23B 1078T 5'-AmMC12 CACCACAAAGAACCCTGA 054 24C 1078delT 5'-AmMC12 ACACCACAAGAACCCTGA 061 25 R334 5'-AmMC12 ATATTTTCCGGAGGATGATT 063 26 R334W 5'-AmMC12 ATATTTTCCAGAGGATGATT 064 27B R347 5'-AmMC12 ACCGCCATGCGCAGAACAA 067 28B R347P 5'-AmMC12 ACCGCCATGGGCAGAACAA 053 29C 711+1G 5'-AmMC12 ATTTGATGAAGTATGTACCTAT 059 30C 711+1G→T 5'-AmMC12 ATTTGATGAATTATGTACCTAT 071 31 G85 5'-AmMC12 TGTTCTATGGAATCTTTTTA 066 32B G85E 5'-AmMC12 ATGTTCTATGAAATCTTTTTA 073 33 3849+10kbC 5'-AmMC12 GTCTTACTCGCCATTTTAAT 077 34 3849+10kbC→T 5'-AmMC12 GTCTTACTCACCATTTTAAT 075 35 A455 5'-AmMC12 CCAGCAACCGCCAACAACTG 011 36D A455E 5'-AmMC12 TCCAGCAACCTCCAACAACTG 036 37 R1162 5'-AmMC12 TAAAGACTCGGCTCACAGAT 060 38 R1162X 5'-AmMC12 TAAAGACTCAGCTCACAGAT 068 39B 3659C 5'-AmMC12 TTGACTTGGTAGGTTTAC 022 40C 3659delC 5'-AmMC12 TTGACTTGTAGGTTTACC 079 41B 2789+5G 5'-AmMC12 TGGAAAGTGAGTATTCCATGTC 074 42D 2789+5G→A 5'-AmMC12 TTGGAAAGTGAATATTCCATGTC 014 43E 2184A 5'-AmMC12 GAAACAAAAAAACAATC 007 44E 2184delA 5'-AmMC12 AGAAACAAAAAACAATC 018 45B 1898+1G 5'-AmMC12 TATTTGAAAGGTATGTTCTTTG 013 (Continued) of microsphere coupling, (5) direct hybridization of biotinylated PCR amplification products to the multiplexed probe-coupled microsphere sets, and (6) results and data analysis. Login to comment
106 Table 3 Reverse Complementary Oligonucleotide Targetsa Target Target sequence Modification Sequence 5' → 3' Standard mutation panel C1b I507 & F508 5'-Biotin AAAATATCATCTTTGGTGTT C2 ΔI507 5'-Biotin AAAGAAAATATCTTTGGTGT C3 ΔF508 5'-Biotin AGAAAATATCATTGGTGTTT C4 W1282 5'-Biotin GGCTTTCCTCCACTGTTGC C5 W1282X 5'-Biotin GGCTTTCCTTCACTGTTGC C6 1717-1G 5'-Biotin TGGAGATGTCCTATTACCAA C7 1717-1G→A 5'-Biotin TGGAGATGTCTTATTACCAA C8 G542 5'-Biotin CCACCTTCTCCAAGAACTAT C9 G542X 5'-Biotin CCACCTTCTCAAAGAACTAT C10 G551 & R553 5'-Biotin CTTGCTCGTTGACCTCCACT C11 G551D 5'-Biotin CTTGCTCGTTGATCTCCACT C12 R553X 5'-Biotin CTTGCTCATTGACCTCCACT C13 R560 5'-Biotin AGTTATTCACCTTGATAAAG C14 R560T 5'-Biotin AGTTATTCACGTTGCTAAAG C15 R117 5'-Biotin CGCGATAGAGCGTTCCTCCT C16 R117H 5'-Biotin CGCGATAGAGTGTTCCTCCT C17 I148 5'-Biotin CTGCATTCCAATGTGATGAA C18 I148T 5'-Biotin CTGCATTCCAGTGTGATGAA C19 621+1G 5'-Biotin GGAAGTATTACCTTCTTATA C20 621+1G→T 5'-Biotin GGAAGTATTAACTTCTTATA C21 N1303 5'-Biotin TTAGAAAAAACTTGGATCCC C22 N1303K 5'-Biotin TTAGAAAAAAGTTGGATCCC C23 1078T 5'-Biotin CTCAGGGTTCTTTGTGGTGT C24 1078delT 5'-Biotin TCTCAGGGTTCTTGTGGTGT C25 R334 5'-Biotin AATCATCCTCCGGAAAATAT C26 R334W 5'-Biotin AATCATCCTCTGGAAAATAT C27 R347 5'-Biotin ATTGTTCTGCGCATGGCGGT C28 R347P 5'-Biotin ATTGTTCTGCCCATGGCGGT C29 711+1G 5'-Biotin TAGGTACATACTTCATCAAA C30 711+1G→T 5'-Biotin TAGGTACATAATTCATCAAA C31 G85 5'-Biotin TAAAAAGATTCCATAGAACA C32 G85E 5'-Biotin TAAAAAGATTTCATAGAACA C33 3849+10kbC 5'-Biotin ATTAAAATGGCGAGTAAGAC C34 3849+10kbC→T 5'-Biotin ATTAAAATGGTGAGTAAGAC C35 A455 5'-Biotin CAGTTGTTGGCGGTTGCTGG C36 A455E 5'-Biotin CAGTTGTTGGAGGTTGCTGG C37 R1162 5'-Biotin ATCTGTGAGCCGAGTCTTTA C38 R1162X 5'-Biotin ATCTGTGAGCTGAGTCTTTA (Continued) Rapid CF Screening by xMAPTM 153 Table 3 (Continued) Target Target sequence Modification Sequence 5' → 3' C39 3659C 5'-Biotin GGTAAACCTACCAAGTCAAC C40 3659delC 5'-Biotin AGGTAAACCTACAAGTCAAC C41 2789+5G 5'-Biotin ACATGGAATACTCACTTTCC C42 2789+5G→A 5'-Biotin ACATGGAATATTCACTTTCC C43 2184A 5'-Biotin AAGATTGTTTTTTTGTTTCT C44 2184delA 5'-Biotin AAGATTGTTTTTTGTTTCTG C45 1898+1G 5'-Biotin AAAGAACATACCTTTCAAAT C46 1898+1G→A 5'-Biotin AAAGAACATATCTTTCAAAT C47 3120+1G 5'-Biotin TTTTTACATACCTGGATGAA C48 3120+1G→A 5'-Biotin TTTTTACATATCTGGATGAA Reflex panel CR2 I506V 5'-Biotin GAAAATGTCATCTTTGGTGT CR3 I507V 5'-Biotin GAAAATATCGTCTTTGGTGT CR4 F508C 5'-Biotin AAAATATCATCTGTGGTGTT CR5 5T 5'-Biotin TCCCTGTTAAAAACACACAC CR6 7T 5'-Biotin CCCTGTTAAAAAAACACACA CR7 9T 5'-Biotin CCTGTTAAAAAAAAACACAC a The position and sequence of the mutation or variation is indicated in bold type. b Target C1 (I507 & F508) is also used in the reflex panel. Login to comment
114 Using a small target DNA (approx 100-300 bp) minimizes the potential for steric hindrance to affect the xMAPTM Table 4 PCR Amplification Primers Size CFTR target Mutation(s) Primer 5' Modification Sequence 5' → 3' (bp) Exon 10 ΔI507, ΔF508, BE10U 5'-Biotin TTCTGTTCTCAGTTTTCCTGG 107 I506V, I507V, E10D None TTGGCATGCTTTGATGACG F508C Exon 20 W1282X E20U None TTGAGACTACTGAACACTGAAGG 126 BE20D 5'-Biotin TTCTGGCTAAGTCCTTTTGC Intron 10 1717-1G→A E11U None TCAGATTGAGCATACTAAAAGTGAC 89 BE11D2 5'-Biotin GAACTATATTGTCTTTCTCTGCAAAC Exon 11 G542X, G551D, E11U2 None AAGTTTGCAGAGAAAGACAATATAG 135 R553X, R560T BE11D 5'-Biotin GAATGACATTTACAGCAAATGC Exon 4 R117H E4U None TTTGTAGGAAGTCACCAAAGC 145 BE4D2 5'-Biotin GAGCAGTGTCCTCACAATAAAGAG Exon 4/intron 4 I148T, E4U2 None CTTCTCTTTATTGTGAGGACACTGC 169 621+1G→T BE4D 5'-Biotin ATGACATTAAAACATGTACGATACAG Exon 21 N1303K BE21U 5'-Biotin TGCTATAGAAAGTATTTATTTTTTCTGG 106 E21D None AGCCTTACCTCATCTGCAAC Exon 7 1078delT, BE7U 5'-Biotin GAACAGAACTGAAACTGACTCG 199 R334W, R347P E7D3 None CAGGGAAATTGCCGAGTG Intron 5 711+1G→T I5U None CAACTTGTTAGTCTCCTTTCC 99 BI5D2 5'-Biotin AGTTGTATAATTTATAACAATAGTGC Exon 3 G85E E3U None CTGGCTTCAAAGAAAAATCC 117 BE3D2 5'-Biotin TGAATGTACAAATGAGATCCTTACC Chromosome 7 3849+10kbC→T BC7U 5'-Biotin GACTTGTCATCTTGATTTCTGG 148 C7D None TTTGGTGCTAGCTGTAATTGC Exon 9 A455E BE9U 5'-Biotin TCACTTCTTGGTACTCCTGTCC 105 E9D None CAAAAGAACTACCTTGCCTGC Exon 19-I R1162X BE19U 5'-Biotin ATTGTGAAATTGTCTGCCATTC 167 E19Da None CAATAATCATAACTTTCGAGAGTTG Exon 19-II 3659delC BE19U2 5'-Biotin TTTAAGTTCATTGACATGCCAAC 91 E19Da None CAATAATCATAACTTTCGAGAGTTG Intron 14B 2789+5G→A I14BU None GTGTCTTGTTCCATTCCAGG 147 BI14BD 5'-Biotin TGGATTACAATACATACAAACATAGTGG Exon 13 2184delA E13U None AGATGCTCCTGTCTCCTGG 126 BE13D 5'-Biotin TGCACAATGGAAAATTTTCGTATAG Intron 12 1898+1G→A I12U None TTAGACTCTCCTTTTGGATACC 110 BI12D 5'-Biotin GTCTTTCTTTTATTTTAGCATGAGC Intron 16 3120+1G→A I16U None ATGACCTTCTGCCTCTTACC 118 BI16D 5'-Biotin ATGAAAACAAAATCACATTTGC Intron 8 5T/7T/9T I8U None TAATGGATCATGGGCCATGTGC 212 BI8D 5'-Biotin ACTGAAGAAGAGGCTGTCATCACC CFTR, cystic fibrosis transmembrane conductance regulator gene. Login to comment
118 Table 5 Genomic DNA Samples CFTR genotype Sourcea Normal/normal Sigma, D6537 ΔF508/normal Patient sample ΔF508/ΔF508 Coriell Cell Repositories, NA04540 ΔI507/normal Coriell Cell Repositories, NA11277 W1282/normal Coriell Cell Repositories, NA11723 1717-1G→A/normal Coriell Cell Repositories, NA12444 G542X/G542X Coriell Cell Repositories, NA11496B G542X/normal Coriell Cell Repositories, NA11497B ΔF508/G551D Coriell Cell Repositories, NA11274 ΔF508/R553X Coriell Cell Repositories, NA07469 G551D/R553X Coriell Cell Repositories, NA11761 ΔF508/R560T Coriell Cell Repositories, NA11284 ΔF508/R117H Coriell Cell Repositories, NA13591 I148T/normal Patient sample ΔF508/621+1G→T Coriell Cell Repositories, NA11281 N1303K/G1349D Coriell Cell Repositories, NA11472A ΔF508/1078delT Patient sample R334W/? Login to comment
276 At higher concentrations of M2, the hybridization efficiency of the exon 11 target was decreased, with a concomitant drop in reporter signal on the G542X-, G551D-, and R553X-specific microsphere sets. Login to comment

[hide] High heterogeneity of CFTR mutations and unexpecte... J Cyst Fibros. 2004 Dec;3(4):265-72. des Georges M, Guittard C, Altieri JP, Templin C, Sarles J, Sarda P, Claustres M
High heterogeneity of CFTR mutations and unexpected low incidence of cystic fibrosis in the Mediterranean France.
J Cyst Fibros. 2004 Dec;3(4):265-72., [PMID:15698946]

Abstract [show]
In this report, we present updated spectrum and frequency of mutations of the CFTR gene that are responsible for cystic fibrosis (CF) in Languedoc-Roussillon (L-R), the southwestern part of France. A total of 75 different mutations were identified by DGGE in 215 families, accounting for 97.6% of CF genes and generating 88 different mutational genotypes. The frequency of p.F508del was 60.23% in L-R versus 67.18% in the whole country and only five other mutations (p.G542X, p.N1303K, p.R334W, c.1717-1G>A, c.711+1G>T) had a frequency higher than 1%. The mutations were scattered over 20 exons or their border. This sample representing only 5.7% of French CF patients contributed to 24% of CFTR mutations reported in France. This is one of the highest molecular allelic heterogeneity reported so far in CF. We also present the result of a neonatal screening program based on a two-tiered approach "IRT/20 mutations/IRT" analysis on blood spots, implemented in France with the aim to improve survival and quality of life of patients diagnosed before clinical onset. This 18-month pilot project showed an unexpected low incidence of CF (1/8885) in South of France, with only six CF children detected among 43,489 neonates born in L-R, and 13 among 125,339 neonates born in Provence-Alpes-Cote-d'Azur (PACA).

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2 The frequency of p.F508del was 60.23% in L-R versus 67.18% in the whole country and only five other mutations (p.G542X, p.N1303K, p.R334W, c.1717-1GNA, c.711+1GNT) had a frequency higher than 1%. Login to comment
38 The 20 most common mutations responsible for CF worldwide were investigated by amplification refractory mutation system (ARMS) and migration on agarose gel (Kit Elucigene CF20, including mutations c.1717-1GNA, p.G542X, p.W1282X, p.N1303K, p.F508del, c.3849+10kbCNT, c.621+1GNT, p.R553X, p.G551D, p.R117H, p.R1162X, p.R334W, p.A455E, c.2183AANG, c.3659delC, c.1078delT, p.I507del, p.R347P, p.S1251N, p.E60X). Login to comment
55 Five other mutations were found with a relative frequency higher than 1%: p.G542X (5.35%), p.N1303K (3.%), p.R334W (1.63%), c.1717-1GNA (1.40%) and c.711+1GNT (1.16%) (Table 1). Login to comment
56 From Fig. 1, it can be seen that mutations p.G542X, p.N1303K, p.R334W and c.711+1GNT are more common in Mediterranean areas than in the whole country. Login to comment
68 of chromosomes (frequency %) p.M1V 1 1 (0.23) p.M1K 1 1 (0.23) c.300delA 3 1 (0.23) p.P67L 3 1 (0.23) c.359insT 3 1 (0.23) p.G85E 3 3 (0.70) c.394delTT 3 1 (0.23) p.Q98R 4 1 (0.23) p.R117H 4 2 (0.47) p.Y122X 4 2 (0.47) p.Y161N 4 1 (0.23) c.621+1GNT intron 4 1 (0.23) c.621+2TNG intron 4 1 (0.23) p.I175V 5 2 (0.47) c.711+1GNT intron 5 5 (1.16) p.L206W 6 3 (0.70) p.Q220X 6 1 (0.23) p.L227R 6 1 (0.23) c.1078delT 7 2 (0.47) p.R334W 7 7 (1.63) p.R347P 7 2 (0.47) c.1215delG 7 1 (0.23) c.T5 intron 8 1 (0.23) p.D443Y 9 1 (0.23) p.I506T 10 1 (0.23) p.I507del 10 4 (0.93) p.F508del 10 259 (60.23) p.F508C 10 1 (0.23) c.1677delTA 10 1 (0.23) c.1717-8GNA intron 10 1 (0.23) c.1717-1GNA intron 10 6 (1.40) p.G542X 11 23 (5.35) p.S549R 11 1 (0.23) p.G551D 11 2 (0.47) p.R553X 11 1 (0.23) c1811+1.6kbANG intron 11 4 (0.93) c.1812-1GNA intron 11 1 (0.23) p.T582I 12 1 (0.23) p.E585X 12 2 (0,47) c.1898+1GNA intron 12 1 (0.23) [c.1898+5GNA ;p.E725K] intron 12 1 (0.23) c.1898+73TNG intron 12 1 (0.23) c.2183AANG 13 4 (0.93) c.2184insA 13 1 (0.23) p.K710X 13 4 (0.93) c.2423delG 13 1 (0.23) p.S776X 13 1 (0.23) c.2493ins8 13 1 (0.23) p.R792X 13 1 (0.23) p.K830X 13 1 (0.23) p.D836Y 14a 1 (0.23) p.W846X1 14a 1 (0.23) c.2711delT 14a 1 (0.23) c.2789+5GNA intron 14b 3 (0.70) p.S945L 15 3 (0.70) p.D993Y 16 1 (0.23) c.3129del4 17a 1 (0.23) c.3195del6 17a 1 (0.23) c.3272-26ANG intron 17a 1 (0.23) [c.3395insA ;pI148T] 17b/4 1 (0,23) p.Y1092X 17b 3 (0.70) Table 1 (continued) Mutation Location exon/intron No. Login to comment
83 Table 2 Genotypes identified by newborn screening in 19 affected babies IRT (ng/ml) Genotypes 118 [p.F508del]+[p.F508del]a 163 [p.F508del]+[p.F508del]a N130 [p.F508del]+[p.F508del]b N130 [p.F508del]+[p.F508del]b N130 [p.F508del]+[p.F508del]b 155 [p.F508del]+[p.F508del]a 166 [p.F508del]+[p.F508del]a 109 [p.F508del]+[p.F508del]a 110 [p.F508del]+[p.F508del]a 136 [p.F508del]+[c.3007delG]a 160 [p.F508del]+[c.2622+1GNA]a 129 [p.F508del]+[c.3850-1GNA]a 151 [p.G542X]+[c.2380del8]a 131 [c.1078delT]+[p.K710X]a N130 [p.I507del]+[p.R334W]b 75 [p.G542X]+[p.R117H ;c1342-6 T7]b MI [p.E1104X]+[p.E1104X]b 84 [p.R117H; c1342-6 T7]+[p.R117H; c1342-6 T7]a 99 [c.2183AANG]+[p.Q220X]a IRT: Immunoreactive trypsinogen (cutoff: 65 ng/ml). Login to comment
87 M. des Georges et al. / Journal of Cystic Fibrosis 3 (2004) 265-272268 in trans of p.G542X, p.K710X in trans of c.1078delT and p.Q220X in trans of c.2183AANG (Table 2). Login to comment
89 The mutation was p.F508del (n=47), p.G542X (n=5), p.N1303K (n=4), p.G551D (n=2), p.R334W (n=2), c.1717- 1NA (n=1), p.I507del (n=1), p.R1162X (n=1), [p.R117H;IVS8-T7] (n=8) or [p.R117H;IVS8-T5] (n=1). Login to comment
131 The panel of 30 mutations (c.1717-1GNA, p.G542X, p.W1282X, p.N1303K, p.F508del, c.3849+10kbCNT, c.621+1GNT, p.R553X, p.G551D, p.R117H, p.R1162X, p.R334W, p.A455E, c.2183AANG, c.3659delC, c.1078delT, p.I507del, p.R347P, p.S1251N, p.E60X, p.Y1092X, c.394delTT, c.1811+1.6kbANG, c.3272-26ANG, c.2789+5GNA, c.3120+1GNA, c.711+ 1GNT, p.G85E, p.Y122X, p.W846X) should account for 83.32% of the CF alleles in L-R and 84.25% in the whole country. Login to comment

[hide] Microsphere bead arrays and sequence validation of... J Mol Diagn. 2004 Nov;6(4):348-55. Hadd AG, Laosinchai-Wolf W, Novak CR, Badgett MR, Isgur LA, Goldrick M, Walkerpeach CR
Microsphere bead arrays and sequence validation of 5/7/9T genotypes for multiplex screening of cystic fibrosis polymorphisms.
J Mol Diagn. 2004 Nov;6(4):348-55., [PMID:15507674]

Abstract [show]
The development of simple and rapid methods for the detection of the common genetic mutations associated with cystic fibrosis (CF) requires access to positive-control samples including the 5/7/9T variants of intron 8. We used PCR and a simple multiplex bead-array assay to identify 5/7/9T control samples from 29 commercially available DNA samples. Unpurified PCR products were directly hybridized to color-coded beads containing allele-specific capture probes for 5/7/9T detection. The performance of the assay was investigated using reverse-complement oligonucleotides, individual PCR products, and multiplex PCR products for 5/7/9T detection within a complex CFTR screening assay. Samples were genotyped by grouping the relative signal intensities from each capture probe. Of 29 commercially available DNA samples analyzed, 2 5T/7T, 2 5T/9T, 9 7T/9T, 11 7T/7T, and 5 9T/9T genotypes were identified. The genotype within each sample group was confirmed by DNA sequencing. The assay was compatible with the analysis of 10 to 1000 ng of genomic DNA isolated from whole blood and allowed for the separate identification of primary CFTR mutations from reflex variants. The correct identification of positive controls demonstrated the utility of a simple bead-array assay and provided accessible samples for assay optimization and for routine quality control in the clinical laboratory.

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197 Intron 8 Genotype by Coriell Number, Characterized CF Mutation and Allele Fraction for 5/7/9T Intron 8 genotype Coriell sample Characterized mutation Allele fraction by probe 5T 7T 9T 7T/7T NA09947 Normal 0.04 0.93 0.03 NA11277 ⌬I507/normal 0.06 0.90 0.04 NA11761 G551D/R553X 0.06 0.92 0.02 NA11859 2789ϩ5GϾA/2789ϩ5GϾA 0.02 0.96 0.02 NA11860 3849ϩ10kbCϾT/3849ϩ10kbCϾT 0.03 0.94 0.03 NA12444 1717-1GϾT/normal 0.06 0.87 0.07 NA12585 R1162X/normal 0.07 0.86 0.08 NA12785 R347P/G551D 0.04 0.92 0.05 NA12960 R334W/normal 0.06 0.92 0.02 NA12961 V520F/normal 0.06 0.89 0.05 NA13033 F508C/normal 0.03 0.93 0.04 9T/9T NA01531 ⌬F508/⌬F508 0.14 0.04 0.82 NA11281 621ϩ1GϾT/⌬F508 0.14 0.04 0.82 NA11283 A455E/⌬F508 0.13 0.05 0.82 NA11290 A455E/621ϩ1GϾT 0.12 0.01 0.87 NA11496 G542X/G542X 0.14 0.05 0.81 5T/7T NA11723 W1282X/normal 0.53 0.44 0.03 NA13032 I506V/normal 0.58 0.39 0.03 5T/9T NA11279 129GϾC/⌬F508 0.51 0.00 0.49 NA13591 R117H/⌬F508 0.52 0.00 0.48 7T/9T NA07441 3120ϩ1GϾA/621ϩ1GϾA 0.08 0.41 0.51 NA07552 R553X/⌬F508 0.09 0.36 0.55 NA07830 556dA/⌬F508 0.11 0.37 0.52 NA11275 3659dC/⌬F508 0.10 0.37 0.53 NA11278 Q493X/⌬F508 0.09 0.38 0.53 NA11280 711ϩ1GϾT/621ϩ1GϾA 0.09 0.37 0.54 NA11282 G85E/621ϩ1GϾA 0.07 0.39 0.53 NA11284 R560T/⌬F508 0.08 0.39 0.52 NA11472 N1303K/G1349D 0.08 0.39 0.54 Figure 3. Login to comment

[hide] CFTR Cl- channel function in native human colon co... Gastroenterology. 2004 Oct;127(4):1085-95. Hirtz S, Gonska T, Seydewitz HH, Thomas J, Greiner P, Kuehr J, Brandis M, Eichler I, Rocha H, Lopes AI, Barreto C, Ramalho A, Amaral MD, Kunzelmann K, Mall M
CFTR Cl- channel function in native human colon correlates with the genotype and phenotype in cystic fibrosis.
Gastroenterology. 2004 Oct;127(4):1085-95., [PMID:15480987]

Abstract [show]
BACKGROUND & AIMS: Cystic fibrosis (CF) is caused by over 1000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and presents with a widely variable phenotype. Genotype-phenotype studies identified CFTR mutations that were associated with pancreatic sufficiency (PS). Residual Cl- channel function was shown for selected PS mutations in heterologous cells. However, the functional consequences of most CFTR mutations in native epithelia are not well established. METHODS: To elucidate the relationships between epithelial CFTR function, CFTR genotype, and patient phenotype, we measured cyclic adenosine monophosphate (cAMP)-mediated Cl- secretion in rectal biopsy specimens from 45 CF patients who had at least 1 non-DeltaF508 mutation carrying a wide spectrum of CFTR mutations. We compared CFTR genotypes and clinical manifestations of CF patients who expressed residual CFTR-mediated Cl- secretion with patients in whom Cl- secretion was absent. RESULTS: Residual anion secretion was detected in 40% of CF patients, and was associated with later disease onset (P < 0.0001), higher frequency of PS (P < 0.0001), and less severe lung disease (P < 0.05). Clinical outcomes correlated with the magnitude of residual CFTR activity, which was in the range of approximately 12%-54% of controls. CONCLUSIONS: Specific CFTR mutations confer residual CFTR function to rectal epithelia, which is related closely to a mild disease phenotype. Quantification of rectal CFTR-mediated Cl- secretion may be a sensitive test to predict the prognosis of CF disease and identify CF patients who would benefit from therapeutic strategies that would increase residual CFTR activity.

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78 Relationship Between the CFTR Genotype and Cl- Channel Function in Native Rectal Epithelia CFTR genotype Number of individuals Sweat Cl-concentration (mmol/L)a cAMP-mediated response Carbachol-induced plateau response or maximal lumen-negative response Isc-cAMP (␮A/cm2) Cl- secretion (% of control) Isc-carbachol (␮A/cm2) Cl- secretion (% of control) Cl- secretion absent R1162X/Q552X 1 71 17.1 0 0.7 0 W1282X/3121-2AϾG 1 112 1.9 0 0.6 0 1898 ϩ 1G Ͼ T/1609delCA 2b 114, 118 25.4, 13.4 0, 0 0, 0.7 0, 0 ⌬F508/Q39X 2b 127, 129 2.6, 4.4 0, 0 1.7, 3.7 0, 0 ⌬F508/G542X 1 102 29.0 0 6.6 0 ⌬F508/R553X 3 112, 102, 109 13.1, 4.5, 23.8 0, 0, 0 1.5, 4.4, 1.0 0, 0, 0 ⌬F508/E585X 1 115 1.4 0 1.1 0 ⌬F508/Q637X 1 100 2.9 0 1.2 0 ⌬F508/Y1092X 1 119 0.0 0 -0.3 0 ⌬F508/120del23c 1 72 20.1 0 3.3 0 ⌬F508/182delT 1 116 10.8 0 5.2 0 ⌬F508/3905insT 2 88, 96 8.4, 5.6 0, 0 2.3, -1.1 0, 1 ⌬F508/V520F 1 68 1.2 0 1.7 0 ⌬F508/A561E 3 113, 146, 100 17.0, 17.0, 16.0 0, 0, 0 2.1, 1.5, 3.7 0, 0, 0 ⌬F508/R1066C 1 138 0.0 0 0.0 0 ⌬F508/N1303K 3 100, 117, 94 1.7, 4.1, 1.5 0, 0, 0 -0.6, 2.2, 0.8 0, 0, 0 A561E/A561E 2 101, 116 6.6, 2.0 0, 0 7.3, 3.3 0, 0 Residual Cl- secretiond G542X/I148N 1 75 -50.1 54 -22.2 12 1898 ϩ 3A Ͼ G/1898 ϩ 3A Ͼ G 1 82 -36.8 39 -12.9 7 ⌬F508/3272-26A Ͼ G 1 116 -17.8 19 -27.2 14 ⌬F508/S108F 1 118 -15.8 17 -12.3 7 ⌬F508/R117H 1 90 -35.9 38 -207.7 109 ⌬F508/Y161Cc 1 44 -35.1 37 -45.9 25 ⌬F508/P205S 1 80 -23.3 25 -10.4 5 ⌬F508/V232D 1 120 -16.9 18 -26.9 14 ⌬F508/R334W 1 92 -22.1 23 -21.1 11 ⌬F508/R334W 1 101 -24.5 26 -37.4 20 ⌬F508/T338I 1 73 -44.4 47 -79.4 42 ⌬F508/G576A 1 40 -16.9 18 -115.5 61 ⌬F508/I1234V 1 113 -13.6 15 -8.6 5 G576A/G85E 1 95 -26.1 28 -61.6 32 F1052V/M1137R 1 47 -36.7 39 -146.6 77 M1101K/M1101K 1 94 -11.1 12 -4.8 3 S1159F/S1159F 1 67 -47.9 51 -38.7 21 N1303K/R334W 1 91 -30.3 32 -47.7 25 NOTE. CFTR Cl- channel function was determined in rectal epithelia from Cl- secretory responses induced by IBMX/forskolin (Isc-cAMP) and after co-activation with carbachol (Isc-carbachol). Login to comment
84 One patient had TG10-9T/TG11-5T with G542X/1148N; the second patient had TG10-9T/TG12-5T with ⌬F508/S108F. Login to comment
101 Functional Classification and Protein Location of CFTR Mutations Mutation type Severe mutations (protein location) Mild mutations (protein location) Missense V520F, A561E (NBD1) G85E (MSD1, TM1) R1066C (MSD2, CL4) S108F, R117H (MSD1, EL1) N1303K (NBD2) I148N, Y161Ca (MSD1, CL1) P205S (MSD1, TM3) V232D (MSD1, TM4) R334W, T338I (MSD1, TM6) G576A (NBD1) I1234V (NBD2) F1052V, M1101K (MSD2, CL4) M1137R (MSD2, TM12) S1159F (pre-NBD2) Splice 1898 ϩ 1G Ͼ T (R domain) 1898 ϩ 3A Ͼ G (R domain) 3121-2A Ͼ G (MSD2, TM9) 3272-26A Ͼ G (MSD2, TM10) Single amino acid deletion ⌬F508 (NBD1) Nonsense Q39X (N-terminus) G542X, Q552X, R553X, E585X (NBD1) Q637X (R domain) Y1092X (MSD2, CL4) R1162X (pre-NBD2) W1282X (NBD2) Frameshift 120del23a 182delT (N-terminus) 1609delCA (NBD1) 3905insT (NBD2) NOTE. Severe mutation, Cl- secretion absent; mild mutation, residual cAMP-mediated Cl- secretion. Login to comment

[hide] Some like it hot: curcumin and CFTR. Trends Mol Med. 2004 Oct;10(10):473-5. Davis PB, Drumm ML
Some like it hot: curcumin and CFTR.
Trends Mol Med. 2004 Oct;10(10):473-5., [PMID:15464445]

Abstract [show]
The activation of mutant forms of the cystic fibrosis transmembrane conductance regulator (CFTR), particularly the most frequent mutant allele (DeltaF508), is a potential strategy for the treatment of the disease cystic fibrosis (CF). Therefore, it is of great interest that curcumin, a component of the spice turmeric, is reported to restore function to this allele, both in heterologous expression systems and in DeltaF508 CF mice. Although other laboratories have not been able to confirm the initial observations, activating DeltaF508 CFTR could have such important therapeutic implications that a thorough investigation of the potential of curcumin is warranted.

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13 The best-studied therapy is gentamicin, which causes read-through of 'stop` codons and has been used to correct the defect for the W1282X, G542X and other similar CF mutations with some success in clinical trials [2]. Login to comment

[hide] Immunohistochemistry of CFTR in native tissues and... J Cyst Fibros. 2004 Aug;3 Suppl 2:37-41. Mendes F, Doucet L, Hinzpeter A, Ferec C, Lipecka J, Fritsch J, Edelman A, Jorna H, Willemsen R, Bot AG, De Jonge HR, Hinnrasky J, Castillon N, Taouil K, Puchelle E, Penque D, Amaral MD
Immunohistochemistry of CFTR in native tissues and primary epithelial cell cultures.
J Cyst Fibros. 2004 Aug;3 Suppl 2:37-41., [PMID:15463923]

Abstract [show]
Studies on CFTR protein expression and localization in native tissues or in primary cultures of human epithelial cells are scarce due to the intrinsic instability of this protein, its low expression in most tissues and also to technical difficulties. However, such data are of the highest importance to understand the pathophysiology of CF. The purpose of this article is to outline several assays for the characterization of primary epithelial cultures and to review different CFTR immunostaining protocols.

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90 Furthermore, tissues from CF patients carrying two nonsense mutations, e.g., R553X, G542X, and W1282X, can represent the golden-standard negative control, as no full-length CFTR protein is produced in these cells. Login to comment
91 Furthermore, tissues from CF patients carrying two nonsense mutations, e.g., R553X, G542X, and W1282X, can represent the golden-standard negative control, as no full-length CFTR protein is produced in these cells. Login to comment

[hide] Cystic fibrosis at the Reunion Island (France): sp... J Cyst Fibros. 2004 Aug;3(3):185-8. Dugueperoux I, Bellis G, Lesure JF, Renouil M, Flodrops H, De Braekeleer M
Cystic fibrosis at the Reunion Island (France): spectrum of mutations and genotype-phenotype for the Y122X mutation.
J Cyst Fibros. 2004 Aug;3(3):185-8., [PMID:15463906]

Abstract [show]
BACKGROUND: The Reunion Island is a French administrative department located in the Indian Ocean between the islands of Madagascar and Mauritius. Its population is known to be at a high risk of cystic fibrosis (CF). METHODS: Data concerning all CF patients born at the Reunion Island was extracted from the French CF Registry. Twenty-eight DeltaF508/DeltaF508, 17 Y122X/DeltaF508, and 11 Y122X/Y122X were included in a genotype-phenotype study. RESULTS: The detection rate of the CFTR mutations was 83% among the CF patients born at the Reunion Island. Three CFTR mutations accounted for 75% of the detected CF alleles at the Reunion Island (DeltaF508, Y122X, and 3120 + 1G-->A.). The DeltaF508/DeltaF508, DeltaF508/Y122X, and Y122X/Y122X genotypes accounted for 60.2% of the CF patients. Patients carrying at least one Y122X mutation were pancreatic insufficient, had high sweat chloride values and significantly lower anthropometric measures. The mean anthropometric values in all three groups were lower that in the whole CF population followed in "continental" France. This may reflect the poor compliance and even the refusal of treatment noted by the clinicians. CONCLUSIONS: The distribution of CFTR mutations could be explained by the history of the Reunion Island: admixture of French settlers, African and Asian populations, founder effect and isolation followed by genetic drift. The Y122X allele appears to be associated with a severe phenotype.

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67 A 7 (4.79) Y122X/Y122X 13 (19.40) G542X 2 (1.37) 3120 + 1G ! Login to comment
76 A 1 (0.68) DeltaF508/ D993Y 1 (1.49) 993del5 1 (0.68) DeltaF508/ G542X 1 (1.49) A455E 1 (0.68) DeltaF508/2183AA ! Login to comment

[hide] Long-range (17.7 kb) allele-specific polymerase ch... J Mol Diagn. 2004 Aug;6(3):264-70. Pont-Kingdon G, Jama M, Miller C, Millson A, Lyon E
Long-range (17.7 kb) allele-specific polymerase chain reaction method for direct haplotyping of R117H and IVS-8 mutations of the cystic fibrosis transmembrane regulator gene.
J Mol Diagn. 2004 Aug;6(3):264-70., [PMID:15269305]

Abstract [show]
Genotyping of genetic polymorphisms is widely used in clinical molecular laboratories to confirm or predict diseases due to single locus mutations. In contrast, very few molecular methods determine the phase or haplotype of two or more mutations that are kilobases apart. In this report, we describe a new method for haplotyping based on long-range allele-specific PCR. Reaction conditions were established to circumvent the incompatibility of using allele-specific primers and a polymerase with proofreading activity. Haplotypes are determined by post-PCR analysis using different detection methods. The clinical application presented here directly determines the phase of two mutations separated by 17.7 kilobases in the cystic fibrosis transmembrane conductance regulator gene. Each mutation, the missense mutation R117H in exon 4 and the 5T polymorphism in intron 8 (IVS-8), have mild phenotypic effect unless they are present on the same chromosome (in cis). If an individual is heterozygous for both R117H and the IVS-8 5T variant, cis/trans testing is required to completely interpret results. The molecular method presented here bypasses the need to perform family studies to establish haplotypes. We propose use of this assay as a reflex clinical test for R117H- 5T-positive samples.

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131 Genotypes and Haplotypes of R117H Samples Sample Genotype Haplotype Interpretation R chromosome (wild type) H chromosome (mutant) 1 R117H-5T/7T R-5T H-7T Trans 2 R117H-5T/7T R-7T H-5T Cis 3 R117H-5T/9T R-9T H-5T Cis 4 R117H-5T/7T R-5T H-7T Trans 5 R117H/Del F508-7T/9T R-9T H-5T Cis 6 R117H-7T/7T failed H-7T na 7 R117H-7T/7T R-7T H-7T na 8 R117H-7T/9T R-9T H-7T na 9 R117H-7T/7T R-7T H-7T na 10 R117H-7T/7T failed H-7T na 11 R117H-7T/7T R-7T H-7T na 12 R117H-7T/7T R-7T H-7T na 13 R117H-7T/7T R-7T H-7T na 14 R117H-7T/9T R-9T H-7T na 15 R117H-7T/9T R-9T H-7T na 16 R117H/DelF508-7T/9T R-9T H-7T na 17 R117H/G542X-7T/9T R-9T H-7T na na, not applicable. Login to comment

[hide] Pancreatitis in hispanic patients with cystic fibr... Clin Gastroenterol Hepatol. 2004 Jun;2(6):504-9. Maisonneuve P, Campbell P 3rd, Durie P, Lowenfels AB
Pancreatitis in hispanic patients with cystic fibrosis carrying the R334W mutation.
Clin Gastroenterol Hepatol. 2004 Jun;2(6):504-9., [PMID:15181620]

Abstract [show]
BACKGROUND & AIMS: Cystic fibrosis (CF) results from abnormal production of sticky mucus, which obstructs many organs. In most cases, the pancreas is severely compromised, but 10%-15% of patients with CF have pancreas sufficiency (PS) and are subject to develop pancreatitis. The aim of this study is to determine which specific genotypes lead to the development of pancreatitis in patients with CF. METHODS: We used prospective data collected by the Cystic Fibrosis Foundation and performed a nested case-control study with all patients who reported at least 1 episode of pancreatitis constituting the cases. We used logistic regression to assess the association between pancreatitis and genotype and the Kaplan-Meier method to estimate the cumulative incidence of pancreatitis for selected genotypes. RESULTS: Three hundred sixty-four of 17,871 genotyped patients with CF (2.0%) reported at least 1 episode of pancreatitis. Only 0.9% of 12,997 patients with genotypes generally associated with pancreas insufficiency reported pancreatitis against 11.9% of 868 patients carrying at least 1 mild CF mutation generally associated with PS. The greatest rate of pancreatitis (19.0%) was observed for patients carrying an R334W mutation: 48% of these 79 patients were Hispanic and 13 patients were living in Puerto Rico. CONCLUSIONS: Of all patients with CF, those carrying an R334W mutation have the greatest risk for developing pancreatitis. This mutation is found mostly in Hispanic patients with CF living in Puerto Rico. There are no current data to determine whether asymptomatic carriers of the R334W mutation are at greater risk for developing pancreatitis or whether this mutation is frequent in Hispanics with idiopathic pancreatitis.

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26 Of Ͼ1000 identified mutations in the CFTR gene, only 25 proven disease-causing mutant alleles (⌬F508, G551D, G542X, R553X, W1282X, R347P, NI303K, R560T, ⌬I507, 1717-1GϾA, A455E, 3120ϩ1GϾA, 621ϩ1GϾT, R117H, 711ϩ1GϾT, R1162X, 3849ϩ10kbCϾT, 2789ϩ5GϾT, R334W, G85E, 1078delT, 1898ϩ1GϾT, 2184delA, 3659delC, and I148T) are recommended by the American College of Medical Genetics for routine diagnostic and carrier testing.16 Most of these are routinely recorded in the CFF registry, but rarer mutations can be recorded if identified by more comprehensive testing. Login to comment
28 Patients were classified according to their genotype: those carrying 2 severe mutations (⌬F508, ⌬I507, G542X, G551D, N1303K, R553X, R560T, R1162X, W1282X, 621ϩ1GϾT, 711ϩ1GϾT, 1717-1GϾA, 2184delA, and 3659delC), which generally are associated with pancreas insufficiency (PI); and those carrying at least 1 mild mutation (3849ϩ10kbCϾT, R117H, 2789ϩ5GϾA, R347P, R334W, and A455E), which are generally associated with PS. Login to comment
67 aPatients with pancreas insufficiency (PI) carrying 2 PI mutations (⌬F508, ⌬I507, G542X, G551D, N1303K, R553X, R560T, R1162X, W1282X, 621ϩ1GϾT, 711ϩ1GϾT, 1717-1GϾA, 2184delA, 3659delC). Login to comment
73 Genotype Distribution of Patients With CF in Puerto Rico and Other States According to Ethnicity Genotypea Hispanics Non-Hispanics Puerto Rico United States United States ⌬F508/any 25 (66) 796 (83) 15,561 (92) R334W/any 13 (34) 25 (3) 41 (0.2) ⌬I507/any 4 (11) 78 (8) 436 (3) G542X/any 2 (5) 88 (9) 754 (4) R553X/any 1 (3) 15 (2) 324 (2) N1303K/any 1 (3) 28 (3) 424 (3) 621ϩ1GϾT/any 1 (3) 9 (1) 314 (2) Not identified/any 18 (47) 269 (28) 3137 (19) NOTE. Values expressed as number (percent). Login to comment
85 Characteristics of 15 Patients With CF Carrying an R334W Mutation Who Developed Pancreatitis Genotype Sex Race Ethnicity Age at CF (yr) CF diagnosis Age (yr)a R334W/⌬F508 Female White Non-Hispanic 4 Respiratory symptoms 38 R334W/⌬F508 Male White Non-Hispanic 23 Respiratory symptoms, nasal polyps 51 R334W/⌬F508 Female White Non-Hispanic 7 Respiratory symptoms 28 R334W/⌬F508 Female White Non-Hispanic 16 Electrolyte imbalance, respiratory symptoms 34 R334W/⌬F508 Male White Hispanic 13 Respiratory symptoms, electrolyte imbalance, failure to thrive Dead 18 R334W/⌬I507 Female White Hispanic 29 Respiratory symptoms, steatorrhea 31 R334W/G542X Female White Hispanic 7 Respiratory symptoms 10 R334W/R553X Female White Hispanic 10 Respiratory symptoms 21 R334W/G85E Female White Hispanic 0 Respiratory symptoms 8 R334W/G85E Female White Hispanic 4 Failure to thrive 10 R334W/R334W Male White Hispanic 4 Meconium ileus 10 R334W/R334W Female White Hispanic 0 Respiratory symptoms 41 R334W/? Login to comment

[hide] Distal intestinal obstruction syndrome in adults w... Clin Gastroenterol Hepatol. 2004 Jun;2(6):498-503. Dray X, Bienvenu T, Desmazes-Dufeu N, Dusser D, Marteau P, Hubert D
Distal intestinal obstruction syndrome in adults with cystic fibrosis.
Clin Gastroenterol Hepatol. 2004 Jun;2(6):498-503., [PMID:15181619]

Abstract [show]
BACKGROUND & AIMS: With the improved survival of patients with cystic fibrosis (CF), gastrointestinal complications become more evident in adults with this condition. The aims of this study were to determine the prevalence and clinical features of distal intestinal obstruction syndrome (DIOS) and its relationship with the cystic fibrosis transmembrane conductance regulator (CFTR) genotype in an adult CF population. METHODS: Cross-sectional study was conducted in an adult CF cohort. RESULTS: Among 171 adults with CF (mean age, 28.9 years), 27 patients (15.8%) reported 43 episodes of DIOS. No significant association was found between DIOS and a history of meconium ileus. The first episode of DIOS occurred in adulthood in 21 cases (77.8%). DIOS recurred in 13 patients (48.1%). All patients who developed DIOS had pancreatic insufficiency. Pulmonary function was significantly more altered in patients with DIOS than in the other patients, but pancreatic insufficiency and age might act as confounding factors. DIOS occurred in 21.9% of patients with a severe CFTR genotype and in only 2.4% of patients with a mild CFTR genotype (P < 0.005). CONCLUSIONS: DIOS is frequent in adults with CF with a severe CFTR genotype and/or advanced-stage pulmonary disease. The relative contributions of malabsorption and impaired intestinal secretion in the development of DIOS are discussed.

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110 CFTR Genotypes of Patients With a History of DIOS CFTR mutations Observations Frequency Mutation classes CFTR genotype ⌬F508/⌬F508 15 55.6% II/II Severe ⌬F508/non-⌬F508 9 33.3% ⌬F508/E60X 1 II/I Severe ⌬F508/G542X 1 II/I Severe ⌬F508/W846X 1 II/I Severe ⌬F508/R851X 1 II/I Severe ⌬F508/2894insAG 2 II/I Severe ⌬F508/⌬I507 1 II/II Severe ⌬F508/G551D 1 II/III Severe ⌬F508/2789ϩ5GϾA 1 II/V Mild Non-⌬F508/non-⌬F508 3 11.1% G542X/G542X 1 I/I Severe W1282X/W1282X 1 I/I Severe 1811ϩ1.6kb AϾG/ni 1 I/undetermined Undetermined Total 27 100.0% ni, not identified. Login to comment

[hide] Glucose intolerance in children with cystic fibros... J Pediatr. 2003 Feb;142(2):128-32. Solomon MP, Wilson DC, Corey M, Kalnins D, Zielenski J, Tsui LC, Pencharz P, Durie P, Sweezey NB
Glucose intolerance in children with cystic fibrosis.
J Pediatr. 2003 Feb;142(2):128-32., [PMID:12584532]

Abstract [show]
OBJECTIVE: To evaluate the relations among glucose intolerance, genotype, and exocrine pancreatic status in patients with cystic fibrosis (CF). STUDY DESIGN: Data on 335 patients <18 years of age were from the Toronto CF database. A modified oral glucose tolerance test was given to 94 patients 10 to 18 years of age without recognized CF-related diabetes. CF transmembrane conductance regulator mutations and exocrine pancreatic status were determined for all patients. RESULTS: CF-related diabetes was clinically recognized in 9 of 335 (2.7%) patients <18 years of age, all of whom were pancreatic insufficient, and 8 of 9 had severe (classes I through III) mutations on both alleles. The ninth patient had unidentified mutations. Although all patients given the oral glucose tolerance test were asymptomatic and had normal fasting blood glucose, 16 of 94 (17%) had impaired glucose tolerance and 4 of 94 (4.3%) had CF-related diabetes without fasting hyperglycemia. Abnormal glucose tolerance was associated exclusively with severe mutations and exocrine pancreatic insufficiency. Glycosylated hemoglobin (HbA(1)C) levels did not correlate with glucose tolerance results. CONCLUSIONS: Screening of pancreatic-insufficient, adolescent patients with CF identified more with abnormal oral glucose tolerance than was suspected clinically and is recommended as a routine practice. HbA(1)C was not useful in screening for CF-related glucose intolerance.

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118 of patients with IGT 2 10 2 0 0 1/1 16 No of patients with CFRD without FH 0 4 0 0 0 0 4 *Genotype class based on mutation with ∆F508: Class I, 621+1G→T, G542X, 441delA, R553X, W1282X, 3120+1G→A, 4016insT, 1154insTC, I1027T; Class II, ∆F508; Class III, G551D, G85E, S549N, L1077P, H199R; Class IV, Class V, 3849+10kbC→T, 5T; Unknown, G85E/-, ∆F508/-; Other, G551D/R506T, W1282X/W1282X. Login to comment

[hide] Therapeutic approaches to repair defects in deltaF... Adv Drug Deliv Rev. 2002 Dec 5;54(11):1395-408. Powell K, Zeitlin PL
Therapeutic approaches to repair defects in deltaF508 CFTR folding and cellular targeting.
Adv Drug Deliv Rev. 2002 Dec 5;54(11):1395-408., [PMID:12458151]

Abstract [show]
The deltaF508 mutation in the cystic fibrosis transmembrane regulator (CFTR) gene is the most common mutation in CF. The mutant CFTR protein is defective with respect to multiple functions including cAMP-regulated chloride conductance, nucleotide transport, and regulatory actions on other ion channels. Since the deltaF508 protein is also temperature-sensitive and unstable during translation and folding in the endoplasmic reticulum (ER), most of the nascent chains are targeted for premature proteolysis from the ER. This paper focuses on the events that occur in the ER during folding and reviews potential targets for therapeutic intervention.

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88 These authors [34] immunoprecipitated G542X, R553X, 621 1 1 G → T, 1717-1 G → A, the immature bands A and B of mutant DF508 CFTR and 3905insT. Login to comment
89 These authors [34] immunoprecipitated G542X, R553X, 621 1 1 G ࢐ T, 1717-1 G ࢐ A, the immature bands A and B of mutant DF508 CFTR and 3905insT. Login to comment

[hide] Genotype and phenotype correlations in patients wi... Gastroenterology. 2002 Dec;123(6):1857-64. Durno C, Corey M, Zielenski J, Tullis E, Tsui LC, Durie P
Genotype and phenotype correlations in patients with cystic fibrosis and pancreatitis.
Gastroenterology. 2002 Dec;123(6):1857-64., [PMID:12454843]

Abstract [show]
BACKGROUND & AIMS: Pancreatitis is known to occur in some patients with cystic fibrosis (CF), but the prevalence, natural history, and genotypic basis are unclear. We examined a well-defined cohort of patients with CF to answer these questions. METHODS: Patients with CF were identified from a computerized database (1966-1996). Chart audit identified all patients with CF and pancreatitis. RESULTS: Among 1075 patients with CF, 937 (87%) were pancreatic insufficient at diagnosis, 28 (3%) were pancreatic sufficient but developed pancreatic insufficiency after diagnosis, and 110 (10%) have remained pancreatic sufficient. No patients with pancreatic insufficiency developed pancreatitis. Nineteen patients (17.3%) with pancreatic sufficiency experienced one or more attacks of pancreatitis. The mean age at diagnosis of pancreatitis was 22.7 +/- 10.3 years (range, 10-35 years), and pancreatitis was recognized before the diagnosis of CF in 6 patients (32%). The diagnosis of CF in pancreatic-sufficient patients, with and without pancreatitis, was established at a significantly older age than in those with pancreatic insufficiency (P < 0.0001). Genotyped patients with pancreatic insufficiency carried 2 severe mutant alleles. All genotyped patients with pancreatic sufficiency and pancreatitis carried at least one mild mutation. No specific genotype was predictive of pancreatitis. CONCLUSIONS: Patients with CF with pancreatic sufficiency carry at least one mild mutant allele and are at a significant risk of developing pancreatitis. Symptoms of pancreatitis may precede the diagnosis of CF. Pancreatitis is associated with an otherwise mild CF phenotype.

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105 CFTR Genotypes Among CF Patients With PS With and Without Pancreatitis Two mutations (n) ⌬F508/R117H (9) ⌬F508/(5T) (6) ⌬F508/3272-26A 3 G (4) ⌬F508/R347H (2) ⌬F508/P574H (2) ⌬F508/875 ϩ 1G Ͼ C (2) ⌬F508/3849 ϩ 10kb C 3 T (1) ⌬F508/A455E (1) ⌬F508/D614G (1) ⌬F508/G85E (1) ⌬F508/R347P (1) ⌬F508/S1251N (1) ⌬F508/⌬F508a (1) ⌬F508/3120G Ͼ A (1) ⌬F508/G551Da (1) G542X/R117H (1) R560T/L206W (1) R117H/R117H (1) R31L/P67L (1) 1461ins4 (AGAT)/G85E (1) G551D/(5T) (1) R1066C/3849 ϩ 10kb C Ͼ T (1) G551D/3849 ϩ 10kb C Ͼ T (1) R334W/R334W (1) R334W/681delC (1) W1282X/3489 ϩ 10kb C Ͼ T (1) One mutation (n) ⌬F508/- (18) L1077P/- (1) W1282X/- (1) M1137V/- (1) G551D/- (1) R347H/- (1) Q30X1/- (1) G1244E/- (1) R117H/- (1) 621 ϩ 2G621 ϩ 1G 3 T/- (1) NOTE. Login to comment
124 of episodes of pancreatitis Genotype 1 0.3 12 21.7 2 ⌬F508/S1251N 2 0.3 34 30.0 1 ⌬F508/R347H 3 4.4 13 42.5 3 / 4 4.4 21 36.5 1 ⌬F508/ 5 7.3 26 40.8 10 ⌬F508/P67L 6 9.6 29 29.9 (D) 1 ⌬F508/ 7 12.0 18 39.9 1 ⌬F508/R347P 8 12.9 37 40.9 2 G542X/D1152H 9 13.0 30 50.3 1 ⌬F508/3849 ϩ 10Kbc Ͼ T 10 14.7 13 21.5 1 DF508/R117H 11 15.6 34 40.8 1 ⌬F508/2789ϩ5G Ͼ T 12 15.6 10 26.0 10 ⌬F508/R117H 13 16.0 10 22.0 14 ⌬F/508/3849 ϩ 10kbC Ͼ T 14 16.0 18 21.2 (D) 1 R1066C/3849 ϩ 10kbC Ͼ T 15 19.9 15 40.8 5 No DNA 16 23.2 19 23.2 15 ⌬F508/11234V 17 24.1 40 47.6 (D) 1 No DNA 18 26.9 25 43.3 12 No DNA 19 27.4 35 50.3 (D) 2 ⌬F508/A455E NOTE. Login to comment

[hide] Cystic fibrosis and related diseases of the pancre... Best Pract Res Clin Gastroenterol. 2002 Jun;16(3):511-26. Naruse S, Kitagawa M, Ishiguro H, Fujiki K, Hayakawa T
Cystic fibrosis and related diseases of the pancreas.
Best Pract Res Clin Gastroenterol. 2002 Jun;16(3):511-26., [PMID:12079272]

Abstract [show]
The discovery of the gene for cystic fibrosis (CF), the cystic fibrosis transmembrane conductance regulator (CFTR), brought about a new era in the study of this disease. Identification of the molecular target has yielded a flood of data that add to our understanding of the pathogenesis, diagnosis and treatment of CF. The CFTR protein is a cAMP-regulated Cl(-) channel with multiple functions in epithelial cells. In the exocrine pancreas the CFTR plays a key role in the apical Cl(-), HCO(3)(-), and water transport in duct cells. The severe loss of functions, caused by mutations of the CFTR gene, leads to pathological lesions of the pancreas. Over 1200 CFTR mutations and polymorphisms have been identified and their diversity may explain the high level of heterogeneity in the CF phenotype. Mutation analyses of the CFTR gene have revealed a spectrum of CFTR-related diseases that do not fit the classical CF picture but are associated with dysfunction of CFTR, such as chronic pancreatitis.

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29 These include regulations of (1) the outwardly rectifying ClÀ channel, a separate class of ClÀ channel regulated by cAMP-dependent PKA and PKC, (2) the epithelial Na channel, (3) the inwardly rectifying K channel, (4) vesicle tracking, and (5) intracellular compartment acidi®cation and protein processing.8 CFTR GENE MUTATIONS Approximately 70% of the mutations in CF patients in Caucasian populations correspond to a speci®c deletion of three base pairs which results in the loss of a phenylalanine at position 508 (DF508) in the CFTR protein.4 Other mutations are rare and vary considerably among dierent ethnic groups.5 The most common 10 mutations are DF508 (66%), G542X (2.4%), G551D (1.6%), N1303K (1.3%), W1282X (1.2%), R553X (0.7%), 621 1G 4 T (0.7%), 1717-1G 4 A (0.6%), R117H (0.3%) and R1162X (0.3%).9 It is not clear how many dierent CF mutations exist in the CFTR gene. Login to comment
62 is observed only when normal CFTR function is less than 1%.13 In general, patients with pancreatic insuciency are homozygous or compound heterozygous for two severe mutations (class I, II or III in Figure 3), such as DF508, DI507, Q493X, G542X, R553X, W1282X, 621 1G 4 T, 1717-1G 4 A, 556delA, 3659delC, I148T, G480C, V520F, G551D, and R560T, whereas the PS phenotype occurs in patients who have one or two mild CFTR mutations, such as R117H, R334W, R347P, A455E, and P574H (class IV or V).5,20 EXOCRINE PANCREAS IN CYSTIC FIBROSIS Pathology of the pancreas in CF There is a spectrum of pancreatic abnormalities in CF irrespective of age.21,22 Pancreatic lesions may be absent in an individual case, but in long-standing CF the pancreas is small, hard and nodular with increased fat and multiple cysts; hence the name `cystic ®brosis of the pancreas'. Login to comment
27 These include regulations of (1) the outwardly rectifying Cl channel, a separate class of Cl channel regulated by cAMP-dependent PKA and PKC, (2) the epithelial NaW channel, (3) the inwardly rectifying KW channel, (4) vesicle traQcking, and (5) intracellular compartment acidi&#ae;cation and protein processing.8 CFTR GENE MUTATIONS Approximately 70% of the mutations in CF patients in Caucasian populations correspond to a speci&#ae;c deletion of three base pairs which results in the loss of a phenylalanine at position 508 (DF508) in the CFTR protein.4 Other mutations are rare and vary considerably among diPerent ethnic groups.5 The most common 10 mutations are DF508 (66%), G542X (2.4%), G551D (1.6%), N1303K (1.3%), W1282X (1.2%), R553X (0.7%), 621 W 1G 4 T (0.7%), 1717-1G 4 A (0.6%), R117H (0.3%) and R1162X (0.3%).9 It is not clear how many diPerent CF mutations exist in the CFTR gene. Login to comment
64 is observed only when normal CFTR function is less than 1%.13 In general, patients with pancreatic insuQciency are homozygous or compound heterozygous for two severe mutations (class I, II or III in Figure 3), such as DF508, DI507, Q493X, G542X, R553X, W1282X, 621 W 1G 4 T, 1717-1G 4 A, 556delA, 3659delC, I148T, G480C, V520F, G551D, and R560T, whereas the PS phenotype occurs in patients who have one or two mild CFTR mutations, such as R117H, R334W, R347P, A455E, and P574H (class IV or V).5,20 EXOCRINE PANCREAS IN CYSTIC FIBROSIS Pathology of the pancreas in CF There is a spectrum of pancreatic abnormalities in CF irrespective of age.21,22 Pancreatic lesions may be absent in an individual case, but in long-standing CF the pancreas is small, hard and nodular with increased fat and multiple cysts; hence the name `cystic &#ae;brosis of the pancreas'. Login to comment

[hide] Relation between the anatomical genital phenotype ... Fertil Steril. 2002 May;77(5):889-96. Robert F, Bey-Omar F, Rollet J, Lapray JF, Morel Y
Relation between the anatomical genital phenotype and cystic fibrosis transmembrane conductance regulator gene mutations in the absence of the vas deferens.
Fertil Steril. 2002 May;77(5):889-96., [PMID:12009340]

Abstract [show]
OBJECTIVE: To study the correlation between genital phenotype and cystic fibrosis genotype in men lacking at least one vas deferens. DESIGN: Prospective study. SETTING: Institut Rhonalpin pour la Reproduction Humaine, Lyon-Bron, France. PATIENT(S): Forty-seven infertile men lacking at least one vas deferens. INTERVENTION(S): All patients were screened for the 13 most common CFTR gene mutations and for the 5-thymidine variant of intron 8. Renal, scrotal, and transrectal ultrasonography were systematically performed. MAIN OUTCOME MEASURE(S): Epididymal and seminal vesicular abnormalities and testicular volume were compared among men with two, one, or no CFTR gene mutation, with or without the 5T allele. RESULTS: Seminal vesicles and the symmetry of epididymal and vesicular abnormalities did not differ between patients with and those without the CFTR gene mutation. Epididymal abnormalities were more frequent in men without the mutation. Testicular volumes were significantly lower in men without the mutation and those with the 5T allele only. CONCLUSION: Men with the CFTR mutation, the 5T allele only, and those without CFTR mutation have few differences in genital phenotype. Low testicular volume is observed in men without the CFTR mutation and those with the 5T allele only.

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60 In 950 unrelated chromosomes, the incidences of ⌬F508, G542X and N1303K mutations were 69%, 4.6% and 2.7% respectively (15, 16; unpublished data). Login to comment
65 Methods used to detect these mutations were [1] heteroduplex formation followed by polyacrylamide gel electrophoresis (⌬F508, ⌬I507, 508C, and 1612delTT in exon 10 and 2183AA3G, 2184delA, and 2347delG in exon 13), [2] digestion with appropriate restriction enzymes, that is, MnlI for W1282X (exon 20) and SspI for 2789ϩ5G3A (exon 14b), and [3] PCR with the modified primers MvaI for G542X (exon 11) and N1303K (exon 21), AvaII for 1717-1G3A (exon 11), and HaII for R117H (exon 4) to create a new restriction site. Login to comment
135 The fetuses had the ⌬F508/⌬F508 and ⌬F508/G542X genetic mutations. Login to comment

[hide] Can a place of origin of the main cystic fibrosis ... Am J Hum Genet. 2002 Jan;70(1):257-64. Epub 2001 Nov 16. Mateu E, Calafell F, Ramos MD, Casals T, Bertranpetit J
Can a place of origin of the main cystic fibrosis mutations be identified?
Am J Hum Genet. 2002 Jan;70(1):257-64. Epub 2001 Nov 16., [PMID:11713719]

Abstract [show]
The genetic background of the mutations that most often cause cystic fibrosis (CF) is different from that of non-CF chromosomes in populations of European origin. It is not known whether these haplotype backgrounds could be found at high frequencies in populations in which CF is, at present, not common; such populations would be candidates for the place of origin of CF mutations. An analysis of haplotypes of CF transmembrane conductance regulator, together with their variation in specific CF chromosomes, in a worldwide survey of normal chromosomes shows (1) a very low frequency or absence of the most common CF haplotypes in all populations analyzed and (2) a strong genetic variability and divergence, among various populations, of the chromosomes that carry disease-causing mutations. The depth of the gene genealogy associated with disease-causing mutations may be greater than that of the evolutionary process that gave rise to present-day human populations. The concept of "population of origin" lacks either spatial or temporal meaning for mutations that are likely to have been present in Europeans before the ethnogenesis of present populations; subsequent population processes may have erased the traces of their geographic origin.

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20 Only four other mutations (G542X, N1303K, G551D, and W1282X) have overall frequencies 11% among the CF chromosomes. Login to comment
22 G542X is common in Mediterranean countries and is present in most of Europe, being most frequent (16.7%) in the Balearic Islands (Estivill et al. 1997). Login to comment
26 In addition, 17 other mutations have frequencies of 0.1%-0.9% (Estiv- Figure 1 Polymorphisms in the CFTR region (IVS1CA, IVS6aGATT, IVS8CA, T854, IVS17bTA, and TUB20), and location of the five most common CF mutations (DF508, G542X, N1303K, G551D, and W1282X). Login to comment
48 OF REPEATS FREQUENCY OF DF508 (n p 148) G542X (n p 56) N1303K (n p 17) 21 .993 1 .941 22 0 0 .059 23 .007 0 0 NOTE.-Numbers of chromosomes studied appear in parentheses. Login to comment
51 Because no disease chromosomes had yet been typed for the more recently described IVS1CA marker, a total of 126 patients (252 chromosomes), who carried the DF508, G542X, and/or N1303K mutations, were typed for this locus. Login to comment
56 IVS6aGATT, IVS8CA, T854, IVS17bTA, and TUB20 allele frequencies for CF chromosomes (with DF508, G542X, N1303K, G551D, and W1282X mutations) were obtained from the literature (Morral et al. 1994, 1996). Login to comment
58 Allele frequencies for the intron 1 CA repeat in CF Table 2 Most Frequent CFTR Haplotype(s) for the Five Most Common CF Mutations CF MUTATION HAPLOTYPE(S) AT MARKER a IVS1CA IVS6aGATT IVS8CA T854 IVS17bTA TUB20 DF508 21 6 23/17 1 31/32 2 G542X 21 6 23 1 33/32 2 N1303K 21 6 23/22/24 1 31 2 G551D NA 7 16 2 7 1 W1282X NA 7 17 2 7 1 a IVS1CA was typed in the present study. Login to comment
62 Table 3 Frequencies, in Normal Chromosomes, of Haplotypes Associated with CF Mutations DF508, G542X, and N1303K POPULATION MEAN FREQUENCY (SE) IN HAPLOTYPE (%)a A B C D Druze 2.4 ‫ע‬ 1.4 0 4 ‫ע‬ 1.7 0 Basque 0 0 4.2 ‫ע‬ 1.4 0 Catalan 0 0 1.4 ‫ע‬ .9 .6 ‫ע‬ .6 Finnish 0 1.6 ‫ע‬ 1.6 3.2 ‫ע‬ 2.2 1.6 ‫ע‬ 1.6 Russian 0 0 1.7 ‫ע‬ 1.7 1.7 ‫ע‬ 1.7 Adygei 0 0 2.0 ‫ע‬ 1.4 0 Japanese 0 0 0 1.2 ‫ע‬ 1.2 NOTE.-The frequency in the populations not listed is zero. Login to comment
66 When all six markers are considered in their chromosomal order (i.e., IVS1CA, IVS6aGATT, IVS8CA, T854, IVS17bTA, and TUB20), these haplotype background groups are: (1) 21-6-(17/22/23/24)-1-(31/32/33)-2, of which the most frequent are 21-6-23-1-31-2 (for DF508 and N1303K mutations) and 21-6-23-1-33-2 (for the G542X mutation) it is evident that these three different CF mutations (which have independent origins) are found in very closely related haplotypes, since they differ only by a few repeat units at the fast-evolving STRP sites; and (2) 7-(16/17)-2-7-1, in which G551D and W1282X are found. Login to comment
72 Haplotypes associated with CF mutations G542X and N1303K are closely related to those of DF508, and the situation is therefore similar. Login to comment
77 Thus, haplotypes found at frequencies of the same order Figure 3 Maximum-likelihood tree of allele frequencies of five loci (IVS6aGATT, IVS8CA, T854, IVS17bTA and TUB20) among normal chromosomes, from worldwide populations, and among CF chromosomes (DF508, G542X, N1303K, G551D and W1282X chromosomes). Login to comment
90 Thus, it appears that DF508, G542X, and N1303K are closely related to each other, as are G551D and W1282X, independently of the population from which chromosomes were sampled. Login to comment
94 Although no population has them in frequencies high enough to suggest a likely place of origin, it should be kept in mind that mutations DF508, G542X, and N1303K are independent unique events and that their occurrence in a similar background gives support to the hypothesis that the three mutations arose in a single population in which these haplotypes were frequent. Login to comment

[hide] Solid phase fluorescent sequencing of the CFTR gen... Methods Mol Biol. 2001;167:63-88. Cuppens H, Cassiman JJ
Solid phase fluorescent sequencing of the CFTR gene.
Methods Mol Biol. 2001;167:63-88., [PMID:11265322]

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17 Depending on the ethnic origin, five to ten mutations, such as 1717-1G A, G542X, G551D, R553X, W1282X, and N1303K, reach rather high frequencies (4). Login to comment

[hide] CFTR mutations in three Latin American countries. Am J Med Genet. 2000 Apr 10;91(4):277-9. Restrepo CM, Pineda L, Rojas-Martinez A, Gutierrez CA, Morales A, Gomez Y, Villalobos MC, Borjas L, Delgado W, Myers A, Barrera-Saldana HA
CFTR mutations in three Latin American countries.
Am J Med Genet. 2000 Apr 10;91(4):277-9., [PMID:10766983]

Abstract [show]
We analyzed 192 cystic fibrosis (CF) alleles in three Latin American countries: Mexico, Colombia, and Venezuela. Mutation screening was performed by polymerase chain reaction (PCR) and a reverse dot blot detection kit that enables determination of 16 of the most common CF mutations worldwide. Mutations were detected in 47.9% of the screened CF alleles. The most prevalent CF allele was DeltaF508 (39. 6%). The remaining 16 non-DeltaF508 detectable mutations represented 8.3% of the CF alleles. Among them, the G542X, N1303K, and 3849+10kb C>T were the most common. Although the frequency of DeltaF508 described here is lower than that reported for Caucasian populations, including in Spain, it is remarkable that mutation prevalences found in this study resemble those observed in Spain. Two of these mutations, G542X and 3849+10kb C>T, that were relevant in this analysis, have a particularly high incidence in Spanish communities. The low frequency of DeltaF508 described here may be explained by the Amerindian, Caucasian, and Black admixture that occurred in Latin America after the discovery of the New World, and also by the probable occurrence of mutations contributed by the original natives, which were undetectable in this analysis.

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5 Among them, the G542X, N1303K, and 3849+10kb C>T were the most common. Login to comment
7 Two of these mutations, G542X and 3849+10kb C>T, that were relevant in this analysis, have a particularly high incidence in Spanish communities. Login to comment
34 The isolated DNA from each patient was amplified by polymerase chain reaction (PCR) using a kit for reverse dot blot detection of 16 common CF mutations: ⌬F508, R553X, G542X, G551D, N1303K, W1282X, R117H, R334W, R347P, A455E, ⌬I507, 1717-1 G>A, R560T, 3849+10kb C>T, 621+1 G>T, S549N [Villalobos-Torres et al., 1997]. Login to comment
43 The next three most common alleles determined in the present study were G542X (4.7% present in the three countries studied), N1303K (1% present in Mexico and Colombia), and 3849+10kb C>T (1% present only in two Mexican CF chromosomes). Login to comment
44 Besides ⌬F508 and G542X, no other CF alleles were detected in the Venezuelan sample. Login to comment
54 G542X mutation, the second most common allele worldwide, has a higher prevalence in Colombia (6.3%). Login to comment
59 Besides the differences in ⌬F508 occurrence, the allele frequencies of G542X and N1303K mutations are similar for Mexico and Colombia, but differ from those found in Venezuela. Login to comment
62 Frequencies and (Chromosome Number) of CF Mutations Detected in Three Latin American Countries CF mutation Mexico % (90) Colombia % (48) Venezuela % (54) Total % (192) ⌬F508 47.8 (43) 35.4 (17) 29.6 (16) 39.6 (76) G542X 4.4 (4) 6.3 (3) 3.7 (2) 4.7 (9) N1303K 1.1 (1) 2.1 (1) 0.0 (0) 1.0 (2) 3849 + 10kb C > T 2.2 (2) 0.0 (0) 0.0 (0) 1.0 (2) W1282X 0.0 (0) 2.1 (1) 0.0 (0) 0.5 (1) 621 + 1 G > T 1.1 (1) 0.0 (0) 0.0 (0) 0.5 (1) S549N 1.1 (1) 0.0 (0) 0.0 (0) 0.5 (1) Non-⌬F508a 10.0 (9) 10.4 (5) 3.7 (2) 8.3 (16) Unknown 42.2 (38) 54.2 (26) 66.7 (36) 52.1 (100) a Detected with the 16 CF mutation panel in this study. Login to comment

[hide] Severe allergic bronchopulmonary aspergillosis in ... Pediatr Pulmonol. 2000 Feb;29(2):155-9. Mussaffi H, Greif J, Kornreich L, Ashkenazi S, Levy Y, Schonfeld T, Blau H
Severe allergic bronchopulmonary aspergillosis in an infant with cystic fibrosis and her asthmatic father.
Pediatr Pulmonol. 2000 Feb;29(2):155-9., [PMID:10639207]

Abstract [show]
An infant with cystic fibrosis and her asthmatic father were diagnosed as suffering from allergic bronchopulmonary aspergillosis (ABPA). Cystic fibrosis was diagnosed in the infant at 6 weeks of age, and gene mutations were W1282X/G542X. She was diagnosed definitively as suffering from ABPA at age 3.5 years, but had suggestive symptoms from age 11 months. This may be the youngest age described to date for ABPA. The child responded well to systemic steroid therapy, but remained steroid-dependent over the next 4 years. Treatment with itraconazole enabled a marked reduction in steroid dosage. The father was an asthmatic, and a heterozygote for the cystic fibrosis transmembrane regulator (CFTR) mutation W1282X. He had a normal sweat test, atopy, and moderate reversible airway obstruction. There was no proven exposure to Aspergillus in the home environment. The importance of considering the diagnosis of ABPA even in infancy, the therapeutic dilemmas, and the possible role of abnormal CFTR function in the development of ABPA are discussed.

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22 Sweat chloride was 101 mEq/L, and her gene mutations were W1282X and G542X. Login to comment

[hide] Detection of CFTR gene mutations in patients suffe... Arch Med Res. 2000 Jan-Feb;31(1):97-100. Kostuch M, Semczuk A, Szarewicz-Adamczyk W, Gasowska-Giszczak U, Wojcierowski J, Kulczycki L
Detection of CFTR gene mutations in patients suffering from chronic bronchitis.
Arch Med Res. 2000 Jan-Feb;31(1):97-100., [PMID:10767489]

Abstract [show]
BACKGROUND: The purpose of the study was to examine cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in patients suffering from chronic bronchitis. METHODS: Thirty-two patients admitted to the Department of Pulmonology, Lublin School of Medicine, Lublin, Poland between 1995 and 1996 due to chronic bronchitis were included in the study. Patients were analyzed for the eight most common mutations of the CFTR gene (DeltaF508, G542X, N1303K, 1717-1(GoA)), W1282X, G551D, R553X, and DeltaI507 by the reverse-hybridization method. RESULTS: CFTR gene mutations were found in five of 32 (16%) patients, all within the DeltaF508 region of the CFTR gene. All positive samples were obtained from patients heterozygous for the DeltaF508 mutation. The presence of the DeltaF508 mutation was considered statistically significant when our study group was compared to the study of Poland's general population (p <0.05 Fisher's exact test). CONCLUSION: Our results suggest there is an increased presence of the DeltaF508 point mutation of the CFTR gene in Polish patients suffering from chronic bronchitis.

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6 Patients were analyzed for the eight most common mutations of the CFTR gene (⌬F508, G542X, N1303K, 1717-1(GoA)), W1282X, G551D, R553X, and ⌬I507 by the reverse-hybridization method. Login to comment
57 These mutations include the following: ⌬F508; G542X; N1303K; 1717-1(GoA); W1282X; G551D; R553X, and ⌬I507. Login to comment
92 Other rare CFTR gene mutations (R553X, G542X, G551D, N1303K, and 621ϩ1G(T)) were excluded in their study. Login to comment
7 Patients were analyzed for the eight most common mutations of the CFTR gene (èc;F508, G542X, N1303K, 1717-1(GoA)), W1282X, G551D, R553X, and èc;I507 by the reverse-hybridization method. Login to comment
91 Other rare CFTR gene mutations (R553X, G542X, G551D, N1303K, and 621af9;1G(T)) were excluded in their study. Login to comment

[hide] Spectrum of CFTR mutations in cystic fibrosis and ... Hum Mutat. 2000;16(2):143-56. Claustres M, Guittard C, Bozon D, Chevalier F, Verlingue C, Ferec C, Girodon E, Cazeneuve C, Bienvenu T, Lalau G, Dumur V, Feldmann D, Bieth E, Blayau M, Clavel C, Creveaux I, Malinge MC, Monnier N, Malzac P, Mittre H, Chomel JC, Bonnefont JP, Iron A, Chery M, Georges MD
Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France.
Hum Mutat. 2000;16(2):143-56., [PMID:10923036]

Abstract [show]
We have collated the results of cystic fibrosis (CF) mutation analysis conducted in 19 laboratories in France. We have analyzed 7, 420 CF alleles, demonstrating a total of 310 different mutations including 24 not reported previously, accounting for 93.56% of CF genes. The most common were F508del (67.18%; range 61-80), G542X (2.86%; range 1-6.7%), N1303K (2.10%; range 0.75-4.6%), and 1717-1G>A (1.31%; range 0-2.8%). Only 11 mutations had relative frequencies >0. 4%, 140 mutations were found on a small number of CF alleles (from 29 to two), and 154 were unique. These data show a clear geographical and/or ethnic variation in the distribution of the most common CF mutations. This spectrum of CF mutations, the largest ever reported in one country, has generated 481 different genotypes. We also investigated a cohort of 800 French men with congenital bilateral absence of the vas deferens (CBAVD) and identified a total of 137 different CFTR mutations. Screening for the most common CF defects in addition to assessment for IVS8-5T allowed us to detect two mutations in 47.63% and one in 24.63% of CBAVD patients. In a subset of 327 CBAVD men who were more extensively investigated through the scanning of coding/flanking sequences, 516 of 654 (78. 90%) alleles were identified, with 15.90% and 70.95% of patients carrying one or two mutations, respectively, and only 13.15% without any detectable CFTR abnormality. The distribution of genotypes, classified according to the expected effect of their mutations on CFTR protein, clearly differed between both populations. CF patients had two severe mutations (87.77%) or one severe and one mild/variable mutation (11.33%), whereas CBAVD men had either a severe and a mild/variable (87.89%) or two mild/variable (11.57%) mutations.

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2 The most common were F508del (67.18%; range 61-80), G542X (2.86%; range 1-6.7%), N1303K (2.10%; range 0.75-4.6%), and 1717-1G>A (1.31%; range 0-2.8%). Login to comment
32 Analysis of more than 43,000 CF chromosomes from different continents has shown that only five mutations have relative world frequencies higher than 1% [F508del (66%), G542X (2.4%), G551D (1.6%), N1303K (1.3%), and W1282X (1.2%)] [The Cystic Fibrosis Genetic Analysis Consortium, 1994]. Login to comment
74 Only three other mutations had relative frequencies ≥1%, G542X (2.86%), N1303K (2.10%), 1717-1G>A (1.31%). Login to comment
80 A trend of decreasing frequency of F508del from Northwest (70%) to Southeast (61%) was generally observed; the second most common mutation was G542X in the Southwest (6.7%) or in the South (5.6%), whereas it was I507del in Normandy (2.7%). Login to comment
84 The most common mutations in this group were F508del (31.01%), 711+1G>T (11.39%), W1282X (6.33%), N1303K (5.7%), G542X (5.06%), and R1162X (3.8%), a distribution which seems different from the global French population. Login to comment
88 Five genotypes are responsible for 57.31% of cases of CF in France: F508del/F508del (47.75 %), F508del/G542X (3.4%), F508del/N1303K (2.7%), F508del/1717-1G>A (2.02%), and F508del/2789+5G>A (1.43%). Login to comment
102 Distribution of 310 CF Mutations in France With Respect to Relative Frequencies (Total Number of CF Chromosomes = 7,420) Group Mutations Number of alleles % Cum. % A F508del 4,985 67.18 G542X 212 2.86 N1303K 156 2.10 73.45 1717-1G>A 97 1.31 B G551D 73 0.98 2789+5G>A 72 0.97 W1282X 68 0.91 R553X 66 0.89 I507del 52 0.70 1078delT 49 0.66 7.47 2183AA>G 48 0.64 711+1G>T 33 0.44 R1162X 33 0.44 Y1092X 30 0.40 3849+10kbC>T 30 0.40 C 12 mutationsa 29 to 15 (239) 0.39-0.20 19 mutationsb 14 to 8 (190) 0.19-0.10 11 mutationsc 7 to 6 (71) 0.09-0.08 11 mutationsd 5 (55) 0.06 10.57 15 mutationse 4 (60) 0.05 23 mutationsf 3 (69) 0.04 50 mutationsg 2 (100) 0.02 D 154 mutationsh 1 (154) 0.01 2.07 6,942 93.56 a 3659delC, R347P, 3272-26A>G, R334W, W846X, 621+1G>T, G85E, R1066C, L206W, 394delTT, 4055+1G>A, R347H. Login to comment
140 Non-F508del Mutations Found as Homozygous in a Sample of 3,710 Patients With Cystic Fibrosis Mutation n 711+1G>T 8 G542X 7 N1303K 7 2183delAA>G 5 W1282X 4 G551D 3 3905insT 3 R334W 2 R347P 2 1078delT 2 1811+1.6kbA>G 2 2113delA 2 Y1092X 2 R1162X 2 306insA 1 E92K 1 G178R 1 L227R 1 1677delTA 1 1717-1G>A 1 1717-8G>A 1 R553X 1 S549R(T>G) 1 R560S 1 V562I 1 Y569D 1 2711delT 1 S945L 1 R1158X 1 I1234V 1 3849+10kbC>T 1 Q1313X 1 del25kb 1 E831X 1 I175V 1 G314V 1 L1077P 1 produce a small quantity of functional protein as a result of a variable proportion of normal CFTR mRNA transcripts in addition to the abnormal ones (class V); 3) they are located in sites known to generate less severe mutants (external loops, residues lining the pore); and/or 4) they have been observed in CF with pancreatic sufficiency, CBAVD, and/or CF-related attenuated phenotypes only. Login to comment
152 Twenty-four non F508del mutations were found associated with the 9T allele: 394delTT, L90S, D110H, R117G, 621+1G>T, V232D, A455E, G542X, R851L, T908N, 2789+5G>A, 2896insAG, H939R, 3007delG, I980K, I1027T, R1066H, A1067T, D1154G, 3737delA, R74W+D1270N, N1303I, N1303K, D1377H. Login to comment
158 Distribution of Mutations Within the CFTR Gene Due to the high frequencies of F508del and G542X, 75.22% of French CF alleles were mutated in exons 10 and 11, encoding for the first nucleotide binding domain of CFTR (NBD1) or their flanking intronic sequences (Fig. 5). Login to comment
171 CFTR Mutation Genotypes Identified Both in Cystic Fibrosis (CF) and in Congenital Bilateral Absence of the Vas Deferens (CBAVD) CF CBAVD F508del/5T 3 143 F508del/2789+5G>A 53 1 F508del/3272-26A>G 17 4 F508del/R117H* 10 39 F508del/R117C 2 2 F508del/L206W 12 4 F508del/R347H 10 5 F508del/R347L 1 1 F508del/D443Y 1 5 F508del/Y569C 1 1 F508del/P574H 3 1 F508del/G628R(G>A) 2 1 F508del/V920M 1 1 F508del/R1070W 2 3 F508del/D1152H 6 8 F508del/S1235R 3 1 F508del/T1246I 1 1 F508del/D1270N+R74W 2 3 F508delN1303I 1 1 3659delC/R347H 1 1 G542X/T338I 2 2 R347H/R1066H 1 1 *The only case with CF whose alleles at IVS8(T)n were reported had mutation R117H associated with a 5T allele. Login to comment
175 1.6kbA>G) is markedly different from the rest of France whereas the Mediterranean coast (G542X) shows similarity between the western and eastern parts,withsomedifferencefromthecentralpart.This study demonstrates that even within one country, wide variations exist among the frequencies of spe- cificmutations.OtherEuropeancountriesshowmore substantial genetic homogeneity than France [Cuppensetal.,1993;Bonizzatoetal.,1995;Schwarz et al., 1995; Tümmler et al., 1996]. Login to comment

[hide] A comparison of fluorescent SSCP and denaturing HP... Hum Mutat. 2000;15(6):556-64. Ellis LA, Taylor CF, Taylor GR
A comparison of fluorescent SSCP and denaturing HPLC for high throughput mutation scanning.
Hum Mutat. 2000;15(6):556-64., [PMID:10862085]

Abstract [show]
We examined 67 different mutations in 16 different amplicons in a comparison of mutation detection by fluorescent single strand conformation polymorphism (F-SSCP) and by denaturing HPLC (DHPLC). F-SSCP was used to analyze fluorescent amplicons with internal size standards and automated fragment analysis (GeneScan, PE Applied Biosystems, Foster City, CA). In DHPLC, unlabelled amplicons were analyzed by reverse phase HPLC with fragment detection by absorbance at 260nm. Both methods had high sensitivity (95-100%) and specificity (100%). Overall, F-SSCP with external temperature control was the more sensitive method, but DHPLC was particularly useful for the rapid analysis of novel fragments.

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97 Comparison of F-SSCP and DHPLC Using a Panel of ABCC7 Mutations Gel condition Location Location 49:1 49:1 49:1 49:1 MDE MDE MDE Capillary DHPLC °C from 5' (bp) from 3' (bp) 15 20 25 35 20 25 35 35 N/A Exon 3 (320bp) E60X 128 192 + + + + + + + + - P67L 150 170 + + + - + + + - + R75X 173 147 + + + + + + + + + R75Q 174 146 + + + - + + + + + G85E 204 116 + + + - + + + + + L88S 213 107 + + + + + + + + + Exon 4 (400bp) 441delA 135 265 + + + + + + + + + D110H 154 246 + + + + + + - + + R117H/H 176 224 + + + + + + + + N/A R117R/H 176 224 + + + + + + + + + L137H 236 164 + + + + + + + + + I148T 261 139 + + + + + + + + + 621+1 (G>T) 309 91 + + + + + + + + + Exon 7 (360bp) R334W 180 180 + + + + + + + - + 1058delC 105 255 + + + + + + + + + 1078delT 125 235 + + + - + + + + + 1138insG 226 134 - + + - + + + + + 1154insTC 202 158 + + + + + + + + + 1161delC 209 151 + + + + + + + + + R347H 220 140 + + + + + + - + + R347P 220 140 + + + - + + + - + A349V 226 134 + + + + + + + + + W356X 248 112 + + + + + + + + + Exon 10 (365bp) M470V 143 222 + + + + + + + + + Q493X 212 153 + + + + + + - + - DelF508 255 110 + + + + + + + + - Del I507 253 112 + + + + + + + + + V520F 293 72 + + - + + - + - + Exon 11 (190bp) 1717-1 (G>A) 54 136 + + + - + + - + + G542X 94 96 + + + - + + - + + S549N 116 74 + + + + + + + + - S549R 117 73 + + + + - - - + + G551D 122 68 + - - - + + + - + R553X 127 63 + + + + + + + + + G551D/R553X + + + + + + + + + R560T 149 41 + + + - - - - - + R560K 149 41 + + + - + + + - + 1811+1 (G>C) 150 40 + + + + + + + + + Exon 12 (250bp) 1898+1(G>A) 167 83 + + + + + + - + + Exon 13a (290bp) C590W 87 203 + + - - + - - + + Exon 13b (405bp) 2184insA 148 257 + + + + + + + - + R709X 220 185 - + - - - - - - + V754M 453 52 + + + + + + + - - Exon 13c (345bp) V754M 65 280 + + + + + + - - + R785X 158 187 + + - - + + - - + Exon 19 (370bp) 3601-17 (T>C) 29 341 - + + - + + + - + R1162X 61 309 + + - - + - - + + 3659delC 105 265 - - - + + + + + + Y1182X 123 247 - + + - + + + - + Exon 20 (370bp) W1282X 186 184 + + + + + + + + + % detected 90 96 86 66 94 88 74 72 90 remainder were detected using DGGE. Login to comment
135 Effect of Amplicon Size on Mutation Detection Rate in ABCC7 Exon 11 SSCP slab gels SSCP 310 DHPLC 49:1, 20°C MDE, 20°C 35°C Exon 11 ABCC7 190bp 490bp 190bp 490bp 190bp 490bp 190bp 490bp 1717-1G>A - - + + + + + + G542X + + + + + - + + S549N + + + + + + - - S549R + + - + + + + - G551D - + + + - - + - R553X + + + + + + + - G551D/R553X + + + + + + + - R560T + - - + - - + + R560K + + + + - - + + 1811+1G>C + + + + + + + + Sensitivity 9/10 8/10 8/10 10/10 7/10 6/10 9/10 5/10 ammonium acetate as an ion-pairing agent has the unintended consequence of modifying the stability of GC and AT base pairs in a similar manner to the effects of tetra-alkyl ammonium salts, tetraethyl ammonium chloride and tetramenthyl ammonium chloride. Login to comment

[hide] Terminal glycosylation in cystic fibrosis. Biochim Biophys Acta. 1999 Oct 8;1455(2-3):241-53. Scanlin TF, Glick MC
Terminal glycosylation in cystic fibrosis.
Biochim Biophys Acta. 1999 Oct 8;1455(2-3):241-53., [PMID:10571016]

Abstract [show]
Cystic fibrosis (CF) is a common genetic disease for which the gene was identified within the last decade. Pulmonary disease predominates in this ultimately fatal disease and current therapy only slows the progression. CF transmembrane regulator (CFTR), the gene product, is an integral membrane glycoprotein that normally functions as a chloride channel in epithelial cells. The most common mutation, deltaF508, results in mislocalization and altered glycosylation of CFTR. Altered fucosylation and sialylation are hallmarks of both membrane and secreted glycoproteins in CF and the focus here is on these investigations. Oligosaccharides from CF membrane glycoproteins have the Lewis x, selectin ligand in terminal positions. In addition, two major bacterial pathogens in CF, Pseudomonas aeruginosa and Haemophilus influenzae, have binding proteins, which recognize fucose in alpha1,3 linkage and asialoglycoconjugates. We speculate that the altered terminal glycosylation of airway epithelial glycoproteins in CF contributes to the chronic infection and robust inflammatory response in the CF lung. Understanding the effects of mutant CFTR on glycosylation may provide further insight into the regulation of glycoconjugate processing as well as therapy for CF.

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650 Some mutations (e.g., G542X) produce a truncated transcript and no protein, while other mutations (e.g., R117H) produce a protein that has impaired conduction properties. Login to comment

[hide] Increased frequency of cystic fibrosis deltaF508 m... Eur Respir J. 1999 Jun;13(6):1281-7. Puechal X, Fajac I, Bienvenu T, Desmazes-Dufeu N, Hubert D, Kaplan JC, Menkes CJ, Dusser DJ
Increased frequency of cystic fibrosis deltaF508 mutation in bronchiectasis associated with rheumatoid arthritis.
Eur Respir J. 1999 Jun;13(6):1281-7., [PMID:10445602]

Abstract [show]
This study investigated the clinical characteristics and the possible involvement of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in patients with symptomatic diffuse bronchiectasis (DB) associated with rheumatoid arthritis (RA). Twenty-six patients with both RA and DB (group RA+DB) and control groups of 29 consecutive patients with RA but no bronchiectasis (group RA) and 29 patients with symptomatic DB of unknown origin (group DB) were prospectively studied. Among the patients of the RA+DB group, four (15.4%) were heterozygous for the CFTR gene deltaF508 mutation, whereas no deltaF508 mutation was found in patients of the RA and the DB groups (both, p<0.05). This frequency of deltaF508 mutation was also higher than the expected frequency (2.8%) in the general European population (p<0.04). Sweat chloride values and nasal potential differences were normal in three out of four patients carrying the deltaF508 mutation. In the RA+DB group, those with deltaF508 mutation had more frequent chronic sinusitis (p<0.05), a trend toward a more severe pulmonary involvement, and a lower value of nasal potential differences (p<0.01) whereas their rheumatic features had no particularity. In the RA+DB group, patients with adult-onset bronchiectasis (including two with deltaF508 mutation) had a greater reduction in total lung capacity (p<0.05) and lower nasal potential differences (p<0.005) than those with childhood-onset bronchiectasis. This study suggests a possible deleterious effect of the cystic fibrosis transmembrane conductance regulator mutated protein in the airways which may predispose to the development and severity of bronchiectasis in patients suffering from rheumatoid arthritis.

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129 In the DB group, two patients carried mutations: one was compound heterozygous (G542X/3849+ 10Kb CRT) and was diagnosed as suffering from an atypical CF with a normal sweat chloride concentration, while the other was heterozygous for the missense mutation G239R with normal values of both sweat chloride concentrations and nasal potential differences. Login to comment
128 In the DB group, two patients carried mutations: one was compound heterozygous (G542X/3849+ 10Kb CRT) and was diagnosed as suffering from an atypical CF with a normal sweat chloride concentration, while the other was heterozygous for the missense mutation G239R with normal values of both sweat chloride concentrations and nasal potential differences. Login to comment

[hide] Respiratory epithelial ion transport in patients w... Eur Respir J. 1999 Jun;13(6):1276-80. Danner I, Boisseau P, Chailleux E, Escande D
Respiratory epithelial ion transport in patients with disseminated bronchiectasis.
Eur Respir J. 1999 Jun;13(6):1276-80., [PMID:10445601]

Abstract [show]
The nosological limits between disseminated bronchiectasis and cystic fibrosis (CF) remain unclear. In patients with isolated congenital bilateral absence of the vas deferens, a forme fruste of the CF disease, a normal baseline nasal transepithelial potential difference (PD) but an impaired response to pharmacological interventions have been reported. The purpose of the present study was to explore ion transport in respiratory epithelium from patients with disseminated bronchiectasis. The PD under both baseline and pharmacological interventions was investigated in 13 healthy subjects, six patients with genetically proven CF and 15 patients with disseminated bronchiectasis as confirmed by computed tomography scan. Baseline PD was similar in the control and bronchiectasis groups but, as expected, was significantly more negative in the CF group. Patients with bronchiectasis responded to pharmacological tests (sequential perfusion with amiloride, chloride-free solution, isoprenaline and uridine triphosphate (UTP) similarly to healthy subjects. In contrast, CF patients exhibited an increased response to amiloride and an impaired response to chloride-free solution and isoprenaline. The data show that patients with disseminated bronchiectasis exhibit normal electrophysiological properties in their nasal epithelium. Nasal transepithelial potential difference including pharmacological tests may appear a valuable diagnostic procedure for cystic fibrosis with disseminated bronchiectasis.

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36 This method allowed the detection of seven common mutations: 621+1GRT, DF508, G542X, N1303K, G551D, 1717-1GRA, and 3659delC [23]. Login to comment

[hide] Genotype-phenotype correlations for the paranasal ... Am J Respir Crit Care Med. 1999 May;159(5 Pt 1):1412-6. Jorissen MB, De Boeck K, Cuppens H
Genotype-phenotype correlations for the paranasal sinuses in cystic fibrosis.
Am J Respir Crit Care Med. 1999 May;159(5 Pt 1):1412-6., [PMID:10228103]

Abstract [show]
Genotype-phenotype correlations in cystic fibrosis (CF) have been found for lung and pancreatic function, but not for paranasal sinus disease. Because such correlations may have pathophysiological and clinical implications, the correlation of mutations, in particular DeltaF508, with paranasal sinus disease was investigated in 113 CF patients with known genotype. The clinical importance of paranasal sinus disease was evaluated using three parameters: polyps, overall clinical severity of upper airway problems, and surgery. Polyps were evaluated by nasal endoscopy and graded on a five-point scale. Four severity groups were distinguished based on history, clinical records, and examination: no upper airway problems; more problems than in control subjects; severe, recurrent or chronic problems; and paranasal sinus surgery cases. DeltaF508 homozygosity correlated with clinical severity (p < 0.02) and with the presence of polyps on endoscopy (p < 0.05). The relative risk for paranasal sinus surgery in DeltaF508 homozygous CF patients was 2.33. In conclusion, there are genotype-phenotype correlations for paranasal sinus disease in CF. DeltaF508 homozygosity is a risk factor for paranasal sinus disease in CF.

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94 The fourth patient had undergone sinus surgery and was ⌬F508 negative (G542X/unidentified). Login to comment
120 of Patients in Surgical Group ⌬F508 Genotype Homozygosity 69 61 22 Compound heterozygosity 33 29 5 Negative 11 10 1 Mutations ⌬F508 171 75.7 27 Non-⌬F508 55 24.3 6 R117H (4) C276X (1) 394delT (1) W401X (2,† ) A455E (1) G542X (4,‡ ) G551D (1) R553X (1) G628R(G→C) (1) Y1092X (1) D1152H (1) S1251N (1) W1282X (3) N1303K (8) W1310X (1) 1717-1G→A (3,† ) 1898ϩ1G→C (1) 2183AA-G (3,†† ) 3659delC (2) 3272-26A→G (2,† ) 4218-insT (2) unknown (11,‡ ) * The genotype and mutations are given for the 113 patients with CF. Login to comment
95 The fourth patient had undergone sinus surgery and was DF508 negative (G542X/unidentified). Login to comment
121 of Patients in Surgical Group DF508 Genotype Homozygosity 69 61 22 Compound heterozygosity 33 29 5 Negative 11 10 1 Mutations DF508 171 75.7 27 Non-DF508 55 24.3 6 R117H (4) C276X (1) 394delT (1) W401X (2,ߤ ) A455E (1) G542X (4,ߥ ) G551D (1) R553X (1) G628R(G࢐C) (1) Y1092X (1) D1152H (1) S1251N (1) W1282X (3) N1303K (8) W1310X (1) 1717-1G࢐A (3,ߤ ) 189811G࢐C (1) 2183AA-G (3,ߤߤ ) 3659delC (2) 3272-26A࢐G (2,ߤ ) 4218-insT (2) unknown (11,ߥ ) * The genotype and mutations are given for the 113 patients with CF. Login to comment

[hide] Sunshine, sweating, and main d'accoucheur. Lancet. 1999 May 1;353(9163):1492. Johnson DW, Parnham A, Herzig K, Wittmann J
Sunshine, sweating, and main d'accoucheur.
Lancet. 1999 May 1;353(9163):1492., [PMID:10232317]

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16 Screening for ten of the common cystic fibrosis transmembrane conductance regulator gene (CFTR) mutations (⌬F508, G551D, R553X, G542X, N1303K, A455E, 621+1, Delta I507 and R117H), showed a single delta F508 mutation. Login to comment

[hide] Cystic fibrosis mutations in heterozygous newborns... Am J Hum Genet. 1999 Jan;64(1):303-4. Castellani C, Benetazzo MG, Bonizzato A, Pignatti PF, Mastella G
Cystic fibrosis mutations in heterozygous newborns with hypertrypsinemia and low sweat chloride.
Am J Hum Genet. 1999 Jan;64(1):303-4., [PMID:9915972]

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16 Am. J. Hum. Genet. 64:303-304, 1999 Table 1 Sweat Chloride Concentration and CFTR Genotypes CASE SWEAT CHLORIDE (mEq/liter) MUTATION Allele 1a Allele 2b 1 10 R1162X 3041-71G/C,c 4002A/Gc 2 14 DF508 3 30 R1162X R117H 4 21 DF508 E527G 5 8 DF508 6 12 N1303K, 2622ϩ14G/Ad 7 6 DF508 8 20 DF508 1716G/Ac 9 16 DF508 10 10 DF508 11 19 R1162X 12 19 N1303K 13 12 G542X 1716G/Ac 14 32 DF508 15 14 DF508 16 26 N1303K 2622ϩ14G/Ac 17 18 DF508 Y301C 18 18 2183AArG a First mutation found, assigned to one gene. Login to comment

[hide] Missense mutations in the cystic fibrosis gene in ... Hum Mutat. 1999;14(6):510-9. Lazaro C, de Cid R, Sunyer J, Soriano J, Gimenez J, Alvarez M, Casals T, Anto JM, Estivill X
Missense mutations in the cystic fibrosis gene in adult patients with asthma.
Hum Mutat. 1999;14(6):510-9., [PMID:10571949]

Abstract [show]
Asthma is a complex genetic disorder that affects 5% of adults and 10% of children worldwide. The complete characterization of the cystic fibrosis transmembrane conductance regulator (CFTR) gene identified missense mutations in 15% of 144 unrelated adult patients with asthma, but in none of 41 subjects from the general population. The four more common mutations were analyzed in an extended sample consisting of 184 individuals from the general population and did not show a significant difference in frequency. The hyperfunctional CFTR M470 allele was detected in 90% of patients with CFTR missense mutations, but in 63% of subjects from the general population and 63% of asthma patients without CFTR mutations. None of the patients with missense mutations had the 5T allele of intron 8 of CFTR, responsible for low CFTR levels, while it was detected in 8% of asthma patients without CFTR mutations and in 9% of subjects from the general population. These findings suggest a putative role for a combination of CFTR missense mutations, including the M470 allele, in the genetic variability of asthma.

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57 TheCFTR gene was analyzed for the two most common mutations in the Spanish population (∆F508 and G542X) and for a DNA variant located in intron 8 of the CFTR gene, IVS8-6(5T) [Chu et al., 1991]. Login to comment

[hide] The DeltaF508 mutation in Ecuador, South America. Hum Mutat. 1999;14(4):348-50. Paz-y-Mino C, Perez JC, Burgos R, Davalos MV, Leone PE
The DeltaF508 mutation in Ecuador, South America.
Hum Mutat. 1999;14(4):348-50., [PMID:10502783]

Abstract [show]
There are few reports about the incidence of the DeltaF508 mutation in Latin American countries. We show the study of the DeltaF508 mutation and the seven most common "European" mutations in 10 Ecuadorian CF affecteds. The incidence of DeltaF508 mutation found was 25% and none of the other seven was detected in our population, which indicates that at least 60% of the mutations in the studied population are different from most common in Europe. Similar data have been reported in other Amerindian populations, therefore it is suggested that Cystic Fibrosis in Ecuador-and other Amerindian countries in Latin America-have a different ethiology than that of Caucasian populations.

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17 In the affecteds who did not show any ∆F508 allele, we searched for the presence of the eight most common "European" mutations (∆F508, G542X, N1303K, 1717-1, W1282X, G551D, R553X, ∆1507) with the test INNO-Lipa. Login to comment
31 However, in this study the G542X and N1303K mutations were found [Villalobos-Torres et al., 1997], although these were not found in Ecuador. Login to comment

[hide] Spectrum of CFTR mutations in the Middle North of ... Hum Mutat. 1999;14(1):89. Telleria JJ, Alonso MJ, Calvo C, Alonso M, Blanco A
Spectrum of CFTR mutations in the Middle North of Spain and identification of a novel mutation (1341G-->A). Mutation in brief no. 252. Online.
Hum Mutat. 1999;14(1):89., [PMID:10447267]

Abstract [show]
We have analyzed 39 unrelated cystic fibrosis (CF) families by denaturing gradient gel electrophoresis (DGGE) and direct sequencing in order to determine the spectrum of CF mutations in our population. This approach has allowed us to detect 72 out of the 78 CF chromosomes (92.3%). The DF508 mutation was found to be present in 51/78 (65.4%) CF chromosomes, in accordance with the predicted Northwest-Southeast gradient within the European population. Another 14 known mutations, and the novel 1341G-->A mutation were identified. Nine out of fifteen non DF508 mutations were present in a single chromosome. The 1341G-->A mutation, found in 2 unrelated patients, is a new mutation associated to severe phenotype, causing pancreatic insufficiency and chronic lung infections. Our data suggest a different distribution of non-DF508 mutations in our population when compared with previous studies carried out in Spanish CF families. Six out of the 14 non-F508 in our study were not present in a recent study carried out in 640 Spanish families with CE These six mutations account for 29.6% non DF508 chromosomes in our sample.

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33 Spectrum of CFTR Mutations Table 1 Spectrum of CFTR mutations identified in the present study compared with data published by Casals et al. (Casals et al. 1997) This study Casals et al. Mutation Exon/Intron n % n % ∆F508 E 10 51 65.4 681 53.2 711+1G→T I 5 3 3.8 22 1.7 G542X E 11 3 3.8 108 8.43 1213delT E 7 2 2.6 0 0 1341G→A E 8 2 2.6 0 0 R1066C E 17b 2 2.6 14 1.09 1717-1G→A I 10 1 1.3 1 0.08 S549R E 11 1 1.3 0 0 V562I E 12 1 1.3 0 0 G576A E 12 1 1.3 0 0 2183AA→G E13 1 1.3 5 0.39 2789+5G→A I 14b 1 1.3 11 0.86 Q890X E 15 1 1.3 13 1.01 3849+1G→A I 19 1 1.3 0 0 N1303K E 21 1 1.3 34 2.65 Other 0 0 391 30.5 Known mutations 72 92.3 1155 90.23 Unknown mutations 6 7.7 125 9.7 DISCUSSION The knowledge of the spectrum of mutations causing CF in any specific geographic region provide useful information to design the best approach in pre and postnatal diagnosis of CF; for the screening of mutations in the population at risk; to stimate the genetic risk etc. Moreover, the response to different therapeutic approaches could vary depending on the CF mutations in any case. Login to comment

[hide] Genetic concerns for the subfertile male in the er... Prenat Diagn. 1998 Dec;18(13):1349-65. Kim ED, Bischoff FZ, Lipshultz LI, Lamb DJ
Genetic concerns for the subfertile male in the era of ICSI.
Prenat Diagn. 1998 Dec;18(13):1349-65., [PMID:9949435]

Abstract [show]
The treatment of severe male factor infertility has seen remarkable advances in the last five years with the introduction and widespread use of intracytoplasmic sperm injection (ICSI). Although ICSI represents one of the most important advances in the treatment of the subfertile male, significant concerns exist regarding the potential for transmission of abnormal genes to the offspring because many of the natural barriers to conception have been bypassed. Because these couples were not able to conceive prior to ICSI, the long-term genetic consequences in these offspring are largely undefined at this time. Genetic abnormalities related to male infertility need to be considered in terms of being (1) causative for male infertility and (2) potentially transmissible to the offspring. Reasons for pursuing a genetic evaluation include (1) establishing a diagnosis, (2) establishing a possible genetic origin, (3) clarifying the pattern of inheritance, and (4) providing information on natural history, variation and expression. The three most common known genetic factors related to male infertility are cystic fibrosis gene mutations leading to congenital absence of the vas deferens, Y-chromosome microdeletions leading to spermatogenic impairment, and karyotype abnormalities. When congenital bilateral absence of the vas deferens with azoospermia is encountered, cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations are commonly the underlying cause. When testicular failure is manifest by azoospermia or severe oligoszoospermia, Y-chromosome microdeletions may be present in approximately 10-15 per cent of otherwise normal appearing men. Karyotyping can uncover potentially transmissible genetic abnormalities in the infertile male including structural chromosomal disorders such as Klinefelter's (classic 47,XXY), mixed gonadal dysgenesis, chromosomal translocations and XYY syndromes. Finally, potential male infertility genes in animal models are reviewed. Without question, advances in clinical and basic research raise scientific and social issues that must be addressed.

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93 Because these previous series had relied only on delta F508 mutations, Scobie et al. (1996) demonstrated the detection of G551D, R553X, G542X, 621+1G>T and delta F508 CFTR gene mutations using a rapid and specific differential amplification system. Login to comment

[hide] Heterogeneity of reproductive tract abnormalities ... Fertil Steril. 1998 Oct;70(4):724-8. Jarvi K, McCallum S, Zielenski J, Durie P, Tullis E, Wilchanski M, Margolis M, Asch M, Ginzburg B, Martin S, Buckspan MB, Tsui LC
Heterogeneity of reproductive tract abnormalities in men with absence of the vas deferens: role of cystic fibrosis transmembrane conductance regulator gene mutations.
Fertil Steril. 1998 Oct;70(4):724-8., [PMID:9797105]

Abstract [show]
OBJECTIVE: To determine if the types of reproductive tract abnormalities linked to absence of the vas deferens varies with the cystic fibrosis transmembrane conductance regulator (CFTR) genotype. DESIGN: Prospective data gathering. SETTING: University infertility clinic. PATIENT(S): Forty-six infertile men with absence of the scrotal vas deferens and no signs of cystic fibrosis. INTERVENTION(S): All had blood taken for CFTR gene analysis, 33 had scrotal ultrasounds, and 25 had transrectal ultrasounds. MAIN OUTCOME MEASURE(S): The frequency of testicular, seminal vesicle, and ampullae of the vas deferens malformations was compared between subgroups of men with two, one, or no CFTR gene mutations. RESULT(S): None (0 of 21) of the men with at least one CFTR gene mutations had normal ampullae of the vas or seminal vesicles bilaterally. Two (50%) of 4 men with no CFTR gene mutations had normal ampullae of the vas deferens bilaterally, and 50% had normal bilateral seminal vesicles (statistically significantly different). There was no correlation between testicular malformations and CFTR genotype. CONCLUSION(S): This study indicates that the severity of the malformations in the testis is unrelated to the CFTR genotype, whereas the frequency and severity of wolffian duct malformations are related directly to the CFTR genotype.

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60 of men Single mutation 5T /Unknown 13 ⌬F508 /Unknown 8 R117H /Unknown 2 W1282X /Unknown 1 4016insT /Unknown 1 N1303K /Unknown 1 Total 26 Two mutations ⌬F508 /5T 4 ⌬F508 /R117H 2 ⌬F508 /R75Q 1 5T /2183AA3G 1 5T /N1303K 1 5T /G542X 1 R117H /G551A 1 R117H /2184insA 1 A455E /3849ϩ10KbC 1 3T Total 13 No mutations 7 Total no. Login to comment

[hide] Evidence for a common ethnic origin of cystic fibr... Am J Hum Genet. 1998 Aug;63(2):656-62. Dork T, El-Harith EH, Stuhrmann M, Macek M Jr, Egan M, Cutting GR, Tzetis M, Kanavakis E, Carles S, Claustres M, Padoa C, Ramsay M, Schmidtke J
Evidence for a common ethnic origin of cystic fibrosis mutation 3120+1G-->A in diverse populations.
Am J Hum Genet. 1998 Aug;63(2):656-62., [PMID:9683582]

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63 This study demonstrates that the 3120ϩ1GrA mutation shares the same extragenic CS.7-KM.19 "risk" haplotype with the other frequent and ancient CF mutations-DF508, N1303K, and G542X (Do¨rk et al. 1992; Morral et al. 1993)-but that it differs from these latter mutations with respect to intragenic CFTR markers. Login to comment

[hide] Cystic fibrosis: a multiple exocrinopathy caused b... Am J Med. 1998 Jun;104(6):576-90. Schwiebert EM, Benos DJ, Fuller CM
Cystic fibrosis: a multiple exocrinopathy caused by dysfunctions in a multifunctional transport protein.
Am J Med. 1998 Jun;104(6):576-90., [PMID:9674722]

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218 The number of missense or point mutations (frequent examples are R117H and G551D), nonsense (frequent examples are G542X and W1282X), and frameshift mutations within CFTR has reached more than 700, according to the CF Genetic Analysis Consortium. Login to comment
223 They include another deletion mutation at amino acid position 507 (⌬I507), several missense mutations (F508C, G551D, G551S, A455E, R553Q, P574H, S549N, A559T), and some nonsense mutations (G542X, R553X, Q493X). Login to comment
299 Examples of such nonsense mutations are G542X in NBF1, W1282X in NBF2, and S1455X in the C-terminus of the CFTR protein. Login to comment

[hide] Mutation frequency of cystic fibrosis transmembran... Fertil Steril. 1998 May;69(5):899-903. Tuerlings JH, Mol B, Kremer JA, Looman M, Meuleman EJ, te Meerman GJ, Buys CH, Merkus HM, Scheffer H
Mutation frequency of cystic fibrosis transmembrane regulator is not increased in oligozoospermic male candidates for intracytoplasmic sperm injection.
Fertil Steril. 1998 May;69(5):899-903., [PMID:9591500]

Abstract [show]
OBJECTIVE: To examine the frequency of anomalies of the vas deferens and the frequency of mutations of the cystic fibrosis transmembrane regulator (CFTR) gene in male candidates for intracytoplasmic sperm injection (ICSI) who had severe oligoasthenoteratozoospermia. DESIGN: The clinical data for male candidates for ICSI were studied. The three most frequent cystic fibrosis (CF)-causing CFTR mutations in the Dutch population (deltaF508, A455E, and G542X) and the three most frequent CFTR mutations potentially causing congenital bilateral absence of the vas deferens (CBAVD) in the Dutch population (deltaF508, R117H, and IVS8-5T) were analyzed. Delta I507 is also detected by the deltaF508 test. Samples of DNA from patients identified as CFTR mutation carriers were subjected to denaturing gradient gel electrophoresis analysis with use of a two-dimensional electrophoretic technique. SETTING: University-based center for reproductive medicine and clinical genetics. PATIENT(S): Male candidates for ICSI who had oligoasthenoteratozoospermia and no history of operative sterilization and refertilization. Males with a chromosomal aberration or a Y-chromosome microdeletion were excluded. INTERVENTION(S): Semen and blood samples were collected from the patients at their first visit to the clinic. MAIN OUTCOME MEASURE(S): Frequency of anomalies of the vas deferens and frequency of mutations of the CFTR gene in male candidates for ICSI who had oligoasthenoteratozoospermia. RESULT(S): None of the patients had abnormalities of the vas deferens at physical examination. In 4 of the 150 chromosomes (75 patients), a CFTR mutation was found, yielding a CFTR mutation frequency of 2.7% (95% confidence interval, 1.0-6.7%). None of the patients had two CFTR mutations. CONCLUSION(S): The frequency of congenital abnormalities of the vas deferens in patients with oligoasthenoteratozoospermia is low. The frequencies of the CFTR mutations identified in this cohort did not differ significantly from the frequencies found in the normal Dutch population.

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2 The three most frequent cystic fibrosis (CF)-causing CFTR mutations in the Dutch population (⌬F508, A455E, and G542X) and the three most frequent CFTR mutations potentially causing congenital bilateral absence of the vas deferens (CBAVD) in the Dutch population (⌬F508, R117H, and IVS8-5T) were analyzed. Login to comment
65 The three most frequent CFTR mutations causing CF in the Dutch population (⌬F508, A455E, and G542X) and the three most frequent CFTR mutations potentially causing congenital bilateral absence of the vas deferens in the Dutch population (⌬F508, R117H, and IVS8-5T) were analyzed. Login to comment
68 Detection of R117H, A455E, and G542X mutations was performed with the use of allele-specific amplification tests, as described by Ferrie et al. (12) for G542X and R117H. Login to comment
92 For the A455E, G542X, and ⌬I507 mutations, the frequency in the normal population was based on the 3.3% carrier frequency and the locally derived relative frequencies of mutations in CF patients (H.S., unpublished data). Login to comment
132 Mutation General population* (95% CI) Patients with OAT† (95% CI) Patients with CBAVD‡ Patients with CF§ ⌬F508 0.013 (0.011-0.015) 0.013 (0.0037-0.047) 0.169 0.777 R117H 0.009 (0.0027-0.030) 0.006 (0.0012-0.037) 0.305 Ͻ0.001 IVS8-5T 0.037 (0.019-0.073) 0.006 (0.0012-0.037) 0.055 ND A455E Ͻ0.001 (ND) 0 (ND) Ͻ0.001 0.026 G542X Ͻ0.001 (ND) 0 (ND) Ͻ0.001 0.015 ⌬I507 Ͻ0.001 (ND) 0 (ND) Ͻ0.001 Ͻ0.001 Total 0.005 (ND)࿣ 0.027 (0.010-0.067) 0.529 0.818 Note: OAT ϭ oligoasthenoteratozoospermia; CBAVD ϭ congenital bilateral absence of the vas deferens; ND ϭ not done. Login to comment
133 * Locally derived frequencies for ⌬F508, R117H, and IVS8-5T (n ϭ 21,544 chromosomes, n ϭ 232 chromosomes, and n ϭ 212 chromosomes, respectively); and theoretical estimates for A455E, G542X, and ⌬I507 based on a 3.3% carrier frequency and regionally derived mutation frequencies (H.S., unpublished data). Login to comment
136 § H.S., unpublished data (n ϭ 726 for ⌬F508, A455E, and G542X; n ϭ 272 for R117H; IVS8-5T not determined). Login to comment

[hide] A mutation in the cystic fibrosis transmembrane co... Hum Mol Genet. 1998 Apr;7(4):729-35. Mickle JE, Macek M Jr, Fulmer-Smentek SB, Egan MM, Schwiebert E, Guggino W, Moss R, Cutting GR
A mutation in the cystic fibrosis transmembrane conductance regulator gene associated with elevated sweat chloride concentrations in the absence of cystic fibrosis.
Hum Mol Genet. 1998 Apr;7(4):729-35., [PMID:9499426]

Abstract [show]
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) have been shown to cause cystic fibrosis (CF) and male infertility due to congenital bilateral absence of the vas deferens. We report the identification of a 6.8 kb deletion (del14a) and a nonsense mutation (S1455X) in the CFTR genes of a mother and her youngest daughter with isolated elevated sweat chloride concentrations. Detailed clinical evaluation of both individuals found no evidence of pulmonary or pancreatic disease characteristic of CF. A second child in this family with classic CF was homozygous for the del14a mutation, indicating that this mutation caused severe CFTR dysfunction. CFTR mRNA transcripts bearing the S1455X mutation were stable in vivo , implying that this allele encoded a truncated version of CFTR missing the last 26 amino acids. Loss of this region did not affect processing of transiently expressed S1455X-CFTR compared with wild-type CFTR. When expressed in CF airway cells, this mutant generated cAMP-activated whole-cell chloride currents similar to wild-type CFTR. Preservation of chloride channel function of S1455X-CFTR was consistent with normal lung and pancreatic function in the mother and her daughter. These data indicate that mutations in CFTR can be associated with elevated sweat chloride concentrations in the absence of the CF phenotype, and suggest a previously unrecognized functional role in the sweat gland for the C-terminus of CFTR.

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151 Mutation analysis Total genomic DNA was assayed for 16 common CFTR mutations (R117H, 621+1G→T, R334W, R349P, A455E, 1717-1G→A, ∆I507, ∆F508, G542X, S549N, G551D, R553X, R560T, 3849+10 kb C→T, W1282X, N1303K) by reverse dot-blot hybridization (46). Login to comment
152 Mutation analysis Total genomic DNA was assayed for 16 common CFTR mutations (R117H, 621+1G࢐T, R334W, R349P, A455E, 1717-1G࢐A, ࢞I507, ࢞F508, G542X, S549N, G551D, R553X, R560T, 3849+10 kb C࢐T, W1282X, N1303K) by reverse dot-blot hybridization (46). Login to comment

[hide] Correlation of sweat chloride concentration with g... Clin Biochem. 1998 Feb;31(1):33-6. De Braekeleer M, Allard C, Leblanc JP, Aubin G, Simard F
Correlation of sweat chloride concentration with genotypes in cystic fibrosis patients in Saguenay Lac-Saint-Jean, Quebec, Canada.
Clin Biochem. 1998 Feb;31(1):33-6., [PMID:9559222]

Abstract [show]
OBJECTIVES: Saguenay Lac-Saint-Jean, a geographically isolated region of northeastern Quebec has a high incidence of cystic fibrosis (CF) and three mutations only account for 94% of the CF chromosomes. The objective of the present study was to determine whether different mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene had different effects upon the sweat chloride concentration. DESIGN AND METHODS: The sweat chloride concentration of 114 patients was measured by quantitative pilocarpine iontophoresis. RESULTS: CF patients carrying the A455E mutation, usually associated with pancreatic sufficiency, had lower sweat chloride concentrations than those carrying mutations associated with pancreatic insufficiency (delta F508 and 621 + 1G-->T). CONCLUSIONS: Our results confirm that mutations resulting in a reduction of the chloride current at the apical membrane of epithelial cells induce lower sweat chloride values. However, there are differences in the chloride current between genotypes, even if they are composed of mutations apparently having the same functional effect.

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69 Simone Aubin, Claudette La- rochelle and Suzanne Mignault from the Clinique de TABLE 2 Distribution of the Mean Sweat Chloride Concentration by Genotype Genotype No. of CF Patients Mean Chloride Concentration (mmol/L) (SD) Pancreatic Status References G542X/⌬F508 128 109 (23) Pl 18 R553X/⌬F508 46 105 (18) Pl 18 N1303K/⌬F508 56 104 (24) Pl 18 W1282X/⌬F508 13 110 (18) Pl 18 1717-1G3A/⌬F508 26 107 (36) Pl 18 621ϩ1G3T/⌬F508 22 100 (20) Pl 18 R117H/⌬F508 20 82 (19) PS 18 ⌬F508/⌬F508 328 106 (22) Pl 18 3849ϩ10kb C3T/⌬F508 6 61 (11) PS 19 3849ϩ10kb C3T/⌬F508 9 41 (12) PS (6) 20 R347P/⌬F508 5 100 (26) Pl 21 R334W/⌬F508 10 108 (19) Pl (6) 22 1811ϩ1.6kb A3C/⌬F508a 17 98 (12) Pl 23 3905insT/⌬F508 7 124 Pl 24 W1282X/W1282X 16 113 (12) Pl 25 W1282X/⌬F508 22 109 (11) Pl 25 G551D/⌬F508 58 101 (16) Pl 26 R1162X/R1162X 9 99 (13) Pl 27 1949del84/⌬F508 4 105 (20) Pl 28 ⌬F508/⌬F508 47 103 (8) Pl This study 621ϩ1G3T/⌬F508 28 103 (7) Pl This study 621ϩ1G3T/A455E 6 94 (11) Pl/PS This study A455E/⌬F508 12 77 (18) Pl/PS This study a Or other 'severe` mutations. Login to comment
71 Simone Aubin, Claudette Larochelle and Suzanne Mignault from the Clinique de TABLE 2 Distribution of the Mean Sweat Chloride Concentration by Genotype Genotype No. of CF Patients Mean Chloride Concentration (mmol/L) (SD) Pancreatic Status References G542X/DF508 128 109 (23) Pl 18 R553X/DF508 46 105 (18) Pl 18 N1303K/DF508 56 104 (24) Pl 18 W1282X/DF508 13 110 (18) Pl 18 1717-1G3A/DF508 26 107 (36) Pl 18 62111G3T/DF508 22 100 (20) Pl 18 R117H/DF508 20 82 (19) PS 18 DF508/DF508 328 106 (22) Pl 18 3849110kb C3T/DF508 6 61 (11) PS 19 3849110kb C3T/DF508 9 41 (12) PS (6) 20 R347P/DF508 5 100 (26) Pl 21 R334W/DF508 10 108 (19) Pl (6) 22 181111.6kb A3C/DF508a 17 98 (12) Pl 23 3905insT/DF508 7 124 Pl 24 W1282X/W1282X 16 113 (12) Pl 25 W1282X/DF508 22 109 (11) Pl 25 G551D/DF508 58 101 (16) Pl 26 R1162X/R1162X 9 99 (13) Pl 27 1949del84/DF508 4 105 (20) Pl 28 DF508/DF508 47 103 (8) Pl This study 62111G3T/DF508 28 103 (7) Pl This study 62111G3T/A455E 6 94 (11) Pl/PS This study A455E/DF508 12 77 (18) Pl/PS This study a Or other 'severe` mutations. Login to comment

[hide] Analysis of the CFTR gene in Turkish cystic fibros... Hum Genet. 1998 Feb;102(2):224-30. Onay T, Topaloglu O, Zielenski J, Gokgoz N, Kayserili H, Camcioglu Y, Cokugras H, Akcakaya N, Apak M, Tsui LC, Kirdar B
Analysis of the CFTR gene in Turkish cystic fibrosis patients: identification of three novel mutations (3172delAC, P1013L and M1028I).
Hum Genet. 1998 Feb;102(2):224-30., [PMID:9521595]

Abstract [show]
In order to determine the spectrum of cystic fibrosis (CF) mutations in the Turkish population, a complete coding region of the cystic fibrosis transmembrane conductance regulator (CFTR) gene including exon-intron boundaries, on 122 unrelated CF chromosomes from 73 Turkish CF families was analysed by denaturing gradient gel electrophoresis and multiplex heteroduplex analysis on MDE gel matrix. In addition to 15 previously reported mutations and 12 polymorphisms, three novel mutations, namely 3172delAC, P1013L and M1028I, were detected. DeltaF508 was found to be present on 18.8% of CF chromosomes. The second most common mutation was 1677delTA, with a frequency of 7.3%, followed by G542X and 2183AA-->G mutations, with frequencies of 4.9%. These four most common mutations in Turkish CF population account for approximately 36% of mutations. This study could only detect 52.5% of disease-causing mutations in this population; 47.5% of CF alleles remain to be identified, reflecting the high molecular heterogeneity of the Turkish population.

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3 The second most common mutation was 1677delTA, with a frequency of 7.3%, followed by G542X and 2183AA→G mutations, with frequencies of 4.9%. Login to comment
26 Other frequent mutations, namely G542X, R560T, S549N or S549I and G551D or R553X, were analysed by the multiplex ARMS method combined with restriction enzyme analysis (Dean et al. 1990; Cutting et al. 1990; Ferrie et al. 1992). Login to comment
41 Three other mutations, namely G542X, N1303K and W1282X, were found on 10 chromosomes and screening of exon 10 by DGGE resulted in the identification of the homozygous presence of 1525-1 G→A in one affected child with a sweat test score of 109 mEq/l. Login to comment
67 Mutations 1677delTA, G542X and 2183AA→G have frequencies greater or equal to approximately 5%, whereas F1052V, 2043delG, D110H, N1303K, L571S and 296+9 A→T have frequencies of 2%. Login to comment
69 Nine homozygous CF genotypes, for mutations ∆F508, 1677delTA, 2183AA→G, G542X, L571S, N1303K, W1282X, 1525-1 G→A and 2043 delG, were observed in 26.0% of cases. Login to comment
75 G542X 11 Gly→Stop at 542 G→T at 1756 6 (4.91) Kerem et al. 1990 5. Login to comment
115 This study reveals the presence of mutations G542X and 2182AA→G with the same frequency (4.9%) in the Turkish population. Login to comment
117 The frequency of G542X is 8.1% in Spain (Cassals et al. 1993), 4% in Italy, 5% in Greece (Nunes et al. 1991) and 13.5% in the Jewish population of Israel (Lerer et al. 1991). Login to comment
140 Nine homozygous CF genotypes, for mutations ∆F508, 1677delTA, 2183AA→G, G542X, L571S, N1303K, W1282X, 1525-1 G→A and 2043delG, were observed in 26.0% of cases, whereas consanguinity was noted in approximately 9.6% of patients. Login to comment

[hide] Genetic diseases of the seminal ducts. Biomed Pharmacother. 1998;52(5):197-203. Meschede D, Dworniczak B, Nieschlag E, Horst J
Genetic diseases of the seminal ducts.
Biomed Pharmacother. 1998;52(5):197-203., [PMID:9755815]

Abstract [show]
Azoospermia due to an obstruction of the genital tract is one of numerous possible pathophysiologic mechanisms underlying male infertility. The blockage of the seminal ducts may be acquired or congenital. Only recently has the strong association between mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and various subtypes of obstructive azoospermia been elucidated. Most patients with congenital bilateral absence of the vas deferens or bilateral ejaculatory duct obstruction are carriers of such mutations. The relationship between abnormal CFTR alleles and unilateral absence of the vas deferens, isolated seminal vesicle anomalies, and Young syndrome is less well characterized and awaits further investigation.

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39 This is the case in homozygotes and compound heterozygotes for AF508 and other common "severe" mutations such as 17 171 G + A, G542X, G55lD, R553X, W1282X or Nl303K. Login to comment
40 This is the case in homozygotes and compound heterozygotes for AF508 and other common "severe" mutations such as 17 171 G + A, G542X, G55lD, R553X, W1282X or Nl303K. Login to comment

[hide] Genetic findings in congenital bilateral aplasia o... Hum Mutat. 1998;11(6):480. de Meeus A, Guittard C, Desgeorges M, Carles S, Demaille J, Claustres M
Genetic findings in congenital bilateral aplasia of vas deferens patients and identification of six novel mutatations. Mutations in brief no. 138. Online.
Hum Mutat. 1998;11(6):480., [PMID:10200050]

Abstract [show]
Congential bilateral aplasia of vas deferens (CBAVD), a form of male sterility, has been suggested to represent a "genital" form of cystic fibrosis (CF), as mutations in the CFTR gene have been identified in most patients with this condition. Interestingly, the 5T allele in intron 8 appeared to be the most frequent mutation associated with CBAVD. However, the molecular basis of CBAVD is not completely understood. We have analysed the complete coding and flanking CFTR sequences by PCR-DGGE in 64 men with CBAVD from southern France with the aim to list any sequence alteration. Fourty-two of the 64 patients (65.6%) had mutations on both copies of the CFTR gene, including one patient with two mutations in the same copy (DF508 + A1067T). The 5T allele was present in 21/64 cases (33%). Six of the 28 different mutations identified in this study had never been described previously, and appeared to be specific to CBAVD (P111L, M244K, A1364V, G544V, 2896insAG,-33G->A).

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83 Phenotype CFTRamutations Intron 8, Poly(T) tract 1 3 crisis of acute pancreatitis F508 / L206W 9/7 2 F508 / L206W 9/9 3 frequent bronchitis F508 / R347H 9/9 4 F508 / R347H 9/9 5 F508 / M244K 9/7 6 F508 / A1364V 9/7 7 F508 / D1152H 9/7 8 chronic sinusitis and bronchitis F508 / D1152H 9/7 9 F508 / R117H 9/7 10 F508 / R117H 9/7 11 F508 / M952I 9/7 12 D443Y / G542X 7/9 13 D443Y / G542X 7/9 14 2184delA / D443Y 7/7 15 2184delA / D443Y 7/7 16 R347H / D443Y 9/7 17 seminal vesicles agenesia R117H / G1349D 7/7 18 R117H / G1244E 7/7 19 N1303K / P111L 9/7 20 chronic sinusitis, nasal polyps W1282X / D1152H 7/7 21 chronic sinusitis R347H / Y1092X 7/7 22 seminal vesicles agnesia 297-3C-GTT / 4279insA 7/7 23 G544V / F508C 7/7 24 D1152H / 2896insAG 7-9 25 F508 / - 9/5 26 F508 / - 9/5 27 F508 / - 9/5 28 F508 / - 9/5 29 F508 / - 9/5 30 chronic sinusitis, bronchitis F508 / - 9/5 31 sinusitis and allergy F508 / - 9/5 32 allergy F508 / - 9/5 33 F508 / - 9/5 34 F508 / - 9/5 35 F508 / - 9/5 36 F508 / - 9/5 37 bronchitis, asthma F508 / - 9/5 38 chronic sinusitis F508+A1067T / - 9/5 39 chronic sinusitis D1152H / - 7/5 40 2184delA / - 7/5 41 R764X / - 7/5 42 711+1G-GTT / - 7/5 43 F508 / - 9/7 44 F508 / - 9/7 45 F508 / - 9/7 46 F508 / - 9/9 47 R553X / - 7/7 48 -33G-GTA / - 7/7 49 K710X / - 7/7 50 - / - 5/5 51 - / - 5/7 52 - / - 5/7 53 - / - 7/7 54 - / - 7/7 55 - / - 7/7 56 - / - 7/7 57 - / - 7/7 58 - / - 7/7 59 - / - 7/7 60 - / - 7/7 61 - / - 7/9 62 - / - 7/9 63 NIDDb - / - 7/9 64 - / - 7/9 a : Cystic Fibrosis Transmembrane Regulator gene b : Non Insulino-Dependant Diabetis References Anguiano A, Oates RD, Amos JA, Dean M, Gerrard B, Stewart C, Maher TA, White MB, Milunsky A (1992) Congenital absence of the vas deferens: a primarily genital form of cystic fibrosis. Login to comment

[hide] High heterogeneity for cystic fibrosis in Spanish ... Hum Genet. 1997 Dec;101(3):365-70. Casals T, Ramos MD, Gimenez J, Larriba S, Nunes V, Estivill X
High heterogeneity for cystic fibrosis in Spanish families: 75 mutations account for 90% of chromosomes.
Hum Genet. 1997 Dec;101(3):365-70., [PMID:9439669]

Abstract [show]
We have analyzed 640 Spanish cystic fibrosis (CF) families for mutations in the CFTR gene by direct mutation analysis, microsatellite haplotypes, denaturing gradient gel electrophoresis, single-strand conformation analysis and direct sequencing. Seventy-five mutations account for 90.2% of CF chromosomes. Among these we have detected seven novel CFTR mutations, including four missense (G85V, T582R, R851L and F1074L), two nonsense (E692X and Q1281X) and one splice site mutation (711+3A-->T). Three variants, two in intronic regions (406-112A/T and 3850-129T/C) and one in the coding region (741C/T) were also identified. Mutations G85V, T582R, R851L, E692X and Q1281X are severe, with lung and pancreatic involvement; 711+3A-->T could be responsible for a pancreatic sufficiency/insufficiency variable phenotype; and F1074L was associated with a mild phenotype. These data demonstrate the highest molecular heterogeneity reported so far in CF, indicating that a wide mutation screening is necessary to characterize 90% of the Spanish CF alleles.

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21 The ∆F508 (Rommens et al. 1990) and G542X (Kerem et al. 1990; Gasparini et al. 1992) mutations were analysed in all patients as they are the most common mutations in the population, 50.6% and 8.0%, re- Teresa Casals · Maria D. Ramos · Javier Giménez · Sara Larriba · Virginia Nunes · Xavier Estivill High heterogeneity for cystic fibrosis in Spanish families: 75 mutations account for 90% of chromosomes Hum Genet (1997) 101:365-370 (c) Springer-Verlag 1997 Received: 3 July 1997 / Accepted: 20 August 1997 ORIGINAL INVESTIGATION T. Casals · M. D. Ramos · J. Giménez · S. Larriba · V. Nunes · X. Login to comment
31 Only ten mutations have a frequency of 1% and above: ∆F508 (53.2%), G542X (8.4%), N1303K (2.6%), 1811+1.6kbA→G (1.8%), 711+1G→T (1.7%), R1162X and R334W (1.6%), R1066C, 1609delCA and Q890X (1.0%). Login to comment
33 Eight mutations have frequencies 366 Table 1 Seventy-five CFTR mutations identified in 640 Spanish families with cystic fibrosis (CF) Mutation Exon/intron CF alleles % ∆F508 E.10 681 53.20 G542X E.11 108 8.43 N1303K E.21 34 2.65 1811+1.6kbA→Ga I.11 24 1.87 711+1G→T I.5 22 1.71 R1162Xa E.19 21 1.64 R334Wa E.7 21 1.64 R1066C E.17b 14 1.09 1609delCAa E.10 13 1.01 Q890X E.15 13 1.01 G85E E.3 12 0.94 712-1G→Ta I.5 11 0.86 2789+5G→A I.14b 11 0.86 ∆I507 E.10 10 0.78 W1282X E.20 10 0.78 2869insGa E.15 9 0.70 L206W E.6a 7 0.54 R709X E.13 7 0.54 621+1G→T I.4 6 0.47 3272-26A→G I.17a 6 0.47 R347H E.7 5 0.39 2183AA→G E.13 5 0.39 K710X E.13 5 0.39 2176insC E.13 5 0.39 3849+10kbC→T I.19 5 0.39 P205Sa E.6a 4 0.31 1078delT E.7 4 0.31 R553X E.11 4 0.31 G551D E.11 4 0.31 1812-1G→Aa I.11 4 0.31 CFdel#1a E.4-7/11-18 4 0.31 V232D E.6a 3 0.23 936delTAa E.6b 3 0.23 1717-8G→A I.10 3 0.23 1949del84 E.13 3 0.23 W1089X E.17b 3 0.23 R347P E.7 3 0.23 del E.3a E.3 2 0.16 R117H E.4 2 0.16 L558S E.11 2 0.16 A561E E.12 2 0.16 2603delT E.13 2 0.16 Y1092X E.17b 2 0.16 Q1100Pa E.17b 2 0.16 M1101K E.17b 2 0.16 delE.19a E.19 2 0.16 G1244E E.20 2 0.16 P5La E.1 1 0.08 Q30Xa E.2 1 0.08 G85Va E.3 1 0.08 E92Ka E.4 1 0.08 A120Ta E.4 1 0.08 I148T E.4 1 0.08 711+3A→Ta I.5 1 0.08 H199Y E.6a 1 0.08 875+1G→A I.6a 1 0.08 Table 1 (continued) Mutation Exon/intron CF alleles % 1717-1G→A I.10 1 0.08 L571S E.12 1 0.08 T582Ra E.12 1 0.08 E585X E.12 1 0.08 1898+3A→G I.12 1 0.08 G673X E.13 1 0.08 E692Xa E.13 1 0.08 R851X E.14a 1 0.08 R851La E.14a 1 0.08 A1006E E.17a 1 0.08 L1065Ra E.17b 1 0.08 F1074La E.17b 1 0.08 R1158X E.19 1 0.08 3667del4a E.19 1 0.08 3860ins31a E.20 1 0.08 3905insT E.20 1 0.08 4005+1G→A I.20 1 0.08 Q1281Xa E.20 1 0.08 Q1313X E.21 1 0.08 Known mutations (75) 1155 90.23 Unknown mutations 125 9.77 a Mutations discovered by the CF group of the Medical and Molecular Genetics Centre - IRO, Barcelona, Spain that range between 0.5% and 0.9%, representing 6.0% of the CF chromosomes. Login to comment
43 The G85V mutation was identified in a patient who carries the G542X mutation on the maternal CF allele. Login to comment

[hide] Novel and characteristic CFTR mutations in Saudi A... J Med Genet. 1997 Dec;34(12):996-9. el-Harith EA, Dork T, Stuhrmann M, Abu-Srair H, al-Shahri A, Keller KM, Lentze MJ, Schmidtke J
Novel and characteristic CFTR mutations in Saudi Arab children with severe cystic fibrosis.
J Med Genet. 1997 Dec;34(12):996-9., [PMID:9429141]

Abstract [show]
More than 600 different CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations have been identified so far that are considered to cause the fatal genetic disorder cystic fibrosis (CF). We have investigated 15 Arab children from 12 families, who were diagnosed as having CF, for mutations in the coding region and in the flanking intron sequences of the CFTR gene. Six different CFTR mutations were identified including two novel mutations, 1548delG in exon 10 and 406-2A-->G in intron 3. Prominent mutations were the splice mutation 3120 + 1G-->A (intron 16) followed by N1303K (exon 21) and 1548delG (exon 10). Most CF children were homozygotes who presented with a severe form of the disease including failure to thrive, recurrent chest infections, particularly with Pseudomonas aeruginosa, and frequent hospital admissions. Identification of the CFTR mutations facilitates molecular investigation of the disease and better understanding of its pathophysiology in Arab children, among whom CF is probably an underdiagnosed disease.

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26 Deletions of two or more base pairs were screened for by electrophoresis using a native 12% polyacrylamide gel. The 20 common CFTR mutations that were screened for were AF508, AI507, 1677delTA, R347P, R347H, R553X, G551D, G542X, N1303K, 3849+1OKbC-8'T, R334W, I336K, 2789+5G-A, 1717-1G-A, 3272- 26A- G, Y1092X, 2143delT, W1282X, RI 17H, and the 5T allele. Login to comment
49 For direct mutation analyses, PCR products were digested with the respective restriction enzyme and the fragments were separated by electrophoresis using a 3% NuSieve/i % SeaKem agarose gel. Mutagenesis primers were designed for some mutations (Ri17H, 1717-lG-A, G542X, 2143delT, 3272-26A-.G, and N1303K) to create artifi- shown). Login to comment

[hide] CFTR gene mutations in men with bilateral ejaculat... Am J Hum Genet. 1997 Nov;61(5):1200-2. Meschede D, Dworniczak B, Behre HM, Kliesch S, Claustres M, Nieschlag E, Horst J
CFTR gene mutations in men with bilateral ejaculatory-duct obstruction and anomalies of the seminal vesicles.
Am J Hum Genet. 1997 Nov;61(5):1200-2., [PMID:9345100]

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23 Direct testing by allele-specific amplification, heteroduplex analysis, or by restriction analysis was performed in all patients, for detection of the following CFTR gene mutations: R117H, R347P, DI507, DF508, 1717-1 GrA, G542X, G551D, R553X, 3849ϩ10 kB, W1282X, and N1303K. Login to comment
22 DNA was isolated from peripheral lymphocytes, and target sequences were amplified by PCR. Direct testing by allele-specific amplification, heteroduplex analysis, or by restriction analysis was performed in all patients, for detection of the following CFTR gene mutations: R117H, R347P, DI507, DF508, 1717-1 GrA, G542X, G551D, R553X, 3849af9;10 kB, W1282X, and N1303K. Login to comment

[hide] Strategies for correcting the delta F508 CFTR prot... J Bioenerg Biomembr. 1997 Oct;29(5):491-502. Brown CR, Hong-Brown LQ, Welch WJ
Strategies for correcting the delta F508 CFTR protein-folding defect.
J Bioenerg Biomembr. 1997 Oct;29(5):491-502., [PMID:9511934]

Abstract [show]
Many human diseases arise as a result of mutations within genes encoding essential proteins. In many cases, the mutations are not so severe as to render the protein biologically inactive. Rather, the mutations oftentimes result in only subtle protein-folding abnormalities. In the case of the CFTR protein, a mutation leading to the loss of a single amino acid is responsible for the diseased state in the majority of individuals with cystic fibrosis. Here the newly synthesized mutant CFTR protein, missing a phenylalanine residue at position 508 (delta F508 CFTR), is unable to transit from the endoplasmic reticulum to the plasma membrane, where it functions as a regulator of chloride transport. All of the available evidence indicate that the newly synthesized delta F508 CFTR protein adopts a slightly altered conformation and therefore is retained at the level of the endoplasmic reticulum, ostensibly by the actions of the cellular quality control system. Because the mutant protein is capable of functioning as a chloride channel, developing ways to elicit its release out of the ER and to the plasma membrane has important clinical implications. Herein, we discuss our recent studies showing that the protein-folding defect associated with the delta F508 CFTR mutation, as well as a number of other temperature-sensitive mutations, can be overcome by strategies designed to influence protein folding inside the cell. Specifically we show that a number of low-molecular-weight compounds, all of which are known to stabilize proteins in their native conformation, are effective in rescuing the folding and/or processing defects associated with different mutations that oftentimes lead to human disease.

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37 Howard and his colleagues have shown that treatment of cells expressing some of these truncation mutants (e.g., G542X and R553X) with different aminoglycoside antibiotics results in "translational read-through" and the production of low levels of a full-length and functional CFTR protein (Howard et al., 1996). Login to comment

[hide] Distinct spectrum of CFTR gene mutations in congen... Hum Genet. 1997 Sep;100(3-4):365-77. Dork T, Dworniczak B, Aulehla-Scholz C, Wieczorek D, Bohm I, Mayerova A, Seydewitz HH, Nieschlag E, Meschede D, Horst J, Pander HJ, Sperling H, Ratjen F, Passarge E, Schmidtke J, Stuhrmann M
Distinct spectrum of CFTR gene mutations in congenital absence of vas deferens.
Hum Genet. 1997 Sep;100(3-4):365-77., [PMID:9272157]

Abstract [show]
Congenital absence of the vas deferens (CAVD) is a frequent cause for obstructive azoospermia and accounts for 1%-2% of male infertility. A high incidence of mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has recently been reported in males with CAVD. We have investigated a cohort of 106 German patients with congenital bilateral or unilateral absence of the vas deferens for mutations in the coding region, flanking intron regions and promotor sequences of the CFTR gene. Of the CAVD patients, 75% carried CFTR mutations or disease-associated CFTR variants, such as the "5T" allele, on both chromosomes. The distribution of mutation genotypes clearly differed from that observed in cystic fibrosis. None of the CAVD patients was homozygous for delta F508 and none was compound heterozygous for delta F508 and a nonsense or frameshift mutation. Instead, homozygosity was found for a few mild missense or splicing mutations, and the majority of CAVD mutations were missense substitutions. Twenty-one German CAVD patients were compound heterozygous for delta F508 and R117H, which was the most frequent CAVD genotype in our study group. Haplotype analysis indicated a common origin for R117H in our population, whereas another frequent CAVD mutation, viz. the "5T allele" was a recurrent mutation on different intragenic haplotypes and multiple ethnic backgrounds. We identified a total of 46 different mutations and variants, of which 15 mutations have not previously been reported. Thirteen novel missense mutations and one unique amino-acid insertion may be confined to the CAVD phenotype. A few splice or missense variants, such as F508C or 1716 G-->A, are proposed here as possible candidate CAVD mutations with an apparently reduced penetrance. Clinical examination of patients with CFTR mutations on both chromosomes revealed elevated sweat chloride concentrations and discrete symptoms of respiratory disease in a subset of patients. Thus, our collaborative study shows that CAVD without renal malformation is a primary genital form of cystic fibrosis in the vast majority of German patients and links the particular expression of clinical symptoms in CAVD with a distinct subset of CFTR mutation genotypes.

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43 This initial screening included the mutations ∆F508, G542X, R553X, G551D, N1303K, 1717-1 G→A, 3272-26 A→G, Y1092X, 2143delT, R347P, R347H, R334W, I336K, R117H, R117C, 2789+5 G→A, 3849+10kB C→T and the "5T" allele, the latter two splice variants being tested according to the instructions of Highsmith et al. (1994) and Chillón et al. (1995). Login to comment
69 Only four other common CF mutations were found in more than one family by this initial screening: 2789+5 G→A on 4 alleles, R347H on 3 alleles, G542X and 3272-26 A→G each on 2 alleles (Table 1). Login to comment
86 The V938G substitution was identified in two unrelated patients, one homozygote with unilateral ab- 368 Table 1A Frequency distribution and haplotypes of CFTR mutations in 106 CAVD patients Mutationa Nucleotide changesb Locationc Frequencyd Haplotypee Referencef 174delA deletion of A at 174 exon 1 1 D3 This study E56K G→A at 298 exon 3 1 B3 This study D58N G→A at 304 exon 3 1 C2 This study D110H G→A at 460 exon 4 2 C2 Dean et al. (1990) R117H G→A at 482 exon 4 24 B6 Dean et al. (1990) A120T G→A at 490 exon 4 1 n.p. Chillón et al. (1994) ̃L138 insertion of CTA after 546 exon 4 1 A2 This study L206W T→G at 749 exon 6a 1 B8 Claustres et al. (1993) M265R T→G at 926 exon 6b 1 A2 Schwarz et al. (pers. comm.) R297W C→T at 1021 exon 7 1 C2 This study 1078delT deletion of T at 1078 exon 7 1 C2 Claustres et al. (1992) R334W C→T at 1132 exon 7 1 B1 Gasparini et al. (1991) R334L G→T at 1133 exon 7 1 D3 This study I336K T→A at 1139 exon 7 1 A2 Cuppens et al. (1993) R347H G→A at 1172 exon 7 3 D1 Cremonesi et al. (1992) L375F A→C at 1257 exon 8 1 B3 Jézéquel et al. (1996) ∆F508 deletion of 3 bp between 1652-1655 exon 10 57 B1 Kerem et al. (1989) G542X G→T at 1756 exon 11 2 B1 Kerem et al. (1990) R553X C→T at 1789 exon 11 1 A4 Cutting et al. (1990) L568F G→T at 1836 exon 12 1 B3 This study 2184insA insertion of A at 2184 exon 13 1 D3 Dörk et al. (1994b) 2789+5 G→A G→A at 2789+5 intron 14b 4 D3 Highsmith et al. (1997) R933S A→T at 2931 exon 15 1 n.p. Login to comment
137 Complex alleles are indicated a One CF allele with R75X and 125G→C b One CBAVD allele with R75Q and R933S c One CBAVD allele with 5T and Q1352H d Two CF alleles with F508C and S1251N e One CF allele with 1716G→A and L619S f G576A and R668C were linked on two CBAVD and three CF alleles, whereas two additional CF alleles carried R668C together with the 3849+10kB C→T mutation (Dörk and Stuhrmann 1995) 371 Table 3 CFTR mutation genotypes in 106 males with CAVD Genotype PolyT Frequency Ethnic descent Diagnosis ∆F508/R117H 9/7 21 German, Austrian 20 CBAVD, 1 CUAVD ∆F508/5T 9/5 9 German, Austrian 8 CBAVD, 1 CUAVD ∆F508/F508C 9/7 3 German CBAVD ∆F508/R347H 9/9 2 German CBAVD ∆F508/1716 G→A 9/7 2 German CBAVD ∆F508/3272-26 A→G 9/7 2 German CBAVD ∆F508/E56K 9/7 1 German CBAVD ∆F508/M265R 9/7 1 German-Portuguese CBAVD ∆F508/R334W 9/9 1 German CBAVD ∆F508/T351S 9/9 1 German CBAVD ∆F508/L375F 9/7 1 Volga German CBAVD ∆F508/G576A & R668C 9/7 1 German CBAVD ∆F508/R933S 9/7 1 German CBAVD ∆F508/L997F 9/9 1 German CBAVD ∆F508/Y1032C 9/7 1 German CBAVD ∆F508/D1152H 9/7 1 German CBAVD ∆F508/K1351E 9/7 1 German CBAVD ∆F508/D1377H 9/7 1 Portuguese CBAVD ∆F508/L1388Q 9/7 1 German CBAVD ∆F508/unknown 9/7 4 German 3 CBAVD, 1 CUAVD 5T/5T 5/5 2 German CBAVD 5T/G542X 5/9 2 German, Turkish CBAVD 5T/D58N 5/7 1 Lebanese CBAVD 5T/̃L138 5/7 1 German-Polish CBAVD 5T/1078delT 5/7 1 German CBAVD 5T/R553X 5/7 1 German CBAVD 5T/2184insA 5/7 1 Turkish CBAVD 5T/D979A 5/7 1 Vietnamese CBAVD 5T/D1152H 5/7 1 Turkish CBAVD 5T/3659delC 5/7 1 German CBAVD 5T/S1235R 5/7 1 Greek CBAVD 5T/W1282X 5/7 1 German CBAVD 5T & Q1352H/ R297W & Q1352H 5/7 1 Vietnamese CBAVD 5T/unknown 5/7 1 German CBAVD R117H/L206W 7/9 1 German CBAVD R117H/2789+5 G→A 7/7 1 German CBAVD R117H/unknown 7/7 1 German CBAVD 2789+5 G→A/2789+5 G→A 7/7 1 Lebanese CBAVD 2789+5 G→A/L973F 7/7 1 German CBAVD V938G/V938G 7/7 1 Greek CBAVD V938G/174delA 7/7 1 German CBAVD D110H/D110H 7/7 1 Turkish CBAVD R334L/I336K 7/7 1 German CBAVD R347H/N1303K 9/9 1 German CBAVD L568F/D1152H 7/7 1 Turkish CBAVD 3272-26 A→G/V1153E 7/7 1 German CBAVD R75Q/unknown 7/7 1 German CBAVD A120T/unknown 9/7 1 German CBAVD 1716G→A/unknown 7/7 1 German CBAVD G576A & R668C/unknown 7/7 1 German CBAVD 2752-15 C→G/unknown 7/7 1 Iranian CBAVD Unknown/unknown 17 German, Turkish 7 CBAVD and 1 CUAVD without observed renal agenesis, 9 CBAVD with renal agenesis allele and the R297W mutation on a homozygous Q1352H background may then reduce CFTR function to a disease-causing level. Login to comment
145 Maldigestion 13 25 5T/D58N 184 99 55 - 14 34 5T/̃L138 177 80 53 - 15 33 5T/1078delT 187 87 56 Recurrent bronchitis 16 31 5T/G542X 181 85 79 - 17 31 5T/2184insA n.d. n.d. 60 Borderline pancreatic sufficiency 18 31 5T/D979A n.d. n.d. 55 Recurrent infections, FEVI 76% 19 29 5T/D1152H n.d. n.d. 57 - 20 32 5T/W1282X 180 76 n.d. Recurrent infections, nasal polyposis 21 37 5T/unknown 180 74 n.d. Nasal polyposis 22 28 D110H/D110H 175 80 n.d Asthma bronchiale, obstipation 23 33 R334L/I336K 170 65 n.d. Recurrent infections, nasal polyposis, maldigestion, salt depletion episodes 24 35 N1303K/R347H 167 77 93 - 25 30 V938G/174delA n.d. n.d. 42 - 26 29 V938G/V938G 197 115 n.d. Asthma bronchiale Fig.2 Spectrum of CFTR mutation genotypes in CF patients (left) and in patients with congenital absence of the vas deferens (right). Login to comment

[hide] Relevance of genetic counselling in couples prior ... Hum Reprod. 1997 Sep;12(9):1909-12. Pauer HU, Hinney B, Michelmann HW, Krasemann EW, Zoll B, Engel W
Relevance of genetic counselling in couples prior to intracytoplasmic sperm injection.
Hum Reprod. 1997 Sep;12(9):1909-12., [PMID:9363704]

Abstract [show]
Since the first reports of successful pregnancies after treatment with intracytoplasmic sperm injection (ICSI) in humans numerous attempts have been made to assess the genetic risks of this highly invasive technique. During the study period (February 1995-November 96), 142 couples were referred to our genetic counselling unit prior to ICSI. In three couples, genetic counselling revealed a high recurrence risk for a monogenic disease (myotonic dystrophy, hereditary ataxia and polycystic kidney disease). In nine out of 128 men (7%) an abnormal karyotype was identified, including three Robertsonian translocations, two reciprocal translocations, three sex chromosome aberrations and one case with centric fission of chromosome no. 7. A total of 14 men refused chromosomal analysis. Only one of the 122 women examined had an abnormal karyotype (47, XXX). Five out of six men with congenital bilateral absence of the vas deferens (CBAVD) had at least one mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Three had mutations in both CFTR alleles, including one case in which the second mutation was the 5T allele. One patient with CBAVD and a single Delta F508 CFTR mutation also had left renal agenesis. In conclusion, we strongly recommend that genetic counselling, chromosomal analysis and, in the case of CBAVD, screening for CFTR mutations should be offered to all couples with a diagnosis of male or idiopathic infertility.

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25 Patients whose history or physical examination showed a possible diagnosis of CBAVD were typed for 19 additional CFTR mutations (e.g. R117H, Introduction R347P, 1717-1, G542X, G551D, R553X, W1282X and N1303K), including an intronic polymorphism (5T allele) which leads to reducedThe development of intracytoplasmic sperm injection (ICSI) for splicing efficiency of the CFTR mRNA (Chillo´n et al., 1995). Login to comment
24 Patients whose history or physical examination showed a possible diagnosis of CBAVD were typed for 19 additional CFTR mutations (e.g. R117H, Introduction R347P, 1717-1, G542X, G551D, R553X, W1282X and N1303K), including an intronic polymorphism (5T allele) which leads to reduced The development of intracytoplasmic sperm injection (ICSI) for splicing efficiency of the CFTR mRNA (Chillo &#b4;n et al., 1995). Login to comment

[hide] A missense cystic fibrosis transmembrane conductan... Pediatrics. 1997 Sep;100(3):E5. Kerem E, Nissim-Rafinia M, Argaman Z, Augarten A, Bentur L, Klar A, Yahav Y, Szeinberg A, Hiba O, Branski D, Corey M, Kerem B
A missense cystic fibrosis transmembrane conductance regulator mutation with variable phenotype.
Pediatrics. 1997 Sep;100(3):E5., [PMID:9271620]

Abstract [show]
OBJECTIVE: Cystic fibrosis (CF) has variable clinical presentation. Disease severity is partially associated with the type of mutation. The aim of this study was to report genotype-phenotype analysis of the G85E mutation. PATIENTS: The phenotype of 12 patients (8 were from the same extended family, and 5 of them were siblings from 2 families) carrying at least one copy of the G85E mutation was evaluated and compared with the phenotype of 40 patients carrying the two severe mutations, W1282X and/or DeltaF508 (group 1), and with 20 patients carrying the splicing mutation, 3849+10kb C->T, which was found to be associated with milder disease (group 2). RESULTS: A high phenotypic variability was found among the patients carrying the G85E mutation. This high variability was found among patients carrying the same genotype and among siblings. All the studied chromosomes carrying the G85E mutation had the 7T variant in the polythymidine tract at the branch/acceptor site in intron 8. Of the G85E patients, 25% had pancreatic sufficiency and none had meconium ileus, compared with 0% and 32%, respectively, of patients from group 1, and 80% and 0%, respectively, from group 2. Two patients carrying the G85E mutation had sweat chloride levels <60 mmol/L whereas all the others had typically elevated levels >80 mmol/L. Compared with group 2, patients carrying the G85E mutation were diagnosed at an earlier age and had higher sweat chloride levels, with mean values similar to group 1 but significantly more variable. Forced expiratory volume in 1 second (FEV1) was similar in the three groups, with no differences in the slope or in age-adjusted mean values of FEV1. The levels of transcripts lacking exon 9 transcribed from the G85E allele measured in 3 patients were 55%, 49%, and 35% and their FEV1 values were 82%, 83%, and 50% predicated, respectively. CONCLUSIONS: The G85E mutation shows variable clinical presentation in all clinical parameters. This variability could be seen among patients carrying on the other chromosome the same CFTR mutation, and also among siblings. This variability is not associated with the level of exon 9 skipping. Thus, the G85E mutation cannot be classified either as a severe or as a mild mutation.

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126 Several genotype-phenotype studies including the CF genotype-phenotype consortium have shown that there are mutations like the ⌬F508,2,6,11 W1282X,3,6 G542X,6 N1303K,4,6 and R533X5,6 in which Ͼ95% of the patients had PI2-10 whereas others like the 3849ϩ10kb C-ϾT,7,8 A455E15 Fig 1. Login to comment

[hide] A new mutation, 3905insT, accounts for 4.8% of 117... Hum Genet. 1997 Aug;100(2):220-3. Hergersberg M, Balakrishnan J, Bettecken T, Chevalier-Porst F, Bragger C, Burger R, Einschenk I, Liechti-Gallati S, Morris M, Schorderet D, Thonney F, Moser H, Malik N
A new mutation, 3905insT, accounts for 4.8% of 1173 CF chromosomes in Switzerland and causes a severe phenotype.
Hum Genet. 1997 Aug;100(2):220-3., [PMID:9254853]

Abstract [show]
We have analysed 1173 cystic fibrosis (CF) chromosomes from Switzerland for eight mutations in the CF transmembrane conductance regulator (CFTR) gene. This permitted the identification of 88.5% of all mutations present. A novel insertion mutation in exon 20 of the CFTR gene, 3905insT, was discovered. This mutation accounted for 4.8% of CFTR gene mutations in Switzerland and has since been identified in other populations of probable Swiss descent. It is associated with a highly variable clinical phenotype but always with pancreatic insufficiency. Haplotype analysis with three intragenic microsatellites in the CFTR gene showed that the mutation is associated with a haplotype rarely identified on other CFTR alleles and, therefore, that the frequency of the mutation in Switzerland is explained by a founder effect of a relatively recent mutation event.

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17 Martin Hergersberg · Jaya Balakrishnan · Thomas Bettecken · Francoise Chevalier-Porst · Christian Brägger · René Burger · Inge Einschenk · Sabina Liechti-Gallati · Michael Morris · Daniel Schorderet · Francine Thonney · Hans Moser · Naseem Malik A new mutation, 3905insT, accounts for 4.8% of 1173 CF chromosomes in Switzerland and causes a severe phenotype Hum Genet (1997) 100:220-223 (c) Springer-Verlag 1997 Received: 17 February 1997 / Accepted: 26 March 1977 ORIGINAL INVESTIGATION M. Hergersberg (౧) · J. Balakrishnan · I. Einschenk Institut für Medizinische Genetik, Universität Zürich, Rämistrasse 74, CH-8001 Zurich, Switzerland Tel.: +411 257 25 35; Fax: +411 262 04 70; e-mail hergie@medgen.unizh.ch T. Bettecken · S. Liechti-Gallati · H. Moser Universitäts-Kinderklinik, Bern, Switzerland F. Chevalier-Porst Hôpital Debrousse, Lyon, France C. Brägger · R. Burger Universitäts-Kinderklinik, Zurich, Switzerland M. Morris Division de Génétique Médicale, Gèneve, Switzerland D. Schorderet · F. Thonney Division Autonome de Génétique Médicale, Lausanne, Switzerland N. Malik Abteilung für Medizinische Genetik, Universitätskinderklinik, Basel, Switzerland Materials and methods Patients and families All blood samples received by the five Swiss University Centres of medical genetics for mutation analysis in the CFTR gene were screened for the eight mutations ∆F508, R553X, 1717-1G→A, G542X, N1303K, W1282X, R347P and 3905insT (Table 1). Login to comment
42 Using this method, the 3905insT mutation was found on 56 (4.8%) of "Swiss" CF chromosomes, but was not detected in more than 400 normal chromosomes, in more than 200 CF chromosomes with the ∆F508 mutation and in numerous CF chromosomes 221 Table 1 The frequency of eight common cystic fibrosis (CF) mutations among 1173 CF mutations in Switzerland Mutation Number of CF Frequency chromosomes (%) ∆F508 841 71.7 3905insT 56 4.8 R553X 43 3.7 1717-1G→A 39 3.3 G542X 23 2.0 N1303K 17 1.4 W1282X 13 1.1 R347P 7 0.6 Other mutations 21 1.9 Total 1060 90.4 Unidentified mutations 113 9. Login to comment

[hide] Detecting CFTR gene mutations by using primer olig... Clin Chem. 1997 Jul;43(7):1151-8. Braun A, Little DP, Koster H
Detecting CFTR gene mutations by using primer oligo base extension and mass spectrometry.
Clin Chem. 1997 Jul;43(7):1151-8., [PMID:9216450]

Abstract [show]
A new method for the reliable identification of localized variations in DNA by detection of associated diagnostic products with matrix-assisted laser desorption ionization time-of-flight mass spectrometry is described. The diagnostic products are generated by the primer oligo base extension (PROBE) reaction, which requires a single detection primer complementary to a region down-stream of a target strand's variable site. On addition of a polymerase, three dNTPs, and the fourth nucleotide in dideoxy form, the primer is extended through the mutation region until the first ddNTP is incorporated; the mass of the extension products determines the composition of the variable site. Tests for five cystic fibrosis mutations, including two exon 11 sites measured in a biplex reaction, and for differentiating between three common alleles of the poly(T) tract at the intron 8 splice acceptor site of the CFTR gene are presented. All experimental steps required for PROBE are amenable to the high degree of automation desirable for a high-through-put diagnostic setting. Furthermore, it requires no fluorescent, chemiluminescent, or radioactive labeling; the mass signals measured offer a far more analytically definitive signal, leading in all cases to high-quality unambiguous and easily interpreted results.

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74 PROBE was also used for simultaneous (biplex) detection of the CFTR gene exon 11 G542X and R553X mutations in two patients. Login to comment
111 a, heterozygous G542X; homozygous wild-type at codon 553. b, homozygous wild-type at codon 542; heterozygous R53X. Login to comment

[hide] Simple triple-label detection of seven cystic fibr... Clin Chem. 1997 Jul;43(7):1142-50. Heinonen P, Iitia A, Torresani T, Lovgren T
Simple triple-label detection of seven cystic fibrosis mutations by time-resolved fluorometry.
Clin Chem. 1997 Jul;43(7):1142-50., [PMID:9216449]

Abstract [show]
We describe a simple hybridization assay performed in microtitration wells with use of DNA probes labeled with three different lanthanide chelates for detection of seven mutations that cause cystic fibrosis. The assay is based on DNA amplification of four fragments containing the mutations (delta F508, G1717-->A, G542X, R553X, 3905 insertion T, W1282X, and N1303K) by PCR, followed by hybridization with short, allele-specific oligonucleotide probes labeled with europium, terbium, or samarium chelates. Because the technology makes it possible to hybridize three DNA probes simultaneously in one reaction, all 14 mutation-related alleles were detected in a total of five reaction wells. Blood spot specimens, obtained from children with cystic fibrosis, their parents, and their siblings, have been assayed, and for all the probes the positive signal-to-noise ratios are > 10. Solution hybridization utilizing triple-label time-resolved fluorometry combined with PCR is a suitable procedure for large-scale screening and automation.

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20 We utilized triple-label time-resolved fluorometry and short, allele-specific oligonucleotide probes to detect ⌬F508, G1717 3A, G542X, R553X, 3905 insertion T 1 Department of Biotechnology, University of Turku, FIN-20520 Turku, Finland. Login to comment
55 of hybridization wells Detection probe 5؅-3؅ sequence and label5؅-Primer 3؅-Primer ⌬F508 w 1 AAGCACAGTGGAAGAATTTC BioCTCTTCTAGTTGGCATGCT 2 Tb-(modC)20ATCATCTTTGGT m 3 Sm-(modC)20TATCAT∧TGGTGT w 1 BioGAGCATACTAAAAGTGACTC BioCATGAATGACATTTACAGCAA 1 Eu-(modC)20ATGTCCTATTAC G1717 3A m 3 Tb-(modC)20TAATAAGACATCT G542X w 1 BioGAGCATACTAAAAGTGACTC BioCATGAATGACATTTACAGCAA 2 Eu-(modC)20GGTCTTGGAGAA m 1 Tb-(modC)20GGTCTTTGAGAA R553X w 1 BioGAGCATACTAAAAGTGACTC BioCATGAATGACATTTACAGCAA 2 Sm-(modC)20GTCAACGAGCAA m 3 Eu-(modC)20TTGCTCATTGAC 3905insT w 2 BioCCTTATAGGTGGGCCTCT BioGCTAAGTCCTTTTGCTCAC 4 Tb-(modC)20AGCTTTTTT*GAG m 5 Sm-(modC)20CTCAAAAAAAGC W1282X w 2 BioCCTTATAGGTGGGCCTCT BioGCTAAGTCCTTTTGCTCAC 4 Eu-(modC)20TTCCTCCACTGT m 5 Eu-(modC)20CAGTGAAGGAAA N1303K w 2 AAGTATTTATTTTTTCTGGAACA BioTTCTTGATCACTCCACTGTT 4 Sm-(modC)20AAAACTTGGATC m 5 Tb-(modC)20AAAAAGTTGGAT Sequences, labels, and design of hybridization wells for wild-type specific (w) and mutant specific (m) detection probes for seven CFTR mutations. Login to comment
85 and type of label Optimal hybridization temperature,b,d °C Optimal probe conc.,b ng/well Hybridization sensitivity (target molecules/well)c,d Cross-reactivity,b,d % Maximal hybridization efficiency,b % ⌬F508 w 19Tb 31 2 4.1 ϫ 107 0.0 6.5 m 19Sm 24 5 1.2 ϫ 108 1.0 13.5 G1717 3A w 18Eu 17 5 5.9 ϫ 107 0.2 10.5 m 19Tb Ͻ10 5 5.6 ϫ 107 0.1 8.0 G542X w 18Eu 18-27 5 6.7 ϫ 107 0.4 5.5 m 19Tb 20 10 8.1 ϫ 107 0.5 2.5 R553X w 9Sm 32 2 3.2 ϫ 108 1.0 8.5 m 16Eu 22 1 2.2 ϫ 107 0.2 14.5 3905insT w 18Tb 25 5 1.8 ϫ 107 2.9 10.5 m 19Sm 22 2 1.8 ϫ 107 6.5 38.0 W1282X w 19Eu 22 1 3.2 ϫ 107 0.4 14.0 m 19Eu 20 2 1.7 ϫ 107 0.5 21.0 N1303K w 9Sm 31 10 6.4 ϫ 107 0.6 8.0 m 13Tb 27 2 3.1 ϫ 107 0.0 11.0 a For sequences, see Table 1. b Determined using 1 ϫ 1011 target molecules/well. Login to comment
88 probes specific for ⌬F508, G1717 3A, G542X, and R553X in wells 1-3, and the fragments amplified in PCR2 with probes specific for 3905insT, W1282X, and N1303K in wells 4 and 5 (Fig. 1). Login to comment
97 The mutations of interest were ⌬F508 in exon 10, G1717 3A, G542X, and R553X in exon 11, 3905insT and W1282X in exon 20, and N1303K in exon 21. Login to comment
110 The hybridization reactions were distributed as follows: wild-type G1717 3A probe and mutant G542X probe were hybridized in well 1, wild-type probes for ⌬F508, G542X, and R553X in well 2, and mutant probes for ⌬F508, G1717 3A, and R553X in well 3. Login to comment
132 The maximal efficiency varied from probe to probe between 2.5% (the mutant G542X probe) and 38% (the mutant 3905insT probe) (Table 2). Login to comment
148 A typical calibration curve for the wild-type G542X probe is shown in Fig. 4. Login to comment
155 Fig. 4 illustrates the cross-reactivity of the wild-type G542X probe with different target concentrations. Login to comment
162 Calibration curve and cross-reaction of wild-type G542X probe. Login to comment
163 Hybridization signals of wild-type G542X probe hybridized with different concentrations of wild-type specific synthetic target (F) and cross-reaction signals with mutant specific synthetic target (ࡗ) (background subtracted). Login to comment
167 Signal-to-noise ratio for wild/mutant ⌬F508 G1717 3A G542X R553X 3905insT W1282X N1303K w m w m w m w m w m w m w m Diagnosis Purified DNA samples 293 1.2 255 1.1 65 1.4 64 2.3 82 1.2 20 1.0 23 1.2 Normal 120 77 218 1.2 57 1.5 57 2.2 76 1.3 24 1.4 18 1.4 ⌬F508/unknown 2.2 166 236 1.1 73 2.7 62 3.0 78 1.4 25 1.6 15 2.0 ⌬F508/⌬F508 141 91 132 17 79 1.5 69 3.0 79 1.4 22 1.7 19 1.6 ⌬F508/G1717 3A 286 1.4 2.5 31 61 2.4 59 2.5 81 1.3 23 1.4 25 1.7 G1717 3A/G1717 3A 280 1.4 61 14 18 13 29 2.8 82 1.2 25 1.0 16 1.5 G1717 3A/G542X 220 1.8 265 1.5 73 1.6 40 36 78 1.3 19 1.4 13 2.0 ⌬F508/R553X 284 2.0 176 1.7 75 4.0 3.0 60 87 1.4 21 1.7 20 1.8 R553X/R553X 264 1.8 226 1.7 76 3.5 37 32 44 17 23 1.1 20 1.5 R553X/3905insT 104 67 214 2.3 46 2.3 48 3.5 44 16 22 1.6 15 1.9 ⌬F508/3905insT 154 97 268 1.5 68 1.5 62 3.2 74 1.8 11 33 21 0.9 ⌬F508/W1282X 310 1.2 294 1.1 83 2.5 73 2.4 78 1.3 18 1.5 6.4 40 N1303K/N1303K Blood spot samples 250 1.2 265 0.9 75 0.9 73 1.3 69 1.3 31 2.2 12 2.7 Normal 69 40 213 1.1 63 1.1 54 1.9 58 1.2 17 55 18 2.4 ⌬F508/W1282X 1.5 138 256 1.2 62 1.1 74 3.7 76 1.5 32 2.1 11 2.7 ⌬F508/⌬F508 96 48 304 1.0 96 12 76 2.2 82 1.5 31 2.9 13 3.2 ⌬F508/G542X Boldface numbers indicate signal-to-noise ratios considered positive. Login to comment
201 Results of DNA samples analyzed for seven CFTR mutations: 16 amplified samples, each plotted for seven mutations, ⌬F508 (ࡗ), G1717 3A (छ), G542X (f), R553X (Ⅺ), 3905insT (F), W1282X (E), and N1303K (‫. Login to comment

[hide] CFTR gene transfer reduces the binding of Pseudomo... Am J Respir Cell Mol Biol. 1997 Jun;16(6):657-63. Davies JC, Stern M, Dewar A, Caplen NJ, Munkonge FM, Pitt T, Sorgi F, Huang L, Bush A, Geddes DM, Alton EW
CFTR gene transfer reduces the binding of Pseudomonas aeruginosa to cystic fibrosis respiratory epithelium.
Am J Respir Cell Mol Biol. 1997 Jun;16(6):657-63., [PMID:9191467]

Abstract [show]
Much of the morbidity and mortality seen in cystic fibrosis (CF) is related to chronic infection of the respiratory tract with Pseudomonas aeruginosa. Some studies have attributed the strong relationship between CF and Pseudomonas colonization to the presence of increased numbers of specific cell-surface receptors, although other work suggests that this relates to the presence of mucus. Several groups are now assessing the use of gene transfer as a novel form of treatment for CF. We have examined whether P. aeruginosa binding to freshly obtained CF respiratory epithelial cells is increased, and have studied the effects of transfer of the CF transmembrane conductance regulator (CFTR) gene on this attachment. Binding of P. aeruginosa to noncultured nasal epithelial cells from both CF patients (n = 31) and healthy controls (n = 15) was studied with scanning electron microscopy. Binding was also assessed for CF cells following transfection with CFTR/liposome complexes. Epifluorescence microscopy was used to assess the effects of gene transfer on chloride fluxes. Adherence of P. aeruginosa directly to the cell surface of CF airway epithelium was significantly (P < 0.001) increased over that in non-CF controls. Liposome-mediated CFTR gene transfer resulted in a significant (P < 0.01) reduction in the numbers of bacteria bound to ciliated epithelial cells. Fluorescence microscopy confirmed correction of the basic chloride defect. Thus, in CF, the absence of normal CFTR results in increased binding of P. aeruginosa to respiratory epithelial cells. This abnormality can be corrected in vitro by restoration of CFTR function. This has important implications both for the pathogenesis of CF and for the future application and assessment of gene therapy for this disease.

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95 Twelve patients were homozygous for the dF508 mutation, six were compound heterozygotes with two identified mutations (d/G542X X 3, d/R347P, d/R117H, d/1717), 11 were heterozygous for dF508 with an unknown mutation, and in 3, the genotype was unknown. Login to comment

[hide] A cystic fibrosis transmembrane conductance regula... Am J Respir Crit Care Med. 1997 Jun;155(6):1914-20. Kerem E, Rave-Harel N, Augarten A, Madgar I, Nissim-Rafinia M, Yahav Y, Goshen R, Bentur L, Rivlin J, Aviram M, Genem A, Chiba-Falek O, Kraemer MR, Simon A, Branski D, Kerem B
A cystic fibrosis transmembrane conductance regulator splice variant with partial penetrance associated with variable cystic fibrosis presentations.
Am J Respir Crit Care Med. 1997 Jun;155(6):1914-20., [PMID:9196095]

Abstract [show]
Some patients express various features of cystic fibrosis (CF) even though essential characteristics of the disease might be absent. Such patients may suffer from respiratory disease without pancreatic insufficiency and normal sweat chloride levels. Others may present as male infertility because of congenital bilateral aplasia of the vas deferens (CBAVD) with no other signs of CF. The 5T allele, a DNA variant in a noncoding region of the cystic fibrosis transmembrane conductance regulator (CFTR) gene that reduces the level of the normal CFTR transcripts, was found in increased frequency among male patients with CBAVD. The purpose of this study was to investigate the possibility that the 5T allele is associated with dysfunction of organs other than the male reproductive system, leading to CF or atypical CF. Analysis of the 5T allele was performed on 148 subjects (29 with CF, 61 with atypical CF, and 58 with CBAVD) carrying 232 chromosomes with unidentified CFTR mutations, and on 142 non-CF chromosomes from healthy subjects of Ashkenazi origin. The frequency of the 5T allele among chromosomes from patients of Jewish Ashkenazi origin with CF and atypical CF (six of 33; 18%) was significantly higher than the frequency in the normal Ashkenazi population (eight of 142; 6%; p = 0.03). Analysis of the clinical presentation of the five patients with CF and the 12 patients with atypical CF carrying the 5T allele indicated that most patients suffered from respiratory disease presenting as asthma like symptoms, nasal polyposis, chronic sinusitis, chronic bronchitis, or bronchiectasis. Six patients had pancreatic insufficiency, two with meconium ileus. Sweat Cl- levels ranged from normal to elevated. Of the six male patients with respiratory disease who were old enough to be evaluated for fertility status, five were fertile and one had pancreatic insufficiency. Among male patients with CBAVD, 41% suffered from respiratory symptoms. Thus, the 5T allele is a variant with partial penetrance causing disease with an extreme variability of clinical presentation: from normal healthy fertile subjects or male patients with CBAVD to those with atypical or typical clinical phenotype of CF.

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70 The frequency of the 5T allele among normal chromosomes was significantly lower than its frequency among chromosomes carried by CF patients and patients with atypical TABLE 1 MUTATIONS AND POLYTHYMIDINE VARIANTS ON THE OTHER CHROMOSOME OF UNRELATED PATIENTS WITH 5T ALLELE Mutation CF and Atypical CF CBAVD Total AF508 4 8 12 W1282X 1 6 7 N1303K 0 2 2 G85E 1 1 2 D1152H 1 0 1 W 1089X 1 0 1 G542X 0 1 1 ST 0 3 3 7T' 7 9 16 9T* 2 2 4 Total 17 32 49 Definition of abbreviations: CF = cystic fibrosis; CBAVD = congenital bilateral aplasia of the vas deferens. Login to comment

[hide] Newborn screening for cystic fibrosis in Wisconsin... Pediatrics. 1997 Jun;99(6):819-24. Gregg RG, Simantel A, Farrell PM, Koscik R, Kosorok MR, Laxova A, Laessig R, Hoffman G, Hassemer D, Mischler EH, Splaingard M
Newborn screening for cystic fibrosis in Wisconsin: comparison of biochemical and molecular methods.
Pediatrics. 1997 Jun;99(6):819-24., [PMID:9164776]

Abstract [show]
OBJECTIVES: To evaluate neonatal screening for cystic fibrosis (CF), including study of the screening procedures and characteristics of false-positive infants, over the past 10 years in Wisconsin. An important objective evolving from the original design has been to compare use of a single-tier immunoreactive trypsinogen (IRT) screening method with that of a two-tier method using IRT and analyses of samples for the most common cystic fibrosis transmembrane regulator (CFTR) (DeltaF508) mutation. We also examined the benefit of including up to 10 additional CFTR mutations in the screening protocol. METHODS: From 1985 to 1994, using either the IRT or IRT/DNA protocol, 220 862 and 104 308 neonates, respectively, were screened for CF. For the IRT protocol, neonates with an IRT >/=180 ng/mL were considered positive, and the standard sweat chloride test was administered to determine CF status. For the IRT/DNA protocol, samples from the original dried-blood specimen on the Guthrie card of neonates with an IRT >/=110 ng/mL were tested for the presence of the DeltaF508 CFTR allele, and if the DNA test revealed one or two DeltaF508 alleles, a sweat test was obtained. RESULTS: Both screening procedures had very high specificity. The sensitivity tended to be higher with the IRT/DNA protocol, but the differences were not statistically significant. The positive predictive value of the IRT/DNA screening protocol was 15.2% compared with 6.4% if the same samples had been screened by the IRT method. Assessment of the false-positive IRT/DNA population revealed that the two-tier method eliminates the disproportionate number of infants with low Apgar scores and also the high prevalence of African-Americans identified previously in our study of newborns with high IRT levels. We found that 55% of DNA-positive CF infants were homozygous for DeltaF508 and 40% had one DeltaF508 allele. Adding analyses for 10 more CFTR mutations has only a small effect on the sensitivity but is likely to add significantly to the cost of screening. CONCLUSIONS: Advantages of the IRT/DNA protocol over IRT analysis include improved positive predictive value, reduction of false-positive infants, and more rapid diagnosis with elimination of recall specimens.

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52 Polyacrylamide gel electrophoresis of PCR-amplified DNA served to identify the ⌬F508 mutation.14 Mutations S549N, R553X, and G551D were screened by PCR amplification of exon 11, followed by restriction enzyme digests that are diagnostic of each mutation. Login to comment
57 Mutations, G542X, W1282X, R117H, R553X, N1303K, 1717-1G3A, R560T, and 621ϩ1G3T were analyzed by the ARMS procedure using published primers and conditions.18 A total of 360 patients were studied by multimutation analysis (80% of the Wisconsin CF population). Login to comment
123 Tested) Frequency (%) Theoretical Cumulative Detection† (%) Patients Missed in One Year‡ ⌬F508§ 513/720 71.25 91.74 1.32 G542X 17/162 3.02 93.38 1.05 W1282X 7/102 1.97 94.36 0.90 R117H 6/101 1.71 95.14 0.77 R553X 11/197 1.61 95.82 0.66 G551D 9/195 1.33 96.35 0.58 N1303K 3/100 0.86 96.67 0.53 1717 - 1G 3 A 2/99 0.58 96.88 0.49 R560T 2/106 0.54 97.06 0.47 621 ϩ 1G 3 T 3/163 0.53 97.25 0.44 S549N 0/196 0.0 97.25 0.44 * Chromosomes were analyzed on blood or cheek cell specimens obtained from 360 patients (80% of the total Wisconsin CF population), all of whom had a positive sweat test. Login to comment
140 They report results of adding DNA testing for the ⌬F508 mutation, and Ranieri et al15 also investigated adding three other CFTR mutations (G542X, R553X, and G551D) to the screen. Login to comment
152 DNA Analysis of Genotyped CF Patients in the US* n Percent ⌬F508 12701 67.7 G542X 403 2.2 G551D 357 1.9 W1282X 240 1.3 N1303K 223 1.2 R553X 157 0.8 3849 ϩ 10kbC 3 T 102 0.5 621 ϩ 1G 3 T 147 0.8 1717 - 1G 3 A 101 0.5 R117H 101 0.5 R334W 36 0.2 ⌬I507 42 0.2 R347P 37 0.2 R560T 23 0.1 R1162X 44 0.2 2789 ϩ 5G 3 A 25 0.1 A455E 16 0.1 3120 ϩ IG 3 A 14 0.0 S549N 12 0.0 711 ϩ IG 3 T 9 0.0 Other 178 0.9 Unidentified 3814 20.3 Total 18782 99.7† Patient Genotypes Allele 1/Allele 2 n % of Genotype ⌬F508/⌬F508 4573 48.7 ⌬F508/Known 1511 16.1 ⌬F508/Unknown 2044 21.8 Known/unknown 310 3.3 Known/known 223 2.4 Unknown/unknown 730 7.8 Total 9391 100.0 *Data from Cystic Fibrosis Registry, 1995; Annual Report. Login to comment
118 Tested) Frequency (%) Theoretical Cumulative Detectionߤ (%) Patients Missed in One Yearߥ DF508&#a7; 513/720 71.25 91.74 1.32 G542X 17/162 3.02 93.38 1.05 W1282X 7/102 1.97 94.36 0.90 R117H 6/101 1.71 95.14 0.77 R553X 11/197 1.61 95.82 0.66 G551D 9/195 1.33 96.35 0.58 N1303K 3/100 0.86 96.67 0.53 1717 2 1G 3 A 2/99 0.58 96.88 0.49 R560T 2/106 0.54 97.06 0.47 621 1 1G 3 T 3/163 0.53 97.25 0.44 S549N 0/196 0.0 97.25 0.44 * Chromosomes were analyzed on blood or cheek cell specimens obtained from 360 patients (80% of the total Wisconsin CF population), all of whom had a positive sweat test. Login to comment
135 They report results of adding DNA testing for the DF508 mutation, and Ranieri et al15 also investigated adding three other CFTR mutations (G542X, R553X, and G551D) to the screen. Login to comment
147 DNA Analysis of Genotyped CF Patients in the US* n Percent DF508 12701 67.7 G542X 403 2.2 G551D 357 1.9 W1282X 240 1.3 N1303K 223 1.2 R553X 157 0.8 3849 1 10kbC 3 T 102 0.5 621 1 1G 3 T 147 0.8 1717 2 1G 3 A 101 0.5 R117H 101 0.5 R334W 36 0.2 DI507 42 0.2 R347P 37 0.2 R560T 23 0.1 R1162X 44 0.2 2789 1 5G 3 A 25 0.1 A455E 16 0.1 3120 1 IG 3 A 14 0.0 S549N 12 0.0 711 1 IG 3 T 9 0.0 Other 178 0.9 Unidentified 3814 20.3 Total 18782 99.7ߤ Patient Genotypes Allele 1/Allele 2 n % of Genotype DF508/DF508 4573 48.7 DF508/Known 1511 16.1 DF508/Unknown 2044 21.8 Known/unknown 310 3.3 Known/known 223 2.4 Unknown/unknown 730 7.8 Total 9391 100.0 *Data from Cystic Fibrosis Registry, 1995; Annual Report. Login to comment

[hide] Identification of common cystic fibrosis mutations... Am J Hum Genet. 1997 May;60(5):1122-7. Macek M Jr, Mackova A, Hamosh A, Hilman BC, Selden RF, Lucotte G, Friedman KJ, Knowles MR, Rosenstein BJ, Cutting GR
Identification of common cystic fibrosis mutations in African-Americans with cystic fibrosis increases the detection rate to 75%.
Am J Hum Genet. 1997 May;60(5):1122-7., [PMID:9150159]

Abstract [show]
Cystic fibrosis (CF)--an autosomal recessive disorder caused by mutations in CF transmembrane conductance regulator (CFTR) and characterized by abnormal chloride conduction across epithelial membranes, leading to chronic lung and exocrine pancreatic disease--is less common in African-Americans than in Caucasians. No large-scale studies of mutation identification and screening in African-American CF patients have been reported, to date. In this study, the entire coding and flanking intronic sequence of the CFTR gene was analyzed by denaturing gradient-gel electrophoresis and sequencing in an index group of 82 African-American CF chromosomes to identify mutations. One novel mutation, 3120+1G-->A, occurred with a frequency of 12.3% and was also detected in a native African patient. To establish frequencies, an additional group of 66 African-American CF chromosomes were screened for mutations identified in two or more African-American patients. Screening for 16 "common Caucasian" mutations identified 52% of CF alleles in African-Americans, while screening for 8 "common African" mutations accounted for an additional 23%. The combined detection rate of 75% was comparable to the sensitivity of mutation analysis in Caucasian CF patients. These results indicate that African-Americans have their own set of "common" CF mutations that originate from the native African population. Inclusion of these "common" mutations substantially improves CF mutation detection rates in African-Americans.

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39 Mutation Analysis All patients were screened for the AF508 mutation and 15 common Caucasian CF mutations using a reverse dot strip hybridization system (Kawasaki et al. 1993) (R117H, 621+1G--T, R334W, R347P, A455E, A1507, 1717-1G-+A, G542X, S549N, GSS1D, R553X, R560T, 3849+10kbC-+T, W1282X, and N1303K) (Welsh et al. 1995) and a deep intron 11 splice-site mutation, 1811+1.6kbA-+G (Chillon et al. 1995). Login to comment
70 Finally, 13 mutations found in one patient each had been previously reported in Caucasian patients (Q98R, R352Q, V520F, 1812-1G--A, G542X, S549N, and Y913C) (Romey et al. 1995; Welsh et al. 1995) or in African-American patients (444delA, G480C, 1342-2delAG [originally reported as 1342-1G--+C], 2307insA, 3662delA, and W1316X) (Cutting et al. 1990b; White et al. 1991; Zielenski et al. Login to comment
86 The most common muta- Table 2 Distribution of CF Mutations in African-American and U.S.-Caucasian CF Patients African-American U.S. Caucasiana Mutation (n= 148) % (n = 8,714) % Caucasian mutations: AF508 71 48 5,769 66.2 R117H 0 0 47 .5 621+1 G--T 0 0 68 .8 R334W 1 .7 7 .1 R347P 0 0 24 .3 A455E 0 0 5 .1 AI507 1 .7 10 .1 1717-1 G-IA 1 .7 39 .5 G542X 1 .7 204 2.3 S549N 1 .7 4 .1 GS51D 1 .7 173 2.0 R553X (Caucasian)b 0 0 87 1.0 R560T 0 0 16 .2 3849+10kb C-T 0 0 51 .6 W1282X 0 0 235 2.7 N1303K 0 0 116 1.3 Subtotal 77 52 6,855 78.7 African-American mutations: 405+3 A-C 2 1.4 ... ... 444delA 1 .7 ... ... G480C 2 1.4 ... ... R553X (African)b 3 2.0 ... ... A559T 3 2.0 ... ... 2307insA 3 2.0 ... ... 3120+1 GC-A 18 12.2 ... ... S1255X 2 1.4 ... ... Subtotal 34 23 ... ... Total 111 75.0 6,855 78.7 NOTE.-Percentages are rounded. Login to comment
40 Mutation Analysis All patients were screened for the AF508 mutation and 15 common Caucasian CF mutations using a reverse dot strip hybridization system (Kawasaki et al. 1993) (R117H, 621+1G--T, R334W, R347P, A455E, A1507, 1717-1G-+A, G542X, S549N, GSS1D, R553X, R560T, 3849+10kbC-+T, W1282X, and N1303K) (Welsh et al. 1995) and a deep intron 11 splice-site mutation, 1811+1.6kbA-+G (Chillon et al. 1995). Login to comment
71 Finally, 13 mutations found in one patient each had been previously reported in Caucasian patients (Q98R, R352Q, V520F, 1812-1G--A, G542X, S549N, and Y913C) (Romey et al. 1995; Welsh et al. 1995) or in African-American patients (444delA, G480C, 1342-2delAG [originally reported as 1342-1G--+C], 2307insA, 3662delA, and W1316X) (Cutting et al. 1990b; White et al. 1991; Zielenski et al. Login to comment
87 The most common muta- Table 2 Distribution of CF Mutations in African-American and U.S.-Caucasian CF Patients African-American U.S. Caucasiana Mutation (n= 148) % (n = 8,714) % Caucasian mutations: AF508 71 48 5,769 66.2 R117H 0 0 47 .5 621+1 G--T 0 0 68 .8 R334W 1 .7 7 .1 R347P 0 0 24 .3 A455E 0 0 5 .1 AI507 1 .7 10 .1 1717-1 G-IA 1 .7 39 .5 G542X 1 .7 204 2.3 S549N 1 .7 4 .1 GS51D 1 .7 173 2.0 R553X (Caucasian)b 0 0 87 1.0 R560T 0 0 16 .2 3849+10kb C-T 0 0 51 .6 W1282X 0 0 235 2.7 N1303K 0 0 116 1.3 Subtotal 77 52 6,855 78.7 African-American mutations: 405+3 A-C 2 1.4 ... ... 444delA 1 .7 ... ... G480C 2 1.4 ... ... R553X (African)b 3 2.0 ... ... A559T 3 2.0 ... ... 2307insA 3 2.0 ... ... 3120+1 GC-A 18 12.2 ... ... S1255X 2 1.4 ... ... Subtotal 34 23 ... ... Total 111 75.0 6,855 78.7 NOTE.-Percentages are rounded. Login to comment

[hide] Relationship between airway ion transport and a mi... Am J Respir Crit Care Med. 1997 May;155(5):1684-9. Walker LC, Venglarik CJ, Aubin G, Weatherly MR, McCarty NA, Lesnick B, Ruiz F, Clancy JP, Sorscher EJ
Relationship between airway ion transport and a mild pulmonary disease mutation in CFTR.
Am J Respir Crit Care Med. 1997 May;155(5):1684-9., [PMID:9154877]

Abstract [show]
Patients with cystic fibrosis (CF) display defects in airway ion transport, but the influence of airway transport phenotype on improved prognosis is not known. We studied airway bioelectric properties in five CF patients with the rare A455E mutation that is associated with mild pulmonary disease. We also evaluated five patients possessing premature truncation mutations (G542X and R553X) for which an association with mild pulmonary disease has not been as well established. We found no evidence in vivo that a mild lung disease mutation in the CF transmembrane regulator gene (CFTR) led to correction or partial correction of: (1) unstimulated Cl- secretion; (2) beta-agonist-activated Cl- secretion; (3) basal sodium reabsorption; or (4) amiloride-sensitive airway sodium transport. Early phase therapeutic trials in CF, including human gene transfer trials, rely heavily on improvements in airway potential difference to identify promising interventions and an improved prognosis. Based on our findings in a naturally occurring group of CF patients with an improved pulmonary prognosis (A455E), one can argue that marked clinical benefit might be possible without any improvement whatsoever in airway bioelectric phenotype. Moreover, if genetic modifiers exist that influence the severity of a particular CFTR mutation (e.g., A455E), these may be independent of human airway Cl-secretion in vivo, since we detected minimal Cl--secretory responses in patients with A455E.

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2 We also evaluated five patients possessing premature truncation mutations (G542X and R553X) for which an association with mild pulmonary disease has not been as well established. Login to comment
20 As a control, we measured airway ion transport in patients with the common R553X and G542X truncation mutations. Login to comment
25 The clinical features of the A455E, R553X, and G542X CF patients we studied are given in Table 1. Login to comment
31 Age (yr) Sex Genotype Pseudomonas Colonization % Predicted FVC 96 Predicted FEV, Sweat C1 (meq/L) PS versus PI* 1 121/2 F A455E/621 + 1G-T + 113 106 106 P1 2 111/2 F A455E/621 + 1G-*T - 109 101 97 PI 3 16 M AF508/A455E - 77 63 84 PS 4 16 F AF508/A455E - 123 120 59 PS 5 151/2 F AF508/A455E + 101 94 54 PS 6 21 M G551D/G542X + 56 26 84 PI 7 18 M AF508/G542X + 90 80 80 PI 8 16 M AF508/G542X + 121 110 132 PI 9 16 F R553X/G542X + 68 46 78 PI 10 18 M AF508/G542X + 51 41 100 PI 1685 * PS = pancreatic sufficient; PI = pancreatic insufficient terfly needle filled with a 4% agar/lactated Ringer's solution) was inserted subcutaneously in the dorsal aspect of the distal forearm or hand. Login to comment
59 RESULTS Table 1 summarizes clinical features of the A455E, G542X, and R553X CF patients we studied. Login to comment
67 Decisions regard- -10 26t5 mV -39*9 mVtt -57±5 mVtt -43*9 mVtt Control G542X or R553X A544E AF508 B 50 40 • C A PD 30 • (mV) • 20 • •• 10 ' • • o - t----------------------------- aj J0 mVtt -27±8 mVtt Control G542X or R553X A455E AF508 C -40 -30 1686 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL. 155 1997 A -60 -50 • t 1 ' PD max -40 I (mV) • • • -30 • • s-20 to mV L - - i--- --- - -- -------loses A Amilo,id Amilonde Amiloride Low CI' Low Cl* fenol lomV L l0 Sea --------------^--------- --------t- - --- -- - --------- l0 mV L ------^--- --------Y- -^ t t lOmV L 10Sa. Login to comment
71 Genotypes of the patients shown include B:A455E/621 + 1 G-*T; C:AF508/A455E; and D:R553X/G542X. Login to comment
77 The remainder of patients with the A455E mutation exhibited no response to low Cl- /isoproterenol, as in the tracing in Figure I.C. Figure 1D shows a tracing from a patient homozygous for truncation mutations within CFTR (G542X/R553X). Login to comment
80 Figure 2 shows a summary of the data from patients and healthy controls in the study, and describes the baseline PD during perfusion with lactated Ringer's solution (Figure 2A), -20 APD (mv) • -10 tl • 0•------'------------------s---------------------------------- --- 10 12t7 mV -2:4 rVtt -4t8 mVtt -5)mVtt Control G542X or R553X A455E AF508 Figure 2. Login to comment
93 In the A455E patients, the maximal PD during perfusion with lactated Ringer's solution and the change after amiloride were well within the range anticipated for the general CF population (Figure 2A and B; [6-21]). Login to comment
94 In the patients with CFTR truncations G542X or R553X, the magnitude of the amiloride-sensitive PD (Figure 2B) was less than in A455E or AF508 patients, although these measurements were still highly useful in discriminating all CF patient groups from individuals without CF (p < 0.01). Login to comment
19 As a control, we measured airway ion transport in patients with the common R553X and G542X truncation mutations. Login to comment
24 The clinical features of the A455E, R553X, and G542X CF patients we studied are given in Table 1. Login to comment
30 Age (yr) Sex Genotype Pseudomonas Colonization % Predicted FVC 96 Predicted FEV, Sweat C1 (meq/L) PS versus PI* 1 121/2 F A455E/621 + 1G-T + 113 106 106 P1 2 111/2 F A455E/621 + 1G-*T - 109 101 97 PI 3 16 M AF508/A455E - 77 63 84 PS 4 16 F AF508/A455E - 123 120 59 PS 5 151/2 F AF508/A455E + 101 94 54 PS 6 21 M G551D/G542X + 56 26 84 PI 7 18 M AF508/G542X + 90 80 80 PI 8 16 M AF508/G542X + 121 110 132 PI 9 16 F R553X/G542X + 68 46 78 PI 10 18 M AF508/G542X + 51 41 100 PI 1685 * PS = pancreatic sufficient; PI = pancreatic insufficient terfly needle filled with a 4% agar/lactated Ringer's solution) was inserted subcutaneously in the dorsal aspect of the distal forearm or hand. Login to comment
58 RESULTS Table 1 summarizes clinical features of the A455E, G542X, and R553X CF patients we studied. Login to comment
66 Decisions regard- -10 26t5 mV -39*9 mVtt -57&#b1;5 mVtt -43*9 mVtt Control G542X or R553X A544E AF508 B 50 40 ߦ C A PD 30 ߦ (mV) ߦ 20 ߦ ߦ ߦ 10 ' ߦ ߦ o - t---------------------------- - aj J0 mVtt -27&#b1;8 mVtt Control G542X or R553X A455E AF508 C -40 -30 1686 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL. 155 1997 A -60 -50 ߦ t 1 ' PD max -40 I (mV) ߦ ߦ ߦ -30 ߦ ߦ s -20 to mV L - - i--- --- - -- -------loses A Amilo,id Amilonde Amiloride Low CI' Low Cl* fenol lomV L l0 Sea --------------^--------- --------t- - --- -- - --------- l0 mV L ------^--- --------Y- -^ t t lOmV L 10Sa. Login to comment
70 Genotypes of the patients shown include B:A455E/621 + 1 G-*T; C:AF508/A455E; and D:R553X/G542X. Login to comment
76 The remainder of patients with the A455E mutation exhibited no response to low Cl- /isoproterenol, as in the tracing in Figure I.C. Figure 1D shows a tracing from a patient homozygous for truncation mutations within CFTR (G542X/R553X). Login to comment
79 Figure 2 shows a summary of the data from patients and healthy controls in the study, and describes the baseline PD during perfusion with lactated Ringer's solution (Figure 2A), -20 APD (mv) ߦ -10 tl ߦ 0ߦ------' ------------------s---------------------------------- --- 10 12t7 mV -2:4 rVtt -4t8 mVtt -5)mVtt Control G542X or R553X A455E AF508 Figure 2. Login to comment

[hide] Chromosomal findings in 150 couples referred for g... Hum Reprod. 1997 May;12(5):930-7. Mau UA, Backert IT, Kaiser P, Kiesel L
Chromosomal findings in 150 couples referred for genetic counselling prior to intracytoplasmic sperm injection.
Hum Reprod. 1997 May;12(5):930-7., [PMID:9194642]

Abstract [show]
A total of 150 infertile couples underwent chromosome analysis and genetic counselling before intracytoplasmic sperm injection (ICSI). Chromosomal abnormalities, including low-level sex chromosome mosaicism, were detected in 12% of the men and an unexpectedly high 6% of the women. Chromosomal abnormalities included gonosomal mosaicism in 13 cases, Robertsonian translocations in four males, autosomal reciprocal translocations in five cases, reciprocal translocation involving a sex chromosome in one case, inversions in three cases and a marker chromosome in one male. Chromosomal variants found in 11 women and 13 men were not included in the above percentages. Couples with a chromosomal aberration in one partner received a second counselling. The different aspects of genetic counselling in these couples are discussed. In conclusion, we recommend genetic counselling and chromosomal analysis of men and women prior to ICSI therapy.

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21 Mutation screening included δI507, δF508, in combination with other abnormalities of the semen 1717-1(G→A), G542X, G551D, R553X, W1282X, N1303K, R347P, (Bourrouillou et al., 1992). Login to comment

[hide] Analysis of 16 cystic fibrosis mutations in Mexica... Am J Med Genet. 1997 Apr 14;69(4):380-2. Villalobos-Torres C, Rojas-Martinez A, Villareal-Castellanos E, Cantu JM, Sanchez-Anzaldo FJ, Saiki RK, Barrera-Saldana HA
Analysis of 16 cystic fibrosis mutations in Mexican patients.
Am J Med Genet. 1997 Apr 14;69(4):380-2., [PMID:9098486]

Abstract [show]
We carried out molecular analysis of 80 chromosomes from 40 unrelated Mexican patients with a diagnosis of cystic fibrosis. The study was performed in two PCR steps: a preliminary one to identify mutation delta F508, the most frequent cause of cystic fibrosis worldwide, and the second a reverse dot-blot with allele-specific oligonucleotide probes to detect 15 additional common mutations in the Caucasian population. A frequency of 45% for delta F508 was found, making it the most common in our sample of Mexican patients. Another five mutations (G542X, 3,849 + 10 kb C-->T, N1303K, SN549N, and 621 + 1 G-->T) were detected, and those accounted for 11.25%. The remaining mutations (43.75%) were undetectable with the methodology used.

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14 According to data from the Cystic Fibrosis Genetic Analysis Consortium [1994] (CFGAC), the most frequent non-⌬F508 mutations are the following: G542X (2.4%), G551D (1.6%), N1303K (1.3%), W1282X (1.2%), R553X (0.7%), 621 + 1 G→T (0.7%), 1717 - 1 G→T (0.6%), R117H (0.3%), R1162X (0.3%), G85E (0.2%), R347P (0.2%), ⌬I507 (0.2%), and 3849 + 10 kb C→T (0.2%). Login to comment
44 All patients with two severe mutations suffered PI (⌬F508/G542X, G542X/S549N, and ⌬F508/⌬F508). Login to comment
47 Of the two G542X/unknown heterozygotes, one had PI and the other had pancreatic sufficiency (PS). Login to comment
54 The G542X mutation is second in frequency after ⌬F508, as has been reported worldwide and for Spain, where it has a frequency of 8.0% [Casals et al., 1993]; this is similar to the 4.8% value reported for Southern Europeans, specifically Italians [Nunes et al., 1991]. Login to comment
60 Mutation Frequency Data and Geographic Distribution of the Mutations Found in 80 Chromosomes From Mexican CF Patients Mutation Northeast n ‫ס‬ 54 Central n ‫ס‬ 16 Western n ‫ס‬ 10 Total n ‫ס‬ 80 CFGAC [1994] (%)n (%) n (%) n (%) n (%) ⌬F508 27 (50) 2 (12.5) 7 (70) 36 (45) 66 G542X 2 (3.7) 2 (12.5) 0 4 (5) 2.4 3849 + 10 kb C→T 1 (1.9) 0 1 (10) 2 (2.5) 0.2 N1303K 0 1 (6.25) 0 1 (1.25) 1.3 S549N 0 1 (6.25) 0 1 (1.25) 0.1 621 + 1 G→T 0 0 1 (10) 1 (1.25) 0.7 Othera 24 (44.4) 10 (62.5) 1 (10) 35 (43.7) Detected 30 (55.6) 6 (37.5) 9 (90) 45 (56.3) a Different from W1282X, R117H, R334W, R347P, A455E, ⌬I507, 1717 - 1 G→T, G551D, R553X, and R560T. Login to comment

[hide] CFTR mutations and IVS8-5T variant in newborns wit... J Med Genet. 1997 Apr;34(4):297-301. Castellani C, Bonizzato A, Mastella G
CFTR mutations and IVS8-5T variant in newborns with hypertrypsinaemia and normal sweat test.
J Med Genet. 1997 Apr;34(4):297-301., [PMID:9138152]

Abstract [show]
Neonates positive for immunoreactive trypsinogen assay (IRT) and negative for sweat test have formerly been found to carry the major cystic fibrosis (CF) mutation, delta F508, much more frequently than the general population. Among the 716 IRT positive newborns detected by a three tier (IRT, mutation analysis plus meconium lactase assay, sweat test) CF screening programme in north eastern Italy during the period January 1993 to March 1996, we found 45 carriers, a number significantly higher than the expected 17 (p < 0.001). We speculated that some of these heterozygotes could actually be affected by a very mild form of CF, and carry on the other chromosome an undetected CFTR mutation or a DNA variant, such as the 5-thymidine allele in intron 8 of the CFTR gene (IVS8-5T). This hypothesis was tested in four samples; group A (the 45 carriers mentioned above), group B (51 non-carrier, IRT positive neonates), group C (50 IRT negative neonates), and group D (90 CF adult female carriers). Chromosomes with IVS8-5T were seven (7.78%) in group A, seven (6.86%) in group B, five (5%) in group C, and four in group D (2.22%). The 5T prevalence in group A was significantly higher (p < 0.05) compared to group D; similarly, a higher (p < 0.05) 5T frequency in group A compared to group C was detected by considering the chromosomes free from CFTR mutations. This study is consistent with previous papers in finding among neonates with high trypsin levels a CF carrier frequency significantly higher than that expected. It is also suggested that in at least some babies raised trypsin levels at birth could be a phenotypic expression of compound heterozygosity for a major CF mutation plus IVS8-5T.

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21 Initially we tested for AF508, Rl 162X, and N1303K, estimated by a cohort study7 to cover 61 % of CF chromosomes in our area; from March 1995 10 other mutations were included (2183AAG, 3849+1OKbCT, G542X, 1717-1GA, R553X, Q552X, G85E, 711+5GA, 3132delTG, Cystic Fibrosis Centre, Ospedale Civile Maggiore, Piazzale Stefani, 37126 Verona, Italy C Castellani A Bonizzato G Mastella Correspondence to: Dr Castellani. Login to comment
62 Sweat IRT Meconium chloride PolyT IRT retest Weight Z Subject Sex (ugll) lactase (Ulg) (mEqlkg) CFTR mutation genotype (pgll) score 1 F 134 0 6 AF508 7/9 67 1.47 2 M 95 0 28 AF508 7/9 3 F 408 0 7 2183AA-*G 7/7 14 -0.29 4 M 150 0.72 16 N1303K 7/9 19 -0.47 5 F 106 Unknown 18 R1162X 7/7 -6.38 6 M 131 0 22 N1303K 9/9 27 -0.54 7 M 106 0 21 AF508 7/9 34 0.11 8 F 100 0 15 AF508 5/9 37 -0.01 9 F 105 0 25 AF508 5/9 51 -0.15 10 F 100 0 24 R1162X 7/7 11 M 266 0 14 AF508 7/9 5 0.20 12 F 103 0 9 AF508 7/9 13 F 105 0 32 AF508 7/9 14 F 268 0 22 AF508 7/9 30 15 M 110 Unknown 33 R1162X 7/7 16 M 174 0 12 AF508 7/9 52 1.87 17 F 100 0 15 AF508 7/9 18 M 140 0 9 AF508 7/9 19 M 98 0 30 AF508 5/9 20 M 110 1.2 10 AF508 7/9 11 0.26 21 F 102 0 20 G542X 7/9 45 0.77 22 F 111 0 16 N1303K 7/9 23 F 100 0 16 AF508 7/9 54 -0.44 24 F 95 0 18 R553X 7/9 25 F 285 0 16 AF508 7/9 20 0.28 26 M 117 0 23 AF508 7/9 101 27 M 115 0 24 AF508 5/9 12 -0.4 28 F 236 0 8 AF508 7/9 21 0.26 29 M 192 0 19 N1303K 7/9 73 0.04 30 M 103 0 39 AF508 5/9 69 31 M 133 0 10 AF508 7/9 65 1.17 32 M 144 0 30 R1162X 7/7 74 -0.83 33 F 123 0 20 AF508 7/9 66 -0.02 34 F 100 Unknown 30 AF508 5/9 65 35 M 134 0 28 AF508 7/9 36 M 294 0.8 12 R553X 7/7 37 M 102 Unknown 32 2789+5G-9A 7/7 38 F 114 0 36 AF508 7/9 39 M 123 0 9 R1162X 7/7 51 -0.36 40 F 118 0 33 AF508 5/9 16 0.18 41 M 134 0 10 AF508 7/9 15 1.36 42 M 97 0 9 AF508 7/9 43 M 98 1.71 31 AF508 7/9 44 F 122 Unknown 23 AF508 7/9 45 F 101 0 23 AF508 9/9 show mild biological abnormalities as early as the first days of life. Login to comment
61 Sweat IRT Meconium chloride PolyT IRT retest Weight Z Subject Sex (ugll) lactase (Ulg) (mEqlkg) CFTR mutation genotype (pgll) score 1 F 134 0 6 AF508 7/9 67 1.47 2 M 95 0 28 AF508 7/9 3 F 408 0 7 2183AA-*G 7/7 14 -0.29 4 M 150 0.72 16 N1303K 7/9 19 -0.47 5 F 106 Unknown 18 R1162X 7/7 -6.38 6 M 131 0 22 N1303K 9/9 27 -0.54 7 M 106 0 21 AF508 7/9 34 0.11 8 F 100 0 15 AF508 5/9 37 -0.01 9 F 105 0 25 AF508 5/9 51 -0.15 10 F 100 0 24 R1162X 7/7 11 M 266 0 14 AF508 7/9 5 0.20 12 F 103 0 9 AF508 7/9 13 F 105 0 32 AF508 7/9 14 F 268 0 22 AF508 7/9 30 15 M 110 Unknown 33 R1162X 7/7 16 M 174 0 12 AF508 7/9 52 1.87 17 F 100 0 15 AF508 7/9 18 M 140 0 9 AF508 7/9 19 M 98 0 30 AF508 5/9 20 M 110 1.2 10 AF508 7/9 11 0.26 21 F 102 0 20 G542X 7/9 45 0.77 22 F 111 0 16 N1303K 7/9 23 F 100 0 16 AF508 7/9 54 -0.44 24 F 95 0 18 R553X 7/9 25 F 285 0 16 AF508 7/9 20 0.28 26 M 117 0 23 AF508 7/9 101 27 M 115 0 24 AF508 5/9 12 -0.4 28 F 236 0 8 AF508 7/9 21 0.26 29 M 192 0 19 N1303K 7/9 73 0.04 30 M 103 0 39 AF508 5/9 69 31 M 133 0 10 AF508 7/9 65 1.17 32 M 144 0 30 R1162X 7/7 74 -0.83 33 F 123 0 20 AF508 7/9 66 -0.02 34 F 100 Unknown 30 AF508 5/9 65 35 M 134 0 28 AF508 7/9 36 M 294 0.8 12 R553X 7/7 37 M 102 Unknown 32 2789+5G-9A 7/7 38 F 114 0 36 AF508 7/9 39 M 123 0 9 R1162X 7/7 51 -0.36 40 F 118 0 33 AF508 5/9 16 0.18 41 M 134 0 10 AF508 7/9 15 1.36 42 M 97 0 9 AF508 7/9 43 M 98 1.71 31 AF508 7/9 44 F 122 Unknown 23 AF508 7/9 45 F 101 0 23 AF508 9/9 show mild biological abnormalities as early as the first days of life. Login to comment

[hide] Estimating the age of alleles by use of intraallel... Am J Hum Genet. 1997 Feb;60(2):447-58. Slatkin M, Rannala B
Estimating the age of alleles by use of intraallelic variability.
Am J Hum Genet. 1997 Feb;60(2):447-58., [PMID:9012419]

Abstract [show]
A method is presented for estimating the age of an allele by use of its frequency and the extent of variation among different copies. The method uses the joint distribution of the number of copies in a population sample and the coalescence times of the intraallelic gene genealogy conditioned on the number of copies. The linear birth-death process is used to approximate the dynamics of a rare allele in a finite population. A maximum-likelihood estimate of the age of the allele is obtained by Monte Carlo integration over the coalescence times. The method is applied to two alleles at the cystic fibrosis (CFTR) locus, deltaF508 and G542X, for which intraallelic variability at three intronic microsatellite loci has been examined. Our results indicate that G542X is somewhat older than deltaF508. Although absolute estimates depend on the mutation rates at the microsatellite loci, our results support the hypothesis that deltaF508 arose < 500 generations (approximately 10,000 years) ago.

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5 The method is applied to two alleles at the cystic fibrosis (CFTR) locus, AF508 and G542X, for which intraallelic variability at three intronic microsatellite loci has been examined. Login to comment
6 Our results indicate that G542X is somewhat older than AF508. Although absolute estimates depend on the mutation rates at the microsatellite loci, our results support the hypothesis that AF508 arose <500 generations (- 10,000 years) ago. Login to comment
46 Finally, we apply this method to estimate the ages of two alleles of the cystic fibrosis (CF) locus, AF508 and G542X. Login to comment
79 Figure 2c shows 1 as a function of t1 with parameters appropriate for a second allele of cystic fibrosis, G542X. Login to comment
83 Solely on the basis of on the observed frequencies of the two alleles, AF508 then appears to be somewhat older than G542X. Login to comment
84 The estimated population frequency of AF508 is higher than that of G542X, and this conclusion agrees with the general view that more frequent alleles are likely to be older. Login to comment
91 With mutation rate g, the number of segregating sites within the allelic class, S is a Poisson distributed random variable with mean pT, where T is the total length of the intraallelic gene genealogy (see, -8 -8.25 -8.5 -8.75 _9 -9.25 -9.5 -9.75 a 2000 2200 2400 2600 2800 3000 -5 -5.25 -5.5 -5.75 e -6 -6.25 -6.5 -6.75 1200 1400 1600 1800 2000 tl 350 400 450 500 550 600 650 tl Figure 2 Log-likelihood (1) of the observed number of copies of the AF508 and G542X mutations of the CF gene as a function of the time of origin of each mutation (tj) measured in units of generations. Login to comment
96 Panel c shows I as a function of t1 for the G542X allele with 4 = 0.005, N = 3 X 108, and f = 0.00014. Login to comment
98 Panel d shows I as a function of t1 for the G542X allele with 4 = 0.02 and the other parameters as given above. Login to comment
223 We can apply our method to another mutation of CFTR, G542X, a nonsense mutation in exon 11, that has been assessed by Casals et al. Login to comment
229 The frequency of G542X is much smaller than AF508, making up 8% of the CF alleles found in a survey of Spain. Login to comment
230 The frequency of G542X varies with location in Spain (Casals et al. 1993), but for the purposes of our analysis we will assume a frequency of .03 x .08 = .0024 in a random sample of the Spanish population. Login to comment
233 (1993) suggest the geographic distribution supports the introduction of G542X from North Africa between 2,000 and 3,000 years ago, which implies that the appropriate population for analysis may be much larger. Login to comment
236 Figure 4 shows the likelihood curves for four combinations of parameter values for G542X. Login to comment
237 These figures, and others not shown, indicate that G542X is somewhat -70 -75 -80 -85 -90 -95 e e - - --L- -13 -14 -15 -16 -17 e a -14r -16 -18 -20 -22 -24 0 100 200 300 400 b 0 50 100 150 200 50 100 150 200 250 tl tl Figure 4 Log-likelihood (1) of the observed number of copies (i = 62) and segregating sites (S = 5) for the G542X allele of CF. Login to comment
246 older than AF508. Although many fewer copies were examined for the G542X allele, the relative amount of intraallelic variability is slightly larger than that found for the AF508 allele. Login to comment
247 The ratio of estimated ages depends on the values of unknown parameters, but, if we assume the same value of 4 for both alleles, then G542X appears to be -1.5-3 times older than AF508. Login to comment
248 If we assume that the value of 4 for G542X is lower than for AF508, reflecting the possibility that AF508 may have a heterozygote advantage that G542X lacks, then the ratio of the ages would be even greater. Login to comment
250 Because G542X is in much lower frequency than AF508, (8) implies that it is younger. Login to comment
80 Figure 2c shows 1 as a function of t1 with parameters appropriate for a second allele of cystic fibrosis, G542X. Login to comment
85 The estimated population frequency of AF508 is higher than that of G542X, and this conclusion agrees with the general view that more frequent alleles are likely to be older. Login to comment
92 With mutation rate g, the number of segregating sites within the allelic class, S is a Poisson distributed random variable with mean pT, where T is the total length of the intraallelic gene genealogy (see, -8 -8.25 -8.5 -8.75 _9 -9.25 -9.5 -9.75 a 2000 2200 2400 2600 2800 3000 -5 -5.25 -5.5 -5.75 e -6 -6.25 -6.5 -6.75 1200 1400 1600 1800 2000 tl 350 400 450 500 550 600 650 tl Figure 2 Log-likelihood (1) of the observed number of copies of the AF508 and G542X mutations of the CF gene as a function of the time of origin of each mutation (tj) measured in units of generations. Login to comment
97 Panel c shows I as a function of t1 for the G542X allele with 4 = 0.005, N = 3 X 108, and f = 0.00014. Login to comment
99 Panel d shows I as a function of t1 for the G542X allele with 4 = 0.02 and the other parameters as given above. Login to comment
224 We can apply our method to another mutation of CFTR, G542X, a nonsense mutation in exon 11, that has been assessed by Casals et al. Login to comment
231 The frequency of G542X varies with location in Spain (Casals et al. 1993), but for the purposes of our analysis we will assume a frequency of .03 x .08 = .0024 in a random sample of the Spanish population. Login to comment
234 (1993) suggest the geographic distribution supports the introduction of G542X from North Africa between 2,000 and 3,000 years ago, which implies that the appropriate population for analysis may be much larger. Login to comment
238 These figures, and others not shown, indicate that G542X is somewhat -70 -75 -80 -85 -90 -95 e e - - - -L- -13 -14 -15 -16 -17 e a -14r -16 -18 -20 -22 -24 0 100 200 300 400 b 0 50 100 150 200 50 100 150 200 250 tl tl Figure 4 Log-likelihood (1) of the observed number of copies (i = 62) and segregating sites (S = 5) for the G542X allele of CF. Login to comment
249 If we assume that the value of 4 for G542X is lower than for AF508, reflecting the possibility that AF508 may have a heterozygote advantage that G542X lacks, then the ratio of the ages would be even greater. Login to comment
251 Because G542X is in much lower frequency than AF508, (8) implies that it is younger. Login to comment

[hide] Genotype-phenotype relationship in 12 patients car... J Med Genet. 1997 Feb;34(2):89-91. Antinolo G, Borrego S, Gili M, Dapena J, Alfageme I, Reina F
Genotype-phenotype relationship in 12 patients carrying cystic fibrosis mutation R334W.
J Med Genet. 1997 Feb;34(2):89-91., [PMID:9039981]

Abstract [show]
We present a phenotype-genotype correlation analysis in 12 patients with cystic fibrosis (CF) carrying the mutation R334W in the CFTR gene. The clinical data obtained for this group were compared with the clinical data of deltaF508/deltaF508 patients. Current age and age at diagnosis were significantly higher in the R334W mutation group (p=0.028 and p=0.0001). We found a lower rate of Pseudomonas aeruginosa colonisation in patients carrying the R334W mutation, although the difference was not found to be statistically significant. However, we found a statistically significant higher age of onset of Pseudomonas aeruginosa colonisation (p=0.0036) in the group of patients with the R334W mutation. Thirty three percent of R334W patients were pancreatic insufficient, significantly lower than the deltaF508/deltaF508 patients (p=0.004). We also found that the weight expressed as a percentage of ideal weight for height was significantly higher in patients with the R334W mutation (p=0.0028).

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19 Patients were diagnosed as either pancreatic sufficient group.bmj.comon October 25, 2012 - Published byjmg.bmj.comDownloaded from Antinolo, Borrego, Gili, Dapena, Alfageme, Reina Table 1 Clinical characteristics of 12 patients with R334Wmutation Patients 1 2 3 4 5 6 7 8 9 10 11 12 Genotype Sex Current age Age at diagnosis Age at first clinical symptoms First clinical symptoms Sweat ClF mEq/l Weight* (%) Chrispin-Norman Schwachman-Kulcycki FEV1 % predicted FVC % predicted Lung colonisation (LC) Age of onset of LC Meconium ileus Dehydration Pancreatic insufficiency (PI) Age of onset ofPI Sterility R334W/ AF508 F 3 y 7 mth 5 mth 3 mth Pulmonary/ pancreatic 90 100 4 90 No R334W/ AF508 M 27 y R334W/ AF508 F 2 y 5 mth 15y 5mth I y 4 mth 5 mth R334W/ AF508 M R334W/ AF508 F R334W/ AF508 M R334W/ R334W/ G542X R1 162X F F 25y 17y 59y 1Oy9 23y mth 20y 2y 49y 5y6mth 15y 4y 3mth 35y 8y I y R334W/ del84 F 3 y 3 mth R334W/ R334W/ R334W/ G542X G542X G542X F F F 27 y 25y 23y 15mth 25y 24y 21y 4mth 5y 17y Pulmonary Dehydration Dehydration Pulmonary Pancreatic Pulmonary Dehydration Dehydration Pulmonary Pulmonary Asymptomatic 90 80 100 105 110 110 100 85 100 100 100 100 100 115 90 89 95 76 89 116 115 121 20 2 23 65 100 95 65 70 95 50 95 85 90 95 24 82 41 44 27 74 85 91 43 91 51 60 43 82 84 97 Yes No No Yes Yes No Yes No No No No - 15y No No Yes Yes Yes Yes 3 mth - - 17y 58y - 14y - - - - No No Yes No No No No No No No Yes Yes Yes No No Yes Yes No No No No No No Yes No No No No Yes No 26y - - - - Yes - Yes - Yes - - - 24y - - - No No No * Weight expressed as a percentage of ideal weight for height. Login to comment
24 Six patients were compound heterozygotes for R334W and AF508, four for R334W and G542X, one for R334W and 1949del84, and one for R334W and R1162X. Login to comment

[hide] Cystic fibrosis mutation frequencies in upstate Ne... Hum Mutat. 1997;10(6):436-42. Shrimpton AE, Borowitz D, Swender P
Cystic fibrosis mutation frequencies in upstate New York.
Hum Mutat. 1997;10(6):436-42., [PMID:9401006]

Abstract [show]
Upstate New York patients (100) with cystic fibrosis (i.e., 200 CF chromosomes), 72 from the CF center in Syracuse and 28 from a Buffalo CF center, were analyzed for their CF-causing mutations using restriction enzyme digest, single-strand conformation analysis (SSCA), and Heteroduplex (HA) analysis. Polymerase chain reaction (PCR) amplified products from all 27 CFTR exons using primers that included flanking intron junction sequence were investigated. More than 120 known cystic fibrosis transmembrane conductance regulator (CFTR) disease-causing mutations were screened. Four novel CFTR disease-causing mutations were identified (N287Y in exon 6b, 1259insA in exon 8, R1070P in exon 17b, and CF?20kbdel14b-18). A detection rate of 96% of the combined Syracuse and Buffalo population CF chromosomes was obtained.

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84 % Comment 3 G85E 1 1 0.5 4 R117H 1 1 0.5 i4 621 + 1,G>T 1 2 3 1.5 5 711 + 1,G>T 1 1 0.5 6b N287Y 1 1 0.5 Novel 7 1154insTC 2 2 1.0 8 1259insA 1 1 0.5 Novel 9 A455E 1 1 0.5 10 Delta F508 109 39 148 74.0 10 1609delCA 1 1 0.5 Spanish i10 1717-1,G>A 3 3 1.5 11 G542X 2 1 3 1.5 11 G551D 3 3 1.5 11 R553X 4 4 2.0 i12 1898+1,G>A 2 2 1.0 13 2143delT 1 1 0.5 13 2184delA+G>A 1 1 0.5 i14 2789+5,G>A 2 2 1.0 17b R1070P 1 1 0.5 Novel 17b Y1092X(C>A) 2 2 1.0 French Canadian (Rozen et al., 1992) 17b CF?20kbdel 14b-18 1 1 0.5 Novel (Shrimpton and Borowitz, 1997) i19 3849+10kb,C>T 1 1 0.5 20 W1282X 2 2 0.5 Ashkenazi 21 N1303K 3 3 6 3.0 Unknown 4/144 4/56 8/200 4.0 AL. 75 and 81 mMol/L. Login to comment

[hide] Missense mutation R1066C in the second transmembra... Hum Mutat. 1997;10(5):387-92. Casals T, Pacheco P, Barreto C, Gimenez J, Ramos MD, Pereira S, Pinheiro JA, Cobos N, Curvelo A, Vazquez C, Rocha H, Seculi JL, Perez E, Dapena J, Carrilho E, Duarte A, Palacio AM, Nunes V, Lavinha J, Estivill X
Missense mutation R1066C in the second transmembrane domain of CFTR causes a severe cystic fibrosis phenotype: study of 19 heterozygous and 2 homozygous patients.
Hum Mutat. 1997;10(5):387-92., [PMID:9375855]

Abstract [show]
We report the clinical features of 21 unrelated cystic fibrosis (CF) patients from Portugal and Spain, who carry the mutation R1066C in the CFTR gene. The current age of the patients was higher in the R1066C/any mutation group (P < 0.01), as compared to the deltaF508/deltaF508 group. Poor values for lung radiological involvement (Chrispin-Norman) and general status (Shwachman-Kulcycki) were observed in the R1066C/any mutation group (P < 0.005 and P < 0.0004). A slightly, but not significantly worse lung function was found in the R1066C/any mutation group when compared with the deltaF508/deltaF508 patients. No significant differences were detected regarding the age at diagnosis, sweat Cl-values, or percentiles of height and weight between the two groups. Neither were significant differences observed regarding sex, meconium ileus (4.7% vs. 11.1%), dehydration (10.5% vs. 14.7%), or pancreatic insufficiency (PI) (100% vs. 97.8%). The proportion of patients with lung colonization by bacterial pathogens was slightly, but not significantly higher in the R1066C/any mutation group (70.0%), as compared with the deltaF508/deltaF508 group (57.5%). Other clinical complications were significantly more frequent in the R1066C/any mutation patients(P < 0.02) than in the deltaF508/deltaF508 group. The two homozygous R1066C/R1066C patients died at the ages of 3 months and 7 years. The data presented in this study clearly demonstrate that the R1066C mutation is responsible for a severe phenotype similar to that observed in homozygous deltaF508 patients. The poor clinical scores and complications of patients with the R1066C mutation are probably related to their slightly longer survival.

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46 Nine patients were heterozygous for five different mutations: R334W, G542X (1 sib deceased), 712-1G→T (1 sib deceased), 711+1G→T (2 sibs deceased), and 3905insT (only one patient for each one of these five mutations is included in Table 1). Login to comment
59 of patients 21 82 Sex: female/male 10/11 36/46 Mean ± SD (no. studied) Mean ± SD (no. studied) Current age-year 11.05 ± 6.93 (21) P < 0.01 7.72 ± 5.26 (82) Age at diagnosis-year 2.57 ± 4.63 (21) 2.22 ± 2.91 (82) Sweat Cl-mEq/l 112.44 ± 28.80 (18) 104.40 ± 15.70 (80) %ile-height 32.19 ± 35.03 (18) 30.70 ± 26.04 (80) %ile-weight 33.68 ± 35.95 (19) 27.99 ± 24.19 (80) Chrispin-Norman 12.50 ± 8.07 (12) P < 0.005 6.60 ± 6.26 (63) Shwachman-Kulcycki 69.46 ± 17.88 (15) P < 0.0004 83.11 ± 11.79 (72) FEV1-% predicted 64.06 ± 22.81 (15) 74.80 ± 23.06 (41) FVC-% predicted 72.20 ± 20.26 (15) 82.31 ± 20.03 (41) No. positive/no. studied (%) No. positive/no. studied (%) Lung colonization with 14/20 (70.0) 46/80 (57.5) bacterial pathogens Meconium ileus 1/21 (4.7) 9/81 (11.1) Dehydration 2/19 (10.5) 10/68 (14.7) Pancreatic insufficiency 21/21 (100.0) 79/81 (97.5) Other clinical features 13/21 (61.9) P < 0.02 29/82 (35.4) a Mutations were: ∆F508 (14 cases), R1066C (2), R334W (1), G542X (1), 712-1G>T (1), 711+1G>T (1), and 3905insT (1); FEV1, forced expiratory volume in 1 sec; FVC, forced vital capacity. Login to comment
73 In all the patients heterozygous for mutation R1066C, the second CF mutation can be considered as severe (∆F508, G542X, 712-1G→T, 711+1G→T, 3905insT, and R334W). Login to comment
93 Brancolini et al. (1995) found three heterozygous patients: R1066H/1717-1G→A, R1066H/ G542X and R1066C/∆F508 all PS. Login to comment

[hide] Novel mutation (A141D) in exon 4 of the CFTR gene ... Hum Mutat. 1997;10(1):86-7. Gouya L, Pascaud O, Munck A, Elion J, Denamur E
Novel mutation (A141D) in exon 4 of the CFTR gene identified in an Algerian patient.
Hum Mutat. 1997;10(1):86-7., [PMID:9222768]

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3 More than 500 mutations have now been identified, but only five mutations have a frequency > 1% worldwide (i.e., ∆F508, G542X, G551D, W1282X, and N1303K) (CFGAC, 1994). Login to comment

[hide] Identification of four novel mutations in the cyst... Hum Mutat. 1997;9(4):368-9. Clavel C, Pennaforte F, Pigeon F, Verlingue C, Birembaut P, Ferec C
Identification of four novel mutations in the cystic fibrosis transmembrane conductance regulator gene: E664X, 2113delA, 306delTAGA, and delta M1140.
Hum Mutat. 1997;9(4):368-9., [PMID:9101301]

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9 In this group, the a508 represents 66%, other common mutations, such as aI507, G542X, R553X, G551D), 1717-lGÃA, R1162X, W1282X, and N1303K, were screened by restriction enzyme assay and account for 11%. Login to comment

[hide] Sensitivity of the denaturing gradient gel electro... Hum Mutat. 1997;9(2):136-47. Macek M Jr, Mercier B, Mackova A, Miller PW, Hamosh A, Ferec C, Cutting GR
Sensitivity of the denaturing gradient gel electrophoresis technique in detection of known mutations and novel Asian mutations in the CFTR gene.
Hum Mutat. 1997;9(2):136-47., [PMID:9067754]

Abstract [show]
More than 500 mutations have been identified in the CFTR gene, making it an excellent system for testing mutation scanning techniques. To assess the sensitivity of denaturing gradient gel electrophoresis (DGGE), we collected a representative group of 202 CFTR mutations. All mutations analyzed were detected by scanning methods other than the DGGE approach evaluated in this study. DGGE analysis was performed on 24 of the 27 exons and their flanking splice site sequences. After optimization, 201 of the 202 control samples produced an altered migration pattern in the region in which an alteration occurred. The remaining sample was sequenced and found not to have the reported mutation. The ability of DGGE to identify novel mutations was evaluated in three Asian CF patients with four unknown CF alleles. Three novel Asian mutations were detected-K166E, L568X, and 3121-2 A-->G (in homozygosity)-accounting for all CF alleles. These results indicate that an optimized DGGE scanning strategy is highly sensitive and specific and can detect 100% of mutations.

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42 621 + 1 GÃT, R334W, R347P, A455E, aI507, aF508, 1717-1 GÃA, G542X, S549N, G551D, R553X, R560T, 3849 + 10kb CÃT, W1282X, and N1303K) was performed using the rapid multiplex reverse dot hybridization system, under conditions provided by Roche Molecular Systems (Alameda, CA) (Kawasaki et al., 1993; Welsh et al., 1995). Login to comment

[hide] Genotype-phenotype relationships in a cohort of ad... Eur Respir J. 1996 Nov;9(11):2207-14. Hubert D, Bienvenu T, Desmazes-Dufeu N, Fajac I, Lacronique J, Matran R, Kaplan JC, Dusser DJ
Genotype-phenotype relationships in a cohort of adult cystic fibrosis patients.
Eur Respir J. 1996 Nov;9(11):2207-14., [PMID:8947061]

Abstract [show]
In cystic fibrosis (CF), relationships between genotype and phenotype have been shown for pancreatic status but not for pulmonary disease. One hundred and ten adult CF patients were classified according to the expected effect of their mutations on cystic fibrosis transmembrane conductance regulator (CFTR) protein: Group 1 (n=48) included deltaF508 homozygotes; Group 2 (n=26), patients with two "severe" mutations and no expected CFTR production; Group 3 (n=17), patients with expected partly functional CFTR corresponding to at least one "mild" mutation; Group 4 (n=19), patients with no mutation identified or only one identified "severe" mutation. As compared to Groups 1 and 2: patients from Groups 3 and 4 had higher arterial oxygen tension (Pa,O2) (9.5+/-1.9 and 9.9+/-1.5 vs 8.8+/-1.5 and 8.3+/-1.7 kPa, respectively p<0.02); and a slower decline in their pulmonary function, estimated by the mean annual loss in forced vital capacity (FVC) (1.2+/-1.0 and 1.5+/-1.1 vs 2.0+/-0.9 and 2.2+/-1.0%, respectively; p<0.01) and in forced expiratory volume in one second (FEV1) (1.7+/-1.1 and 1.9+/-1.3 vs 2.6+/-1.0 and 2.8+/-1.0%, respectively; p<0.005). They had fewer episodes of colonization of the airways by Pseudomonas aeruginosa, and colonization occurred at a more advanced age (median age 25 and 19 vs 15 and 17 yrs, respectively; p<0.01) and required fewer intravenous antibiotic courses (p<0.01). Pancreatic insufficiency was less frequent in Groups 3 (23%) and 4 (63%) than in Groups 1 (100%) and 2 (96%). This study suggests that the phenotype of adult cystic fibrosis patients, including the severity of the lung disease, is related to the severity of the cystic fibrosis transmembrane conductance regulator mutations.

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32 Only three patients had a normal sweat test: two related patients, who were compound heterozygotes for the G542X and 3849+10 kb cytosine (C)→thymine (T) mutations, and one patient who was compound heterozygote for the R1070Q and D1152H mutations. Login to comment
65 The ∆F508 mutation was found in 134 chromosomes (61%), the mutation G542X in nine (4%), whereas all the other mutations were rare, since they were found in no more than 1% of the cohort. Login to comment
77 - Genotype of the 110 CF patients: details of the CF mutations and classification into four groups Genotype Genotype Pts groups n 1 ∆F508/∆F508 48* 2 ∆F508/G542X 6 ∆F508/E827X 3† ∆F508/R553X 2 ∆F508/W1282X 2 ∆F508/E595X 1 ∆F508/E60X 1 ∆F508/W846X 1 ∆F508/1078delT 1 ∆F508/2143delT 1 ∆F508/2347delG 1 ∆F508/3659delC 1 ∆F508/4382delA 1 ∆F508/2183 AA→G 1 ∆F508/1717-1 G→A 1 ∆F508/1811+1.6 kb A→G 1 E595X/Y1092X 1 1717-1 G→A/1078delT 1 3 ∆F508/I336K 1 ∆F508/G27E 1 ∆F508/D192N 1 ∆F508//I980K 1 ∆F508/P205S 1 ∆F508/2789+5 G→A 1 ∆F508/3272-26 A→G 1 G542X/3849+10 kb C→T 2‡ G542X/2789+5 G→A 1 W361R/297-3 C→T 1 G551D/1717-1 G→A 1 N1303H/2183 AA→G 1 2789+5 G→A/2183 AA→G 1 R1070Q/D1152H 1 R1070Q/unidentified 1 S1251N/unidentified 1 4 ∆F508/unidentified 7 ∆I507/unidentified 2 1811+1.6 kb A→G/unidentified 1 1161delC/unidentified 1 unidentified/unidentified 8 *: two patients are brothers; †: three brothers; ‡: two sisters. Login to comment
105 - Characteristics of patients with pancreatic sufficiency and FEV1 <30% of predicted value Age at P. aeruginosa Genotype Age diagnosis FVC FEV1 colonization yrs yrs % pred % pred Group 2 ∆F508/4382del A 27 18 43 21 Yes Group 3 ∆F508/3272-26 G→A 21 8 32 28 Yes G542X/3849+10kb C→T 35 32 53 25 Yes W361R/297-3 C→T 45 25 25 12 Yes Group 4 unid/unid 30 15 38 27 Yes unid: unidentified. For further definitions see legends to tables 1 and 3. Login to comment
107 - Characteristics of patients with FEV1 >70% of predicted value Age at P. aeruginosa PI Hepatic Genotype Age diagnosis FVC FEV1 colonization cirrhosis yrs yrs % pred % pred Group 1 ∆F508/∆F508 18 <1 83 75 Yes Yes No ∆F508/∆F508 19 8 88 72 Yes Yes Yes ∆F508/∆F508 24 <1 87 84 Yes Yes No ∆F508/∆F508 25 13 85 82 Yes Yes No ∆F508/∆F508 37 34 90 83 No Yes No Group 2 ∆F508/E827X 18 <1 82 76 Yes Yes Yes ∆F508/W846X 29 27 101 95 No Yes No ∆F508/W1282X 31 28 91 77 No Yes No Group 3 2789+5 G→A/G542X 18 2 107 103 No No No 2789+5 G→A/2183 AA→G 36 34 93 87 No No No ∆F508/G27E 39 28 115 78 No No No Group 4 ∆I507/unid 18 <1 103 103 No Yes No unid/unid 26 5 89 77 No Yes No unid/unid 39 38 96 87 No No No unid/unid 40 38 110 106 No No No PI: pancreatic insufficiency; unid: unidentified. For further definitions see legend to tables 1 and 3. and the nature of pulmonary infection are very different among patients. Login to comment
116 In the latter study, most of the compound heterozygotes for ∆F508 were associated with another mutation (G542X, R553X, W1282X, 1717- 1G→A, 621+1G→T) that corresponded to Group 2. Login to comment

[hide] Heterogeneity of phenotype in two cystic fibrosis ... J Med Genet. 1996 Aug;33(8):711-3. Parad RB
Heterogeneity of phenotype in two cystic fibrosis patients homozygous for the CFTR exon 11 mutation G551D.
J Med Genet. 1996 Aug;33(8):711-3., [PMID:8863168]

Abstract [show]
In the heterozygous state, the cystic fibrosis transmembrane conductance regulator (CFTR) exon 11 mutation G551D has been described as "severe," causing pancreatic insufficiency. Two cystic fibrosis (CF) patients homozygous for this mutation showed a mild rather than severe pancreatic phenotype and a variable pulmonary phenotype.

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10 R B Parad Received 27 December 1995 Revised version accepted for publication 15 March 1996 Methods Cheekbrush DNA for CFTR mutation analysis was collected and prepared according to Richards et al.1 CFTR mutation analysis was performed for 12 mutations (AF508, G551D, G542X, 621 + 1G->T, AI507, 1717-1G-4A, R117H, N1303K, W1282X, R560T, R553X, 3849 + 1Okb C-+T). Login to comment
53 Lanes are paired to show both normal and abnormal alleles con; mutations 621 + JG-*T (normal band lane 1, abnormal band lane 2), G551D (abnormal band lane 1, normal band lane 2), G542X (abnormal band lane 1, n band lane 2), and AF508 (normal band lane 1, abnormal band lane 2) at these first pair oflanes shows the ARMS pattern ofa GSSJDIN heterozygote. Login to comment
60 Recent studies have shown present (but diminished) chloride conduct- iction for ance, and absent CFTR inhibitory regulation G542X of the outwardly rectifying chloride channel. Login to comment

[hide] A mild variant of cystic fibrosis. Thorax. 1996 Aug;51 Suppl 2:S51-4. Alton EW
A mild variant of cystic fibrosis.
Thorax. 1996 Aug;51 Suppl 2:S51-4., [PMID:8869353]

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41 Examples include G542X, a mutation with a frequency of around 3% worldwide. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Am J Hum Genet. 1996 Jul;59(1):45-51. Miller PW, Hamosh A, Macek M Jr, Greenberger PA, MacLean J, Walden SM, Slavin RG, Cutting GR
Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in allergic bronchopulmonary aspergillosis.
Am J Hum Genet. 1996 Jul;59(1):45-51., [PMID:8659542]

Abstract [show]
The etiology of allergic bronchopulmonary aspergillosis (ABPA) is not well understood. A clinical phenotype resembling the pulmonary disease seen in cystic fibrosis (CF) patients can occur in some individuals with ABPA. Reports of familial occurrence of ABPA and increased incidence in CF patients suggest a possible genetic basis for the disease. To test this possibility, the entire coding region of the cystic fibrosis transmembrane regulator (CFTR) gene was analyzed in 11 individuals who met strict criteria for the diagnosis of ABPA and had normal sweat electrolytes (< or = 40 mmol/liter). One patient carried two CF mutations (deltaF508/R347H), and five were found to carry one CF mutation (four deltaF508; one R117H). The frequency of the deltaF508 mutation in patients with ABPA was significantly higher than in 53 Caucasian patients with chronic bronchitis (P < .0003) and the general population (P < .003). These results suggest that CFTR plays an etiologic role in a subset of ABPA patients.

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62 Mutation Analysis Genomic DNA samples from both patient groups were screened for six of the most common CF mutations-AF508, G542X, GS51D, R553X, W1282X, and N1303K-by using reverse allele-specific oligonucleotide (ASO) analysis (Erlich et al. 1991). Login to comment
85 One patient with normal sweat electrolytes and pseudomonas colonization had the G542X mutation on one allele. Login to comment

[hide] In vivo analysis of fluid transport in cystic fibr... Am J Physiol. 1996 May;270(5 Pt 1):C1326-35. Zhang Y, Yankaskas J, Wilson J, Engelhardt JF
In vivo analysis of fluid transport in cystic fibrosis airway epithelia of bronchial xenografts.
Am J Physiol. 1996 May;270(5 Pt 1):C1326-35., [PMID:8967432]

Abstract [show]
An in vivo human bronchial xenograft model system was used to simultaneously analyze electrolyte and fluid transport defects in fully differentiated human cystic fibrosis (CF) and non-CF proximal airways. CF airways demonstrated three discernible defects when compared with non-CF, including 1) a lack of adenosine 3',5'-cylic monophosphate (cAMP)-inducible Cl- secretion, 2) a fourfold higher basal fluid absorption rate, and 3) an altered regulation of fluid absorption in response to amiloride-stimulated changes in Na+ transport. A unique finding in this study demonstrated that treatment of epithelia with amiloride led to a greater than threefold decrease in the rate of fluid absorption in CF tissues as contrasted to a greater than threefold increase in the rate of fluid absorption in non-CF tissues. The removal of apical Na+ from amiloride-treated non-CF xenografts was capable of ablating this amiloride-induced increase in fluid absorption. In light of the recent interactions demonstrated between CF transmembrane conductance regulator (CFTR) and the rat epithelial, amiloride-sensitive Na+ channel, these findings implicate additional complexities between the Na+ conductance pathways and fluid transport in normal and CF proximal airways. Such findings suggest that CFTR may also regulate amiloride-insensitive Na+ channels.

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77 Genotypes for CF tissues included l-AF50NAF508, l-AF508/ G542X, 2-AF508/unknown, l-R553X/unknown, and Reservoir (7oQN Fluid Apparatus Subcutaneous Buffer A graded scale Buffer B (mm) detachment detachment perfusion point 1 Pump subcutaneous Potential Difference Apparatus Fig. 2. Login to comment
83 1-G542X/G%lD. Login to comment

[hide] Cystic fibrosis heterozygote screening in 5,161 pr... Am J Hum Genet. 1996 Apr;58(4):823-35. Witt DR, Schaefer C, Hallam P, Wi S, Blumberg B, Fishbach A, Holtzman J, Kornfeld S, Lee R, Nemzer L, Palmer R
Cystic fibrosis heterozygote screening in 5,161 pregnant women.
Am J Hum Genet. 1996 Apr;58(4):823-35., [PMID:8644747]

Abstract [show]
A screening program for cystic fibrosis (CF) heterozygotes was conducted in a large HMO prenatal population, to evaluate the level of interest among eligible patients, the effectiveness of prescreening education, attitudes toward the screening process, psychological effects, and utilization of prenatal diagnosis and its outcomes. The heterozygote identification rate and frequency of specific CFTR mutations were also assessed. Identified carriers were offered genetic counseling and testing of male partners. Prenatal diagnosis was offered if both parents were identified as carriers. A total of 5,161 women underwent carrier testing; 947 others completed survey instruments only. The acceptance rate of screening was high (78%), and pretest education by videotape was generally effective. Adverse psychological effects were not reported. Participants generally found screening to be desirable and useful. Screening identified 142 female heterozygotes, 109 couples in which the male partner was not a carrier, and 7 high-risk couples. The incidence of R117H mutations was much higher than expected. The number of identified carriers was much lower in Hispanics than in Caucasians. We conclude that large-scale prenatal screening for CF heterozygotes in the absence of a family history of CF is an acceptable method for identifying couples at risk for affected fetuses. Sufficient pretest education can be accomplished efficiently, test insensitivity is well accepted, adverse psychological events are not observed, and general patient satisfaction is high.

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69 Lab group A was screened for the 6 most common mutations (F508, G542X, G551D, R553X, W1282X, and N1303K); lab group B was screened for 12 mutations, including the 6 most common and an additional 6 less common alleles (R117H, 621+1, I507, 1717G-A, R560T, and S549N). Login to comment
142 Table 7 CFTR Mutations in Screened Women NUMBER (%) wITH MUTATIONa GROUP ETHNiciTY F508 G542X GS5lD R553X W1282X N1303K R117H 621+1 1507 1717G-A R560T S549N TOTAL A Caucasian 26 (81) 5 (16) 1 (3) NA NA NA NA NA NA 32 Hispanic 2 (100) NA NA NA NA NA NA 2 B Caucasian 63b (65) 4 (4) 2 (3) 1 (1) 2 (2) 4 (4) 16b (16) 4 (4) 1 (1) 97b Hispanic 7 (88) 1 (12) 8 Caucasian/ Hispanic 2 (50) 1 (25) 1 (25) 4 'NA = not applicable. Login to comment
172 Factors that potentially contributed to the high rate of acceptance in this study included incorporation of pretest education into an ex- Table 8 Results of Prenatal Diagnosis in High-Risk Pregnancies CFTR MUTATIONSa COUPLE PRENATAL DIAGNOSIS Mother Father Fetus 1 No (miscarriage) GSS1D R117H N/GSS1D (abortus) 2 No (miscarriage) F508 1717G-A NA 3 No (miscarriage) F508 RS60T NA Yes (second pregnancy) N/N 4 Yes G542X F508 N/N S Yes F508 F508 N/F508 6 Yes (twins) F508 F508 F508/F508, F508/F508 7 Yes F508 F508 N/N 8b Yes F508 NA N/F508 9b Yes N1303K NA N/N1303K a NA = not available; and N = normal. Login to comment

[hide] Survey of cystic fibrosis transmembrane conductanc... Dig Dis Sci. 1996 Mar;41(3):540-2. McGill JM, Williams DM, Hunt CM
Survey of cystic fibrosis transmembrane conductance regulator genotypes in primary sclerosing cholangitis.
Dig Dis Sci. 1996 Mar;41(3):540-2., [PMID:8617131]

Abstract [show]
A variety of cholestatic liver diseases appear to primarily affect the biliary epithelium, including cystic fibrosis (CF). CF results from a defect in the chloride channel protein, cystic fibrosis transmembrane conductance regulator (CFTR). Although the majority of CF patients have a genomic deletion in deltaF508, other mutations of CFTR may result in less severe clinical presentations and outcomes. Recently, CFTR has been shown to be involved in secretin-stimulated choleresis in intrahepatic bile duct epithelial cells. Cholestasis in cystic fibrosis appears to result from defective chloride transport across the biliary epithelium and is the only cholestatic disease of bile ducts for which a cellular defect has been identified. Primary sclerosing cholangitis (PSC) is a cholestatic disease with histological and cholangiographic features similar to CF. The purpose of this pilot study was to explore whether there is an increased prevalence of CFTR mutations. Two patients exhibited mutations in one allele, yielding a carrier rate of 10.6%, not statistically different from the general U.S. population carrier rate of 4%.

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33 In total, 32 mutations were evaluated, which represent 90% of the most common mutations (t4): AF508 G542X G551D W1282X 3905insT NI303K 3849+ 10kbC--~T R553X 621+ IG--*T 1717- IG--,A lt)78delT 2789+5G---~A 3849+4A--~G 711+ IG---oT R1162X 1898+IG----~A R117H 3659delC G85E 2184delA A1507 R347P Y1092X R560T A455E R334W Y122X S549R(T---~G) Q493X V520F $549N R347H Patient Selection. Login to comment

[hide] Identification of the five most common cystic fibr... Mol Hum Reprod. 1996 Mar;2(3):203-7. Scobie G, Woodroffe B, Fishel S, Kalsheker N
Identification of the five most common cystic fibrosis mutations in single cells using a rapid and specific differential amplification system.
Mol Hum Reprod. 1996 Mar;2(3):203-7., [PMID:9238680]

Abstract [show]
We describe a rapid and specific differential amplification system which can detect five of the most common cystic fibrosis mutations from a single cell. In the first round of the polymerase chain reaction (PCR), regions of exons 4, 10 and 11 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene containing the mutations delta F508, G551D, R553X, G542X and 621+1G > T were co-amplified in a single multiplex PCR. To identify potential contamination, we included external amplification primers for the polymorphic human tyrosine hydroxylase (HUMTH01) locus as a fingerprint for the sample. In the second round of PCR, detection of any of the five mutations was achieved using the amplification refractory mutation system (ARMS) in two separate reactions, each containing nested amplification primers for either wild type or mutant sequence. A separate second round PCR for the fingerprinting was performed with nested HUMTH01 primers. Using this procedure we have successfully and accurately detected five cystic fibrosis mutations in 30 single cells with a failed amplification rate of 7% and a contamination rate of 4.6% and that PCR failure or possible contamination will also be easily detected. This procedure allows detection of the five most common point mutations and small deletions responsible for cystic fibrosis from a single cell in < 8 h which could be applicable to preimplantation diagnosis in human embryos.

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2 In the first round of the polymerase chain reaction (PCR), regions of exons 4, 10 and 11 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene containing the mutations AF508, G551D, R553X, G542X and 621 + 1 O T were co-amplified in a single multiplex PCR. Login to comment
11 Although this mutation accounts for ~50-80% of cases (CF Genetic Analysis Consortium, 1990), depending on the population being studied, in the UK around 10% of the remainder present with one or two of the four other most common mutations, these being the G551D Gly-Asp, R553X Arg-Stop (Cutting et al, 1990), the G542X Gly-Stop (Kerem et al, 1990) and the G-T splice mutation 621 + 1 O T (the first intronic base in the splice donor site flanking the 3' end of exon 4) (Zielenski et al, 1991). Login to comment
22 Using this procedure, we have shown it is possible to genotype a single cell for any of the five most common mutations responsible for CF, which when used in parallel 621 + IOT AFSOS A B G551D0U53X) G542X Cystic fibrosis diagnosis in single cells 200 bp 4S 4AS S21 + 10 >T 10S 11S 10AS AFSOS 11 AS R553X QS51D L G54ZX Figure 2. Login to comment
23 Schematic diagram of external CFTR primers for exons 4, 10 and 11 and the relative positions of the cystic fibrosis mutations 621 + 1 O T , AF508, G542X, R553D and G551X. Login to comment
36 CF cell lines Four lymphoblastoid cell lines with genotypes AF5O8/AF5O8, G542X/ G542X, G551D/R553X and 621+ l>G/AF508 were purchased from Coriell Cell Repositories (Camden, NJ, USA) and one cell line with genotype 621 + lG>T/normal was purchased from European Collection of Animal Cell Cultures (Porton Down, UK). Login to comment
54 Table I. Sequences of pnmary and secondary polymerase chain reaction (PCR) primers including ARMS kit primers and their relevant uM concentrations (compared to a standard concentration of 0.86 nM per reaction) CFTR Exon Sequence Size (bp) Primary PCR primers Exon 4 Exon 10 Exon II HUMTH01 5'- CAAGTCTTATTTCAAAGTACCAAG 5'- CAGCTCACTACCTAATTTATGACA 5'- AAGTGAATCCTGAGCGTGATTTGATAATGA 5'- CACAGTAGCTTACCCATAGAGGAAACATAA 5'- TATTTAATGATCATTCATGACATTT 5'- TAAAGCAATAGAGAAATGTCTGTA 5'- ATTCAAAGGGTATCTGGGCTCTGG 5'- GTGGGCTGAAAAGCTCCCGATTAT 479 380 425 179-203 Mutation and sequence Size (bp) Tube Concentration Secondary PCR primers AF508 C: 5'- GACTTCACTTCTAATGATGATTATGGGAGA N: 5'- GTATCTATATTCATCATAGGAAACACCAC M: 5'- GTATCTATATTCATCATAGGAAACACCATT Exon II C: 5'- TAAAATTTCAGCAATGTTGTTTTTGACC G551D/R553X N: 5'- GCTAAAGAAATTCTTGCTCGTTGCC M: 5'- AGCTAAAGAAATTCTTGCTCGTTGCT G542X N: 5'- ACTCAGTGTGATTCCACCTTCTAC M: 5'- CACTCAGTGTGATTCCACCTTCTCA 621 + 1 O T C: 5'- TCACATATGGTATGACCCTCTATATAAACT N: 5'- TGCCATGGGGCCTGTGCAAGGAAGTATTCC M: 5'- TGCCATGGGGCCTGTGCAAGGAAGTATTCA HUMTHOI 5'- TGATTCCCATTGGCCTGTTCCTCC 5'- TGGCCCACACAGTCCCCTGTACAC 160 157 285 286 256 257 380 380 123-147 A/B A B A/B I A B A A/B A B 1.0 1.0 2.0 2.0 2.0 1.0 1.0 1.0 0.5 0.5 0.5 C = common primer, N = normal primer, M = mutant primer. Login to comment
56 Figure 3 shows the detection of individual AF508, G551D, R553X and G542X, 621 + 1 O T mutations using the CF ARMS kit after primary amplification of individual CFTR exons containing these mutations, and the corresponding fingerprint from the same cells (Figure 4). Login to comment
63 One drawback of using single exon 1 2 3 4 5 6 7 8 9 10 0 X 621+1G>T G551D/R553X G542X AF508 Figure 3. Login to comment
65 Tubes A and B for a normal genotype (lanes I and 2), then tubes A and B for the mutations: AF5O8/AF5O8 (lanes 3 and 4), 621 + lOT/normal (lanes 5 and 6), G551D/R553X (lanes 7 and 8), and G542X/G542X (lanes 9 and 10). Login to comment
69 Lanes 1-5 are cells containing the mutations AF508/AF508, 621 + lG>T/normal, G551D/R553X, G542X/G542X and 621 + 1OT/AF508 respectively. Login to comment
71 1 2 3 4 5 6 7 8 9 10 11 12 621+1G>T G551D/R553X G542X AF508 Figure 5. Login to comment
72 Detection of the same five mutations using a multiplex containing external primers for exons 4, 10 and 11, and subsequent genotyping using the ARMS kit Tubes A and B for a normal genotype (lanes 1 and 2), then tubes A and B for the mutations AF5O8/AF5O8 (lanes 3 and 4), 621+ lG>17normal (lanes 5 and 6), G551D/R553X (lanes 7 and 8), G542X/G542X (lanes 9 and 10) and 621 + 1OT/AF508 (lanes 11 and 12). Login to comment
86 The ARMS procedure used here has this advantage in that it is capable of detecting the 621 + 1 O T , AF508, G551D, R553X and the G542X mutations within 6-8 h from a single cell. Login to comment

[hide] Mutation characterization of CFTR gene in 206 Nort... Hum Mutat. 1996;8(4):340-7. Hughes DJ, Hill AJ, Macek M Jr, Redmond AO, Nevin NC, Graham CA
Mutation characterization of CFTR gene in 206 Northern Irish CF families: thirty mutations, including two novel, account for approximately 94% of CF chromosomes.
Hum Mutat. 1996;8(4):340-7., [PMID:8956039]

Abstract [show]
A variety of mutation detection techniques, including restriction endonuclease digestion, allele specific oligonucleotides, and automated fluorescent sequencing, were used in the identification of 15 CFTR mutations representing 86.7% of CF chromosomes in 206 Northern Irish cystic fibrosis (CF) families. A systematic analysis of the 27 exons and intron/exon boundaries of the CFTR gene was performed using denaturing gradient gel electrophoresis (DGGE) in an attempt to characterise the 55 unknown CF mutations in 51 patients. Twenty different mutations were detected by DGGE on 30 chromosomes accounting for a further 7.3% of CF alleles. Fifteen of these mutations had not previously been found in Northern Ireland, and two are novel, M1I(G > T) and V562L. In total, 30 CFTR mutations account for 93.9% of the 412 Northern Irish CF chromosomes tested. The three major CF mutations in Northern Ireland are delta F508, G551D, and R117H with respective frequencies of 68.0%, 5.1%, and 4.1%. The efficacy of the DGGE technique was proven by the detection of 77 out of 77 control variants from all the CFTR exons. DGGE is a highly efficient and sensitive method for mutation screening especially in large genes where the mutation spectrum is known to be heterogeneous.

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45 Prior to the DGGE study a variety of methods (see Tables 1 and 2) were used to screen for CFTR mutations, including single mutation assaysfor AF508, AI507, G55lD, G542X, R553X, R117H, R560T, 621+lG>T, and 3849+10kbC>T. Login to comment
53 35%) PAGE (278) Kerem et al.. 1989AF508 G551D R117H R560T G542X 621+1G>T A1507 E60X 3659delC R553X 3120G>A 1l54insTC 2789+5G>A N1303K MlI(G>T) QW P67L 557delT 711+3A>G L206W R297Q V520F V562L Y563N Y917C R1162X 3849G>A 3849 +10kbC>T 3850-1GBA W1282X 280 21 17 12 9 9 7 3 2 1 68.0 5.1 4.1 2.9 2.2 2.2 1.7 0.7 0.5 0.24 17-32-13 (38;27%j 17-31-13(24,17%) 16-07-17 16-30-13 plus14 rare haplotypes (29) 16-07-17 23-33-13 (4) 22-31-13 (2) 21-31-13 17-07-17 (5) 16-31-13 16-35-13 17-58-13 17-35-13 16-07-17 17-07-17 23-29-13 (1) 23-31-13 (1) 16-07-17 16-31-13 16-07-17 15-29-13 16-33-13 16-07-17 17-07-17 16-07-17 16-07-17 16-30-13 16-32-17 17-31-13 16-31-14 16-46-13 16-30-14 17-07-17 DGGE(2) ' RD ASO's (11) DGGE(6) RD AR (8) DGGE (1) RD PAGE (5) DGGE (2) SEQ SEQ (2) DGGE (1) RD DGGE DGGE DGGE SEQ DGGE DGGE DGGE SEQ DGGE DGGE SEQ DGGE DGGE DGGE DGGE DGGE SEQ RD DGGE DGGE Cutting et al.. 1990 Dean et al.. 1990 Kerem et al., 1990 Kerem et al.. 1990 Zielenski et al., 1991 Kerem et al.. 1990 Malone et al., CFGAC Kerem et al., 1990 Cutting et al., 1990 Zielenski et al., CFGAC lannuzzi et al., 1991 Highsmith et al., 1990 Osborne et al., 1991 this study Savov et al., 1994 Hamosh et al., CFGAC Graham et al., 1992 Petreska et al., CFGAC Claustres et al., 1993 Graham et al., 1991 Jones et al.. 1992 this study Kerem et al.. 1990 Edkins & Creegan, CFGAC Gasparini et al., 1991 Cutting et al.. 1992 Highsmith et al., 1994 Audriizet et al., 1993 Vidaud et al., 1990 "Numbers in parentheses after the microsatellite haplotypes refer to the number of alleles haplotyped when not all of the available chromosomeswere typed. Login to comment
77 R553X, G542X. Login to comment
99 G542X (2.2%), 621+1G>T (2.2%), and A1507 (1.7%); the next seven mutations were found in two or three chromosomes, with the remaining 16 mutations being identified only once. Login to comment
108 There were four mutations, R117H, AF508, AI507, and G542X, on 11 CF alleles that were missed due to poor PCR amplification in their original screening methods (Table 1) but were detected using DGGE. Login to comment
120 A commercially available reverse dotblot assay (Innogenetics) is presently used in our laboratory for routine screening of eight CF mutations, of which seven are found in this population; AF508, AI507, G551D, G542X, R553X, N1303K, and W1282X and account for 78% of CFTR defects with additional mutations R560T, 621+1G>T and Rl17H screened using individual assays. Login to comment

[hide] Fluorescent multiplex microsatellites used to defi... Hum Mutat. 1996;8(3):229-35. Hughes D, Wallace A, Taylor J, Tassabehji M, McMahon R, Hill A, Nevin N, Graham C
Fluorescent multiplex microsatellites used to define haplotypes associated with 75 CFTR mutations from the UK on 437 CF chromosomes.
Hum Mutat. 1996;8(3):229-35., [PMID:8889582]

Abstract [show]
The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene contains three highly informative microsatellites: IVS8CA, IVS17bTA, and IVS17bCA. Their analysis improves prenatal/ carrier diagnosis and generates haplotypes from CF chromosomes that are strongly associated with specific mutations. Microsatellite haplotypes were defined for 75 CFTR mutations carried on 437 CF chromosomes (220 for delta F508, 217 for other mutations) from Northern Ireland and three English regions: the North-West, East Anglia, and the South. Fluorescently labelled microsatellites were amplified in a triplex PCR reaction and typed using an ABI 373A fluorescent fragment analyser. These mutations cover all the common and most of the rare CF defects found in the UK, and their corresponding haplotypes and geographic region are tabulated here. Ancient mutations, delta F508, G542X, N1303K, were associated with several related haplotypes due to slippage during replication, whereas other common mutations were associated with the one respective haplotype (e.g., G551D and R560T with 16-7-17, R117H with 16-30-13, 621 + 1G > T with 21-31-13, 3659delC with 16-35-13). This simple, fast, and automated method for fluorescent typing of these haplotypes will help to direct mutation screening for uncharacterised CF chromosomes.

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6 Ancient mutations, AF508, G542X, N1303K, were associated with several related haplotypes due to slippage during replication, whereas other common mutations were associated with the one respective haplotype (e.g., G551D and R560T with 16-7-17, R117H with 16-30-13,621+1G>T with 21-31-13,3659delC with 16-35-13). Login to comment
43 AF508, G542X, and N1303K are ancient CFTR mutations that are the first, second, and fourth most prevalent in Northern Europe, with respective frequenciesof -70%, 2%, and 1% (CFGAC, 1994). Login to comment
50 There are six associated haplotypes with G542X whose ancestral haplotype probably is 23-33-13, as it is found most frequentlyhere and in Spain (Morral et al., 1993). Login to comment
74 CF 8CA-17bTA-17bCA Mutation chromosomes % Normal Laboratoryb Reference' HaplotVpe 1)15-29-13 557delT Nl Graham et al.. 1992 21 16-07-17 MU (G>T) 3) 16-24-13 4) 16-25-13 5) 16-29-13 6) 16-30-13 7) 16-30-14 8) 16-31-13 9) 16-31-14 10) 16-32-13 12) 16-33-13 13) 16-34-13 14) 16-35-13 11)16-32-17 15)1645-13 16) 1646-13 17) 1646-14 19) 17-07-17 18)16-53-13 20)17-29-14 21) 17-31-13 22) 17-32-13 23) 17-35-13 24) 17-51-11 25) 17-55-13 27) 17-58-13 28) 21-31-13 29) 22-31-13 31)23-22-17 26) 17-56-13 30) 22-33-13 32) 23-29-13 33)23-31-13 34)23-32-13 35)23-33-13 36)23-34-13 37) 23-36-13 38)24-22-17 39) 24-31-13 182delT P67L R75X L206W 1154insTC 146linsAGAT Q493x V520F 1717-1G>A G551D R560T V562L R709X S1196X L1254X R1283M G85E 2184insA 711+lG>T 3495delA 4279insA SlOR L88S R117C R117H G178R 1717-1G>A Y563N W1098R G1123R 3850- 1G>A E6OX %%deIT 1138insG R34P 2183AA>G 2184delA R1158X 1078delT R1162X 3849G>A Q141W R347P Y917C G2iX 711+3A>G 441delA 3130de115 3659delC 1898+1G>A R709X 2711delT R1158X E92K 3849+lOkbC>T 2118delAACT 4048insCC 296+1 2 T S Q22OX R297Q A1507 2789+5G>A 3120+1G>A W128W 1811+lG>C AF508 E831X R116W AF508 W846X1 3120G>A R785X R553X R553X R553X 621+1G>T G542X G542X Y1182X N1303K AF508 G54W 3041delG 1525-1G>A N1303K G542X G542X G542X 394delTT R709X N1303K 1 1 1 2 1 1 4 2 3 4 2 26 8 1 1 1 1 1 8 1 1 1 1 1 1 1 19 1 2 1 1 1 1 7 1 1 2 1 1 2 1 1 1 1 1 1 1 1 2 1 1 7 4 1 2 1 1 2 1 1 4 Asian 1 2 1Asian 5 4 i Afro-Caribbean 5 1 42 (19%) 1 1 57 (26%) 1 2 1 1 1 2 12 2 11.4 0.4 4.9 16.3 1.1 3.8 1.9 10.6 2.3 1.5 2.3 1.5 2.7 4.5 0.4 0.8 0.8 0.4 0.8 0.4 1 2 1 7 1 1 1Asian 1 1.5 0.8 0.8 NI G NI, M M NI NI. Login to comment
82 MicrosatelliteHaplotypesfor 16 Most Common CFTR Mutations in UK Population Mutation frequency(%)b Haplotype chromosomes Normal Laboratory" AF508 75 23-31-13 81 0.4 NI,M, G, C 17-32-13 57 0.8 NI,M, G, C 17-31-13 42 0.8 N1,M, G, C G551D 3 16-07-17 26 11.4 NI,M, G, C G542X 2 23-33-13 7 1.5 NI,C ApproximateUK 8CA-17bTA-17bCA No.CF % 22-31-13 1 NI 22-33-13 1 G 23-31-13 1 G 23-32-13 1 N1 23-34-13 1 0.8 C 621+1G>T 1 21-31-13 12 NI N1303K 0.5 23-29-13 2 NI. Login to comment

[hide] Haplotype analysis of 94 cystic fibrosis mutations... Hum Mutat. 1996;8(2):149-59. Morral N, Dork T, Llevadot R, Dziadek V, Mercier B, Ferec C, Costes B, Girodon E, Zielenski J, Tsui LC, Tummler B, Estivill X
Haplotype analysis of 94 cystic fibrosis mutations with seven polymorphic CFTR DNA markers.
Hum Mutat. 1996;8(2):149-59., [PMID:8844213]

Abstract [show]
We have analyzed 416 normal and 467 chromosomes carrying 94 different cystic fibrosis (CF) mutations with polymorphic genetic markers J44, IVS6aGATT, IVS8CA, T854, IVS17BTA, IVS17BCA, and TUB20. The number of mutations found with each haplotype is proportional to its frequency among normal chromosomes, suggesting that there is no preferential haplotype in which mutations arise and thus excluding possible selection for specific haplotypes. While many common mutations in the worldwide CF population showed absence of haplotype variation, indicating their recent origins, some mutations were associated with more than one haplotype. The most common CF mutations, delta F508, G542X, and N1303K, showed the highest number of slippage events at microsatellites, suggesting that they are the most ancient CF mutations. Recurrence was probably the case for 9 CF mutations (R117H, H199Y, R347YH, R347P, L558S, 2184insA, 3272-26A-->G, R1162X, and 3849 + 10kbC-->T). This analysis of 94 CF mutations should facilitate mutation screening and provides useful data for studies on population genetics of CF.

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9 Only four other mutations, G542X, G551D, N1303K, and W1282X, are rel- ativelyfrequent in the world Caucasoid population, and each has an overall frequency of 1-2.5% (CFGAC, 1994).Another 19mutations are found in 0.1-0.7% of chromosomes, but their distribution is variable, and in many cases they are only present in some populations (CFGAC, 1994). Login to comment
34 The high number of haplotypes associated with mutations AF508, G542X, and N1303K suggests that they are the most ancient CF mutations in the population. Login to comment
85 Other haplotypes that were less commonon normal chromosomes(16-44-13, 16-35-13, 16-33-13, and 16-29-13)were each associatedwith only one CF mutation. Several mutations were associated with more than one haplotype apparentlyas the result of slippage at one of the microsatellites IVS8CA, IVS17BTA, and IVS17BCA: AF508, G542X, N1303K, R553X, Q552X, 2869insG, L1077P, 7H, and R1162X (Table 3). Login to comment
90 Mutations AF508, G542X, and N1303K were associated with severalmicrosatellite haplotypes as the result of slippage at one of the three microsat- Y R334W, 1811+1.6kbA+G, 711+IG-T, R34- ellites, giving additional support to an ancient origin of these mutations (Morral et al., 1993a, 1994a). Login to comment
106 (1992) Dork et al. (1994a) Malone et al. (personal communication) Claustreset al. (1992) Ferec et al. (1992) Fanen et al. (1992) lvaschenko et al. (1991) T. Dork (personal communication) Dean et al. (1990) Dork et al. (1994a) Ferec et al. (1992) Bozon et al. (1994) Costes et al. (personal communication) Fanen et al. (1992) Audrezet et al. (personal communication) Zielenski et al. (1991a) Zielenski et al. (1991a) Granell et al. (1992) Highsmith et al. (1990) Mercier et al. (1993b) Vidaud et al. (1990) Fanen et al. (1992) Fanen et al. (1992) Dork et al. (1994b) (continued) HAPLOTYPESFOR 94 CF MUTATIONS TABLE2. CFTR HaplotvpesforDiallelic and Multiallelic DNA Markers for 94 CF Mutations"(Continued) ~~ ~ J44-GAIT- 8CA-17BTA- No. of TSU-TUB20 17BCA Mutation chromosomes % Normal Laboratory Reference 1-6-1-2 (9.1%) 1-6-2-2 (8.9%) 1-7-1-2 (3.4%) 1-7-2-2 (2.6%) 2-7-1-1 (1.2%) 2-7-2-2 (0.7%) 17-7-16 16-7-18 16-7-17 15-7-17 24-31-13 23-52-13 23-34-13 23-33-14 23-33-13 23-32-13 23-31-13 23-30-13 23-21-19 23-18-13 22-35-13 22-31-13 22-30-13 21-31-13 19-33-13 18-45-13 18-37-13 18-35-13 17-57-11 17-55-13 17-55-11 17-54-11 17-53-11 17-52-11 17-51-11 17-33-13 16-46-13 16-45-13 16-44-13 16-42-13 16-35-13 16-30-13 16-30-13 16-7-17 16-21-19 L107% L1077P 24ldelAT L719X A1507 3849+10kbC-T 2184insA 2991de132 G551D 1154insTC V520F R560T 4114ATA+lT 3667de14 435insA Q414X C225R Q39X N1303K R1162X H199Y G542X G542X w1204x R347H G542X AF50gb N1303K 2143delT 3849f 10kbC-T N1303K 681delC R347H A455E N1303K A120T 621+1 h T 574delA 1221delCT F311L R560K R553X R533X R553X Q552X R553X Q552X R116W R553X 1898+5 h T 3272-26A-G 1717-1hA 1342-2A-C A1507 2869insG 2869insG E92X 4374+1 h T 2183AA-G R117H 1609delCA I336K W1063X 1 1 1 1 6 1 3 1 1 22 17 1 1 1 1 1 1 1 1 1 1 1 1 1 17 1 1 4 157 7 1 2 2 1 1 2 2 1 9 1 1 1 1 1 1 6 1 1 1 2 1 3 2 1 3 1 1 1 4 2 4 1 1 - - 10.33 1.45 - - 0.48 1.45 - 0.24 1.45 0.24 - - - - 0.24 0.48 - - - - - - 0.49 0.48 - 0.24 0.24 0.24 - - - - - 0.72 0.24 0.72 - t h fP h b.fb,fP h b,fp.t t h b.fb.fp,h,t b.fb.fp,h,t t t t h b h h fP h fP fb b fP b.fb,fP,h.t fP fb b,fP,t b.fb,fp,h,t b.fb,h h h h,t t fb t b b b.fb.t fP fb fb tb h fP h h t t b h t h b b h h b,fb,h fP.h b h fP fP Bozon et al. (1994) Fanen et al. (1992) Dork et al. (1994a) Kerem et al. (1990) Dork et al. (1994~) Cutting et al. (1990) Kerem et al. (1990) lannuui et d. Login to comment
131 Data based on the evolution of two other common mutations G542X and N1303K have shown that they could also be very ancient (more than 30,000 years old) (Morral et al., 1993a). Login to comment
133 Therefore, on normal and on AF508, G542X and N1303K chromosomes, a large microsatellite haplotype variability has been generated, supporting a very ancient origin for these chromosomes. Login to comment

[hide] Cystic fibrosis mutation detection by hybridizatio... Hum Mutat. 1996;7(3):244-55. Cronin MT, Fucini RV, Kim SM, Masino RS, Wespi RM, Miyada CG
Cystic fibrosis mutation detection by hybridization to light-generated DNA probe arrays.
Hum Mutat. 1996;7(3):244-55., [PMID:8829658]

Abstract [show]
We have combined photochemistry and photolithography with solid-phase DNA synthesis chemistry to form a new technology that makes high density oligonucleotide probe array synthesis possible. Hybridization to these two-dimensional arrays containing hundreds or thousands of oligonucleotide probes provides a powerful DNA sequence analysis tool. Two types of light-generated DNA probe arrays have been used to test for a variety of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. One array, made up of 428 probes, was designed to scan through the length of CFTR exon 11 and identify differences from the wild type reference sequence. The second type of array contained 1480 probes chosen to detect known deletions, insertions, or base substitution mutations. The validity of the probe arrays was established by hybridizing them with fluorescently labeled control oligonucleotide targets. Characterized mutant CFTR genomic DNA samples were then used to further test probe array hybridization specificity. Finally, ten unknown patient samples were genotyped using the CFTR probe array assay. The genotype assignments were identical to those obtained by PCR product restriction fragment analysis. Our results show that light-generated DNA probe arrays are highly effective in analyzing complex mutation and polymorphism patterns in a relatively large gene such as CFTR.

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203 Figure 5 shows a typical image from this experiment made from CHO sample nine which had two exon 11 mutations, G542X and G551D. Login to comment
238 Cystic Fibrosis Mutation-Specific DNA Probe Array" Mutation Exon and column Tested Subarrayhow G85E R117H I148T 621 -+ l(G+T) 711 + 1(G+T) R334W R347H R347P 1078 delT A455E G480C Q493X A1507 F508C AF508 V520F G542X S549R(T-+ G) G551D Q552X R553X A559T R560T 1898 + l(G-,A) 2184 del A 2789 + 5(G+ A) R1066C L1077P Y1092X R1162X 3659 del C 1717-1(& A) 3272 - 26(A+ G) 3 4 4 in 4 in 5 7 7 7 7 9 10 10 10 10 10 10 in 10 11 11 11 11 11 11 11 in 12 13 in 14b in 17a 17b 17b 17b 19 19 * * * * * * * * * * * * * * * * * * * * * * * * * * * * 3849 + lOkb C-, T in 19 9,3 W1282X 20 994 3905insT 20 10.1 * N1303K 21 10,2 * * * "Row and column locations for each of the mutation specific,40 probe sets included in the specialized probe array design. Login to comment
248 Results From Unknown Patient Sample CFTR Genotyping" Sample CHO 1 CHO 2 CHO 3 CHO 4 CHO 5 CHO 6 CHO 7 CHO 8 CHO 9 CHO 10 Exon 10 genotype Wild-type AF508 Wild-type Wild-type AF508 Wild-type Wild-type AF508 Wild-type Wild-type Exon 11genotype Wild-type G542X Wild-Type Wild-Type G551D R553X G542X R553X G542WG551D Wild-type "Results summary from blind analysis of ten patient samples for CFTR exon 10and 11 mutations. Login to comment
265 Image of a specialized mutation array hybridized with exon lO/exon 11targets prepared from a compound heterozygote for exon 11 mutations G542X and G551D (Children`s Hospital of Oakland, Sample 9). Login to comment
268 G542X: 5'TAGTTClTTGAGAAGGT3`.Wild type: 5'GAGTGGAGGTCAACGAG3`. Login to comment
280 All except G542X and G551D display homozygous wild-type hybridization patterns. Other hybridization signals that appear as incomplete hybridization patterns compared with examples in Figure 3result from cross-hybridization with probes partially complementary to the target. Login to comment

[hide] Identification of three novel mutations in the cys... Hum Mutat. 1996;7(4):376-7. Bienvenu T, Chertkoff L, Beldjord C, Segal E, Carniglia L, Barreiro C, Kaplan JC
Identification of three novel mutations in the cystic fibrosis transmembrane conductance regulator gene in Argentinian CF patients.
Hum Mutat. 1996;7(4):376-7., [PMID:8723695]

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42 In addition, none of the most common CF mutations (G542X, G551D, W1282X, N1303K, R553X, 1717-1G>A, R1162X, 81507) were present in this series. Login to comment

[hide] Analysis of five CFTR mutations in Hungarian cysti... J Inherit Metab Dis. 1996;19(3):378. Nemeth K, Fekete G, Kiss E, Varadi A, Holics K, Ujhelyi R
Analysis of five CFTR mutations in Hungarian cystic fibrosis patients.
J Inherit Metab Dis. 1996;19(3):378., [PMID:8803789]

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5 Budapest; 3Children's Hospital Helm Pill, Budapest, Hungary *Correspondence Four hundred and fifty-eight cystic fibrosis (CF; McKusick 219700) patients, aged 6 months to 19 years old were screened for mutations AF508, G542X, G551D, R553X and N1303K. Login to comment
14 Mutation G542X was identified in 14 (3.0%), mutation R553X in 6 probands (1.3%); 7 patients (1.5%) were heterozygous for mutation N1303K. Login to comment

[hide] Methods for screening in cystic fibrosis. Methods Mol Med. 1996;5:99-119. Schwarz M, Malone G
Methods for screening in cystic fibrosis.
Methods Mol Med. 1996;5:99-119., [PMID:21374513]

Abstract [show]
Cystic fibrosis (CF) is the most common lethal autosomal recessive disorder in Whites, with an incidence of approx 1 m 2500 live births and a carrier frequency of approx 1 in 25. Since the discovery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene m 1989 (1-3), molecular genetics laboratories throughout the world have endeavored to identify the mutations present in their population of CF-bearing chromosomes. Since the entire CFTR gene and its intron-exon boundaries have been sequenced, mutation analysis in CF has become relatively simple, although time consuming. Generally, a number of different methods are applied to mutation analysis, but all involve an imtial step of amplification of part of the gene by polymerase chain reaction (PCR) (4), or a derivative of it, such as amplification refractory mutation system (ARMS) (5).

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10 Molecular D/agnoss of GenetIc Diseases Edlted by R Elles Humana Press Inc , Totowa, NJ 99 Table1 RelativeFrequenciesoftheCommonCFMutationsintheUnitedKingdom(S),NorthAmerica, andNorthernandSouthernEurope(7)" NorthNorthernSouthern UKAmericaEuropeEurope MutationExonNo.%No.%No.%No%References AF508 G551D G542X 621+l(G>T) 1717-l(G>A) sR117H =R553x 1898+l(G>A) N1303K R560T AI507 G85E 1154insTC V520F W1282X E60X 3659delC 1078delT 10738775.32690066.114,86670.28400755.033 113023.082061.973561.68370.518 111651.682342.244392.082593.569 intron4910.931541.48970.46370.51IO intron10560.57440.421600.76650.899 4450.46610.586202930.04II 11450.46960.921650.7844068 intron12450.4620.02410.191001412 21450.461301.252090.991792.4613 11410.42240.2340019009 10300.31200.19570.2750.079,14 3210.211601530014140.19II 7190.19n/an/an/an/an/an/a15 10170.17n/an/an/an/an/an/a16 20170.172452.351200.57430.5917 3160.16n/an/an/an/an/an/aMaloneb 19140.14140.133901810.019 790.0910.015302520.318 S549N1180.0850.051800920038 Q493X1070.07n/an/adan/adan/a9 R347P760.06260.25550.26240.3311 3849+10kb(C>T)intron1950.05570.55230.1180.1119 A455E930.03270.26350.17009 %/a,Datanotavadable. Login to comment
82 For example the mutations AEl15 and K1060T, aswell asAF508, G542X, and RI 17H have been seenm CBAVD (29). Login to comment
193 Odd-numbered tracks contain the ARMS products for the normal alleles of 621 + l(G > T) and AF508, plus the mutant alleles of G55 1D and G542X. Login to comment
194 Even-numbered tracks contain products for the normal alleles of G55 1D and G542X and the mutant alleles of 62 1 + 1(G > T) and AF508. Tracks 1 and 2 are from an individual who is negative for all four mutations, as are tracks 3 and 4. Login to comment
198 Tracks 13 and 14 are from a compound heterozygote of G542X and AF508. Tracks 15 and 16 are a negative (no DNA) control and track 17 is DNA molecular weight marker VI (Boehringer Mannheim). Login to comment

[hide] Four adult patients with the missense mutation L20... Hum Genet. 1995 Dec;96(6):717-20. Desgeorges M, Rodier M, Piot M, Demaille J, Claustres M
Four adult patients with the missense mutation L206W and a mild cystic fibrosis phenotype.
Hum Genet. 1995 Dec;96(6):717-20., [PMID:8522333]

Abstract [show]
We report molecular and clinical analyses in four unrelated patients with cystic fibrosis (CF) with compound heterozygosity for the L206W mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR). This uncommon missense mutation (frequency less than 1% in a sample of 336 CF chromosomes from Southern France) replaces a leucine by a tryptophan residue in the middle of the third transmembrane domain of CFTR. On the basis of the clinical features presented by the four patients, we postulate that the L206W might be associated with pancreatic sufficiency and residual transmembrane transport of chloride in lung.

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24 Open symbols, and J~ 0 halJ:filled symbols, healthy individuals filled symbols, CF (cystic fibrosis) G542X G542X G542X G542X L206W 1.,206W AI507 S L206W L206W A1507 O L206W AF508 AF508 V #, g g g AF508 L206W L206W AF508 AF508 while working in the sun in the hot and arid climate of southern France, he presented with two severe acute dehydration episodes, with excessive sweating and salt crystals on the skin. Login to comment
58 The alleles AF508 and AI507, located in NBD1, affect the processing of the CFTR, which fails to translocate to the correct cellular location at the apical membrane; the nonsense mutation G542X produces premature termination of translation, resulting in an unstable mRNA and no detectable protein (Welsh and Smith 1993). Login to comment
61 The CFTR protein with 206W might retain some residual function in respiratory and digestive tissues, exercising a "dominant" effect on the null alleles G542X, AI507 or AF508 and allowing the occurrence of mild phenotypes of CE In the two male patients reported here clinical expression of CF occurs only when the need for heat dissipation is tremendously increased and depends on evaporative heat loss. Login to comment

[hide] The Irish cystic fibrosis database. J Med Genet. 1995 Dec;32(12):972-5. Cashman SM, Patino A, Delgado MG, Byrne L, Denham B, De Arce M
The Irish cystic fibrosis database.
J Med Genet. 1995 Dec;32(12):972-5., [PMID:8825927]

Abstract [show]
We have found records of 1014 Irish cystic fibrosis patients alive by December 1994, belonging to 883 families. Prevalence in the population is 1/3475 and incidence at birth 1/1461, with a gene frequency of 2.6%. Twenty percent of the patients are aged over 20 years, but at present survival rate falls rapidly after that age. We have identified 85% of the mutations on the CFTR gene in a sample of 29% of the families (506 CF chromosomes). Mutation delta F508 is found in 72% of Irish CF chromosomes, G551D in 6.9%, and R117H in 2%. These are the highest frequencies reported for the latter two mutations world wide. Another seven mutations are found in an additional 4% of CF families. We present new microsatellite haplotype data that could be useful for genetic counselling of CF families bearing some of the 15% of CF mutations still unidentified, and comment on possible uses of our database.

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39 Care Table 2 Haplotypes associated with the most common CF mutations in Ireland, as well as with Irish CF chromosomes with unidentified mutations and non-CF chromosomes Mutation NmlNcf % X/Kt Mt Microsatellite§ No chrsll AF508 367/506 72-5 A 1 See table 3 2 B 2 116 C - 0 D 2 2 G551D 35/506 6-9 B 2 16-7-17 20 R117H 10/506 2-0 C 1 16-30-13 5 C 1 16-31-13 1 G542X 5/506 1-0 B 2 17-32-13 2 621 + 1G-T 4/506 0-8 B 2 21-7-17 2 B 2 21-31-13 2 R560T 4/506 0-8 D 2 16-7-17 1 D 2 16-31-17 1 1717-1G-A 3/506 0-6 C 1 16-32-13 2 A 2 17-32-13 1 N1303K 2/506 0-4 B 2 23-29-13 2 3659delC 2/506 0-4 C 1 16-35-13 2 AI507 2/506 0-4 ND ND R352Q 1/506 0-2 C 1 16-31-13 1 R553X 0/506 0 0 ND ND 1078delT 0/506 0.0 ND ND Total identified 435/506 85-6 Unidentified 71/506 14-4 A 1 See table 3 3 A 2 5 B 2 17 C 1 5 D 2 8 Normal A 1 See table 3 16 A 2 1 B 2 3 C 1 13 C 2 1 D 2 2 * Nm = number of chromosomes bearing the mutation. Login to comment
69 For instance, table 2 shows that mutations G542X, 621 + 1G-T, N1303K, 3659delC, R560T, and Ri 17H are associated with the same haplotypes, or with haplotypes probably derived from these by slippage of the DNA polymerase, as has been seen in patients from other European populations.7'0 Since those haplotypes are relatively rare in normal chromosomes (table 3), the constancy of the association has been explained as suggesting identity by descent of all chromosomes bearing the same mutations.9 Haplotype 16-7-17 is found in CF non-AF508 chromosomes more frequently than would be expected from its frequency in normal chromosomes (p<0 01, table 3), suggesting perhaps that another fairly common CF mutation may be associated with this haplotype in Ireland. Login to comment
38 Care Table 2 Haplotypes associated with the most common CF mutations in Ireland, as well as with Irish CF chromosomes with unidentified mutations and non-CF chromosomes Mutation NmlNcf % X/Kt Mt Microsatellite&#a7; No chrsll AF508 367/506 72-5 A 1 See table 3 2 B 2 116 C - 0 D 2 2 G551D 35/506 6-9 B 2 16-7-17 20 R117H 10/506 2-0 C 1 16-30-13 5 C 1 16-31-13 1 G542X 5/506 1-0 B 2 17-32-13 2 621 + 1G-T 4/506 0-8 B 2 21-7-17 2 B 2 21-31-13 2 R560T 4/506 0-8 D 2 16-7-17 1 D 2 16-31-17 1 1717-1G-A 3/506 0-6 C 1 16-32-13 2 A 2 17-32-13 1 N1303K 2/506 0-4 B 2 23-29-13 2 3659delC 2/506 0-4 C 1 16-35-13 2 AI507 2/506 0-4 ND ND R352Q 1/506 0-2 C 1 16-31-13 1 R553X 0/506 0 0 ND ND 1078delT 0/506 0.0 ND ND Total identified 435/506 85-6 Unidentified 71/506 14-4 A 1 See table 3 3 A 2 5 B 2 17 C 1 5 D 2 8 Normal A 1 See table 3 16 A 2 1 B 2 3 C 1 13 C 2 1 D 2 2 * Nm = number of chromosomes bearing the mutation. Login to comment

[hide] Genetic and clinical features of patients with cys... Thorax. 1995 Dec;50(12):1301-4. Gan KH, Geus WP, Bakker W, Lamers CB, Heijerman HG
Genetic and clinical features of patients with cystic fibrosis diagnosed after the age of 16 years.
Thorax. 1995 Dec;50(12):1301-4., [PMID:8553305]

Abstract [show]
BACKGROUND: Cystic fibrosis is usually diagnosed in childhood, but a number of patients are not diagnosed until adulthood. The aim of this study was to investigate whether patients diagnosed at an older age had a different genetic constitution, manifestations of disease, and prognosis from those diagnosed at an early age. METHODS: Clinical data and results of lung function tests and DNA analysis of 143 adult patients with cystic fibrosis were entered into a computerised database. Patients diagnosed before their 16th birthday (early diagnosis, ED) were compared with those diagnosed at 16 years of age or older (late diagnosis, LD). RESULTS: Mean age of diagnosis of the ED group was 4.6 years compared with 27.7 years for the LD group. Mean (SD) percentage predicted pulmonary function was better for the LD group than for the ED group: forced expiratory volume in one second (FEV1) 72.5 (31.1)% and 52.0 (24.8)%, and forced vital capacity (FVC) 89.8 (25.7)% and 71.9 (23.0)%, respectively. Colonisation with Pseudomonas aeruginosa was present in 70% of the ED group and 24% of the LD group. In the ED group 81% had pancreatic insufficiency compared with only 12% of the LD group. None of the LD group was homozygous for delta F508 compared with 58% of the ED group. In the LD group 72% were compound AF508 heterozygotes and 28% had two non-delta F508 mutations. CONCLUSIONS: Among this group of 143 adult patients with cystic fibrosis late diagnosis is caused mainly by delayed expression and mild progression of clinical symptoms. Late diagnosis is associated with milder pulmonary disease, less pancreatic insufficiency, and different cystic fibrosis mutations. Since mortality in cystic fibrosis depends on the progression of pulmonary disease, patients with a late diagnosis have a better prognosis than those diagnosed early.

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41 DNA was analysed for the following mutations: E60X, R117H, A455E, AI507, AF508, G542X, S549N, G550X, G551D, R553X, R560T, R1162X, S1251N, W1282X, N1303K, 621 + 1G-+T, 1717-1G--+A. These mutations represent 80% ofthe expected cystic fibrosis mutations in The Netherlands. Login to comment
65 Table 3 CFTR mutations in 278 chromosomes of adult cystic fibrosis patients Early diagnosis Late diagnosis (n= 118) (n=25) n % n % AF508s 175 74-2 18 36-0 A455Em 12 5-1 14 28-0 1717-15 6 2-5 1 2-0 G542X' 4 1-7 - W1282X1 3 1-3 - R553X' 1 04 1 2-0 S1251N 2 0-8 - N1303K' 1 0 4 - E60X 1 0-4 3 6-0 Not identified 31 13-2 13 26-0 Total 236 50 Mutations not found: RI 17H, AI507, S549N, G550X, G551D, R560T, R1162X, 621+1G-+T. Login to comment

[hide] Neonatal screening for cystic fibrosis: result of ... Hum Genet. 1995 Nov;96(5):542-8. Ferec C, Verlingue C, Parent P, Morin JF, Codet JP, Rault G, Dagorne M, Lemoigne A, Journel H, Roussey M, et al.
Neonatal screening for cystic fibrosis: result of a pilot study using both immunoreactive trypsinogen and cystic fibrosis gene mutation analyses.
Hum Genet. 1995 Nov;96(5):542-8., [PMID:8530001]

Abstract [show]
We have evaluated a two-tier neonatal cystic fibrosis (CF) screening of immunoreactive trypsinogen (IRT) followed by CFTR gene mutation analysis using a systematic scanning of exons 7, 10, and 11, and, if necessary, by direct DNA sequencing. Over an 18-month period we screened 32,300 neonates born in the western part of Britanny. The first tier, involving IRT screening at 3 days of age, utilizes a low elevation of the trypsinogen level (600 ng/ml), which is highly sensitive. The second tier, which corresponds to the exhaustive screening for mutations in three exons of the gene, is highly specific for this population (Britanny). The false positive rate is very low, and no false negatives have been reported to date. This strategy has allowed the identification of five novel alleles (V322A, V317A, 1806 del A, R553G, G544S).

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82 {17bi DI507 [ Y569X W846X 2789+5G->A ,' $492F i ] i I G551D 2622+1 G->A Y1092X 1717-1 G->A E827X A1067T G542X 2183 AA->G R1066H R560K 2184 ins A 3320,ins 5 R553G R1070W 1806 del A & 4005+1G->A W1282X ] i "- Exons Fig.2 Distribution of the different mutations (except AF508) of the CFTR gene in Brittany Table 1 Mutations and genotypes in newborns Genotypes of newborns Number Sweat test AF508/AF508 7 + > 90 AF508/1806 del A 1 + > 90 R553G/G551D 1 Borderline (60) AF508/G551D 1 + > 90 AF508/R1070W 1 40 AF508/G542X 1 + > 90 AF508/G149R 1 45 Total 13 Mutations found in heterozygote newborns AF508 31 R560K 1 1078 del T 1 G544S l G542X 1 V317A 1 R347H 1 V322A 1 Total 38 gene. Login to comment

[hide] Correlation of sweat chloride concentration with c... J Pediatr. 1995 Nov;127(5):705-10. Wilschanski M, Zielenski J, Markiewicz D, Tsui LC, Corey M, Levison H, Durie PR
Correlation of sweat chloride concentration with classes of the cystic fibrosis transmembrane conductance regulator gene mutations.
J Pediatr. 1995 Nov;127(5):705-10., [PMID:7472820]

Abstract [show]
OBJECTIVE: To compare differences in epithelial chloride conductance according to class of mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. METHODS: We evaluated the relationship between the functional classes of CFTR mutations and chloride conductance using the first diagnostic sweat chloride concentration in a large cystic fibrosis (CF) population. RESULTS: There was no difference in sweat chloride value value between classes of CFTR mutations that produce no protein (class I), fail to reach the apical membrane because of defective processing (class II), or produce protein that fails to respond to cyclic adenosine monophosphate (class III). Those mutations that produce a cyclic adenosine monophosphate-responsive channel with reduced conductance (class IV) were associated with a significantly lower, intermediate sweat chloride value. However, patients with the mutations that cause reduced synthesis or partially defective processing of normal CFTR (class V) had sweat chloride concentrations similar to those in classes I to III. CONCLUSION: Studies of differences in chloride conductance between functional classes of CFTR mutations provide insight into phenotypic expression of the disease.

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13 Abnormal function of the channel results in aber- cAMP CF CFTR NBF PI PS RT-PCR Cyclicadenosinemonophosphate Cysticfibrosis Cysticfibrosistransmembraneconductance regulator Nucleotidebindingfold Pancreaticinsufficiency Pancreaticsufficiency Restrictiontransferasepolymerasechainreaction rant chloride conductance across the apical membrane of epithelial cells in a variety of organs.l-5 Approximately 70% of the CF chromosomes harbor a three base-pair deletionre- 705 0 Wilschanski et al. The Journal of Pediatrics November 1995 I II Ill IV V Normal Nonsense Missense G542X Missense Missense Missense A455E Frameshift AA deletion G551D R117H Alternative 394delTT AF508 splicing Splice junction 3849+10kbC~T 1717-1G~A (a) (b) (c) (d) (e) (f) Figure. Login to comment
43 Defined mutations (each mutation cited in references 8, 23, and 24; numerals in parentheses indicate number of patients): Nonsense mutations-----class I: Frameshift mutations---class I: Splice site mutations-class I: Missense mutations---class HI: Missense mutations---class IV: Partially defective processing---class V: Alternative spficing-----classV: R1162X (3), Y1092X (3), G542X (21), Q552X (2), Q493X (2), w1282x (2), E1104X (1), R553X (6), E585X (l), (all PI) 3659delC (5), 2184delA (4), 4010de14 (1), 556delA (1), 3002delG (1) 3905insT (1), 4016insT (3), 1154insTC (l), 441delA (1), 2184insA (2), 1078delT (1), 4326delTC (3) (all PI) I717-1G--~A (4), 621+lG--*T (10), 711+IG--~T (3), 875+1G-+C (2), 3120+IG-~A (1) (18 PI, 2 PS) G551D (25), N1303K (7), R560T (8), I148T (1), G85E (3), A559T (1), L1077P (2), T1234V (1), (47 PI, 1 PS) R117H (10), R347H (3), R347P (1), D614G (1), S1251N (2), (all PS) P574H (2), A455E (2), (all PS) 3272-26A-+G (4), 3849+10KbC---~T (2), 3120G-+A (1), (all PS) analysis, we further grouped the patients according to the molecular consequences conferred by the CFTR alleles. Login to comment
109 More recently, a multicenter study reported no significant differences in sweat chloride levels in 79 compound heterozygotes carrying the mutations G55ID with AF508 (class III), in comparison with those homozygous for AF508.21 In addition, no significantdifferences in sweat chloride values could be detected between those who were homozygous for AF508 and those who had other common "severe" mutations, including the nonsense mutations (G542X, R553X, and W1282X), missense mutation (N1303K), and splice site mutations (621 + 1G--->Tand 1717 - 1G--~A).22 In the latter study there was a significant difference in sweat chloride concentration between a group heterozygous for the mild missense mutation (AF508/R117H) and the reference group (AF508/AF508).22 These data were limited by the range of mutations and were defined by genotype rather than functional class, but the results are in complete agreement with the findings of the present study. Login to comment
12 Abnormal function of the channel results in aber- cAMP CF CFTR NBF PI PS RT-PCR Cyclicadenosinemonophosphate Cysticfibrosis Cysticfibrosistransmembraneconductance regulator Nucleotidebindingfold Pancreaticinsufficiency Pancreaticsufficiency Restrictiontransferasepolymerasechainreaction rant chloride conductance across the apical membrane of epithelial cells in a variety of organs.l-5 Approximately 70% of the CF chromosomes harbor a three base-pair deletionre- 705 0 Wilschanski et al. The Journal of Pediatrics November 1995 I II Ill IV V Normal Nonsense Missense G542X Missense Missense Missense A455E Frameshift AA deletion G551D R117H Alternative 394delTT AF508 splicing Splice junction 3849+10kbC~T 1717-1G~A (a) (b) (c) (d) (e) (f) Figure. Login to comment

[hide] CFTR gene variant for patients with congenital abs... Am J Hum Genet. 1995 Oct;57(4):958-60. Zielenski J, Patrizio P, Corey M, Handelin B, Markiewicz D, Asch R, Tsui LC
CFTR gene variant for patients with congenital absence of vas deferens.
Am J Hum Genet. 1995 Oct;57(4):958-60., [PMID:7573058]

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21 More recently, CFTR alleles Letters to the Editor Table I CFTR Mutations Detected in the CBAVD Patients Number of Percentage Genotype Patients of Total AF508 IVS8/ST 16 W1282X IVS8/5T 9 AF508 R117H(7T) 4 N1303K IVS8/5T 2 IVS8/ST IVS8/5T 2 AF508 R117C 1 AF508 D1152H 1............ 1 58.6 AF508 S50Y 1 R553X R117H(7T) 1 R117H(7T) R117H(7T) 1 G542X IVS8/5T 1 1717-1G-+A IVS8/ST 1 1525-1G-A IVS8/5T 1 IVS8/5T Unknown 4 AF508 Unknown 4. Login to comment
22 W1282X Unknown 2 R553X Unknown ............ 20.0 4173delC Unknown1 1 D614G Unknown 1 1716+12T- C Unknown 1 J Unknown Unknown ............ .15 21.4 NOTE.-The known CFTR mutations screened included AF508, G542X, GSS1D, N1303K, R553X, W1282X, AI507, 1717-1G-A, R560T, S549N, 621+1G--T, and R117H. Login to comment

[hide] Screening Young syndrome patients for CFTR mutatio... Am J Respir Crit Care Med. 1995 Oct;152(4 Pt 1):1353-7. Friedman KJ, Teichtahl H, De Kretser DM, Temple-Smith P, Southwick GJ, Silverman LM, Highsmith WE Jr, Boucher RC, Knowles MR
Screening Young syndrome patients for CFTR mutations.
Am J Respir Crit Care Med. 1995 Oct;152(4 Pt 1):1353-7., [PMID:7551394]

Abstract [show]
Young syndrome is characterized by obstructive azoospermia associated with chronic sinobronchial disease of an infectious nature, but normal sweat-gland and pancreatic function as well as normal nasal potential differences. Congenital bilateral absence of the vas deferens (CBAVD) in some patients arises from mutations within the cystic fibrosis (CF) transmembrane regulator (CFTR) gene. Because of some similarities between Young syndrome, CF, and CBAVD, we evaluated 13 patients with Young syndrome, including screening for more than 30 different mutations within the CFTR gene. The mean age of the patients was 43 yr (range, 32 to 50 yr), and all were of northern European extraction. The sweat chloride concentration was normal in all patients (mean = 29 mEq/L; range, 8 to 43 mEq/L). Most had intermittent bronchial and sinus infections, but none was chronically colonized with Staphylococcus aureus or Pseudomonas aeruginosa. The FEV1 was normal or only mildly reduced in most patients (mean = 74%; range, 48 to 100% predicted). Of 26 Young syndrome chromosomes, we identified one with the recognized CF mutation delta F508. The incidence of CFTR mutations (1 in 26) did not differ significantly from the expected carrier frequency in this population. In summary, it is unlikely that the typical Young syndrome patient has a clinical disease associated with CFTR mutation on both alleles.

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78 Of the 13 Young syndrome patients, we identified one (Patient 5) who was het- CBAVD Dl152H D1270N G576A* R75Q* P67L Rl17H 3849 + 10 KB C > T G551S Rl17H Pancreatic Sufficient, Moderate Pulmonary Symptoms, Normal Sweat Chloride Concentrations Pancreatic Sufficient, Moderate Pulmonary Symptoms R347P 2789 + 5 G > A R334W G85E R347H R347L Rl17H G91R A455E S945L Y563N Q1291H R297Q R352Q L1065P 3850-3 T > G F1286S 3849 + 10 KB C > T TABLE 1 CFTR MUTATION SCREENING PANEL Severe M508 G551D R553X N1303K W1282X G542X 1717-1 G > A ~1507 R560T 3659deiC 621 + 1 G > T S549N TABLE 2 CLINICAL FEATURES OF YOUNG SYNDROME PATIENTS Patient Age Sweat CI- FEV, Paranasal Sputum No. Login to comment

[hide] Mutation analysis of ten exons of the CFTR gene in... Hum Genet. 1995 Sep;96(3):364-6. Kanavakis E, Tzetis M, Antoniadi T, Traeger-Synodinos J, Doudounakis S, Adam G, Matsaniotis N, Kattamis C
Mutation analysis of ten exons of the CFTR gene in Greek cystic fibrosis patients: characterization of 74.5% of CF alleles including one novel mutation.
Hum Genet. 1995 Sep;96(3):364-6., [PMID:7544320]

Abstract [show]
To initiate the complete characterization of mutations in the CFTR gene in Greek cystic fibrosis (CF) patients, we screened 184 patients for six relatively common mutations (delta F 508, G542X, G551D, 621 + 1 G-->T, N1303K, W1282X) using allele-specific hybridization and, in addition, analyzed exons 4, 5, 7, 8, 10, 11, 17b, 19, 20 and 21 using the method of denaturing gradient gel electrophoresis (DGGE). Six mutations accounted for 65.9% of the CF alleles in Greek patients, of which the delta F 508 mutation had a frequency of 52.7%. A further 15 previously described mutations accounted for another 8.3% CF alleles and one previously undescribed mutation (3272-4A-->G) was found in one chromosome. The W1282X mutation was not detected at all. Thus, so far, we have identified 21 mutations in the CFTR gene in Greek CF patients, accounting for 74.5% of the CF alleles.

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0 Hum Genet (1995) 96:364-366 9 Springer-Verlag 1995 Emmanuel Kanavakis 9Maria Tzetis 9Thalia Antoniadi Joanne Traeger-Synodinos - Stavros Doudounakis George Adam 9Nikos Matsaniotis 9Christos Kattamis Mutation analysis of ten exons of the CFTR gene in Greek cystic fibrosis patients: characterization of 74.5% of CF alleles including one novel mutation Received: 15 November 1994 / Revised: 20 February 1995 Abstract To initiate the complete characterization of mutations in the CFTR gene in Greek cystic fibrosis (CF) patients, we screened 184 patients for six relatively common mutations (zXF 508, G542X, G551D, 621+1 G-)T, N 1303K, W 1282X) using allele-specific hybridization and, in addition, analyzed exons 4, 5, 7, 8, 10, 11, 17b, 19, 20 and 21 using the method of denaturing gradient gel electrophoresis (DGGE). Login to comment
10 Allele-specific hybridization (ASO) The regions encompassing the mutations of interest (AF 508, G542X, G551D, 621+1 G ~ T, N1303K and WI282X) were amplified by the polymerase chain reaction (PCR) as described in Zielenski et al. (1991). Login to comment
15 Results and discussion Studies in southern European populations disclosed that the most frequent CF alleles are AF 508, G542X, G551 D, Fig. 1 Sequencing of the region of the cystic fibrosis transmembrane conductance regulator (CFTR)gene showing the A---~Gtran- sition in intron 17a, 4 bp 5" to the acceptor splice site (3272-4A--*G) 621+1 G-+T, W1282X, and N1303K (Tsui 1992; Abeliovich et al. 1992; Casals et al. 1993; Kazazian 1994). Login to comment
26 (ASO allele-specific hybridization, DGGE denaturing gradient gel electrophoresis, Seq direct genomic sequencing) Mutation Method Number of Percentage positive alleles AF 508 621+lG---~T G542X N1303K Rll7H R334W 574delA 3272-26A---~G R1158X 1677delTA R1070Q G551D G 1244V R553X 444delA 3849+4A---)G 457-TAT--)G 4010delTATT 4040delA W361R 3272-4A--)Ga Known Unknown Total number alleles ASO, DGGE ASO, DGGE ASO, DGGE ASO, DGGE DGGE, Seq DGGE, Seq DGGE, Seq DGGE, Seq DGGE, Seq DGGE DGGE, Seq ASO, DGGE DGGE, Sec DGGE, Sec DGGE, Sec DGGE, Sec DGGE, Sec DGGE, Sec DGGE, Sec DGGE, Sec DGGE, Sec 194 52.7 17 4.6 16 4.3 14 3.8 4 1.1 4 1.1 3 0.8 3 0.8 3 0.8 3 0.8 2 0.5 2 0.5 1 0.3 1 O.3 1 0.3 1 0.3 1 0.3 1 0.3 1 0.3 1 0.3 1 0.3 274 74.5% 94 25.5% 368 aNovel mRNA splicing mutations Acknowledgements This work was supported by the Greek Ministry of Health, the Hellenic Cystic Fibrosis Association and the Bodosakis Foundation. Login to comment

[hide] Search for mutations in pancreatic sufficient cyst... Hum Genet. 1995 Sep;96(3):312-8. Brancolini V, Cremonesi L, Belloni E, Pappalardo E, Bordoni R, Seia M, Russo S, Padoan R, Giunta A, Ferrari M
Search for mutations in pancreatic sufficient cystic fibrosis Italian patients: detection of 90% of molecular defects and identification of three novel mutations.
Hum Genet. 1995 Sep;96(3):312-8., [PMID:7544319]

Abstract [show]
A cohort of 31 cystic fibrosis patients showing pancreatic sufficiency and bearing an unidentified mutation on at least one chromosome was analyzed through denaturing gradient gel electrophoresis of the whole coding region of the cystic fibrosis transmembrane conductance regulator gene, including intron-exon boundaries. Three new and 19 previously described mutations were detected. The combination of these with known mutations detected by other methods, allowed the characterization of mutations on 56/62 (90.3%) chromosomes. Among those identified, 17 can be considered responsible for pancreatic sufficiency, since they were found in patients carrying a severe mutation on the other chromosome. Among these presumed mild mutations, eight were detected more than once, R352Q being the most frequent in this sample (4.83%). Intragenic microsatellite analysis revealed that the six chromosomes still bearing unidentified mutations are associated with five different haplotypes. This may indicate that these chromosomes bear different mutations, rarely occurring among cystic fibrosis patients, further underlying the molecular heterogeneity of the genetic defects present in patients having pancreatic sufficiency.

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21 We have studied a cohort of 31 Italian patients with PS using firstly traditional methods to screen for mutations which predominate in the Italian population [AF508, G542X (Kerem et al. 1990b), N1303K (Osborne et al. 1991), 1717-1G--~A (Guillermit et al. 1990) and W1282X (Vidaud et al. 1990)], secondly denaturing gradient gel electrophoresis (DGGE) analysis of the entire coding part of the CFTR gene, thirdly testing for the presence of the two mutations [1811+ 1.2kbA--+G (Chillon et al., personal communication to the CF Genetic Analysis Consortium) and 3849+10kbC-+T (Highsmith et al. 1994)] located in non-coding portions of the gene, which were not detectable by DGGE, and finally intragenic microsatellites [IVS8/GT (Morral et al. 1991), IVS17b/TA and IVS17b/CA (Zielenski et al. 1991b)] mapping. Login to comment
33 Mutation detection Screening for mutations which predominate in our population: AF508, G542X, N1303K, 1717-1G---~A,W1282X, and of the two intronic mutations 3849+10kbC--+T and 1811+l.2kbA---~G was carried out as previously described (Ballabio et al. 1990;Friedman et al. 1991; Cremonesi et al. 1991; Vidaud et al. 1990; Highsmith et al. 1994; Chillon et al., personal communication to the CF Genetic Analysis Consortium). Login to comment
38 Table 1 PCR primers and conditions for denaturing gradient gel electrophoresis analysis of exons 1 and 9 (for exon 9 two different PCR products were analyzed under different conditions in order to detect all possible base changes) Exon PCR primers 5"---~3" Annealing Denaturing Electrophoresis temperature (~C) range time (h) 1 TAGGTCTTTGGCATTAGGAG 54 40%-90% 6 (55GC)CCAAACCCAACCCATA CACAC (35GC)TGAAAATATCTGACAA 45 10%-60% 6 ACTC CCTTCCAGCACTACAAACTA (37GC)AACAGGGATTTGGGG 50 10%-60% 6 AATTA AACTAGAAAAAAAAAGAGA Results Screening for predominant mutations A preliminary screening for mutations being predominant in our population was carried out in our series of patients showing PS, revealing the presence of AF508 on 19 (30.6%) chromosomes, 1717-1G---~A and G542X on 2 (3.22%). Login to comment
39 In a previous study the overall frequencies of mutations in the whole sample population referring to our Center had been evaluated on a sample of 1018 CF chromosomes having the following frequencies: AF508:516 (50.7%) chromosomes, G542X: 52 (5.1%), 1717-1G--->A: 41 (4.0%), N1303K: 35 (3.4%), WI282X: 14 (1.4%) (our unpublished results). Login to comment
70 (UN yet unidentified mutation) Patient Genotype after Genotype at the end number preliminary screening of the analysis UN/UN M1V/4382delA 1717-1G---~A/UN 1717-1G---~A/R1066H AF508/UN AF508/D579G UN/UN M1V/UN AF508/UN AF508/UN UN/UN T338I/R1158X UN/UN G85E/71 I+5G---~A UN/UN D1152H/UN AF508/UN AF508/UN AF508/UN AF508/3849+ 10kbC---~T UN/UN 711+3A---~G/UN AF508/UN AF508/F1052V UN/UN R352Q/W57G UN/UN 1898+3A----~G/UN AF508/UN AF508/711+5G--~A G542X/UN G542X/DI 152H AF508/UN AF508/E193K 1717-1G---~A/UN 1717-1G---~A/2789+5A---)G AF508/UN AF508/G1349D AF508/UN AF508/G85E AF508/UN AF508/R347P AF508/UN AF508/R352Q AF508/UN AF508/R352Q AF508/UN AF508/S549N G542X/UN G542X/R1066H AF508/UN AF508/T338I AF508/UN AF508/R334W AF508/UN AF508/R334W AF508/UN AF508/S1251N AF508/UN AF508/R1066C AF508/UN AF508/D579G results) while the remaining three haplotypes had been found in association with other rare mutations, which were excluded by DGGE analysis in these patients (Table 3). Login to comment
85 In total, among the mutations detected in our PS patients, 17 (D579G, E193K, F1052V, 711+5G---~A, G1349D, G85E, R347R R352Q, $549N, 2789+5A---~G, D1152H, R1066H, R334W, T338I, 3849+10kbC---~T, S1251N, R1066C) have been detected in compound heterozygosity with a mutation already classified as severe (AF508, 1717-1G--~A, G542X) and thus can be considered as presumably mild. Login to comment
89 The results of this search showed, as expected, a different distribution of classical severe mutations (AF508, G542X, 1717-1G-+A, N1303K, W1282X) in patients with PS as compared to the overall CF population (37.1% against 67.4%). Login to comment
93 Screening for only eight presumed mild mutations (R352Q, R1066H, G85E, Dl152H, 711+5G---~A, T338I, R334W and D579G) in addition to the predominant four severe mutations (AF508, G542X, 1717-1G-+A and N1303K), would have allowed the identification of 64.5% of the molecular defects in our patients having PS. Login to comment

[hide] Identification of two novel mutations (296 + 1G-C ... Mol Cell Probes. 1995 Aug;9(4):283-5. Tzetis M, Kanavakis E, Antoniadi T, Traeger-Synodinos J, Doudounakis S, Adam G, Kattamis C
Identification of two novel mutations (296 + 1G-C and A46D) in exon 2 of the CFTR gene in Greek cystic fibrosis patients.
Mol Cell Probes. 1995 Aug;9(4):283-5., [PMID:7477025]

Abstract [show]
Two novel CFTR mutations were detected in Greek cystic fibrosis patients. One was a missense mutation, A46D, and the other a splice mutation, 296 + 1G-C. Neither was detected on normal chromosomes.

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4 Since the identification of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein and the initial characterization of the predominant mutation in the Caucasian population, AF508,'-~ more than 400 other mutations have been reported.4 Mutations have been described in all of the exons and neighbouring intronic sequences of the CFTR gene, with highest concentration of the more common mutations occuring in exons coding for NBF1.s To determine the type and frequency of cystic fibrosis (CF) mutations in the Greek population we screened 184 patientsfor the most common mutations (,4F508, G542X, G551D, 621 +IG>T, N1303K and W1282X) by ASO hybridization and then proceeded to analyseexons2, 4, 5, 6a, 6b, 7, 8, 9, 10, 11,17b, 19, 20, and 21 by denaturing gradient gel electrophoresis (DGGE) asdescribed.6The DNA sequence of samples showing a shift in mobility was determined after as- symetric PCR as describedZ The six most common mutations accounted for 66-9% of the CF alleles in Greek patients, of which /IF508 had a frequency of 52.7%. Login to comment
8 One was a compound heterozygote A46D/G542X and the other a compound heterozygote with the 621 +IG-T mutation. Login to comment
12 1 2 3 CFTR genotype A46D/G542X A46D/621 +IG-T 296+1G-C/ unknown Sex Female Female Male Date of birth 1983 1965 1967 Age at diagnosis 4 years 4.5 years 26 years Sweat test chloride conc. Login to comment

[hide] A cystic fibrosis mutation associated with mild lu... N Engl J Med. 1995 Jul 13;333(2):95-9. Gan KH, Veeze HJ, van den Ouweland AM, Halley DJ, Scheffer H, van der Hout A, Overbeek SE, de Jongste JC, Bakker W, Heijerman HG
A cystic fibrosis mutation associated with mild lung disease.
N Engl J Med. 1995 Jul 13;333(2):95-9., [PMID:7539891]

Abstract [show]
BACKGROUND: Cystic fibrosis is the most common lethal autosomal recessive disorder among whites. Among Dutch patients with cystic fibrosis, delta F508 is the most common mutation and A455E the second most common mutation of the cystic fibrosis transmembrane conductance regulator gene on chromosome 7. A455E is associated with preserved pancreatic function and residual secretion of chloride across membranes. We investigated whether it is also associated with less severe pulmonary disease in patients with cystic fibrosis. METHODS: A total of 33 patients with compound heterozygosity for the A455E mutation were matched according to age and sex with patients who were homozygous for the delta F508 mutation. The pairs were analyzed with respect to the following outcome variables: age at diagnosis, pulmonary-function values, and the frequency of pseudomonas colonization, pancreatic sufficiency, and diabetes mellitus. RESULTS: Cystic fibrosis was diagnosed at a later age in the patients with the A455E mutation than in the delta F508 homozygotes (mean age at diagnosis, 15.0 vs. 3.1 years; P < 0.001). Fewer patients with the A455E mutation had pancreatic insufficiency (21.2 percent vs. 93.9 percent, P < 0.001), and none had diabetes mellitus (0 percent vs. 27.3 percent, P = 0.004). Forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) were significantly higher in the patients with the A455E mutation (mean FEV1, 73.9 percent of the predicted value vs. 54.3 percent of the predicted value; P = 0.002; mean FVC, 88.7 percent of the predicted value vs. 76.3 percent of the predicted value; P = 0.04). Fewer patients with the A455E mutation were colonized with Pseudomonas aeruginosa (33.3 percent vs. 60.6 percent, P = 0.02). CONCLUSIONS: A455E is a common mutation causing cystic fibrosis in the Netherlands. Although several mutations are known to be associated with less severe pancreatic disease, our findings demonstrate a correlation between the A455E mutation and mild pulmonary disease. Because mortality in this disease depends primarily on the progression of pulmonary disease, patients with the A455E mutation have a better prognosis than patients who are homozygous for the delta F508 mutation.

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34 Among the patients screened, 151 were found to be homozygous for the ⌬F508 mutation and 39 were found to have compound heterozygosity for the A455E mutation. Login to comment
35 In the A455E compound heterozygotes, the following mutations were found on the other allele: ⌬F508 (27 patients), 1717-1G→A (4 patients), E60X (4 patients), G542X (2 patients), R553X (1 patient), and an unknown mutation (1 patient). Login to comment
69 †The following genotypes were identified: A455E/⌬F508 (25 patients), A455E/E60X (4), A455E/G542X (2), A455E/R553X (1), and A455E/1717-1G→A (1). Login to comment
70 ߤThe following genotypes were identified: A455E/F508 (25 patients), A455E/E60X (4), A455E/G542X (2), A455E/R553X (1), and A455E/1717-1G࢐A (1). Login to comment

[hide] Double mutant alleles: are they rare? Hum Mol Genet. 1995 Jul;4(7):1169-71. Savov A, Angelicheva D, Balassopoulou A, Jordanova A, Noussia-Arvanitakis S, Kalaydjieva L
Double mutant alleles: are they rare?
Hum Mol Genet. 1995 Jul;4(7):1169-71., [PMID:8528204]

Abstract [show]
The presence of two different mutations carried by the same CF allele has been demonstrated in four out of 44 Bulgarian CF patients during a systematic search of the entire coding sequence of the CFTR gene. Two of the double mutant alleles include one nonsense and one missense mutation and although the nonsense mutation can be considered to be the main defect, the amino acid substitutions are good candidates for disease-causing mutations as well. One double mutant carries two missense mutations whose contribution to the CF phenotype is difficult to evaluate. The findings suggest that double mutant alleles may be more common than expected and could account for some of the problems in phenotype-genotype correlations. Such alleles may have important implications for molecular diagnosis and genetic counselling.

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44 S912L and G1244V A C->T transition at nucleotide position 2867 in exon 15 and a G->T transversion at position 3863 in exon 20 of the CFTR gene were found in a patient who also carries G542X. Login to comment

[hide] High incidence of delta I507 mutation of the CFTR ... J Med Genet. 1995 Jul;32(7):577. Mittre H, Leymarie P, Leporrier N
High incidence of delta I507 mutation of the CFTR gene in a limited area of the north west of France.
J Med Genet. 1995 Jul;32(7):577., [PMID:7562977]

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5 The other most common mutations observed in our study with a frequency higher than 1% were: G551D (2 4%), G542X (2-4%), 574delA (1-2%), 3659delC (1-2%), and N1303K (1-2%). Login to comment

[hide] The impact of newborn screening on cystic fibrosis... J Med Genet. 1995 Jul;32(7):537-42. Balnaves ME, Bonacquisto L, Francis I, Glazner J, Forrest S
The impact of newborn screening on cystic fibrosis testing in Victoria, Australia.
J Med Genet. 1995 Jul;32(7):537-42., [PMID:7562966]

Abstract [show]
Newborn screening for cystic fibrosis (CF) by examining the levels of immunoreactive trypsinogen was introduced in Victoria in 1989. This was modified by the addition of testing for the common CF gene mutation, delta F508, in 1990. Problems with the first newborn screening protocol were overcome with the addition of the DNA test as there was no need to contact the majority of families, there was a reduced number of sweat tests, and less anxiety was experienced by parents. The mode of diagnosis changed from failure to thrive, steatorrhoea, rectal prolapse, and family history to diagnosis through newborn screening. Newborn screening dramatically reduced the time of diagnosis of CF to approximately six weeks or less in the majority of cases. Since the introduction of newborn screening, the uptake of prenatal diagnosis in CF families has increased two and a quarter fold.

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121 Three hundred and fifty six people (to 30.9.93) have been tested for AF508 and three exon 11 mutations: G542X, G551D, and R553X. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Lancet. 1995 Jun 17;345(8964):1578. Jarvi K, Zielenski J, Wilschanski M, Durie P, Buckspan M, Tullis E, Markiewicz D, Tsui LC
Cystic fibrosis transmembrane conductance regulator and obstructive azoospermia.
Lancet. 1995 Jun 17;345(8964):1578., [PMID:7540706]

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18 We screened for 6 CF mutations (AF508, G551D, G542X, W1283X, N1303K, R117H), representing the most common mutations detected previously in men with CBAVD, and determined the CFTR gene variants of R75Q and T tract length of intron 8. Login to comment

[hide] CFTR haplotype analysis reveals genetic heterogene... Am J Hum Genet. 1995 Jun;56(6):1359-66. Rave-Harel N, Madgar I, Goshen R, Nissim-Rafinia M, Ziadni A, Rahat A, Chiba O, Kalman YM, Brautbar C, Levinson D, et al.
CFTR haplotype analysis reveals genetic heterogeneity in the etiology of congenital bilateral aplasia of the vas deferens.
Am J Hum Genet. 1995 Jun;56(6):1359-66., [PMID:7539210]

Abstract [show]
Congenital bilateral aplasia of the vas deferens (CBAVD) was suggested to be a mild form of cystic fibrosis (CF). Mutation analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in males with CBAVD revealed that in some males CBAVD is caused by two defective CFTR alleles. The genetic basis of CBAVD in the other males and its association with CF remained unclear. We undertook this study to test the hypothesis of commonality of CBAVD and CF by haplotype analysis, in the CFTR locus, of males suffering from CBAVD and of their families. According to the hypothesis of commonality of CBAVD and CF, two brothers with CBAVD are expected to carry the same two CFTR alleles, while their fertile brothers are expected to carry at least one different allele. Eleven families were studied, of which two families, with unidentified CFTR mutations, did not support this hypothesis. In these families two brothers with CBAVD inherited different CFTR alleles. Their fertile brothers inherited the same CFTR alleles as their brothers with CBAVD. These results provide evidence for genetic heterogeneity in CBAVD. Though in some families CBAVD is associated with two CFTR mutations, we suggest that in others it is caused by other mechanisms, such as mutations at other loci or homozygosity or heterozygosity for partially penetrant CFTR mutations.

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38 The entire studied group of males with CBAVD was tested for 16 CFTR mutations, using DNA-PCR amplification (Saiki et al. 1985, 1988), followed by specific tests as described elsewhere: AF508 (Rommens et al. 1990); W1282X (Shoshani et al. 1992a); G542X, S549R, S549I, and 1717-1G-+A, by direct sequencing of exon 11, using oligonucleotide primers (Zielenski et al. 1991b); N1303K (Osborne et al. 1991); 3849+10Kb C&-T (Highsmith et al. 1994); W1089X and 4010delTATT (Shoshani et al. Login to comment
58 Mutation Analysis Fourteen CF-R mutations that were elsewhere identified among the Israeli CF patient population (Kerem et al. 1994) were analyzed: W1282X, AF508, N1303K, G542X, 3849+10Kb C--T, S549R, S549I, W1089X, 4010delTATT, G85E, 1717-1G--A, D1152H, 405+1G--A, and Q359K1T360K. Login to comment
59 Mutation Analysis Fourteen CF-R mutations that were elsewhere identified among the Israeli CF patient population (Kerem et al. 1994) were analyzed: W1282X, AF508, N1303K, G542X, 3849+10Kb C--T, S549R, S549I, W1089X, 4010delTATT, G85E, 1717-1G--A, D1152H, 405+1G--A, and Q359K1T360K. Login to comment

[hide] Structural analysis of CFTR gene in congenital bil... Clin Chem. 1995 Jun;41(6 Pt 1):833-5. Jezequel P, Dorval I, Fergelot P, Chauvel B, Le Treut A, Le Gall JY, Le Lannou D, Blayau M
Structural analysis of CFTR gene in congenital bilateral absence of vas deferens.
Clin Chem. 1995 Jun;41(6 Pt 1):833-5., [PMID:7539342]

Abstract [show]
Congenital bilateral absence of the vas deferens (CBAVD) is found in most males with cystic fibrosis (CF), but this malformation can be observed without any pulmonary or digestive features. We have analyzed 13 exons of the CF gene in a cohort of 25 CBAVD patients. Among the 50 chromosomes studied, 24 mutations were identified: delta F508 (14 cases), R117H (7 cases), R1070W (2 cases), 621 + 1 G --> T (1 case), and A1067V (1 case). Except for delta F508, the most frequent mutations (R117H, R1070W) were not observed in the CF group (109 patients) studied in our laboratory. We discuss the significance of these results.

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46 SF508/ SF508 SF508 / N1303K AF508/ G551D SF508 / 3272-26G--*A SF508 / 1078 delT F508/Y1092X SF508 / Ai507 F5O8 / G542X SF508 / 621+1G-T F508 / 3898 insC SF508 / 574 delA AF508 / G85E SF508 / W1282X N1303K/F311L G551D/F311L R553X I? Login to comment
47 N1303K/? Login to comment
72 However, none of the most frequent mutations described in our CF population (G551D, N1303K, 3272-26G--A, G542X) was identified in our CBAVD cohort. Login to comment
73 However, none of the most frequent mutations described in our CF population (G551D, N1303K, 3272-26G--A, G542X) was identified in our CBAVD cohort. Login to comment

[hide] Mutations in the cystic fibrosis gene in patients ... N Engl J Med. 1995 Jun 1;332(22):1475-80. Chillon M, Casals T, Mercier B, Bassas L, Lissens W, Silber S, Romey MC, Ruiz-Romero J, Verlingue C, Claustres M, et al.
Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens.
N Engl J Med. 1995 Jun 1;332(22):1475-80., [PMID:7739684]

Abstract [show]
BACKGROUND: Congenital bilateral absence of the vas deferens (CBAVD) is a form of male infertility in which mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been identified. The molecular basis of CBAVD is not completely understood. Although patients with cystic fibrosis have mutations in both copies of the CFTR gene, most patients with CBAVD have mutations in only one copy of the gene. METHODS: To investigate CBAVD at the molecular level, we have characterized the mutations in the CFTR gene in 102 patients with this condition. None had clinical manifestations of cystic fibrosis. We also analyzed a DNA variant (the 5T allele) in a noncoding region of CFTR that causes reduced levels of the normal CFTR protein. Parents of patients with cystic fibrosis, patients with types of infertility other than CBAVD, and normal subjects were studied as controls. RESULTS: Nineteen of the 102 patients with CBAVD had mutations in both copies of the CFTR gene, and none of them had the 5T allele. Fifty-four patients had a mutation in one copy of CFTR, and 34 of them (63 percent) had the 5T allele in the other CFTR gene. In 29 patients no CFTR mutations were found, but 7 of them (24 percent) had the 5T allele. In contrast, the frequency of this allele in the general population was about 5 percent. CONCLUSIONS: Most patients with CBAVD have mutations in the CFTR gene. The combination of the 5T allele in one copy of the CFTR gene with a cystic fibrosis mutation in the other copy is the most common cause of CBAVD: The 5T allele mutation has a wide range of clinical presentations, occurring in patients with CBAVD or moderate forms of cystic fibrosis and in fertile men.

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74 OF PATIENTS POLYT GENOTYPE† ⌬F508/R668C ⌬F508/D1152H ⌬F508/D1270N ⌬F508/R75L ⌬F508/R117H ⌬F508/L206W ⌬F508/R258G ⌬F508/S1235R ⌬F508/R347H ⌬F508/R347H R117H/G1349D R117H/712-1G192;T G149R/R668C R347H/R1066H R553X/R668C R1070W/2869insG ⌬F508/- G542X/- W1282X/- R334W/- K1060T/- R1162X/- N1303K/- A800G/- ⌬F508/- ⌬F508/- ⌬F508/- ⌬E115/- R117H/- R347H/- G542X/- R553X/- 1677delTA/- 2184delA/- 2789ϩ5G→Α/- S1235R/- W1282X/- -/- -/- -/- -/- 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 22 4 3 1 1 1 1 1 7 1 1 1 1 2 1 1 1 1 1 1 1 3 3 1 19 9T/7T 9T/7T 9T/7T 9T/7T 9T/7T 9T/9T 9T/7T 9T/7T 9T/7T 9T/9T 7T/7T 7T/9T 9T/7T 9T/7T 7T/7T 7T/7T 9T/5T 9T/5T 7T/5T 7T/5T 7T/5T 7T/5T 9T/5T 5T/5T 9T/7T 9T/9T 7T/7T 7T/7T 7T/7T 9T/7T 9T/7T 7T/7T 7T/7T 7T/7T 7T/7T 7T/9T 7T/7T 9T/5T 7T/5T 5T/5T 7T/7T -/- 3 7T/9T *Data were obtained from the Spanish population analyzed in this study. Login to comment
101 Four fathers who were carriers of cystic fibrosis had one CFTR gene with the 5T allele and the other with a severe cystic fibrosis mutation (G542X, N1303K, 1812-1G→A, or 936delTA). Login to comment

[hide] Evaluation of repeat administration of a replicati... Hum Gene Ther. 1995 May;6(5):667-703. Crystal RG, Mastrangeli A, Sanders A, Cooke J, King T, Gilbert F, Henschke C, Pascal W, Herena J, Harvey BG, et al.
Evaluation of repeat administration of a replication deficient, recombinant adenovirus containing the normal cystic fibrosis transmembrane conductance regulator cDNA to the airways of individuals with cystic fibrosis.
Hum Gene Ther. 1995 May;6(5):667-703., [PMID:7578402]

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112 G55 ID, S5491, A455E, and G542X account for 10-20% of ttie non-delta F508 mutations (33). Login to comment
116 "NuU" mutations, including frameshift, nonsense, and splicing mutations, have been found in compound heterozygotes [e.g., G542X/S1255X or R553X/W1316X]. Login to comment

[hide] Fluorescent multiplex microsatellites used to iden... Hum Genet. 1995 Apr;95(4):462-4. Hughes D, Hill A, Redmond A, Nevin N, Graham C
Fluorescent multiplex microsatellites used to identify haplotype associations with 15 CFTR mutations in 124 Northern Irish CF families.
Hum Genet. 1995 Apr;95(4):462-4., [PMID:7535745]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator gene contains three highly informative microsatellites; IVS8CA, IVS17BTA and IVS17BCA. Fluorescent multiplexes of these microsatellites were assayed in 124 CF families carrying 15 different CFTR mutations, from N. Ireland.

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23 Each mutation that has been found more than once is linked to a single haplotype, except AF508 and G542X. Login to comment
25 Nine haplotypes having specific CF mutations are not present in normal chromosomes Mutation Alleles tested (%) AF508 41 (34.2) 32 (26.7) 22 (18.3) G551D 16 R560T 08 621 + 1G > T 08 Rll7H 05 G542X 03 02 E60X 02 M507 02 R297Q 01 R553X 01 3849 G > A 01 N1303K 01 3659delC 01 557delT 01 Q2X 01 Frequency Haplotype of mutation in % 8AC 17AT 17AC 463 % in normal chromosomes 58.0 23 31 13 - 17 32 13 01 17 31 13 - 4.0 16 07 17 03 2.5 16 07 17 03 1.7 21 31 13 - 2.1 16 30 13 16 1.7 23 33 13 01 22 31 13 - 0.6 16 31 13 03 0.8 17 07 17 08 0.2 17 07 17 08 0.2 17 58 13 - 0.2 16 31 14 - 0.4 23 31 13 - 0.2 16 35 13 03 0.2 15 29 13 - 0.2 23 34 13 01 Table 2 Frequent haplotypes generated from normal and uncharacterised CF chromosomes in N. Ireland. Login to comment

[hide] Analysis of the complete coding region of the CFTR... Hum Genet. 1995 Apr;95(4):397-402. Bonizzato A, Bisceglia L, Marigo C, Nicolis E, Bombieri C, Castellani C, Borgo G, Zelante L, Mastella G, Cabrini G, et al.
Analysis of the complete coding region of the CFTR gene in a cohort of CF patients from north-eastern Italy: identification of 90% of the mutations.
Hum Genet. 1995 Apr;95(4):397-402., [PMID:7535742]

Abstract [show]
A complete coding-region analysis on 225 cystic fibrosis (CF) chromosomes from a cohort that includes all the affected subjects born in two North-Eastern Italian regions over eight years was performed. In a previous study, we identified mutations on 166/225 (73.8%) CF chromosomes after screening for 62 mutations. To characterise the remaining 59 CF chromosomes, we carried out automated direct DNA sequencing (exons 9 and 13), RNA single-strand conformation polymorphism (exons 1-8 and 10-12) and denaturing gradient gel electrophoresis (exons 14a-24) of the 27 exons and flanking regions of the CF transmembrane conductance regulator gene. We identified 22 mutations, four of which are novel, viz. 711 + 5G-->A, R709X, 3132delTG and 2790-2A-->G, and we characterised 90.2% (203/225) of the CF chromosomes. Taking advantage of the homogeneity of the sample, an evaluation of the most important clinical parameters, assessed at the age of 12 years, is presented. We confirm some previously reported genotype-phenotype correlations and we report a new nonsense mutation (R709X) associated with a pancreatic sufficient phenotype.

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35 Table 1 CF mutations identified in this cohort study (225 chromosomes from Veneto and Trentino Alto-Adige) n Number of CF chromosomes, Cum fi cumulative fraction, wnovel mutation identified during this study " Cystic Fibrosis Genetic Analysis Consortium, personal comunication Table 2 DNA sequence variations identified in this cohort study (w Novel sequence variation identified during this study a Cystic Fibrosis Genetic Analysis Consortium, personal comunication Mutation Exon n % Cure fr References AF508 l0 107 47.56 47.56 Kerem et al. 1989 R1162X 19 22 9.78 57.33 Gasparini et al. 1991 2183AA----~G 13 21 9.33 66.67 Bozon et al. 1994 N1303K 21 9 4.00 70.67 Osborne et al. t991 G542X 11 6 2.67 73.33 Kerem et al. 1990 711+5G--~A intron 5 6 2.67 76.00 w 1717 1G--~A intron 10 5 2.22 78.22 Kerem et al. 1990 G85E 3 3 1.33 79.56 Zielenski et al. 1991~' R553X 11 3 1.33 80.89 Cutting et al. 1990 2789+5G--~A intron 14b 3 1.33 82.22 Highsmith* Q552X 11 3 1.33 83.56 Devoto et al. 1991 621+lG---~T intron 4 2 0.89 84.44 Zielenski et al. 1991b W1282X 20 2 0.89 85.33 Vidaud et al. 1990 3132delTG 17a 2 0.89 86.22 w 2790-2A---~G intron 14b 2 0.89 87.11 w 457TAT--)G 4 1 0.44 87.56 Ravnik-Glavac et al. 1993 R347P 7 1 0.44 88.00 Dean et al. 1990 G551D 11 .1 0.44 88.44 Cutting et al. 1990 1717-8G-+A intron 10 1 0.44 88.89 Savov et al. 1994 3849+ 10KbC--)T intron 19 1 0.44 89.33 Highsmith* R709X 13 1 0.44 89.78 w 1898+3A---~G intron 12 1 0.44 90.22 Cremonesi et al. 1992 Identified 203 90.22 Unidentified 22 9.78 Variatioh Exon References 1540 A orG Met or Val at 470 10 Kerem et al. 1990 1898+152 T or A intron 12 Chillon et al. 1991 2134 C or T Arg or Cys at 668 13 Fanen et al. 1992 2694 T or G No change Thr at 854 14a Zielenski et al. 199 lb 2752-22 A or G intron 14a w 3601-65 C or A intron 18 Dork et al. 199l 4029 A or G No change Thr at 1299 21 Fanen et al. 1992 4404 C or T No change Tyr at 1424 24 ShoshanP 711 +5G--+A This mutation was found in the splice donor site flanking the 3' end of exon 5. Login to comment

[hide] Increased incidence of cystic fibrosis gene mutati... Hum Mol Genet. 1995 Apr;4(4):635-9. Pignatti PF, Bombieri C, Marigo C, Benetazzo M, Luisetti M
Increased incidence of cystic fibrosis gene mutations in adults with disseminated bronchiectasis.
Hum Mol Genet. 1995 Apr;4(4):635-9., [PMID:7543317]

Abstract [show]
In order to identify a possible hereditary predisposition to the development of obstructive pulmonary disease of unknown origin, we have looked for the presence of Cystic Fibrosis Transmembrane Regulator (CFTR) gene mutations in unrelated patients with no signs of Cystic Fibrosis (CF). We screened for 70 common mutations, and also for rare mutations by denaturing gradient gel electrophoresis analysis. In this search, different CFTR gene mutations (R75Q, delta F508, R1066C, M1137V and 3667ins4) were found in five out of 16 adult Italian patients with disseminated bronchiectasis, a significant increase over the expected frequency of carriers. Moreover, three rare CFTR gene DNA polymorphisms (G576A, R668C, and 2736 A-->G), not deemed to be the cause of CF, were found in two patients, one of which was a compound heterozygote with R1066C. These results indicate that CFTR gene mutations, and perhaps also DNA polymorphisms, may be involved in the etiopathogenesis of at least some cases of bronchiectasis.

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25 RESULTS Common CF mutations All the study subjects were initially typed with respect to some CFTR mutations known to be present in CF patients in the North East Italian population: AF508, R1162X, 2183AA->G, NI303K, G542X, 711 + 5G->A, 1717-1 G^>A, 1717-8G->A, G85E, R553X, 2789 + 5 G->A, Q552X, 621 + 1 G->T, W1282X, 3132delTG, 2790-2A->G, 457 TAT->G, R347P, G551D, 1898 + 3A->G and 3849 + 10 kbC^T. Login to comment
31 List of CFTR gene mutations and DNA polymorphisms screened Mutations R75Q/X/L, G85E, 394deITT 457TAT->G, R117H 621 + 1G->T 711 + 5G->A L206W 875 + 40 A->G 936 del TA 1001 + 11C->T R334W, R347 P/H/L, 1154insTC A455E, V456F DF5O8 1717-IG->A, 1717-8G->A G542X, G551D, Q552X, R553X P574H 1898 + 3A->G 2183 AA->G, 2184delA, R709X D836Y, 2694 T/G 2752-22 A/G 2789 + 5 G->A, 2790-2 A-»G Q890X 3041-71 G/C 3132delTG 3271 + 18 C-»T, 3272-26 A->G H1054D, G1061R, R1066C/H, A1067T, H1085R, Y1092X, 3320 ins5 D1152H R1162X, 3667ins4, 3737delA, 11234V 3849 + 10 kb C-»T, 3850-1 G-»A SI25IN, S1255P, 3905insT, 3898insC, D127ON, W1282X, R1283M, 4002 A/G 4005 + 1 G-»A N1303 K/H, 4029 A/G D1377H Q1411 X 4404 C/T, 4521 G/A Location e 3 e 4 i 4 i 5 e 6a i 6a e 6b i 6b e 7 e 9 e 10 i 10 e 11 e 12 i 12 e 13 e 14a i 14a i 14b e 15 i 15 e 17a i 17a e 17b e 18 e 19 i 19 e 20 i 20 e2l e 22 e 23 e24 Listing is in order of location along the CFTR gene, e = exon; i = intron. Login to comment
124 Reverse dot blot analysis was used for detecting the following mutations: A F508, G542X, G55ID, R553X, R1162X, W1282X, N1303K (Roche Molecular Systems, kindly provided by Dr R.Saiki). Login to comment

[hide] A novel donor splice site in intron 11 of the CFTR... Am J Hum Genet. 1995 Mar;56(3):623-9. Chillon M, Dork T, Casals T, Gimenez J, Fonknechten N, Will K, Ramos D, Nunes V, Estivill X
A novel donor splice site in intron 11 of the CFTR gene, created by mutation 1811+1.6kbA-->G, produces a new exon: high frequency in Spanish cystic fibrosis chromosomes and association with severe phenotype.
Am J Hum Genet. 1995 Mar;56(3):623-9., [PMID:7534040]

Abstract [show]
mRNA analysis of the cystic fibrosis transmembrane regulator (CFTR) gene in tissues of cystic fibrosis (CF) patients has allowed us to detect a cryptic exon. The new exon involves 49 base pairs between exons 11 and 12 and is due to a point mutation (1811+1.6kbA-->G) that creates a new donor splice site in intron 11. Semiquantitative mRNA analysis showed that 1811+1.6kbA-->G-mRNA was 5-10-fold less abundant than delta F508 mRNA. Mutation 1811+1.6kbA-->G was found in 21 Spanish and 1 German CF chromosomes, making it the fourth-most-frequent mutation (2%) in the Spanish population. Individuals with genotype delta F508/1811+1.6kbA-->G have only 1%-3% of normal CFTR mRNA. This loss of 97% of normal CFTR mRNA must be responsible for the pancreatic insufficiency and for the severe CF phenotype in these patients.

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67 To test this hypothesis, mRNA studies were performed in one Spanish CF patient (heterozygous for G542X and 3601-11G/C) and one German CF patient (heterozygous for SF508 and 3601-11lG/C). Login to comment
70 However, an extra band was detected between exons 10 and 12 in the G542X/3601-11lG/C patient. Login to comment
76 In the initial cDNA analysis of the G542X/1811+1.6kbA--G patient, we detected both the 49-bp insertion and the G542X allele, while in the AF508/1811+l.6kbA-oG patient, we mainly detected the AF508 allele, since RNA from the 1811 +1.6kbA-*G allele was 5-10-fold less abundant than the AF508 allele (fig. 3). Login to comment
111 Three CF patients carried mutation P205S on the other chromosome, which is associated with a mild CF phenotype and pancreatic sufficiency (Chillon et al. 1993). Login to comment
112 Seventeen other patients were carriers of a known severe CF mutation (AI507, AF508, 1609delCA, G542X, K71OX, or N1303K) on the other chromosome. Login to comment
125 of patients ...................... 17 3 82 Age (years) ...................... 9.1 ± 5.8 12 ± 5.3 7.8 ± 5.2 Age at diagnosis (years) ............... 2.8 ± 4.0 7.1 ± 5.2 2.2 ± 2.8 Sweat chloride (mmolIL) ............. 98 ± 11.7 100 ± 10 104.4 ± 15.7 FEV1 (% predicted)d .................... 65 ± 24.8 70.8 ± 12.8 74.8 ± 23.1 Shwachman score' ...................... 74.5 ± 12.3 86.6 ± 2.3 83.1 ± 11.8 Pancreatic sufficiency ................... 0/19 (0%) 3/3 (100%) 1/82 (1.2%) a A1507, AF508, 1609delCA, G542X, K710X, and N1303K. Login to comment
150 We could detect the two mRNA species in the G542X/1811 +1.6kbA-+G patient, since both mutations lead to a reduction in levels of mRNA. Login to comment
124 of patients ...................... 17 3 82 Age (years) ...................... 9.1 &#b1; 5.8 12 &#b1; 5.3 7.8 &#b1; 5.2 Age at diagnosis (years) ............... 2.8 &#b1; 4.0 7.1 &#b1; 5.2 2.2 &#b1; 2.8 Sweat chloride (mmolIL) ............. 98 &#b1; 11.7 100 &#b1; 10 104.4 &#b1; 15.7 FEV1 (% predicted)d .................... 65 &#b1; 24.8 70.8 &#b1; 12.8 74.8 &#b1; 23.1 Shwachman score' ...................... 74.5 &#b1; 12.3 86.6 &#b1; 2.3 83.1 &#b1; 11.8 Pancreatic sufficiency ................... 0/19 (0%) 3/3 (100%) 1/82 (1.2%) a A1507, AF508, 1609delCA, G542X, K710X, and N1303K. Login to comment
149 We could detect the two mRNA species in the G542X/1811 +1.6kbA-+G patient, since both mutations lead to a reduction in levels of mRNA. Login to comment

[hide] Common CFTR mutations are not likely to predispose... Hum Genet. 1995 Feb;95(2):226-8. Artlich A, Boysen A, Bunge S, Entzian P, Schlaak M, Schwinger E
Common CFTR mutations are not likely to predispose to chronic bronchitis in northern Germany.
Hum Genet. 1995 Feb;95(2):226-8., [PMID:7532152]

Abstract [show]
The frequency of six common mutations in the cystic fibrosis transmembrane conductance regulator gene was studied in 100 patients hospitalized with chronic bronchitis. Only one patient with chronic bronchitis and diffuse bronchiectasis was heterozygous for the common delta F508 mutation. R553X, G542X, G551D, N1303K and 621 + 1G-->T were not detected. This result is not significantly different from the frequency of cystic fibrosis carriers in Northern Europe. Predisposition of heterozygotes for chronic bronchitis is therefore unlikely.

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2 R553X, G542X, G551D, N1303K and 621 + 1G--->T were not detected. Login to comment
10 We have analyzed CF carrier frequency and the frequency of the more common CFTR mutations AF508, R553X, G551D, G542D, G542X, N1303K and 621 + 1G--+T by examination of 100 patients with chronic bronchitis. Login to comment
20 Sequences from exons 4, 10, l l and 21 were amplified by the polymerase chain reaction (PCR) according to the published protocols in order to search for mutations AF508, G542X, R553X, G551D, N1303K and 621 +IG---~T (Rommens et al. 1990; Cutting et al. 1990; Kerem et al. 1990;Osborne et al. 1991; Zielenski et al. 1991). Login to comment
22 Primers F: TTGCAGAGAAAGA- CAATATAGTTCCT and R: GCACAGATTCTGAGTAACCAT- AATC generate a 296 bp product with a BstI site destroyed by G542X. Login to comment

[hide] Extensive analysis of 40 infertile patients with c... Hum Genet. 1995 Feb;95(2):205-11. Casals T, Bassas L, Ruiz-Romero J, Chillon M, Gimenez J, Ramos MD, Tapia G, Narvaez H, Nunes V, Estivill X
Extensive analysis of 40 infertile patients with congenital absence of the vas deferens: in 50% of cases only one CFTR allele could be detected.
Hum Genet. 1995 Feb;95(2):205-11., [PMID:7532150]

Abstract [show]
Mutations in the cystic fibrosis (CF) conductance transmembrane regulator (CFTR) gene have been detected in patients with CF and in males with infertility attributable to congenital bilateral absence of the vas deferens (CBAVD). Thirty individuals with CBAVD and 10 with congenital unilateral absence of the vas deferens (CUAVD) were analyzed by single-strand conformation analysis and denaturing gradient gel electrophoresis for mutations in most of the CFTR gene. All 40 individuals were pancreatic sufficient, but twenty patients had recurrent or sporadic respiratory infections, asthma/asthmatic bronchitis, and/or rhino-sinusitis. Agenesia or displasia of one or both seminal vesicles was detected in 30 men and other urogenital malformations were present in six subjects. Among the 40 samples, we identified 13 different CFTR mutations, two of which were previously unknown. One new mutation in exon 4 was the deletion of glutamic acid at codon 115 (delta E115). A second new mutation was found in exon 17b, viz., an A --> C substitution at position 3311, changing lysine to threonine at codon 1060 (K1060T). CFTR mutations were detected in 22 out of 30 (73.3%) CBAVD patients and in one out of 10 (10%) CUAVD individuals, showing a significantly lower incidence of CFTR mutations in CBAVD/CUAVD patients (P << 0.0001), compared with that found in the CF patient population. Only three CBAVD patients were found with more than one CFTR mutation (delta F508/L206W, delta F508/R74W + D1270N, R117H/712-1G --> T), highlighting L206W, R74W/D1270N, and R117H as benign CF mutations. Sweat electrolyte values were increased in 76.6% of CBAVD patients, but three individuals without CFTR mutations had normal sweat electrolyte levels (10% of the total CBAVD patients), suggesting that factors other than CFTR mutations are involved in CBAVD. The failure to identify a second mutation in exons and their flanking regions of the CFTR gene suggests that these mutations could be located in introns or in the promoter region of CFTR. Such mutations could result in CFTR levels below the minimum 6%-10% necessary for normal protein function.

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41 Genomic DNA from CBAVD and CUAVD subjects was first analyzed for the two most common mutations in the Spanish population, viz., AF508 and G542X (Casals et al. 1993). Login to comment
56 The two most frequent CF mutations in the Spanish population (AF508 and G542X) were found on 14 chromosomes (10 AF508 and 4 G542X). Login to comment
59 Direct sequencing of these two abnormal fragments identified mutation R ll7H, a known Table 1 Semen analysis of patients with CAVD, given as the mean (range) CBAVD CUAVD (n = 27) (n = 10) Sperm (x 106/ml) 0 10.6 (0-90) Seminal volume (ml) 0.9 (0.2-3.1) 2.5 (0.4 5.4) pH 6.7 (6.0-8.0) 7.3 (6.4-7.7) Fructose (retool/l) 2.6 (0-9) 10.3 (3-) '~Citrate (mmol/l) 77.5 (11-188) 48.6 (36-88) ~Reference values: fructose, 8 28 retool/l; citrate, 10 35 retool/1 Table 2 CFTR mutation analysis in 30 CBAVD and 10 CUAVD patients (CBAVD congenital bilateral absence of the vas deferens, CUAVD congenital unilateral absence of the vas deferens, ND not determined, - absence of mutations, RRI recurrent respiratory infection, R rhinitis, RS rhino-sinusitis, BR.ASTH bronchitis asthmatic) Table 3 Congenital malformations associated with CAVD in 40 patients 207 Patient Age Phenotype Sweat test Mutation Other clinical (years) (mEq/l) features 1 37 CBAVD 108 1677delTA 2 28 CBAVD 50 G542X 3 28 CBAVD 118 - 4 33 CBAVD 90 AF508/L206W RRI, R 5 26 CBAVD 118 R117H/712-1G-+T 6 42 CBAVD 66 - RS 7 31 CBAVD 170 AF508 R 8 27 CBAVD 100 AF508/R74W + D1270N RRI, R 9 32 CBAVD 74 AE115 RS 10 35 CBAVD 90 - Nasal polyps 11 33 CBAVD 78 KI060T RI, family history 12 45 CBAVD 150 R334W RS 13 42 CBAVD 60 - 14 40 CBAVD 110 R 1070W RS 15 29 CBAVD 110 G542X 16 37 CBAVD 80 R117H RI, RS, BR.ASTH 17 37 CBAVD 85 - Asthma 18 46 CBAVD 15 R1162X 19 37 CBAVD 110 AF508 RS, diarrhoea 20 42 CBAVD 45 2789+5G--)A RI 21 49 CBAVD 95 AF508 22 36 CBAVD 70 AF508 RRI, RS 23 42 CBAVD 90 - 24 15 CBAVD 150 AF508 25 26 CBAVD 60 - 26 39 CBAVD 100 AF508 RRI, RS 27 33 CBAVD 57 AF508 RRI 28 33 CBAVD 80 G542X 29 34 CBAVD 78 - 30 32 CBAVD 113 G542X 31 33 CUAVD ND AF508 RS, pancreatitis 32 37 CUAVD ND - 33 31 CUAVD 77 - BR.ASTH 34 39 CUAVD ND - 35 40 CUAVD 40 - 36 33 CUAVD 59 - 37 40 CUAVD 90 - 38 47 CUAVD 40 - RRI 39 39 CUAVD 50 - 40 35 CUAVD 100 - No. Login to comment
103 In 13 cases, the mutations are known to be associated with severe CF (AF508, G542X, Rl162X and 1677delTA), whereas in five cases, the phenotypic effect of the mutation is still unknown (AEll5, K1060T, R334W, R1070W, and 2789 + 5G---)A); in one case (Rll7H), the mutation is known to result in mild CE Of these mutations, R334W seems to cause pancreatic insufficiency with a variable age of onset (X. Estivill, in press), whereas mutation 2789 + 5G--)A (W. E. Jr. High- smith, personal communication to the CFGAC) has frequently been found in adult CF patients and is probably involved in a mild phenotype (T. Casals et al. unpublished). Login to comment
141 In: Striver CR, Beaudet AL, Sly WS, Valle D (eds) The metabolic basis of inherited disease, 6th edn. McGraw-Hill, New York, pp 2649-2680 Casals T, Nunes V, Palacio A, Gimdnez J, Gaona A, Ibfifiez N, Morral N, Estivill X (1993) Cystic fibrosis in Spain: high frequency o1"mutation G542X in the Mediterranean coastal area. Login to comment

[hide] Two CF patients, one homozygous for the 621 + 1G >... J Med Genet. 1995 Feb;32(2):158. Cheadle JP, Meredith AL, Millar-Jones L, Goodchild MC
Two CF patients, one homozygous for the 621 + 1G > T splice mutation, the other homozygous for the 1898 + 1G > A splice mutation.
J Med Genet. 1995 Feb;32(2):158., [PMID:7539080]

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5 To date, investigators have described homozygotes for G542X,2 R553X,3 G85E,4 S549N,5 Rl17H,6 2184delA,7 R1162X,8 and W128X.9 We report here two patients, one homozygous for 621 + 1G>T, the other homozygous for 1898 + 1G>A. Login to comment

[hide] Binding of Pseudomonas aeruginosa to respiratory e... J Pediatr. 1995 Feb;126(2):230-3. Zar H, Saiman L, Quittell L, Prince A
Binding of Pseudomonas aeruginosa to respiratory epithelial cells from patients with various mutations in the cystic fibrosis transmembrane regulator.
J Pediatr. 1995 Feb;126(2):230-3., [PMID:7531240]

Abstract [show]
OBJECTIVE: To determine whether there is an association between mutations of the cystic fibrosis transmembrane regulator (CFTR) and the predilection of patients with cystic fibrosis (CF) for Pseudomonas aeruginosa infection. METHOD: We quantified the adherence of P. aeruginosa PA01, labeled with sulfur 35-methionine, to epithelial monolayers derived from nasal scrapings of patients with specific CFTR mutations, and of carriers and normal subjects. RESULTS: Adherence of P. aeruginosa to epithelial cells from patients with CF was significantly greater than to cells from either carriers (t = 2.94; p = 0.009) or normal subjects (t = 3.32; p = 0.004). Adherence to epithelial cells from patients with CF who were homozygous for the delta F508 mutation ranged from 12% to 35% (mean, 23.7%) of the added inoculum, which was significantly greater than the binding to cells from patients with other mutations, which ranged from 3% to 18% (mean, 9.4%; t = 3.71; p = 0.002), from heterozygote carriers (3% to 11%; mean, 7.9%; t = 4.87; p = 0.002), or from normal subjects (2% to 10%: mean, 7.0%; t = 5.21; p = 0.002). CONCLUSION: Adherence to P. aeruginosa can be correlated with homozygosity for the delta 508 mutation; CFTR dysfunction may be one of the factors involved in the pathogenesis of pulmonary infection in CF.

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50 (yr) Sex Genotype Mutation defect disease* Kulczycki score (%) 1 13 F AF508/AF508 Processing Mild 80 35 2 21 F AF508/2xF508 Processing Mild 83 33 3 13 M 2xF508/AF508 Processing Mild 87 26 4 15 M AF508/AF508 Processing Mild 87 22 5 15 F AF508/AF508 Processing Moderate 60 21 6 28 M AF508/AF508 Processing Moderate 50 17 7 15 F AF508/AF508 Processing Moderate 60 12 8 29 M AF508/3849 + 10 Processing/protein synthesis Moderate 55 7 9 43 F WI282X/W1282X Protein synthesis Moderate 60 3 10 13 F 2xF508/W1282X Processing/protein synthesis Mild 88 13 11 16 M Unknown Unknown Mild 82 6 12 22 F AF508/G551D Processing/regulation Mild 80 4 13 19 M AF508/G551D Processing/regulation Mild 80 18 14 12 M G542X/3849 + 10 Protein synthesis Mild 87 18 15 60 M W1282X/unknown Protein synthesis Moderate 75 5 *Severityofpulmonarydiseasewasmeasuredwiththe Shwachman-Kulczyckiscoringsystem. binding, there was no significant correlation between pulmonary status and the quantity of adherent bacteria. Login to comment

[hide] Is congenital bilateral absence of vas deferens a ... Am J Hum Genet. 1995 Jan;56(1):272-7. Mercier B, Verlingue C, Lissens W, Silber SJ, Novelli G, Bonduelle M, Audrezet MP, Ferec C
Is congenital bilateral absence of vas deferens a primary form of cystic fibrosis? Analyses of the CFTR gene in 67 patients.
Am J Hum Genet. 1995 Jan;56(1):272-7., [PMID:7529962]

Abstract [show]
Congenital bilateral absence of the vas deferens (CBAVD) is an important cause of sterility in men. Although the genetic basis of this condition is still unclear, it has been shown recently that some of these patients carry mutations in their cystic fibrosis transmembrane conductance regulator (CFTR) genes. To extend this observation, we have analyzed the entire coding sequence of the CFTR gene in a cohort of 67 men with CBAVD, who are otherwise healthy. We have identified four novel missense mutations (A800G, G149R, R258G, and E193K). We have shown that 42% of subjects were carriers of one CFTR allele and that 24% are compound heterozygous for CFTR alleles. Thus, we have been unable to identify 76% of these patients as carrying two CFTR mutations. Furthermore, we have described the segregation of CFTR haplotypes in the family of one CBAVD male; in this family are two male siblings, with identical CFTR loci but displaying different phenotypes, one of them being fertile and the other sterile. The data presented in this family, indicating a discordance between the CBAVD phenotype and a marked carrier (delta F508) chromosome, support the involvement of another gene(s), in the etiology of CBAVD.

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63 We identified 28 AF508, 5 W1282X, 1 G542X, 1 R553X, and 2 N1303K mutations. Login to comment
77 of Patients Genotypea 1 AF508 + (G628R + S1235R) 1 AF508 + (R74W + D1270N) 2 AF508 + R668C 4 AF508 + R117H 1 AF508 + R258G 1 AF508 + R75L 1 E193K + N1303K 1 R347H + R1066H 1 R117C + W1282X 1 R553X + R668C 1 G149R + R668C 1 R117H+R117H 18 AF508/unidentified 4 W1282X/unidentified 1 G542X/unidentified 1 N1303K/unidentified 1 S1235R/unidentified 1 R347H/unidentified 1 A800G/unidentified 1 F1052V/unidentified 23 unidentified/unidentified a In parentheses are the two mutations located on the same haplotype. Login to comment

[hide] Cystic fibrosis: genotypic and phenotypic variatio... Annu Rev Genet. 1995;29:777-807. Zielenski J, Tsui LC
Cystic fibrosis: genotypic and phenotypic variations.
Annu Rev Genet. 1995;29:777-807., [PMID:8825494]

Abstract [show]
Cystic fibrosis (CF) is a common genetic disorder in the Caucasian population. The gene was identified in 1989 on the basis of its map location on chromosome 7. The encoded gene product, named cystic fibrosis transmembrane conductance regulator (CFTR), corresponds to a cAMP-regulated chloride channel found almost exclusively in the secretory epithelial cells. Although the major mutation that results in a single amino acid deletion (F508) accounts for 70% of the disease alleles, more than 550 additional mutant alleles of different forms have been detected. Many of these mutations can be divided into five general classes in terms of their demonstrated or presumed molecular consequences. In addition, a good correlation has been found between CFTR genotype and one of the clinical variables--pancreatic function status. An unexpected finding, however, is the documentation of CFTR mutations in patients with atypical CF disease presentations, including congenital absence of vas deferens and several pulmonary diseases. Thus, the implication of CFTR mutation is more profound than CF alone.

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545 Even the second most common mutation (G542X) has only a relative frequency of about 2.4% (Table 1). Login to comment
580 For example, many different microsatellite alleles have been found associated with AF508, G542X, and N1303K (123). Login to comment
628 This strategy may also be applied to patients with other Normal II III IV V No synthesis Block in Block in Altered Reduced processing regulation conductance synthesis Nonsense Missense G542X Missense Missense Missense A455E Frameshift N1303K G551D R117H 394deiTT AA deletion Alternative L1F508 R347P splicing Splice junction 1717-1G-->A 3849+1OkbC-->T Figure 3 Molecular consequence of different classes ofCF mutations. Login to comment
679 Table 3 Atypical (non-CF) diseases associated with the CFTR gene Common manifestations Disease shared with CF CBAVD absence of vas deferens (bilateral CUAVD absence of vas deferens (unilateral) Obstructive azoospermia azoosperma Diffuse bronchiectasis abnormal dilatation of bronchi Bronchiectasis with elevated abnormal dilatation of bronchi and sweat CI- high levels of sweat chloride Allergic bronchopulmonary allergic asthma, tenacious sputum, aspergillosis mucus plugs Chronic pseudomonas bron- chronic sinusitis, nasal polyposis chitis Chronic bronchial abnormal mucous secretion hypersecretion Nasal polyposis nasal polyps Neonatal transitory hyper- high levels of immunoreactive tryp- trypsinemia sin (IRT) Fraction of patients with at least one CFTR mutation (%) Reference 80/\02 (78)" 31 51168 (75)' 207a 6/14 (43)b 1 1 8 8/17 (47)' 93 6/10 (6W 13 6/48 (l2.5)e 161 9/28 (32)" 136 5/16 (3 1)1 78 6/1 1 (54)e 1 19 2/10 (20)e 1 1 9 6/65 (9.2)f 65 7/1 12 (6.2)g 22 9/149 (6)f 106 • The numbers are based on comprehensive screening of CFfR mutations (including IVS8 : 5T) by a variety of methods; btesting of three mutations (�F508, RI I7H and R75Q; '-�F508, G55 1O, G542X, W1282X, N1303K, RI 17H and IVS8 : 5T;d direct sequencing of exons encoding NBFI; ' the most common CFTR mutations (unspecified); f �F508 only: "eight mutations (�F508, �I507, DlIOH, RII7H, 621 + IG .... T, N1303K, G5SID, and R553X). Login to comment
993 CFTR nonsense mutations G542X and WI282X associated with severe reduction of CFTR mRNA in nasal epithelial cells. Hum. Mol. Genet. 1 :542-44 85. Hamosh A, Trapnell BC, Zeitlin PL, Montrose RC, Rosenstein BJ, et aI. 1991. Login to comment
1376 A cystic fibrosis patient with 6F508, G542X and a deletion at the D7S8 locus. Login to comment

[hide] Complete detection of mutations in cystic fibrosis... Hum Genet. 1994 Dec;94(6):629-32. Mercier B, Raguenes O, Estivill X, Morral N, Kaplan GC, McClure M, Grebe TA, Kessler D, Pignatti PF, Marigo C, et al.
Complete detection of mutations in cystic fibrosis patients of Native American origin.
Hum Genet. 1994 Dec;94(6):629-32., [PMID:7527370]

Abstract [show]
An increased incidence of cystic fibrosis (CF) has been reported in some populations of Native Americans of the Southwest such as the Pueblo, which is a genetic isolate. As the most common mutation found in Caucasians (delta F508) was absent and only one chromosome carried the G542X mutation, we decided to analyze the entire coding sequence of the CFTR gene in eight Pueblo CF patients. We have identified four different mutations: G542X, R1162X, 3849+10kbC-->T, and D648V that account for these 16 haplotypes. The R1162X was found on 11 chromosomes. Using intragenic microsatellites, we have compared the haplotypes of those chromosomes to those of Italian origin where the R1162X mutation was initially reported. These haplotypes turned out to be identical, suggesting a common origin and an admixture with Italian or Spanish settlers, followed by typical founder effect. In contrast the 3849+10kbC-->T mutation, which was found on three chromosomes, is associated with different haplotypes than those on chromosomes carrying the same mutation in Caucasians. A novel mutation, D648V, observed on one chromosome has not been found outside the Pueblo population.

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2 As the most common mutation found in Caucasians (AF508) was absent and only one chromosome carried the G542X mutation, we decided to analyze the entire coding sequence of the CFTR gene in eight Pueblo CF patients. Login to comment
3 We have identified four different mutations: G542X, Rl162X, 3849+10kbC---~T, and D648V that account for these 16 haplotypes. Login to comment
18 The other mutations most frequently found in Caucasians, such as G551D, R553X, N1303K, or W1282X, were also excluded and only one patient was found to carry the G542X. Login to comment
47 First, the change to valine instead of aspartic acid would introduce a conformational change in the R domain, a region of the protein that plays a major Table 1 Clinical and mutations data of these Native American cystic fibrosis (CF) patients Patient Nation Pancreatic Weight Height Haplosufficient (PS) (%ile) (%ile) type Pancreatic insufficient (PI) Mutations 021-l Pueblo PI 20 10 CC Zuni 022-1 Pueblo P1 7.5 20 CC Zuni 023-1 Pueblo PI 75 50 CC Zuni 024-1 Pueblo PI 40 10 CC Zuni 028-1 Pueblo PI 7.7 5 CC Zuni 029-1 Pueblo PS 2 17.5 AC Zuni 006-1 Pueblo PS 8.5 45 AC Jemez 008-1 Pueblo Santo PS > 95 > 95 AB Domingo R1162X R1162X R1162X R1162X R1162X R1162X D648V G542X R1162X R1162X R1162X R1162X R1162X 3849+ 10kbC---~T 3849+ I0kbC----~T 3849+ 10kbC--)T Fig. 1 Sequencing data of the D648V. Login to comment
66 The fourth mutation, G542X, was present on a single chromosome. Login to comment

[hide] Identification of seven rather infrequent and one ... Hum Mol Genet. 1994 Dec;3(12):2249-50. Teng H, Cuppens H, De Boeck C, Cassiman JJ
Identification of seven rather infrequent and one novel CFTR mutation in the Belgian population.
Hum Mol Genet. 1994 Dec;3(12):2249-50., [PMID:7881429]

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3 Five mutations had a frequency higher than 1%: AF508 (72.5%), G542X (5.5%), N1303K (3.5%), 1717-1G-A (2.5%) and S1251N (2.0%). Login to comment

[hide] Independent origins of cystic fibrosis mutations R... Am J Hum Genet. 1994 Nov;55(5):890-8. Morral N, Llevadot R, Casals T, Gasparini P, Macek M Jr, Dork T, Estivill X
Independent origins of cystic fibrosis mutations R334W, R347P, R1162X, and 3849 + 10kbC-->T provide evidence of mutation recurrence in the CFTR gene.
Am J Hum Genet. 1994 Nov;55(5):890-8., [PMID:7526685]

Abstract [show]
Microsatellite analysis of chromosomes carrying particular cystic fibrosis mutations has shown different haplotypes in four cases: R334W, R347P, R1162X, and 3849 + 10kbC-->T. To investigate the possibility of recurrence of these mutations, analysis of intra- and extragenic markers flanking these mutations has been performed. Recurrence is the most plausible explanation, as it becomes necessary to postulate either double recombinations or single recombinations in conjunction with slippage at one or more microsatellite loci, to explain the combination of mutations and microsatellites if the mutations arose only once. Also in support of recurrence, mutations R334W, R347P, R1162X, and 3849 + 10kbC-->T involve CpG dinucleotides, which are known to have an increased mutation rate. Although only 15.7% of point mutations in the coding sequence of CFTR have occurred at CpG dinucleotides, approximately half of these CpG sites have mutated at least once. Specific nucleotide positions of the coding region of CFTR, distinct from CpG sequences, also seem to have a higher mutation rate, and so it is possible that the mutations observed are recurrent. G-->A transitions are the most common change found in those positions involved in more than one mutational event in CFTR.

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16 Of all the other mutations, only G542X (Kerem et al. 1990), G551D (Cutting et al. 1990), N1303K (Osborne et al. 1991), and W1282X (Vidaud et al. 1990) have a frequency of 1%-2.5% in the worldwide population (Cystic Fibrosis Genetic Analysis Consortium, in press). Login to comment
105 Mutations that have a relatively high worldwide frequency (AF508, G542X, and N1303K) and that are also ancient have a small proportion of chromosomes associated with haplotypes different from the original, which can be explained by slippage of the DNA polymerase at one microsatellite during replication. Login to comment

[hide] Detection of more than 50 different CFTR mutations... Hum Genet. 1994 Nov;94(5):533-42. Dork T, Mekus F, Schmidt K, Bosshammer J, Fislage R, Heuer T, Dziadek V, Neumann T, Kalin N, Wulbrand U, et al.
Detection of more than 50 different CFTR mutations in a large group of German cystic fibrosis patients.
Hum Genet. 1994 Nov;94(5):533-42., [PMID:7525450]

Abstract [show]
We have conducted a comprehensive study of the molecular basis of cystic fibrosis (CF) in 350 German CF patients. A screening approach based on single-strand conformation analysis and direct sequencing of genomic polymerase chain reaction products has allowed us to detect the molecular defects on 95.4% of the CF chromosomes within the coding region and splice sites of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The spectrum of sequence changes comprises 54 different mutations, including 17 missense mutations, 14 nonsense mutations, 11 frameshift mutations, 10 splice site variants and two amino acid deletions. Eleven of these mutations have not previously been described. Our results reflect the marked mutational heterogeneity of CF in a large sample of patients from a non-isolated population.

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49 A few other mutations known to be frequent (e.g. NI303K, R553X, G542X, G551D, 2789+5 G-+A and 3849+10 kB C--+T) were then screened by specific direct methods as previously described (D6rk et al. 1992 a). Login to comment
77 Table 1 Frequency distribution and haplotypes of CFTR mutations in 700 German CF chromosomes Mutation~ Nucleotide changesb Locationc Frequencyd Haplotype~ Referencef Q39x C--~T at 247 Exon 2 1 (0.1%) D3 Cutting et al. (1992) E60X G-+T at 310 Exon 3 1 (0.1%) A2 Malone et al. (*) R75X C--+T at 355 Exon 3 1 (0.1%) C2 This study 405+1 G---~A G-+A at 405+1 Intron 3 1 (0.1%) C2 D6rk et al. (1993c) E92X G--~T at 406 Exon 4 2 (0.3%) B2 Will et al. (1994) R117C C---~Tat 481 Exon 4 1 (0.1%) C2 This study R117H G--+A at 482 Exon 4 2 (0.3%) B6 Dean et al. (1990) 621+1 G--+T G--+T at 621+1 Intron 4 1 (0.1%) B1 Zielenski et al. (1991b) H199Y C--+T at 727 Exon 6a 1 (0.1%) A2 This study (*) 1078delT Deletion of T at 1078 Exon 7 4 (0.6%) C2 Claustres et al. (1992) R334W C-~T at 1132 Exon 7 2 (0.3%) BI Gasparini et al. (1991) 1336K T-->A at 1139 Exon 7 3 (0.4%) A2 Cuppens et al. (1993) R347P G--+C at 1172 Exon 7 11 (1.6%) A2, C2 Dean et al. (1990) 1342-2 A--+C A--+C at 1342-2 Intron 8 3 (0.4%) A4 D/3rk et al. (1993b) Q414X C--+T at 1372 Exon 9 1 (0.1%) D3 D6rk et al. (1994a) A455E C-+A at 1496 Exon 9 1 (0.1%) BI Kerem et al. (1990) V456F G--~T at 1498 Exon 9 1 (0.1%) B3 D6rk et al. (1994a) A1507 Deletion of 3 bp between 1648-1653 Exon 10 1 (0.1%) D5 Kerem et al. (1990) AF508 Deletion of 3 bp between 1652-1655 Exon 10 504 (72.0%) B1, DI, B7 Kerem et al. (1989) 1717-1 G--+A G--+A at 1717-1 lntron 10 6 (0.9%) B3 Kerem et al. (1990) G542X G--+T at 1756 Exon 11 10 (1.4%) B1 Kerem et al. (1990) G551D G--+A at 1784 Exon 11 7 (l.0%) B3 Cutting et al. (1990) Q552X C-+T at 1786 Exon 11 1 (0.1%) A4 Devoto et al. (1991) R553X C--+T at 1789 Exon 11 16 (2.3%) A4, B4, D3 Cutting et al. (1990) L558S T--+C at 1805 Exon 11 1 (0.1%) C2 Maggio et al. (*) 1811+I.6kBA-+G A--+Gat 1811+l.6kB lntron 11 1 (0.1%) A2 Chillonetal. Login to comment
120 There are, however, only six additional CFTR mutations with a frequency of approximately 1% or more of the CF chromosomes; two nonsense mutations, G542X and R553X, and the missense mutations G551D and NI303K were predominantly seen in severely affected patients, whereas the transmembrane missense mutation R347P and the splice mutation 3849 + 10 kB C---~T Table 2 Rare sequence variants in the CFTR promoter and coding region Sequence variant Nucleotide change Location Frequency Associated mutatiow' Reference 125 G--+C G--~C at 125 Promoter 1 (0.1%) R75X F508C T--~G at 1655 Exon 10 2 (0.3%) S1251N 1716 G---)A G---~Aat 1716 Exon 10 1 (0.1%) L619S R553Q G-~A at 1790 Exon I 1 I (0.1%) * R668C C--~T at 2134 Exon 13 1 (0.1%) 3849+10 kB C--eT 3030 G---~A G--+A at 3030 Exon 15 1 (0.1%) 405+1 G--~A I1027 T T--~C at 3212 Exon 17a 2 (0.3%) * 3417 A-+T A--->Tat 3417 Exon 17b 1 (0.1%) Unknown 4002 A--eG A--~G at 4002 Exon 20 2 (0.3%) Unknown Cutting et al. (1992) Kobayashi et al. (1990) Kerem et al. (1990) D6rk et al. ( 1991) Fanen et al. (1992) Chillon et al. (1992) Fanen et al. (1992) This study Ferec et al. (1992) ~'Marked (*) sequence variations were present on AF508 chromosomes were the most frequent in pancreas-sufficient patients. Login to comment

[hide] High-density multiplex detection of nucleic acid s... Nucleic Acids Res. 1994 Oct 25;22(21):4527-34. Grossman PD, Bloch W, Brinson E, Chang CC, Eggerding FA, Fung S, Iovannisci DM, Woo S, Winn-Deen ES
High-density multiplex detection of nucleic acid sequences: oligonucleotide ligation assay and sequence-coded separation.
Nucleic Acids Res. 1994 Oct 25;22(21):4527-34., [PMID:7526344]

Abstract [show]
We describe a non-isotopic, semi-automated method for large-scale multiplex analysis of nucleic acid sequences, using the cystic fibrosis transmembrane regulator (CFTR) gene as an example. Products of a multiplex oligonucleotide ligation assay (OLA) are resolved electrophoretically from one another and from unligated probes under denaturing conditions with fluorescence detection. One ligation probe for each OLA target carries a fluorescent tag, while the other probe carries an oligomeric non-nucleotide mobility modifier. Each OLA product has a unique electrophoretic mobility determined by the ligated oligonucleotides and the mobility-modifier oligomer arbitrarily assigned (coded) to its target. The mobility range for practical mobility modifiers is much wider than the accessible range from unmodified ligated oligonucleotides of practical length. Each mobility modifier is built from phosphoramidite monomers in a stepwise manner on its associated oligonucleotide using an automated synthesizer. The resulting mobility modifiers lower the probe-target duplex Tm by less than 3 degrees C and retard probe-target annealing by less than 50%, with negligible effect on OLA yield and specificity. This method is especially useful for allelic discrimination in highly polymorphic genes such as CFTR.

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50 These included GM07224 (normal), GM11496B (G542X homozygote), and GM11274 (D551/AF508). Login to comment
55 Probes for multiplex CF OLA assay CF Locus S549R2-WT S549R2-MUT S549R2-COM S549N WT S549N-MUT S549N-COM G542-WT G542-MUT G542X-COM R553-WT R553X-MUT R553-COM G551-WT G55ID-MUT G55ICOM W1282-WT W1282X-MUT W1282-COM R560T-WT R560T-MUT R560-COM 1717-WT 1717-MUT 1717-COM 3905-WT 39O51NST-MUT 3905-COM Sequence (5'-3') (HEO)2-TTGCTCGTTGACCTCCA (HEO)3-TTGCTCGTTGACCTCCC PO4-CTCAGTGTGATTCCACCT-FAM (HEO)4-TGCTCGTTGACCTCCAC (HEO)5-TGCTCGTTGACCTCCAT PO4-TCAGTGTGATTCCACCTTC-FAM (HEO)6-GTGATTCCACCTTCTCC (HEO)7-GTGATTCCACCTTCTCA PO4-AAGAACTATATTGTCTTTCTCT-FAM (HEO)8-TGCTAAAGAAATTCTTGCTCG (HEO)9-TTGCTAAAGAAATTCTTGCTCA PO4-TTGACCTCCACTCAGTGTGA-FAM (HEO)IO-TAAAGAAATTCTTGCTCGTTGAC (HEO)11-TAAAGAAATTCTTGCTCGTTGAT PO4-CTCCACTCAGTGTGATTCCA-FAM (HEO) 12-TATCACTCCAA AGGCTTTCCTC (HEO) 13-TATCACTCCAAAGGCTTTCCTT PO4-CACTGTTGCAAAGTTATTGAATCC-FAM (HEO)14-TAGACCAATAATTAGTTATTCACC (HEO)15-TAGACCAATAATTAGTTATTCACG PO4-TTGCTAAAGAAATTCTTGCTCG-FAM (HEO) 16-TCTGCAAACTTGG AGATGTCC (HEO) 17-TCTGCAAACTTGGAGATGTCT PO4-TATTACCAAAAATAGAAAATTAGAGA-FAM (HEO) 18-A AGAGTACTTTGTTATCAGCTTTTTT (HEO)19-AAGAGTACTnUTIATCAGCTnTnT PO4-GAGACTACTGAACACTGAAGGAG-FAM Oligonucleotide ligation assay kinetics study For the study of ligation reaction kinetics, probe concentrations were 5 nM, and oligonucleotide target concentration was 0.5 nM. Login to comment
157 Variations in OLA product yield seen in Figure 4 can more likely be attributed to the high degree of overlap of the probes used in the exon 11 mutational hotspot (G542X, S549N, S549R2, G551D, R553X, R560T). Login to comment

[hide] Association of pancreatic adenocarcinoma, mild lun... Clin Chem. 1994 Oct;40(10):1972-4. Tsongalis GJ, Faber G, Dalldorf FG, Friedman KJ, Silverman LM, Yankaskas JR
Association of pancreatic adenocarcinoma, mild lung disease, and delta F508 mutation in a cystic fibrosis patient.
Clin Chem. 1994 Oct;40(10):1972-4., [PMID:7522998]

Abstract [show]
A case of adenocarcinoma of the pancreas and mild lung disease in a 39-year-old man homozygous for the delta F508 cystic fibrosis mutation is presented. Cystic fibrosis is the most common lethal genetic disease in Caucasians, and is most commonly associated with severe obstructive lung disease. To our knowledge, this is only the fifth case of adenocarcinoma of the pancreas in a CF patient to be reported and the first case for which molecular data are available. The rare incidence of this type of malignancy in the general population suggests a possible association of CF with this malignant disease.

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44 CorrelatIon of phenotype and genotype of CFTR mutations Key phenotypic Lung disease SweatC1 Exocnne pancreas function Vasdeferens Associated CFTR mutations Pancreatic InsuffIcIent Pancreatic sufficient Normalsweat C1 Severe Less severe Relatively mild Elevated Elevated Normal Insufficient Sufficient Sufficient Absent Absent Absent SF508, G542X, R553X, G5510, Ni 303K, Wi 282X, RI 17H, and others 2789 + 5G>A, R117H, R334W, R347P, A455E, P574H, S945L, G85E, and others G551S, R117H, 3849 + 10kb C>T, and others Congenitalabsence of the vas deferens None Normal or elevated Sufficient Absent F508C, Ri 17H, Di D1152H, and others FIg. 2. Login to comment

[hide] A cluster of cystic fibrosis mutations in exon 17b... J Med Genet. 1994 Sep;31(9):731-4. Mercier B, Lissens W, Novelli G, Kalaydjieva L, de Arce M, Kapranov N, Canki Klain N, Estivill X, Palacio A, Cashman S, et al.
A cluster of cystic fibrosis mutations in exon 17b of the CFTR gene: a site for rare mutations.
J Med Genet. 1994 Sep;31(9):731-4., [PMID:7529319]

Abstract [show]
Intensive screening has improved our understanding of the profile of mutations in the CFTR gene in which more than 400 mutations have been detected to date. In collaboration with several European laboratories we are involved in such analysis. We have identified 14 new mutations in exon 17b of CFTR, having analysed 780 CF chromosomes, and have compared the frequency of mutations in this exon with that of other regions of the CFTR gene. The results obtained indicate an accumulation of mutations, not only in regions encoding the two nucleotide binding folds, but also in those encoding transmembrane domains of the CFTR gene, in particular exon 17b.

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51 All the most frequent mutations reported are located in the exons encoding the NBFs: AF508 inexon 10, G542X, G551D, and 1717-1 G--A in exon 11, and R1162X in exon 19. Login to comment

[hide] Identification of three novel mutations in the CFT... Hum Genet. 1994 Aug;94(2):154-8. Grade K, Grunewald I, Graupner I, Behrens F, Coutelle C
Identification of three novel mutations in the CFTR gene using temperature-optimized non-radioactive conditions for SSCP analysis.
Hum Genet. 1994 Aug;94(2):154-8., [PMID:7519167]

Abstract [show]
Optimal temperature conditions for the detection of 28 known mutations on 15 exons of the human cystic fibrosis transmembrane conductance regulator gene by single strand conformation polymorphism analysis using the Diagen TGGE Apparatus were established. This procedure was applied to the detection of unknown mutations in 58 non-deltaF508 chromosomes. Three novel mutations, -471del3 (5' flanking region), 3171insC (exon 17a) and 4700(T)8/9 (3' non-translated region) of the CFTR gene were found. Mutation 3171insC occurred in conjunction with the delta F508 mutation on the other allele of a child presenting with severe pathology. Mutation -471del3 has so far only been found in one healthy individual and her father, and 4700(T)8/9 is a DNA sequence polymorphism.

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7 A further 10% (approximately) are attributable to the presence of the mutations R347P, G542X, G551D and R553X (Coutelle et al. 1992). Login to comment
24 G542X G551D R553X G576A Splice rout. Login to comment
36 Lanes 1, 7, wild type; lane 2, G542X/normal; lane 3, G551D/normal; lane 4, G551D/ G551D; lane 5, R553X/normal; lane 6, 1717G--+A/normal Case history of the patient with the 317 linsC mutation Among the patients carrying one non-deltaFs0~mutation, we found one girl with the genotype 3171insC/deltaFs0s. Login to comment

[hide] Premature translation termination mutations are ef... J Biol Chem. 1994 Jul 8;269(27):17802-8. Fearon K, McClendon V, Bonetti B, Bedwell DM
Premature translation termination mutations are efficiently suppressed in a highly conserved region of yeast Ste6p, a member of the ATP-binding cassette (ABC) transporter family.
J Biol Chem. 1994 Jul 8;269(27):17802-8., [PMID:7517933]

Abstract [show]
The requirements for efficient translation termination are incompletely understood. Since the local context surrounding stop codons can influence the efficiency of translation termination, premature termination codons introduced by random mutation may not always terminate at the optimal efficiencies expected of naturally occurring stop codons. To investigate whether this could result in physiologically significant levels of read through, we examined the suppression of premature translation termination mutations within a sequence motif of the yeast Ste6 protein (Ste6p) that is highly conserved among members of the ATP-binding cassette (ABC) transporter family. The human cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in individuals with the disease cystic fibrosis, is also a member of this protein family. The mutations examined in Ste6p were chosen because a premature termination codon at the corresponding residue of CFTR has previously been reported to cause less severe pulmonary involvement than some missense mutations, suggesting that low level suppression of this stop codon could be occurring. Our results indicate that these premature stop codons in Ste6p can be suppressed at frequencies as high as 10%. Characterization of this phenomenon using a beta-galactosidase read through assay system showed that a limited sequence context surrounding this site contained information that was sufficient to cause suppression of translation termination. Amino acid sequence analysis of the full-length translation products produced by read through of an amber codon demonstrated that termination suppression was mediated by near-cognate tRNA mispairing that resulted in the insertion of tyrosine, lysine, or tryptophan.

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35 CFTR was identified as the protein that isdefective in thedisease cystic fibrosis (CF)(13-15).Currently, over 230 CFTR mutations havebeen found that cause CF (16).Among this largecollection of mutations aretwo that result in in-frameopal (UGA) termination codons at glycine codon 542 (G542X) andarginine codon 553 (R553X) of CFTR (see Fig. 1).Both of these mutationsoccur near a highly conserved LSGGQ sequence motif that is shared by all members of the ABC transporter family. Login to comment

[hide] Analysis of mutations and alternative splicing pat... Hum Mol Genet. 1994 Jul;3(7):1141-6. Hull J, Shackleton S, Harris A
Analysis of mutations and alternative splicing patterns in the CFTR gene using mRNA derived from nasal epithelial cells.
Hum Mol Genet. 1994 Jul;3(7):1141-6., [PMID:7526925]

Abstract [show]
Ten to fifteen percent of CF chromosomes carry mutations which are not detected by routine screening of the CFTR gene for known mutations. Many techniques have been used to screen the CFTR gene for these remaining mutations. Most of the methods use genomic DNA, and since the CFTR gene contains 27 exons, are necessarily labour intensive. We have screened the entire coding region of CFTR, by chemical cleavage of 7 overlapping segments of amplified cDNA. Using this method we have identified 4 sequence changes which had not been detected by screening genomic DNA, and successfully detected 10 out of 13 known mutations. In addition, we have identified 8 alternatively spliced forms of CFTR mRNA, 4 of which have not been described previously. These include transcripts lacking a) exon 3, b) exons 2 + 3, c) exons 9 + 12, and d) the final 357 bp of exon 15 as a result of use of the cryptic splice donor site CA2863/GTTCGT).

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33 Of the 13 known sequence changes, 9 (G85E (8), E92K (10), Q220X (11), AF508 (1), G542X (12), G551D (13), 3659delC (12), W1282X (14), 4271delC (11)) were readily identified by •To whom correspondence should be addressed A-6 \ B c ~~i r D t 1 F 1 2 3 4 5 Ga 6b 7 8 9 1 0 1 1 1 2 13 14i 14bl 516 17a 17bl S 19 202122 23 24 MSD 1 NBF 1 R domain MSD 2 NBF 2 Figure 1. Login to comment
37 Autoradiograph showing cleavage products for the 3659delC mutation (577 bases) in the E fragment AF5O8 (634 + 267 bases), G542X (164 bases) and G551D (136 bases) mutations in the B fragment. Login to comment
43 An autoradiograph showing the labeled cleavage products for the AF508, G542X, G551D, and 3659delC mutations is seen in Figure 2. Login to comment
137 They had been screened for the AF5O8, G542X, G551D, R553X and 621 + 1G-T mutations by the clinical genetics service at the Churchill Hospital, Oxford, and they did not carry these mutations. Login to comment

[hide] Evaluation of laboratory methods for cystic fibros... J Med Genet. 1994 Jul;31(7):545-50. Miedzybrodzka ZH, Yin Z, Kelly KF, Haites NE
Evaluation of laboratory methods for cystic fibrosis carrier screening: reliability, sensitivity, specificity, and costs.
J Med Genet. 1994 Jul;31(7):545-50., [PMID:7525964]

Abstract [show]
We report a comparative evaluation of three different laboratory methods for screening large numbers of mouthwash DNA samples for common cystic fibrosis mutations. Sensitivity, specificity, and costs of ARMS (allele refractory mutation detection system), dot blotting, and a deletion/digest/PAGE method (multiplex PCR of exons 10 and 11, digest with HincII followed by polyacrylamide gel electrophoresis (PAGE)) were assessed. ARMS was the most reliable and sensitive method and so was considered more suitable than the cheaper deletion/digest/PAGE. As well as being less reliable than ARMS, the dot blotting method assessed was considerably more costly. ARMS was the best laboratory method for CF screening tested.

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8 Standard methods of CF mutation analysis include deletion detection using polyacrylamide gel electrophoresis (PAGE) of PCR product (for example, AF508 and AI507), restriction enzyme digestion ofPCR product (for example, G551D, R553X, 621 + 1(G-.T)), and probing of PCR product with labelled allele specific oligonucleotides (ASOs) (for example, G542X). Login to comment
12 Methods The Cellmark ARMS system tests simultaneously for the mutations AF508, G551D, G542X, and 621 + 1(G--T).5 PCR is performed in two tubes, one tube containing mutation specific primers for G551D, G542X, and normal site specific primers for AF508 and 621 + 1(G-+T), the second containing mutation specific primers for AF508 and 621 + 1(G-4T) and normal site specific primers for G551D and G542X. Login to comment
19 We chose to evaluate dot blotting by using the Inno-LiPA CF2 kit as an example (Innogenetics).9 This kit detects the mutations AF508, AI507, G551D, G542X, N1303K, W1282Xm 1717-1,G-A, and R553X.4 12"1 Mouthwash DNA preparation (5 p1) and Taq polymerase are added to the PCR reagents provided, with biotin dUTP incorporated into PCR product as a label. Login to comment
26 2A + B normal sample: lane A indicates normal sequence at AF508 and 621 + 1, G-T mutation sites, lane B indicates normal sequence at G542X and G551D mutation sites. Login to comment
30 8A + B G542X/621 + 1, G-T compound heterozygote. Login to comment
32 12A + B G542X carrier. Login to comment
39 Strip 12 is AF508/G542X compound heterozygote, 14 is AF508IG551D compound heterozygote, 15 is 1717-1,G--A carrier, 16 is A1507 carrier, 17 is W1282X carrier. Login to comment
84 The G542X mutation specific band appeared, although it was fainter than its control. Login to comment
85 When the test was repeated on two occasions, no mutation specific band was present and direct sequencing confirmed that DNA tested had the wild type sequence at the G542X site. Login to comment
97 About half of the reported failures occurred in the first two tests in a batch, with faint extra bands in the mutant G551D and G542X positions. Login to comment

[hide] Mutation analysis in 600 French cystic fibrosis pa... J Med Genet. 1994 Jul;31(7):541-4. Chevalier-Porst F, Bonardot AM, Gilly R, Chazalette JP, Mathieu M, Bozon D
Mutation analysis in 600 French cystic fibrosis patients.
J Med Genet. 1994 Jul;31(7):541-4., [PMID:7525963]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) gene of 600 unrelated cystic fibrosis (CF) patients living in France (excluding Brittany) was screened for 105 different mutations. This analysis resulted in the identification of 86% of the CF alleles and complete genotyping of 76% of the patients. The most frequent mutations in this population after delta F508 (69% of the CF chromosomes) are G542X (3.3%), N1303K (1.8%), W1282X (1.5%), 1717-1G-->A (1.3%), 2184delA + 2183 A-->G (0.9%), and R553X (0.8%).

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2 The most frequent mutations in this population after AF508 (69% of the CF chromosomes) are G542X (3-3%), N1303K (1P8%), W1282X (1P5%), 1717-lG-.A (1P3%), 2184delA+2183 A-+G (0 9%), and R553X (0-8%). Login to comment
23 The most frequent mutations after AF508 are G542X (33%), N1303K (1*8%), W1282X (1 5%), 1717-1G-+A (1 3%), 2184delA+ 2183A-4G (09%), and R553X (08%). Login to comment
57 For the other partner, who has an initial risk of being a carrier of 1 in 25, the screening ofthe seven mutations AF508, G542X, N1303K, W1282X, 1717-1G-+A, 2184delA+2183A-.G, and R553X allows a better estimation ofthis risk; it drops to 1 in 120 if this screening is negative. Login to comment

[hide] Identification of three novel mutations (457 TAT--... Hum Genet. 1994 Jun;93(6):659-62. Audrezet MP, Canki-Klain N, Mercier B, Bracar D, Verlingue C, Ferec C
Identification of three novel mutations (457 TAT-->G, D192G, Q685X) in the Slovenian CF patients.
Hum Genet. 1994 Jun;93(6):659-62., [PMID:7516305]

Abstract [show]
Chromosomes from a cohort of 60 Slovenian families, corresponding to the 121 cystic fibrosis (CF) chromosomes available, were fully scanned for mutations in the coding sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene (The 60 families yielded 121 CF alleles because the mother of one patient was also affected). This corresponds to the 27 exons and intron/exon boundaries that have been studied in chromosomes carrying unidentified alleles. As a result of this survey 84% of the alleles are now clearly identified and we describe in this paper three novel mutations (457 TAT-->G, D192G, and Q685X).

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40 She carries the G542X mutation on the other chromosome (Table 1, CF-53). Login to comment
50 Some clinical data of patients with the three novel mutations Patient Exon Mutation Age of Age at Chloride sweat Pancreatic Lung Pseudomonas onset diagnostic test mmol/per 1 function disease aeruginosa CF-53 4 457 TAT--~G Birth 4 months 90.9-167.0 Insufficient Severe Yes 11 G542X CF-67 5 D192G 2 months 3 months 58.8-106.7 Insufficient Moderate No 10 AF508 CF-52 13 Q685X 3 months 6 years 113.4-177.2 Insufficient Severe Yes 10 AF508 Table 2. Login to comment
51 Mutations identified in the population of Slovenia Number of chromosomes Mutations 661 83 AF508 4 G542X 5 R1162X 2 3905 ins T 2 I148T 1 Q552X 1 Q685X 1 S4X 1 457 TAT---~G 1 D192G 1 R1066H 19 Unidentified 121 Exons Frequencies References 10 68.60% Kerem et al. (1989) 11 3.30% Kerem et al. (1990) 19 4.10% Gasparini et al. (1991) 20 1.65% Personal Communication 4 1.65% Personal Communication 11 0.85% Devoto et al. (1991) 13 0.85% This study 1 0.85% Glavac et al. (1993) 4 0.85% This study and Glavac et al. (1993) 5 0.85% This study 17b 0.85% Ftrec et al. (1992) 15.70% by 11 mutations, occurring in 9 exons of the gene (1, 4, 5, 10, 11, 13, 17b, 19, and 20). Login to comment
54 A study performed by Dabovic et al. (1992) in a cohort of 54 families, each with one living CF child, from Serbia, Montenegro, Bosnia, and Herzegovina showed that the AF508 accounts for 70% (76/108 CF chromosomes) of CFTR genes and the G542X, for 4% (4/108). Login to comment
63 The distribution of mutations in our study differs from that observed in other populations of the Mediterranean area where, besides the AF508, two mutations, G542X and R1162X, have a frequency greater than 1% (Nunes et al. 1991). Login to comment

[hide] Novel cystic fibrosis mutation associated with mil... Hum Genet. 1994 May;93(5):529-32. Boteva K, Papageorgiou E, Georgiou C, Angastiniotis M, Middleton LT, Constantinou-Deltas CD
Novel cystic fibrosis mutation associated with mild disease in Cypriot patients.
Hum Genet. 1994 May;93(5):529-32., [PMID:7513296]

Abstract [show]
Cyprus is an island in the eastern Mediterranean basin inhabited by people of Caucasian extraction, mostly Greek-Cypriots. The most common inherited disease among Caucasians is cystic fibrosis (CF). Although no careful scientific study had ever been done the impression was that CF was extremely rare among the Greek-Cypriots, with an incidence estimated at around 1:30,000. About 2 years ago, we introduced molecular diagnostic methodology in an effort to assist clinicians in safer diagnosis of patients presenting with atypical CF symptomatology, and also for testing the hypothesis that mutations that cause milder phenotypes might be responsible for misdiagnosis or for missing entirely some cases of CF. Initial screening for delta F508 revealed that it is indeed rare in the general population. Further screening of suspected CF patients revealed a novel mutation that converted leucine at position 346 to proline (L346P) in two unrelated families. The second CF mutation was delta F508 and 1677delTA in the two families respectively, both reportedly associated with severe phenotypes. Yet our patients did not present with typical CF pictures possibly because of the dominant nature of this novel mild mutation in exon 7. Symptoms included failure to thrive, chest infections and electrolyte disturbances. These findings raise the possibility that Cyprus might have been spared very severe CF phenotypes but not cystic fibrosis transmembrane conductance regulator (CFTR) mutations.

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39 Those mutations were 621+lG>T, G542X, G551D, R553X, W1282X and R1283M, and the methodology used is the Amplification Refractory Mutation System (Newton et al. 1989). Login to comment

[hide] Sensitivity of single-strand conformation polymorp... Hum Mol Genet. 1994 May;3(5):801-7. Ravnik-Glavac M, Glavac D, Dean M
Sensitivity of single-strand conformation polymorphism and heteroduplex method for mutation detection in the cystic fibrosis gene.
Hum Mol Genet. 1994 May;3(5):801-7., [PMID:7521710]

Abstract [show]
The gene responsible for cystic fibrosis (CF) contains 27 coding exons and more than 300 independent mutations have been identified. An efficient and optimized strategy is required to identify additional mutations and/or to screen patient samples for the presence of known mutations. We have tested several different conditions for performing single-stranded conformation polymorphism (SSCP) analysis in order to determine the efficiency of the method and to identify the optimum conditions for mutation detection. Each exon and corresponding exon boundaries were amplified. A panel of 134 known CF mutations were used to test the efficiency of detection of mutations. The SSCP conditions were varied by altering the percentage and cross-linking of the acrylamide, employing MDE (an acrylamide substitute), and by adding sucrose and glycerol. The presence of heteroduplexes could be detected on most gels and in some cases contributed to the ability to distinguish certain mutations. Each analysis condition detected 75-98% of the mutations, and all of the mutations could be detected by at least one condition. Therefore, an optimized SSCP analysis can be used to efficiently screen for mutations in a large gene.

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121 1078delT (35), L327R (Ravnik-Glavac a al., unpublished), R334W (36), D36K (31), R347L (26), R347P (14), A349V (26), R352Q (30), 1221delCT (34); Exon 8: W401X (31), 1342-1G-C (25); Exon 9: G458V (37), 1525 -1G-A (38); Exon 10: S492F (34), Q493X (39), 1609delCA (40,17), deltaI507 (39,41), deltaF5O8 (3), 1717-1G-A (39,42); Exon 11: G542X (39), S549N, G551D, R553X (43), R553Q (44), A559T (43), R560K (Fine et al., pers. comm.), R560T (39); Exon 12: Y563N (39), 1833delT (Schwartz et al., pers. comm.), P574H (39), 1898 + 1G-C (31), 1898+3A-G (Ferrari et al., pers. comm.); Exon 13: G628R(G-C) (31), Q685X (Firec et al., pers. comm.), K716X (26), L719X (Dork etal., pers. comm.), 2522insC (15), 2556insAT (45), E827X (34); Exon 14a: E831X (Ffrec et al., pers. comm.), R851X (29), 2721delll (31), C866Y (Audrezet et al., pers. comm.); Exon 14b: 2789+5G-A (Highsmith et al., pers. comm.); Exon 15: 2907denT (21), 2991del32 (Dark and TQmmler, pers. comm.), G970R (31); Exon 16: S977P, 3100insA (D6rk et al., pers. comm.); Exon 17a: I1005R (Dork and TQmmler, pers. comm.), 3272-1G-A (46); Exon 17b: H1054D (F6rec et al., pers. comm.), G1061R (Fdrec et al., pers. comm.), 332Oins5, R1066H, A1067T (34), R1066L (Fe"rec etal., pers. comm.), R1070Q (46), E1104X (Zielenski el al., pers. comm.), 3359delCT (46), L1077P (Bozon « a/., pers. comm.), H1085R (46), Y1092X (Bozon etal., pers. comm.), W1098R, M1101K (Zielenski et al., pers. comm.); Exon 18: D1152H (Highsmith et al., pers. comm.); Exon 19:R1162X (36), 3659delC (39), 3662delA (25), 3667del4 (Chillon et al., pers. comm.), 3737ddA (35), 3821ddT (15), I1234V (35), S1235R (31), Q1238X (26), 3849G-A (25), 385O-3T-G (38); Exon20:3860ins31 (Chillon etal., pers. comm.), S1255X (47), 3898insC (26), 3905insT (Malik et al., pers. comm.), D127ON (48), W1282X (49), Q1291R (Dork et al., pers. comm.), Exon 21: N1303H (35), N13O3K (50), W1316X (43); Exon 22: 11328L/4116delA (Dork and TQmmler, pers. comm.), E1371X (25); Exon 23: 4374+ 1G-T (38); Exon 24: 4382delA (Claustres et al., pers. comm.). Login to comment

[hide] Analysis of the whole CFTR coding regions and spli... Hum Genet. 1994 Apr;93(4):467-70. Culard JF, Desgeorges M, Costa P, Laussel M, Razakatzara G, Navratil H, Demaille J, Claustres M
Analysis of the whole CFTR coding regions and splice junctions in azoospermic men with congenital bilateral aplasia of epididymis or vas deferens.
Hum Genet. 1994 Apr;93(4):467-70., [PMID:7513294]

Abstract [show]
Several recent studies have demonstrated the presence of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in healthy males with infertility caused by congenital absence of the vas deferens (CBAVD), previously recognized as an idiopathic genetic condition distinct from CF. In order to document further the genetic commonality of these two disorders, we undertook a double screening of the entire coding and flanking sequences of the CFTR gene, by using single-strand conformational polymorphism analysis and denaturing gradient gel electrophoresis in 12 unrelated infertile men with abnormalities of the vas deferens and/or epididymis. This strategy allowed us to identify 11 DNA sequence alterations considered as CF-causing mutations and several variations. Despite this double analysis, only two patients out of eight with CBAVD could be demonstrated as compound heterozygotes for CF mutations.

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31 Two CFTR mutations were found in patients not described as CBAVD: the stop mutation G542X (Kerem et al. 1990) in a case with congenital unilateral epididymis and vas deferens aplasia (CUAVD) (patient no. 4), and the missense mutation S1235R (Cuppens et al. CF Consortium) in a patient with congenital bilateral aplasia of the tail of epididymis (CBAE) (patient no. 7). Login to comment
33 CFTR mutations identified in 12 patients lacking an epididymis or vas deferens Patient Age Mutations Exon XV2c/KM19 (years) haplotypesa CBAVD 1 43 Unknown B Unknown A CBAVD 6 33 p AF508/A1067T 10/17b B m Unknown A CBAVD 8 32 p Unknown A m AF508 10 B CBAVD 9 27 p R347H 7 C m AF508 10 B CBAVD 11 31 m Unknown D p 2184delA+A---~G at 2183 13 B CBAVD 12 30 Unknown B Unknown B CBAVD 13 32 p AF508 10 B m Unknown A CBAVD 14 AF508 10 B R347H 7 C CUAVD 4 35 m G542X 11 B p Unknown D CBAE 2 28 Unknown C Unknown D CBAE 7 34 p Unknown B m S1235R 19 A CBAE 10 30 Unknown B Unknown C CBAVD, Congenital bilateral aplasia of the vas deferens; CUAVD, congenital unilateral aplasia of the vas deferens; CBAE, congenital bilateral aplasia of the epididymis; p, paternal chromosome; m, maternal chromosome The four haplotypes defined by the CFTR-linked polymorphic restriction sites XV-2c and KM-19 are as follows: A, 1-1; B, -2; C, 2-1; D, 2-2 (the absence of the restriction site for each polymorphism is defined as allele "1", and the presence of the site as allele "2") Table . Login to comment

[hide] Analysis of the CFTR gene confirms the high geneti... Hum Genet. 1994 Apr;93(4):447-51. Chillon M, Casals T, Gimenez J, Ramos MD, Palacio A, Morral N, Estivill X, Nunes V
Analysis of the CFTR gene confirms the high genetic heterogeneity of the Spanish population: 43 mutations account for only 78% of CF chromosomes.
Hum Genet. 1994 Apr;93(4):447-51., [PMID:7513293]

Abstract [show]
We have analysed 972 unrelated Spanish cystic fibrosis patients for 70 known mutations. Analysis was performed on exons 1, 2, 3, 4, 5, 6a, 6b, 7, 10, 11, 12, 13, 14a, 14b, 15, 16, 17b, 18, 19, 20 and 21 of the cystic fibrosis transmembrane regulator gene using single strand conformation polymorphism analysis and denaturing gradient gel electrophoresis. The major mutation delta F508 accounts for 50.6% of CF chromosomes, whereas another 42 mutations account for 27.6% of CF chromosomes, with 21.8% of Spanish CF chromosomes remaining uncharacterized. At present, we have identified 36 mutations that have frequency of less than 1% and that are spread over 15 different exons. This indicates that, in the Spanish population, with the exception of delta F508 (50.6%) and G542X (8%), the mutations are not concentrated in a few exons of the gene nor are there any predominating mutations. This high degree of genetic heterogeneity is mainly a result of the different ethnic groups that have populated Spain and of the maintenance of separated population sets (Basques, Arab-Andalusian, Mediterranean, Canarian and Gallician). The high proportion of CF chromosomes still unidentified (21.8%) together with association analysis with intragenic markers suggest that at least 100 different mutations causing CF are present in our population.

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5 This indicates that, in the Spanish population, with the exception of AF508 (50.6%) and G542X (8%), the mutations are not concentrated in a few exons of the gene nor are there any predominating mutations. Login to comment
22 We have previously analysed the three most frequent mutations in our population (AF508, G542X and N1303K), and the association between CF Spanish chromosomes and their intragenic microsatellites IVSCA8-IVS17BTA-IVS 17BCA (Nunes et al. 1991; Casals et al. 1992; Morral et al. 1993; Chilldn et al. 1993). Login to comment
30 There are seven mutations with a frequency higher than 1%, viz. AF508 (50.6%), G542X (8.0%), N1303K (2.3%), 3601-111G---~C (1.9%), Rl162X (1.8%, 711+IG--+T (1.2%) and R334W (1.1%), whereas the other 36 mutations have a frequency lower than 1% and account for only about 11% of CF chromosomes. Login to comment
34 A non-uniform distribution had previously been observed in the Spanish population for the AF508 and G542X mutations (Casals et al. 1993) but this had not been shown for any other mutations. Login to comment
37 When we analysed the Spanish CF population in subsets (Fig. 1), we found typical mutations; in the Mediterranean subset, G542X and N1303K (11.8% and 4.5%, respectively); in the Arab-Andalusian subset, 3601-111G---~ C (5.6%); in the Gallician subset, Rl162X and 711+1 G---~T (8.5% and 6.4%, respectively); in the Canary islands, because of the founder effect, we observed only a few mutations but with high frequencies, whereas we only found the AF508 mutation and none of the other six most common mutations in the Basques. Login to comment
40 Frequencies of CF mutations in the Spanish population Mutation Exon/intron N~chro- % mosomes Known (43) 760 78.18 AF508 Exon 10 492 50.61 G542X Exon 11 78 8.02 N1303K Exon 21 23 2.36 3601-111 G---~C Intron 18 19 1.95 R1162X Exon 19 18 1.85 711+1 G---~T Exon 5 12 1.23 R334W Exon 7 11 1.13 1609 del CA Exon 10 9 0.92 G85E Exon 3 8 0.82 2789+5 G---~A Intron 14b 7 0.72 2869 ins G Exon 15 7 0.72 R1066C Exon 17b 7 0.72 W1282X Exon 20 6 0.62 AI507 Exon 10 5 0.51 3272-26 A---~G Intron 17a 5 0.51 G551D Exon 11 4 0.41 1812-1 G---~A Intron 11 4 0.41 2184 de! Login to comment
42 Thus, of the seven most common mutations, they only have the G542X 4/47=8.5% MUTATION )/39=0% N1303K 0/47=0% MUTATION D/39=0% 3601-111G>CMUTATION )/47=0% _oI~=O% 10/260:3.8% )4" 26/220=11.8% I~7/49=14.3%~Qr ~lP,m. Login to comment
43 5/260=1.9%41~4," p- I0/220=4,5% 1/49:2.0%CT !/248=1.2% 13/'232=5.6% ~=0% R1162XMUTATION t/47=8.S% ~I3/49--6.1%(I" f-it*" 711+1G>T 5/47=6A% MUTATION 0~9=0% <7" Fig.1.DistributionoftheG542X,N1303K,3601-111G---~C,R1162X,7tI+IG--)Tand R334WmutationsinthedifferentsubsetsoftheSpanishpopulation.Onlythe615chro- mosomesforwhichtheethnicorigincouldbeestablishedhavebeenincluded.Subsets withahighfrequencyforagivenmutationareshaded.TheMediterraneansubsethasa R334WMUTATION 1/47=2.1% highfrequencyforG542XandN1303K;theArab-Andalusiansubsetfor3601-111G---) C;theGalliciansubsetforG542X,R1162Xand711+1G---)C;theCanariansubsetfor G542X,RlI62XandR334W,whereasnoneofthesesixmutationsispresentinthe Basquesubset4~ ~D AF508 mutation (76.9% compared with 50.6% for the rest of Spain). Login to comment
46 The Mediterranean subset The Mediterranean subset shows high frequencies of the mutations G542X and N1303K. Login to comment
48 The G542X mutation has been postulated to have arisen in a population of Semitic origin (Phoenicians and Jews) in the Neolithic Age, and to have been introduced into Spain, via the Mediterranean Sea, by Phoenicians (Casals et al. 1993); this would explain its high frequency in this subset (11.8%) with respect to the rest of Spain (3.8%) (~2 = 10.1, P < 0.001). Login to comment
59 Thus, we have found AF508 (46.9%), G542X (14.3%), Rl162X (6.1%), R334W (4.0%) and N1303K (2.0%) but we have not found 3601-111G--->C, 711+IG-~T or any of the other 36 less frequent mutations. Login to comment
69 There are seven mutations with a frequency higher than 1% (AF508, G542X, N1303K, 3601-111G-->C, Rl162X, 711+IG---~T and R334W), whereas another 36 less frequent mutations account for only 11% of the CF chromosomes. Login to comment
70 These results show that the Spanish population [like other Southern and Mediterranean European populations (Nunes et al. 1991)] has a marked genetic heterogeneity, since the CF mutations are not confined to a few exons and there are no predominant mutations (except for AF508 and G542X). Login to comment
75 Thus, the Basque subset has the AF508 mutation but none of the other 6 mutations; the Mediterranean subset has a high frequency of G542X and N1303K (as in South European populations); the Arab-Andalusian subset has a high frequency of 3601-111G---~C, which we propose has an Arab origin; the Canarian Islands has a founder effect, where we find high frequencies of the G542X, Rl162X and R334W mutations, and the Gallician subset has a high frequency of G542X, R1162X and 711+1G---~C. Login to comment

[hide] Retrospective study of the cystic fibrosis transme... Hum Genet. 1994 Apr;93(4):429-34. Verlingue C, Mercier B, Lecoq I, Audrezet MP, Laroche D, Travert G, Ferec C
Retrospective study of the cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in Guthrie cards from a large cohort of neonatal screening for cystic fibrosis.
Hum Genet. 1994 Apr;93(4):429-34., [PMID:7513292]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes a cAMP-activated chloride channel, and in individuals with both alleles of the gene mutated, symptoms of CF disease are manifest. With more than 300 mutations so far described in the gene the profile of mutant alleles in a population is specific to its ethnic origin. For an analysis with an unbiased recruitment of the CF alleles in neonates of similar origin (Normandy, France), we have retrospectively analyzed the Guthrie cards of affected newborns, diagnosed by the immunoreactive trypsinogen (IRT) assay. Analysis of the 27 exons of the CFTR gene using a GC clamp denaturing gradient gel electrophoresis (DGGE) assay has enabled us to identify over 96% of the mutated alleles. Two of these were novel mutations. We would like to propose this strategy as an efficient method of retrospective molecular genetic diagnosis that can be performed wherever Guthrie cards can be obtained. Knowledge of rare alleles could be a prerequisite for CF therapy in the future.

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68 CFI'R mutations characterized by denaturing gradient gel electrophoresis and DNA sequencing from Guthrie cards of 98 children with cystic fibrosis 431 Number of Mutations Exons Fre- References chromo- quencies somes 129 AF508 10 65.8 Kerem et al. 1989 5 G551D 11 2.8 Cutting et al. 1990 3 2183 AA---~G 13 1.7 unpublished data 3 N1303K 21 1.7 Osborne et al. 1991 3 G542X 11 1.7 Kerem et ai.1990 2 E92K 4 1.1 Nunes et al. 1993 2 I148T 4 1.1 unpublished data 2 574 del A 4 1.1 Fanen et a1.1992 2 1078 del T 7 1.1 Claustres et a1.1992 2 E585X 12 1.1 Cremonesi et al. 1992 2 2789 + 5 G--->A intron 14b 1.1 unpublisheddata 2 3659 del C 19 l. Login to comment

[hide] Heterogeneity in the severity of cystic fibrosis a... Hum Genet. 1994 Apr;93(4):364-8. Dean M, Santis G
Heterogeneity in the severity of cystic fibrosis and the role of CFTR gene mutations.
Hum Genet. 1994 Apr;93(4):364-8., [PMID:7513291]

Abstract [show]
Cystic fibrosis is a common, fatal disorder caused by abnormalities in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR encodes a chloride channel that regulates secretion in many exocrine tissues. The presentation of cystic fibrosis is highly variable as measured by the age of onset of disease, the presence of pancreatic insufficiency, or the progression of lung disease. Over 400 mutations in the CFTR gene have been described in cystic fibrosis patients and considerable effort has focused on the correlation between specific mutations and genotypes and clinical characteristics. Individual tissues display variation in their sensitivity to CFTR mutations. The vas deferens is functionally disrupted in nearly all males, whereas mild and severe pancreatic involvement is determined by the patient's genotype. The severity of pulmonary disease is poorly correlated with genotype, suggesting that there are other important genetic and/or environmental factors that contribute to lung infections and the subsequent disruption of lung function.

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57 These studies found no significant differences in % FEV1 or age of onset of chronic sputum colonization by Pseudomonas aeruginosa between AF508/G551D, AF508/G542X, AF508/ R553X, AF508/W1282X, AF508/N1303K, AF508/1717- 1G-A, AF508/621+lG-T compound heterozygotes and AF508 homozygotes (Hamosh et al. 1991; Cystic Fibrosis Genotype Analysis Consortium 1990). Login to comment

[hide] Two novel mutations in the CFTR gene: W1089X in ex... Hum Mol Genet. 1994 Apr;3(4):657-8. Shoshani T, Augarten A, Yahav J, Gazit E, Kerem B
Two novel mutations in the CFTR gene: W1089X in exon 17B and 4010delTATT in exon 21.
Hum Mol Genet. 1994 Apr;3(4):657-8., [PMID:7520798]

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7 Together with the AF5O8 mutation and three other less common mutations (G542X, N1303K and 3849 + 10 Kb C-T) CFFM Figure 1. Login to comment

[hide] CFTR haplotype backgrounds on normal and mutant CF... Hum Mol Genet. 1994 Apr;3(4):607-14. Cuppens H, Teng H, Raeymaekers P, De Boeck C, Cassiman JJ
CFTR haplotype backgrounds on normal and mutant CFTR genes.
Hum Mol Genet. 1994 Apr;3(4):607-14., [PMID:7520797]

Abstract [show]
Ten polymorphic loci, located in a 1 Mb interval across the cystic fibrosis locus, were analyzed on normal and mutant CFTR genes. A different distribution of haplotype backgrounds among normal and mutant CFTR genes was observed. With exception of the D7S8 locus, the three most common mutations, delta F508, G542X and N1303K, were found on an identical haplotype background. In agreement with the observed linkage equilibrium between the Q1463Q and D7S8 loci, both alleles at the D7S8 locus were found on delta F508 CFTR genes. However, the G542X and N1303K mutations, which have been estimated to be at least 35000 years old, were found to be associated with a single allele at the D7S8 locus. Absence of recombination between the D7S8 and Q1463Q loci was also observed on normal CFTR genes with this haplotype background. At the Tn locus in intron 8, allele 9 known to result in very efficient splicing was associated with the most frequent mutations. At the M470V locus, located in a conserved region of the first nucleotide binding fold, the amino acid methionine was found to be associated with the frequent mutations, in particular with mutations located in one of the two nucleotide binding folds which are generally known as severe mutations with regard to exocrine pancreatic function. On mutant CFTR gene, this locus was in complete association with the centromeric D9 locus, in the absence of a complete association with the intervening loci.(ABSTRACT TRUNCATED AT 250 WORDS)

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4 With exception of the D7S8 locus, the three most common mutations, AF508, G542X and N1303K, were found on an identical haplotype background. Login to comment
6 However, the G542X and N1303K mutations, which have been estimated to be at least 35000 years old, were found to be associated with a single allele at the D7S8 locus. Login to comment
34 Distribution of alleles at 10 polymorphic loci Locus Allele Normal Mutant Mutations XV2c KM19 D9 1 2 1 2 1 2 58 (0.492) 60 (0.508) 84 (0.622) 51 (0.378) 78 (0.586) 55 (0.414) 146 (0.918) 13 (0.082) 19(0.109) 156 (0.891) 15 (0.085) 161 (0.915) 1001 + llC/T Tn 115 (0.927) R 9 (0.073) 5 7 (0.057) 7 102 (0.836) 9 13 (0.107) M470V C 62 (0.496) R 63 (0.504) 1898+15 2T/A C 84(0.641) R 47 (0.359) T854T Q1463Q D7S8 C 82 (0.636) R 47 (0.364) C 90 (0.692) R 40 (0.308) 1 38 (0.317) 2 82 (0.683) 33 (0.192) 139 (0.808) 0 (0.000) 32 (0.190) 136 (0.810) 156 (0.902) 17 (0.098) 163 (0.926) 13 (0.074) 162 (0.926) 13 (0.074) 162 (0.931) 12 (0.069) 91 (0.569) 69 (0.431) E60X, 622-2A-C, A455E, AF508 (98.3%), 1717-1G-A, G542X, 0.479 63.54 G628R(G-C)/S1235R,2183AA-G, G970R, W1282X, N1303K p<10~ G458V, AI5O7, AF508 (1.7%), 1898 + 1G-C, E73OX, 3272-26A-G, W1310X, 4218insT, UA, UB, UC I336K, W401X, 2T2ldelll, Y1092X, 3659delC, S1251N: not included (5%) E60X, 622-2A-C, W401X, G458V, AF5O8 (1.6%), 1898+ 1G-C, -0.541 90.63 G628R(G-Q/S1235R, E730X, G970R, 3272-26A-G (50.0%), p<10" Y1092X, 3659delC, S1251N, W1310X, UB, UTC A455E, AI507, AF5O8 (98.4%), 1717- 1G-A, G542X, 2183AA-G, 3272-26A-G (50.0%), W1282X, N13O3K, 4218insT, UA 1336K, 2721delU: not included (1%) E60X, 622-2A-C, W401X, G458V, 1898 +1G-C, E730X, G970R, -0.541 90.46 Y1092X, 3659delC, S1251N, W1310X, UB, UC p<10" A455E, AI507, AF508, 1717- 1G-A, G542X, G628R(G-Q/S1235R, 2183AA-G, 3272-26A-G, W1282X, N13O3K, 4218insT, UA I336K, 2721delll: not included (1%) E60X, 622-2A-C, I336K, W401X, G458V, AI507, 1717- 1G-A, -0.726 155.94 1898 + 1G-C, G628R(G-C)/S1235R, 2183AA-G, E730X, 2721delll, p< 10" G970R, 3272-26A-G, Y1092X, 3659delC, S1251N, W1282X, W1310X, 4218insT, UA, UB, UC A455E, AF5O8, G542X, N13O3K E60X, 622-2A-C, I336K, W401X, G458V, AI507, 1717-1G-A, 1898 + 1G-C, G628R(G-C)/S1235R, 2183AA-G, E730X, 2721delll, G970R, 3272-26A-G, Y1092X, 3659delC, S1251N, W1282X, W1310X, 4218insT, UA, UB, UC A455E, AF5O8, G542X, N13O3K A455E, AI5O7, AF508, 1717-1G-A, G542X, G628R(G-Q/S1235R, 2183AA-G, 3272-26A-G, W1282X, N13O3K, 4218insT, UA E60X, 622-2A-C, W401X, G458V, 1898 + 1G-C, E730X, G970R, Y1092X, 3659delC, S1251N, W1310X, UB, UC 1336K, midclll: not included (1%) E60X, 622-2A-C, W401X, A455E, G458V, AF508 (99.2%), G542X, 1898 + 1G-C, 2183AA-G, E730X, G970R, Y1092X, 3659delC, S1251N, N1303K, W1310X, UB, UC AI507, AF5O8 (0.8%), 1717-1G-A, G628R(G-Q/S1235R, 3272-26A-G, W1282X, 4218insT, UA I336K, 2721delU: not included (1%) E60X, 622-2A-C, W401X, A455E, G458V, AF508 (99.2%), G542X, 1898+1G-C, 2183AA-G, E730X, G970R, Y1092X, 3659delC,S1251N, N13O3K, W1310X, UB, UC AI507, AF508 (0.8%), 1717-1G-A, G628R(G-C)/S1235R, 3272-26A-G, W1282X, 4218insT, UA 1336K, midelll: not included (1%) E60X, 622-2A-C, W401X, A455E, G458V, AF5O8 (99.2%), G542X, G628R(G-Q/S1235R, 2183AA-G, E730X, G970R, Y1092X, 3659delC, S1251N,N1303K, W1310X, UC AI507, AF5O8 (0.8%), 1717-1G-A, 1898 + 1G-C, 3272-26A-G, W1282X, 4218insT 1336K, 2721del11. Login to comment
35 UA, UB: not included (2%) A455E, AF508 (61.2%), 1717-1G-A (66.7%), G542X, G628R(G-C)/S1235R, 3272-26A-G, S1251N, W1282X, W1310X E60X, 622-2A-C, W401X, G458V, AJ507, AF5O8 (38.8%), 1717- 1G-A (33.3%), 1898 +1G-C, 2183AA-G, E730X, G970R, Y1092X, 3659delC, N13O3K, 4218insT, UA, UB, UC 1336K, 2721delll: not included (1%) -0.694 139.81 p<10~ 0.452 60.83 p<10" 0.355 38.77 p<10" 0.360 39.44 p<10~7 0.314 29.91 0.250 17.54 p<10"4 The observed CFTR genes associated with a particular allele are given, proportions are given between brackets. Not all the mutations were informative for each of the tested loci, which were therefore not included. For the Tn locus the standardized linkage disequilibrium coefficient was calculated for the group of the non-T9 alleles and the T9 alleles. Login to comment
37 Standardized linkage disequilibrium coefficient at ten polymorphic loci for all mutant CFTR genes and for individual AF5O8, G542X and N13O3K CFTR genes. Login to comment
45 At the D7S8 locus, a higher value of A was found for G542X CFTR genes than for AF508 CFTR genes. Login to comment
46 All G542X CFTR genes (9 genes studied) were associated with allele 1, while only about 60% of AF508 CFTR genes (116 genes studied) were associated with this allele. Login to comment
59 These mutations are also the most common mutations worldwide, such as the G542X, "N1303K, 1717- 1G-A and W1282X mutations. Login to comment
72 Extragenic (XV2c/KM19/D9) haplotypes Haplotype Normal Mutant Mutations 111 211 121 112 212 122 222 23 (0.204) 43 (0.381) 2 (0.018) 6 (0.053) 0 (0.000) 22 (0.195) 17 (0.150) 4 (0.026) E60X, 622-2A-C, G970R 6 (0.039) G458V, 1898+1G-C, E73OX, W1310X, UB, UC 0 (0.000) 3 (0.019) AF5O8 (1.7%), G628R(G-Q/S1235R 1 (0.006) 3272-26A-G (50.0%) 134 (0.870) A455E, AF508 (96.5%), 1717-1G-A, G542X, 2183AA-G, W1282X, N13O3K 6 (0.039) AI507, AF508 (1.8%), 3272-26A-G (50.0%), 4218insT, UA p<10"3 p<10"7 p<10~7 p<10"2 The observed CFTR genes associated with a particular haplotype are given, proportions are given between brackets. Login to comment
95 With the exception of the D7S8 locus, the three most common mutations AF508, G542X, and N1303K shared the same haplotype background. Login to comment
103 CFTR haplocypes I II ma mb rv V VI Haplotype C7RCCC 211C7RCCC1 111C7RCCC1 /11C7RCCC1 211C7RCCC2 111C7RCCC2 /11C7RCCC2 122C7RCCC2 121C7RCCC2 C5CRRR 122C5CRRR1 211C5CRRR2 222C5CRRR2 C7CRRR 122C7CRRR1 222C7CRRR1 212C7CRRR1 122C7CRRR2 222C7CRRR2 122C7CRRR/ C9CRRR 211C9CRRR1 R9CCCC 122R9CCCC1 222R9CCCC1 /22R9CCCC1 112R9CCCC1 122R9CCCC2 222R9CCCC2 112R9CCCC2 R9CRRR 122R9CRRR1 C7CRRC 112C7CRRC1 112C7CRRC2 C9CRRC 211C9CRRC1 C7CCCC 211C7CCCC1 222C7CCCC1 122C7CCCC2 C7RCCR 211C7RCCR2 Normal 0.524 (43) 0.085 0.073 0.195 0.146 0.012 0.012 0.049 (4) 0.012 0.024 0.012 0.220 (18) 0.024 0.073 0.000 0.073 0.049 0.012 (1) 0.012 0.073 (6) 0.000 0.000 0.000 0.061 0.012 0.000 0.000 (0) 0.000 0.061 (5) 0.000 0.061 0.012 (1) 0.012 0.037 (3) 0.012 0.024 0.000 0.012 (1) 0.012 Mutant 0.080 (IS) 0.005 0.000 0.020 0.015 0.020 0.020 0.000 0.000 0.000 (0) 0.000 0.000 0.000 Mutations p<10"7 W1310X S1251N G458V, E730X, UC E60X, 622-2A-C, G970R W401X, Y1092X, 3659delC 0.055 (9) p<10"2 0.017 0.005 0.005 0.008 0.010 0.010 0.000 (0) 0.000 1717-1G-A (66.7%) 50.0% of 3272-26A-G 50.0% of 3272-26A-G 1717-1G-A(33.3%) AI507, 4218insT W1282X 0.819 (130) p<10~7 0.466 0.007 0.010 0.007 0.312 0.007 0.007 0.005 (1) 0.005 0.005 (1) 0.005 0.000 0.000 (0) 0.000 0.010 (2) 0.000 0.000 0.010 0.005 (1) 0.005 56.7% of AF508, G542X 1% of AF5O8 A455E 1% of AF5O8 38.1% of AF508, N1303K 1.0% of AF5O8 1% of AF508 1% of AF508 G628R(G-Q/S1235R 2183AA-G 1898+1G-C The proportion of CFTR genes associated with a particular haplotype, and the mutations found to be associated with that haplotype are given. Login to comment
123 The three most common CFTR mutations share a similar haplotype background The 3 most common mutations, AF5O8, G542X, N1303K, shared a very similar haplotype background. Login to comment
127 A complete association of allele 1 with G542X CFTR genes (Fisher Exact Test, p < 10~4 ) was found. Login to comment
128 Given the facts that the Q1463Q and D7S8 loci are in linkage equilibrium (ANorma] = -0.032, AMua« = -0.018) and that the G542X mutation has been estimated to be at least 35000 years old (16), this complete association is surprising. Login to comment
131 Absence of recombination between the Q1463Q and D7S8 loci was also observed among particular normal CFTR genes: no normal CFTR genes having haplotype background Ilia (haplotype background ma was associated with the AF508, G542X and N1303K mutations), and certain genes with haplotype background IV (Table 4), carried allele 1 at the D7S8 locus. Login to comment

[hide] Mutation detection by mismatch binding protein, Mu... Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2674-8. Lishanski A, Ostrander EA, Rine J
Mutation detection by mismatch binding protein, MutS, in amplified DNA: application to the cystic fibrosis gene.
Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2674-8., [PMID:7511817]

Abstract [show]
An experimental strategy for detecting heterozygosity in genomic DNA has been developed based on preferential binding of Escherichia coli MutS protein to DNA molecules containing mismatched bases. The binding was detected by a gel mobility-shift assay. This approach was tested by using as a model the most commonly occurring mutations within the cystic fibrosis (CFTR) gene. Genomic DNA samples were amplified with 5'-end-labeled primers that bracket the site of the delta F508 3-bp deletion in exon 10 of the CFTR gene. The renatured PCR products from homozygotes produced homoduplexes; the PCR products from heterozygotes produced heteroduplexes and homoduplexes (1:1). MutS protein bound more strongly to heteroduplexes that correspond to heterozygous carriers of delta F508 and contain a CTT or a GAA loop in one of the strands than to homoduplexes corresponding to homozygotes. The ability of MutS protein to detect heteroduplexes in PCR-amplified DNA extended to fragments approximately 500 bp long. The method was also able to detect carriers of the point mutations in exon 11 of the CFTR gene by a preferential binding of MutS to single-base mismatches in PCR-amplified DNA.

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49 a Mutation Location PCR primers (5' 4 3') AF508 exon 10 (1) CTCAGTTTTCCTGGATTATGCC (2) TGGCATGCTTIGATGACG (1) CTCAG1TTICCTGGATTATGCC (3) CTAACCGATTGMTATGGAGCC (4) CMGTGMTCCTGAGCGTGA (3) CTAACCGATTGAATATGGAGCC (5) GCAGAGTACCTGAAACAGGA (6) CATrCACAGTAGCTrACCCA R553X exon 11 (7) GCCTTTCAAATCAGATTGAGC (9) GACA1TTACAGCAAATGCTTGC (10) CAACTGTGGTTAAAGCAATAGTGT (11) GCACAGATTCTGAGTAACCATMT G551 D exon 11 (7) GCC1TTCAAATTCAGATTGAGC (9) GACA1TUACAGCAAATGCTTGC G542X exon 11 (7) GCCTTTCAAATTCAGATTGAGC (8) TGCTCGTTGACCTCCACTC b Exon 10 490 bp I 340 bp 200 bp 100 bp 100 200 PCR Lesion Mismatches product formed size upon (bp) reannealing of PCR product 100 3 bp deletion CTT + GAA (Phe deletion) bulges 200 340 491 203 C 4 T (Arg m 4 Stop) 425 203 G 4 A (Gly 5544 Asp) 141 G 4 T (Gly 542 4 Stop) 300 C/A + T/G single-base mismatches G/T + A/C single-base mismatches G/A + T/C single-base mismatches 400 AF508 LExon11 100 200 300 400 425 bp L 203 bp 141 bp G542X G51 D R553X FIG. 1. Login to comment
111 Similar results were obtained with G542X and G551D, as described in Fig. la (data not shown). Login to comment
136 Similar results were also obtained forthe G542X and G551D point mutations using 141- and 203-bp PCR products, respectively (data not shown). Login to comment

[hide] Genetic analysis of Hispanic individuals with cyst... Am J Hum Genet. 1994 Mar;54(3):443-6. Grebe TA, Seltzer WK, DeMarchi J, Silva DK, Doane WW, Gozal D, Richter SF, Bowman CM, Norman RA, Rhodes SN, et al.
Genetic analysis of Hispanic individuals with cystic fibrosis.
Am J Hum Genet. 1994 Mar;54(3):443-6., [PMID:7509564]

Abstract [show]
We have performed molecular genetic analyses of Hispanic individuals with cystic fibrosis (CF) in the southwestern United States. Of 129 CF chromosomes analyzed, only 46% (59/129) carry delta F508. The G542X mutation was found on 5% (7/129) of CF chromosomes. The 3849 + 10kbC-->T mutation, detected primarily in Ashkenazi Jews, was present on 2% (3/129). R1162X and R334W, mutations identified in Spain and Italy, each occurred on 1.6% (2/129) of CF chromosomes. W1282X and R553X were each detected once. G551D and N1303K were not found. Overall, screening for 22 or more mutations resulted in detection of only 58% of CF transmembrane conductance regulator gene mutations among Hispanic individuals. Analysis of KM19/XV2c haplotypes revealed an unusual distribution. Although the majority of delta F508 mutations are on chromosomes of B haplotypes, the other CF mutations are on A and C haplotypes at higher-than-expected frequencies. These genetic analyses demonstrate significant differences between Hispanic individuals with CF and those of the general North American population. Assessment of carrier/affected risk in Hispanic CF individuals cannot, therefore, be based on the mutation frequencies found through studies of the general population but must be adjusted to better reflect the genetic makeup of this ethnic group. Further studies are necessary to identify the causative mutation(s) in this population and to better delineate genotype/phenotype correlations. These will enable counselors to provide more accurate genetic counseling.

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No. Sentence Comment
45 The following CFTR gene mutations were identified by published methods: AF508 (Rommens et al. 1990); G542X (Kerem et al. 1990); GS51D and R553X (Cutting et al. 1990); R1162X (Gasparini et al. 1991); W1282X (Vidaud et al. 1990); N1303K (Osborne et al. 1991); 3849 +lOkbC- T (Highsmith et al., submitted); and R117H, Y122X, 1148T, 621+1G-*oT, 711+1G- T, G314E, 1078AT, R334W, R347P, Q493X, A1507, V520F, 1717 -1G-oA, R560T, and 3569AC (J. DeMarchi et al., submitted). Login to comment
46 Alternatively, the G542X, G551D, R553X, and N1303K mutations were assayed by the method of Ng et al. Login to comment
52 .................. 67.1 59/129 (46) G542X .................. 3.4 7/129 (5.4) 3849+10kbC--T ......... Unknown 3/129 (2.3) G551D .................. 2.4 0/129 (0) R553X .................. 1.3 1/129 (.8) R1162 .................. .85b 2/129 (1.6) R334W .................. <1 2/129 (1.6) W1282X ................. 2.1 1/129 (.8) Otherc .................. 65 0/129 (0) Undetected ............... 15 54/129 (42) a CF Consortium 1992, unpublished data. Login to comment
54 COther = A1507, 621+1G- T, R117H, N1303K, 711+1G-*.T, 1717-1G-.A, R560T, Y122X, 1148T, G314E, 1078AT, R347P, Q493X, V520F, and 3659AC. Login to comment
56 The G542X mutation was found in 5.4% of Hispanic CF chromosomes, similar to the 3% frequency in the general population. Login to comment
66 (%) OF HAPLOTYPES CHROMOSOME TYPE A B C D TOTAL CF: AF508 ............ 2 (4) 36 (72) 5 (10) 7 (14) 50 Non-AF508a ....... 12 (29) 14 (33) 14 (33) 2 (5) 42 Normal" ............. 22 (49) 5 (11) 16 (34) 4 (9) 47 a Includes chromosomes carrying either G542X, RI 162X, W1282X, R334W, R553X, 3849+10kbC-*oT, or an unidentified mutation." Login to comment
85 This has been observed elsewhere (European Working Group in CF Genetics 1990), and several mutations, including G542X and GSS1D, have been identified on chromosomes of this haplotype (Kerem et al. 1990). Login to comment
47 The following CFTR gene mutations were identified by published methods: AF508 (Rommens et al. 1990); G542X (Kerem et al. 1990); GS51D and R553X (Cutting et al. 1990); R1162X (Gasparini et al. 1991); W1282X (Vidaud et al. 1990); N1303K (Osborne et al. 1991); 3849 +lOkbC-T (Highsmith et al., submitted); and R117H, Y122X, 1148T, 621+1G-*oT, 711+1G-T, G314E, 1078AT, R334W, R347P, Q493X, A1507, V520F, 1717 -1G-oA, R560T, and 3569AC (J. DeMarchi et al., submitted). Login to comment
48 Alternatively, the G542X, G551D, R553X, and N1303K mutations were assayed by the method of Ng et al. Login to comment
58 The G542X mutation was found in 5.4% of Hispanic CF chromosomes, similar to the 3% frequency in the general population. Login to comment
68 (%) OF HAPLOTYPES CHROMOSOME TYPE A B C D TOTAL CF: AF508 ............ 2 (4) 36 (72) 5 (10) 7 (14) 50 Non-AF508a ....... 12 (29) 14 (33) 14 (33) 2 (5) 42 Normal" ............. 22 (49) 5 (11) 16 (34) 4 (9) 47 a Includes chromosomes carrying either G542X, RI 162X, W1282X, R334W, R553X, 3849+10kbC-*oT, or an unidentified mutation. " Login to comment
87 This has been observed elsewhere (European Working Group in CF Genetics 1990), and several mutations, including G542X and GSS1D, have been identified on chromosomes of this haplotype (Kerem et al. 1990). Login to comment

[hide] G1244V: a novel missense mutation in exon 20 of th... Hum Mol Genet. 1994 Mar;3(3):513-4. Savov A, Jordanova A, Gavrilov D, Angelicheva D, Kalaydjieva L
G1244V: a novel missense mutation in exon 20 of the CFTR gene in a Bulgarian cystic fibrosis patient.
Hum Mol Genet. 1994 Mar;3(3):513-4., [PMID:7516777]

Abstract [show]

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17 The male patient who carries this mutation is of Bulgarian ethnic origin and is a compound heterozygote for G1244V and G542X. Login to comment

[hide] Cystic fibrosis in a low-incidence population: two... Hum Genet. 1994 Feb;93(2):162-6. Kere J, Estivill X, Chillon M, Morral N, Nunes V, Norio R, Savilahti E, de la Chapelle A
Cystic fibrosis in a low-incidence population: two major mutations in Finland.
Hum Genet. 1994 Feb;93(2):162-6., [PMID:7509311]

Abstract [show]
The incidence of cystic fibrosis (CF) in Finland, 1:25,000 newborn, is one of the lowest in Caucasian populations. The delta F508 mutation accounts for 18/40 (45%) of CF chromosomes in Finland. Other mutations were therefore sought among the remaining 55%. Twelve out of 40 chromosomes (30%) were found to carry 394delTT, whereas G542X and 3732delA were each detected in one chromosome. Eight mutations remained unidentified using a testing panel for 26 mutations. Mutation 394delTT was associated exclusively with haplotype 23-36-13. Five unknown mutations were associated with different haplotypes for microsatellite markers, whereas three shared the same haplotype. Most delta F508 mutations and all unidentified mutations originated from regions of old and dense settlement in the coastal regions, whereas 394delTT was geographically clustered and enriched in a rural location, consistent with a local founder effect. The remote location of Finland and her population history give a plausible explanation for the rarity of CF in Finland.

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29 The haplotype associations of AF508 an G542X are similar to those found in other European populations (Casals et al 1992, 1993; Mortal et al. 1993). Login to comment
30 G542X and 3732delA (Tsui et al. 1992) were each identified in one chromosome, and the remaining eight mutations were not identified. Login to comment
59 Cystic fibrosis mutations and associated haplotypes in Finnish CF families CF PI/PS metD metH PT-3 XV2c CS7 KM19 MP6d-9 Mutations Microsatellites H-80 J3.11 chromo- MI/N TaqI TaqI BanII TaqI Hhal Pstl MspI IVS8/IVS 17TA/ PstI Mspl some IVS 17CA 1 pat PI, N l 1 1 1 2 2 2 394delTT 23/36/13 ND 2 l mat PI, N 1 2 1 l 2 2 2 394delTT 23/36/13 ND 1 2 pat PI, MI 1 2 1, 2 1 2 2 2 394delTT 23136/13 1, 2 1 2 mat PI, MI 1 1 1, 2 1 2 2 2 AF508 17/31/13 1, 2 1 3 pat PI, MI 1 1 2 l 1 l 1 Unknown 16/33/13 2 2 3 mat PI, MI 1 1 2 1 2 2 2 AF508 17/31/13 2 2 4 pat PI, N l 2 2 1 2 2 2 AF508 17/3 l/13 2 1 4 mat PI, N 1 1 2 1 2 2 2 G542X 23/33/13 1 1 5 pat PI, N 1 1 2 1 2 2 2 AF508 -/-/- 2 2 5 mat PI, N 1 1 2 1 2 2 2 AF508 -/-/- 2 2 6 pat PI, MI 1 2 1 1 2 2 2 AF508 23/31/13 2 2 6 mat PI, MI 1 1 l 1 2 2 2 394delTT 23/36/13 1 2 7 pat PI, N 1 1 2 1 1 1 1 Unknown 16/33/13 2 2 7 mat PI, N 1 2 2 1 2 2 2 394delTT 23/36/13 l 1 8 pat P1, N 1 2 1 1 2 2 2 394delTT 23/36/13 1 1 8 mat P1, N 1 2 1 1 2 2 2 394delTT 23/36/13 1 1 9 pat P1, MI 1 1 2 1 1 1 l Unknown 16/33/13 2 2 9 mat PI, MI I 2 1 1 2 2 2 394delTT 23/36/13 l 1 10 pat P1, N 1 1 1 1 2 1 2 Unknown 17/31/11 2 2 10 mat P1, N 1 1 2 1 2 2 2 AF508 17/31/13 2 1 i 1 pat PI, N 2 1 1 1 2 2 2 AF508 23/30/13 2 2 11 mat P1, N 1 1 2 1 2 2 2 AF508 17/31/13 2 1 12 pat PI, N 2 1 2 1 1, 2 1, 2 1, 2 AF508 17/32/13 2 2 12 mat PI, N 1 1 2 1 1, 2 1, 2 1, 2 Unknown 16/07/17 1 1 13 pat PI, N 1 1 2 l, 2 1, 2 l, 2 l, 2 AF508 23/31/13 2 2 13 mat PI, N 2 1 2 1, 2 l, 2 l, 2 1, 2 Unknown 16/35/13 2 2 14 pat PI, MI 1 2 1 1 2 2 2 394delTT 23/36/13 1 1 14 mat PI, MI 1 l 2 1 2 2 2 AF508 17/31/13 2 1 15 pat PS, N 1, 2 1, 2 2 1, 2 2 2 2 Unknown 15/07/17 2 15 mat PS, N 1, 2 1, 2 2 1, 2 2 2 2 AF508 17/07/13 2 1,2 1,2 16 pat PI, N 1 1 2 1 2 2 2 AF508 17/31/13 2 1 16 mat PI, N 1 1 1 1 2 2 2 AF508 17/31/13 2 1 17 pat PI, N 1 2 1 1 2 2 2 394delTT 23/36/13 1 1 17 mat PI, N 1 2 1 1 2 2 2 394delTT 23/36/13 1 1 18 pat PI, N 1 1 2 1 2 2 2 AF508 17/31/13 2 1 18 mat PI, N 1 1 2 1 2 2 2 AF508 17/31/13 2 1 19 pat PI, N ND ND 2 1, 2 2 2 2 394delTT 23/36/13 ND 19 mat PI, N ND ND 2 l, 2 1 l 2 Unknown 16/31/13 ND 1,2 1,2 20 pat PI, N ND 1 ND ND ND ND ND AF508 23/31/13 ND 2 20 mat PI, N ND l ND ND ND ND ND 3732delA 17/35/13 ND 2 pat, Paternal allele; mat, maternal allele; PUPS, pancreatic insufficiency/sufficiency; MI/N, meconium ileus/normal; ND, not determined 9 AF508 ~) 394delTT (~ G542X (~ Unknown, same Io C) Unknown, different haplotypes r IO | A D '* | Oo .g 9 Fig.2. Login to comment
82 Am J Hum Genet 339:119-120 Casals T, Nunes V, Palacio A, Gimtnez J, Gaona A, Ib~fiez N, Morral N, Estivill X (1993) Cystic fibrosis in Spain: high frequency of mutation G542X in the Mediterranean coastal area. Login to comment

[hide] 394delTT: a Nordic cystic fibrosis mutation. Hum Genet. 1994 Feb;93(2):157-61. Schwartz M, Anvret M, Claustres M, Eiken HG, Eiklid K, Schaedel C, Stolpe L, Tranebjaerg L
394delTT: a Nordic cystic fibrosis mutation.
Hum Genet. 1994 Feb;93(2):157-61., [PMID:7509310]

Abstract [show]
In a systematic screening for mutations in the gene encoding the cystic fibrosis transmembrane regulator among Danish cystic fibrosis (CF) patients, we identified a mutation in exon 3 (394delTT); this mutation was found to be relatively common in Denmark. We therefore screened for 394delTT in Sweden and Norway, where it turned out to be the second most frequent mutation, accounting for 4% of all CF mutations. It also occurs with a high frequency in Finland, but has not been found in larger surveys of mutations in the CFTR gene. Thus, 394delTT seems to be a specific Nordic CF mutation.

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9 A few of the non-AF508 mutations are found in more than one population at a frequency of 1-5%, viz. W1282X (Abeliovich et al. 1992), G551D and G542X (Cutting et al. 1990, 1992) and N1303K (Osborne et al. 1991), but most of the others are rare or private mutations. Login to comment
10 Correspondence to: M. Schwartz, Section of Clinical Genetics, Rigshospitalet 4062, Blegdamsvej 9, DK-2100 Copenhagen, Denmark Screening for AF508 and a few other common mutations, i.e. G542X, N1303K, 621+lG--->T and G551D, in the Danish CF population showed that these mutations account for 90% of all CF disease genes (Schwartz et al. 1992), a fact that has been used in a recent screening programme for carriers of CF among pregnant women (Schwartz et al. 1993). Login to comment
70 Other CF mutations Very few of the previously reported most "common" mutations (G551D, G542X, R553X, N1303K, 1717-1G---~ T, and W1282X) were identified on the "Nordic" CF chro- Discussion More than 200 CF mutations have been reported (Ysui 1992a) since the cloning of the CFTR gene. Login to comment
88 394delTT should therefore be regarded as one of the most common CF mutations in these countries, as common as G542X, G551D and W1282X are in other populations. Login to comment

[hide] Exon 9 of the CFTR gene: splice site haplotypes an... Hum Genet. 1994 Jan;93(1):67-73. Dork T, Fislage R, Neumann T, Wulf B, Tummler B
Exon 9 of the CFTR gene: splice site haplotypes and cystic fibrosis mutations.
Hum Genet. 1994 Jan;93(1):67-73., [PMID:7505767]

Abstract [show]
The alternatively spliced exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene codes for the initial part of the amino-terminal nucleotide-binding fold of CFTR. A unique feature of the acceptor splice site preceding this exon is a variable length polymorphism within the polypyrimidine tract influencing the extent of exon 9 skipping in CFTR mRNA. We investigated this repeat for its relationship to CFTR mutations and intragenic markers on 200 chromosomes from German patients with cystic fibrosis (CF). Four frequent length variations were strongly associated with the four predominant haplotypes previously defined by intragenic marker dimorphisms. One of these alleles displayed absolute linkage disequilibrium to the major CF mutation delta F508. Other frequent CFTR mutations were linked to one particular splice site haplotype indicating that differential exon 9 skipping contributes little to the clinical heterogeneity among CF patients with an identical mutation. We also identified a novel missense mutation (V456F) and a novel nonsense mutation (Q414X) within the coding region of exon 9. The missense mutation V456F adjacent to Walker motif A was present in a pancreas-sufficient CF patient. In contrast, the pancreas-insufficient Q414X/delta F508 compound heterozygote suffered from a severe form of the disease, indicating that alternative splicing of exon 9 does not overcome the deleterious effect of a stop codon with this exon.

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61 Association of (TG),Tm alleles with CFTR mutations (TG),Tm CFTR mutationsa (TG)llT7 E60X, E92X, R117C, 1078delT, R347P, R553X, 2184delA, 2184insA, I1005R, 3272-26A--~G, L1059X, Y1092X, R1162X, 3659delC, 3850-3T-oG, S1251N Q39X, R117H, Q414X, V456F, AI507, 1717-1G--~A, G551D, 2043delG, 2183AA---~G, 2184insA, 2789 + 5 G---~A,3272-26A---~G, R1066C, L1077P, 3849 + l0 kB C---~T,4374 + 1 G---~T 621 + 1 G---~T,R334W, A455E, AF508, G542X, 2143delT, 3849 + 10 kB C---~T,NI303K 405 + 1 G----~A,1342-2 A---~C,R553X (TG)IoT7 (TG)10T9 (TG)12T7 a References are compiled in Tsui (1992), except for 2143delT (Dtrk et al. 1992b), 3850-3 T---~G,4374 + 1 G---~T,1342-2 A---~C (Dtrk et al. 1993a, b), Q414X, V456F (this work), 405 + 1 G---~A, E92X, R117C, 2184delA, 2184insA, I1005R, L1059X (T. Login to comment
158 J Clin Invest 88:1880-1885 Hamosh A, Rosenstein BJ, Cutting GR (1992) CFTR nonsense mutations G542X and W1282X associated with severe reduction of CFTR mRNA in nasal epithelial cells. Login to comment

[hide] Identification of six novel mutations in the CFTR ... Hum Mol Genet. 1994 Jan;3(1):57-60. Savov A, Mercier B, Kalaydjieva L, Ferec C
Identification of six novel mutations in the CFTR gene of patients from Bulgaria by screening the twenty seven exons and exon/intron boundaries using DGGE and direct DNA sequencing.
Hum Mol Genet. 1994 Jan;3(1):57-60., [PMID:7512860]

Abstract [show]
The CFTR gene, in which more than 300 mutations have been described, displays a spectrum of mutations which varies according to ethnic and geographic origin of patients. In this paper we report an exhaustive study of the 27 exons and exon/intron boundaries of a sample of 35 CF patients from Bulgaria which is situated in the south east of Europe. We have used denaturing gradient gel electrophoresis assay followed by DNA sequencing and we report the identification of six previously undescribed CFTR alleles.

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22 Preliminary studies on the molecular basis of CF in Bulgaria have shown that AF5O8 accounts for about 55% of the mutant alleles (9) with the N13O3K accounting for 6% (10), the G542X for 5% and three additional mutations including 1677 del TA, R1070Q and R347P for about 8%. Login to comment
74 The most common mutations of the CFTR gene (AF508, N1303K, G542X, 1677 del TA, R347P, R1070Q) have been in a first step identified and in our ongoing effort to identify the other mutations, we have fully scanned the entire coding sequence of 35 CF patients. Login to comment

[hide] Severe pulmonary and digestive disease in a cystic... J Med Genet. 1994 Jan;31(1):84-5. Desgeorges M, Laussel M, Rollin B, Demaille J, Claustres M
Severe pulmonary and digestive disease in a cystic fibrosis child homozygous for G542X.
J Med Genet. 1994 Jan;31(1):84-5., [PMID:7512145]

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43 Using the SSCP technique" to study the DNA extracted from the patient and her family, we detected then identified the stop mutation G542X in the two CFTR genes transmitted to the affected child. Login to comment
45 The stop mutation G542X is predicted to result in decreased levels of mutant messenger RNA"2 and in a truncated CFTR protein from NBD-1,' suppressing 63% of the molecule. Login to comment
46 However, the homozygous patients for G542X previously reported had mild pulmonary disease,""5'° which would imply alternative splicing mechanisms suppressing the effect of the stop mutation in some tissues. Login to comment
47 Contrasting with these reports, we present the second case ofa child homozygous for G542X with severe pancreatic and lung disease. Login to comment
98 Also the intrafamilial heterogeneity of cystic fibrosis, perhaps because of other modifying loci, makes it plausible that 85 group.bmj.comon October 25, 2012 - Published byjmg.bmj.comDownloaded from doi: 10.1136/jmg.31.1.84-a 1994 31: 84-85J Med Genet M Desgeorges, M Laussel, B Rollin, et al. cystic fibrosis child homozygous for G542X. Login to comment

[hide] Detection of 98.5% of the mutations in 200 Belgian... Genomics. 1993 Dec;18(3):693-7. Cuppens H, Marynen P, De Boeck C, Cassiman JJ
Detection of 98.5% of the mutations in 200 Belgian cystic fibrosis alleles by reverse dot-blot and sequencing of the complete coding region and exon/intron junctions of the CFTR gene.
Genomics. 1993 Dec;18(3):693-7., [PMID:7508414]

Abstract [show]
We have previously shown that about 85% of the mutations in 194 Belgian cystic fibrosis alleles could be detected by a reverse dot-blot assay. In the present study, 50 Belgian chromosomes were analyzed for mutations in the cystic fibrosis transmembrane conductance regulator gene by means of direct solid phase automatic sequencing of PCR products of individual exons. Twenty-six disease mutations and 14 polymorphisms were found. Twelve of these mutations and 3 polymorphisms were not described before. With the exception of one mutant allele carrying two mutations, these mutations were the only mutations found in the complete coding region and their exon/intron boundaries. The total sensitivity of mutant CF alleles that could be identified was 98.5%. Given the heterogeneity of these mutations, most of them very rare, CFTR mutation screening still remains rather complex in our population, and population screening, whether desirable or not, does not appear to be technically feasible with the methods currently available.

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14 Two of these CF alleles were derived from an individual homozygous for the G542X mutation, having a rather mild form of cystic fibrosis, in which only a limited number of exons of the CFTR gene had been analyzed (6). Login to comment
36 Five mutations had a frequency higher than 1% (Table 1) AF508 (72.5%), G542X (5.5%), N1303K (3.5%), 1717-1G --~ A (2.5%), and $1251N (2.0%). Login to comment
43 TABLE 1 Mutations (and Their Frequencies) Identified in This Study Predicted amino Mutation Nucleotide change~ acid change Location Frequencyb Reference E60X G --~ T at 310 (TAGATAGCT) Glu --~ Stop at 60 Malone et al. in (21); this study G --~ A at 482 (GAACACTCT) (8) A --~ C at 622-2 (TTTTCGACT) This study T --~ A at 1139 (AAAAAATTC) This study G --~ A at 1335 (TCTGAGAGG) This study C --~ A at 1496 (TTGGAGGTT) (14) G -~ T at 1505 (GCTGTATCC) (6) Deletion of ATC from 1651 (14); Schwarz et al. (TATC_TTTG) in (21) Deletion of CTT from 1653 145 (13) (TCAT_TGGT) G --~ A at 1717-1 (AATAAGACA) G --~ T at 1756 (TCTTTGAGA) G --~ C at 1898 + 1 (AAAGCTATG) G --~ C at 2014 (TTATCGGAC Deletion of A at 2184; A --~ G at 2183 (AAAAG CAAT) G --~ T at 2320 (TGATTAGCC Deletion of 11 nucleotides from 2721 (TGCT_TAGT) G --~ C at 3040 (AGCACGTAC A --~ G at 3272-26 (TGCAGTGTT) C --~ A at 3408 (TGTAACTGT) Deletion of C at 3659 (CCTA_CAAG) T --~ G at 3837 (TAAGGCCTG G --* A at 3884 (AAGAATACT G --~ A at 3978 (AGTGAAGGA' C --~ G at 4041 (AAAAGTTGG G -~ A at 4061 (CAGTAGAGT Insertion of T after 4218 (CAGTTAAGG) R117H 622-2A --~ C I336K W401X A455E G458V AI507 AF508 1717-1G -~ A G542X 1898+ 1G-~C G628R(G -~ C) 2184delA plus A -~ G at 2183 E730X 2721de111 G970R 3272-26A --~ G Y1092X 3659delc $1235R $1251N W1282X N1303K W1310X 4218insT Exon 3 2 (1.0%) Arg --~ His at 117 Exon 4 c 3' splice signal Intron 4 1 (0.5%) Ile -~ Lys at 336 Exon 7 1 (0.5%) Trp --~ Stop at 401 Exon 8 2 (1.0%) Ala --~ Glu at 455 Exon 9 2 (1.0%) Gly --* Val at 458 Exon 9 1 (0.5%) Deletion of Ile 507 Exon 10 1 (0.5%) Deletion of Phe 508 Exon 10 (72.5%) 3' splice signal Intron 10 5 (2.5%) Gly --* Stop at 542 Exon 11 11 (5.5%) 5' splice signal Intron 12 1 (0.5%) Gly -~ Arg at 628 Exon 13 1 (0.5%) Frameshift Exon 13 2 (1.0%) Glu --~ Stop at 730 Exon 13 1 (0.5%) Frameshift Exon 14a I (0.5%) Gly --~ Arg at 970 Exon 15 1 (0.5%) 5' splice signal? Login to comment
48 The G628R(G --~ C) and $1235R mutations were found on a single allele; the W1310X allele and one 2184delA (plus A --~ G at 2183) allele were found on a CFTR gene from Turkish descent. Login to comment
49 For each type mutation, at least one allele was completely sequenced: 14 for AF508, 3 for G542X, 2 for 1717-1G -~ A, 1 for A455E, 1 for G458V, 1 for AI507, 1 for W1282X, 1 for N1303K, and for the remainder, the total number of alleles that were found in this study. Login to comment
94 No additional mutations, except for some polymorphisms, in the CF patient homozygous for the G542X mutation, as well as in her CF cousin, who was a compound heterozygote for the G458V and G542X mutations (6), could be found. Login to comment
104 The G458V allele carried seven thymidines in its poly- (T) tract, which suggests that the proportion of 9- CFTR transcripts of this allele will not be very low. Login to comment
105 The fact that the G458V/G542X individual has CF suggests that the alternatively spliced 9- CFTR mRNA transcript cannot compensate for the basic defect of the CFTR protein in this CF patient. Login to comment
35 Five mutations had a frequency higher than 1% (Table 1) AF508 (72.5%), G542X (5.5%), N1303K (3.5%), 1717-1G --~ A (2.5%), and $1251N (2.0%). Login to comment
42 TABLE 1 Mutations (and Their Frequencies) Identified in This Study Predicted amino Mutation Nucleotide change~ acid change Location Frequencyb Reference E60X G --~ T at 310 (TAGATAGCT) Glu --~ Stop at 60 Malone et al. in (21); this study G --~ A at 482 (GAACACTCT) (8) A --~ C at 622-2 (TTTTCGACT) This study T --~ A at 1139 (AAAAAATTC) This study G --~ A at 1335 (TCTGAGAGG) This study C --~ A at 1496 (TTGGAGGTT) (14) G -~ T at 1505 (GCTGTATCC) (6) Deletion of ATC from 1651 (14); Schwarz et al. (TATC_TTTG) in (21) Deletion of CTT from 1653 145 (13) (TCAT_TGGT) G --~ A at 1717-1 (AATAAGACA) G --~ T at 1756 (TCTTTGAGA) G --~ C at 1898 + 1 (AAAGCTATG) G --~ C at 2014 (TTATCGGAC Deletion of A at 2184; A --~ G at 2183 (AAAAG CAAT) G --~ T at 2320 (TGATTAGCC Deletion of 11 nucleotides from 2721 (TGCT_TAGT) G --~ C at 3040 (AGCACGTAC A --~ G at 3272-26 (TGCAGTGTT) C --~ A at 3408 (TGTAACTGT) Deletion of C at 3659 (CCTA_CAAG) T --~ G at 3837 (TAAGGCCTG G --* A at 3884 (AAGAATACT G --~ A at 3978 (AGTGAAGGA' C --~ G at 4041 (AAAAGTTGG G -~ A at 4061 (CAGTAGAGT Insertion of T after 4218 (CAGTTAAGG) R117H 622-2A --~ C I336K W401X A455E G458V AI507 AF508 1717-1G -~ A G542X 1898+ 1G-~C G628R(G -~ C) 2184delA plus A -~ G at 2183 E730X 2721de111 G970R 3272-26A --~ G Y1092X 3659delc $1235R $1251N W1282X N1303K W1310X 4218insT Exon 3 2 (1.0%) Arg --~ His at 117 Exon 4 c 3' splice signal Intron 4 1 (0.5%) Ile -~ Lys at 336 Exon 7 1 (0.5%) Trp --~ Stop at 401 Exon 8 2 (1.0%) Ala --~ Glu at 455 Exon 9 2 (1.0%) Gly --* Val at 458 Exon 9 1 (0.5%) Deletion of Ile 507 Exon 10 1 (0.5%) Deletion of Phe 508 Exon 10 (72.5%) 3' splice signal Intron 10 5 (2.5%) Gly --* Stop at 542 Exon 11 11 (5.5%) 5' splice signal Intron 12 1 (0.5%) Gly -~ Arg at 628 Exon 13 1 (0.5%) Frameshift Exon 13 2 (1.0%) Glu --~ Stop at 730 Exon 13 1 (0.5%) Frameshift Exon 14a I (0.5%) Gly --~ Arg at 970 Exon 15 1 (0.5%) 5' splice signal? Login to comment
93 No additional mutations, except for some polymorphisms, in the CF patient homozygous for the G542X mutation, as well as in her CF cousin, who was a compound heterozygote for the G458V and G542X mutations (6), could be found. Login to comment

[hide] In frame deletion (delta F311) within a short trin... Hum Mol Genet. 1993 Dec;2(12):2173-4. Meitinger T, Golla A, Dorner C, Deufel A, Aulehla-Scholz C, Bohm I, Reinhardt D, Deufel T
In frame deletion (delta F311) within a short trinucleotide repeat of the first transmembrane region of the cystic fibrosis gene.
Hum Mol Genet. 1993 Dec;2(12):2173-4., [PMID:7509232]

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4 Analysing CF chromosomes in a sample of 227 CF patients from South Germany by screening for known mutations (AF5O8, G542X, G551D, R553X) and further investigation of samples with unresolved chromosomes by SSCP and direct sequencing, we identified a novel in-frame deletion occurring within a short trinucleotide repeat in exon 7 of the CFTR gene. Login to comment

[hide] Extended haplotype analysis of cystic fibrosis mut... Hum Genet. 1993 Oct 1;92(3):289-95. Sereth H, Shoshani T, Bashan N, Kerem BS
Extended haplotype analysis of cystic fibrosis mutations and its implications for the selective advantage hypothesis.
Hum Genet. 1993 Oct 1;92(3):289-95., [PMID:7691712]

Abstract [show]
The major cystic fibrosis (CF) mutation, delta F508, is associated with one haplotype (B) determined by the two polymorphic markers, XV2C and KM19. This haplotype is rare (15%) among non-CF chromosomes. Its frequency among non-delta F508 CF chromosomes is 50% with variation between populations. One hypothesis for the high frequency of CF haplotype B chromosomes suggests that there was a selective advantage for CF mutations on this specific "background" as a result of epistatic selection at other closely linked loci. Since the XV2C and KM19 markers are located 200 kb 5' to the CF gene and span only 60 kb, an extended haplotype analysis was needed to test this hypothesis. Haplotypes were determined for 183 CF and 120 non-CF Israeli chromosomes at the XV2C and KM19 loci and at three intragenic polymorphic sites (GATT in intron 6A, TUB18 in intron 19, and 24M in exon 24). Among the studied chromosomes the frequency of non-delta F508 CF chromosomes associated with haplotype B was 70% (88% among Ashkenazi CF chromosomes). Nine mutations (delta F508, W1282X, G542X, N1303K, 3849 + 10 kb C-->T, Q359K/T360K, S549I, S549R, and 1717-1G-->A) were identified among the studied chromosomes. These mutations accounted for 96% of CF chromosomes of Ashkenazi origin. Haplotype B was associated with seven of these (delta F508, W1282X, G542X, N1303K, Q359K/T360K, S549R, and 1717-1G-->A). The extended haplotype analysis revealed that in five of the seven mutations associated with the haplotype B, 97% of the chromosomes shared the same intragenic haplotype, 212. The variation found in 3% of the chromosomes was only in the GATT repeat. Two mutations, W1282X and 1717-1G-->A, were associated with a completely different intragenic haplotype, 121. The results of this study indicate that grouping of CF chromosome by haplotype analysis spanning a small extragenic region might not be sufficient. In addition, the results of the extended haplotype analysis indicate that all the studied CF chromosomes that carry the same mutation derived from the same origin. Furthermore, the results indicate that the majority of the CF mutations are associated with the same extended haplotype, supporting the selective advantage hypothesis.

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43 The G542X, $5491, 549R and 1717- IG--+A, and N1303K mutations, were detected by PCR and subsequent allele-specific oligonucleotide (ASO) hybridization as previously described (Kerem et al. 1990; Osborne et al. 1991). Login to comment
51 Distribution of CF and normal chromosomes associated with the extended extra- and intragenic DNA polymorphic markers a 291 Mutation Haplotype 2.3A GATT TUB 18 JG2E1 24M Ethnic origin Ash- Non- Arab kena- Ash- zim kenazim AF508 Total: B 2 1 2 12 19 12 B 1 1 2 1 B (2) 1 2 4 B (2) (1) 2 1 B 2 (1) (2) 2 B 2 (1) 2 1 (B) 2 (1) 2 1 D 2 1 2 1 22 19 13 W1282X Total: B 1 2 1 40 B (1) 2 1 5 B 1 (2) 1 1 B 1 (2) (1) 3 B (1) (2) (1) 3 B 2 2 1 1 53 1 2 Q359K/T360K b Total: B 2 1 2 5 B (2) 1 2 1 B 1 1 2 1 7 N1303K Total: B 2 1 2 2 B 2 1 (2) 2 (B) (2) (1) (2) 1 3 2 G542X B 2 1 2 6 1 2 $549R B 2 1 2 2 1717-1G---)A B 1 2 1 1 $549I A 2 1 2 2 3849+10kb C--*T C 1 1 2 4 1 (C) 1 1 2 1 C 2 1 2 1 Total: 6 1 Unknown B 2 1 2 1 3 2 B (2) 1 2 1 B (2) (1) (2) 1 B 1 1 2 3 A 1 1 2 1 5 1 (A) (1) 1 2 1 C 1 1 2 2 8 2 (C) 1 (1) (2) 1 C 2 1 2 2 2 D 1 2 1 1 D 2 1 2 3 Total: 5 21 14 Total CF: 95 50 38 Table 2 (continued) Mutation Haplotype Ethnic origin 2.3A GATT TUBI8 24M Ash- Non- Arab JG2EI kena- Ash- zim kenazim Normal Total: B 2 1 2 2 1 1 B 1 l 2 3 1 B l 2 1 1 A 2 1 2 7 2 2 A I 1 2 12 8 6 A 2 2 l 1 A 1 2 2 1 A 1 2 1 I C 2 1 2 3 1 C 1 1 2 20 13 1I C 2 1 I 1 C 2 2 2 1 C 1 2 1 3 2 D 2 I 2 1 1 D 1 1 2 2 1 D 2 1 I I D 2 2 1 1 D 1 2 1 2 5 2 57 37 26 Alleles that could not be phased are shown in parentheses b All the chromosomes carrying the Q359K/T360K mutation are of Georgian origin in the respective flanking introns of the CF gene (Kerem et al. 1990; Zielenski et al. 1991). Login to comment
67 Eight additional mutations: WI282X, G542X, N1303K, 3849+10kb C---)T, Q359K/T360K, $549I, $549R, and 1717-1G-->A were identified among the studied chromosomes and have all been described elsewhere (Vidaud et al. 1990; Kerem et al. 1990; Osborne et al. 1991; Tsui 1992; Shoshani et al. 1992a, b). Login to comment
75 This extended haplotype analysis revealed that 75 of 77 (97%) of the chromosomes carrying five of the seven mutations associated with haplotype B, (F508, G542X, N1303K, Q359K/T360K, $549R) share the same intragenic haplotype, 212. Login to comment
84 The studied chromosomes included all the chromosomes carrying the Q359K/T360K, G542X, $549R, N1303K, and 1717-1G---~A mutations, 12 chromosomes carrying the W1282X mutations, and 14 chromosomes carrying the AF508 mutation. Login to comment
111 The F508, W1282X, G542X, N1303K, and 1717-1G--+A mutations were found to be associated with the same intragenic haplotype in both ethnic groups suggesting that chromosomes carrying these mutations derive from a common origin. Login to comment
121 In a previous study (Shoshani et al. 1992) we have shown that the identification of 92% of CF chromosomes of Ashkenazi origin is possible by detection of only 5 mutations (W1282X, F508, G542X, NI303K, and 1717-1G--~A). Login to comment

[hide] Nasal epithelial ion transport and genetic analysi... Hum Mol Genet. 1993 Oct;2(10):1605-9. Osborne LR, Lynch M, Middleton PG, Alton EW, Geddes DM, Pryor JP, Hodson ME, Santis GK
Nasal epithelial ion transport and genetic analysis of infertile men with congenital bilateral absence of the vas deferens.
Hum Mol Genet. 1993 Oct;2(10):1605-9., [PMID:7505692]

Abstract [show]
It has been suggested that congenital bilateral absence of the vas deferens (CBAVD), an important cause of male infertility, is a variant of cystic fibrosis (CF). This study describes a defect in chloride conductance across the nasal epithelium of subjects with CBAVD which is dissimilar to that found in patients with CF. It also demonstrates normal sodium transport across the nasal epithelium in these men, in contrast to patients with CF who exhibit increased sodium absorption. The increased frequency of CFTR mutations in these men implicates the CFTR gene in the pathogenesis of this disorder. Genetic analysis of men with CBAVD who were heterozygous for a known CFTR mutation failed to identify a second mutation within any of the exons or introns of the CFTR gene. These results demonstrate that most men presenting with CBAVD are not compound heterozygotes for mutations within the CFTR gene and can be distinguished from individuals with atypical or asymptomatic CF on the basis of the bioelectric properties of their nasal epithelium. We postulate that mutations in the promoter region or at other regulatory sites of the CFTR gene may be responsible for the CBAVD phenotype in a proportion of cases.

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25 Nasal potential difference, sweat sodium concentration and CF genotype in subjects with CBAVD, CF patients and controls CF genotype Control cohort CBAVD cohort CF cohort N/N« AF5O8/R347H SM9N/R1070Q AF508/Other R553X/N*> Unknown/Unknowtf AF 5«/AF5O8 AFjQj/Other AF508/R347P AF50e/G542X AFjQg/RllTH AF5O8/G85E AF5Q8/R553X AFJO8/G551D AF5O8A^520F AFJO8/S549N AF^/DIjQ, N1303K/Other G542X/R117H AFJ08/R334W Other/Other Subjects 50 1 1 1 6 1 16 25 18 3 2 1 1 1 2 1 1 1 3 1 1 3 Mean (range) sweat Na+ concentration (mmol/1) 50 (27-78) 88 N/A 94 57 (47-70) 36 49 (32-76) 126(80-162) 99 (80-128) 108 (99-115) 128 (118-137) 95 107 130 122 (116-128) 90 80 118 96 (92-99) 84 123 108(83-130) Mean (range) nasal potential difference (-mV) 21 (8-30) 31 N/A 34 23 (13-29) -15 20 (-13-28) 45 (32-58) 41 (33-61) 50 (36-77) 43 (33-52) 51 42 39 60 (50-71) 32 37 41 38 (36-40) 32 34 44(31-57) N = non-CF chromosome Other = uncharacterised CF chromosome N/A not available • with CF carrier frequency of 1/20-1/25, it is likely that 2 or 3 of these individuals will be carriers. Login to comment
116 DNA studies Genomk DNA from subjects with CBAVD was extracted from whole blood and initially analysed for four common CFTR mutations (AFjog, G551D, G542X and 621 + 1 G-T) using the amplification refractory mutation system (ARMS, Cellmark Diagnostics UK)29 . Login to comment

[hide] Genotype analysis of adult cystic fibrosis patient... Hum Mol Genet. 1993 Oct;2(10):1557-60. Ferec C, Verlingue C, Guillermit H, Quere I, Raguenes O, Feigelson J, Audrezet MP, Moullier P, Mercier B
Genotype analysis of adult cystic fibrosis patients.
Hum Mol Genet. 1993 Oct;2(10):1557-60., [PMID:7505690]

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To assess the relationship between the genotype and phenotype of adult CF patients we have selected from a group of 512 CF patients attending centres in France, all these of greater than 35 years. We have analysed the entire coding sequence of their CFTR genes. The complete genotype was determined in 7 of the 8 patients and clinical data regarding pancreatic, respiratory and reproductive function were carefully evaluated. All these patients are compound heterozygote, seven carrying the delta F508 and one the G542X on one allele. The other allele carried is: (i) a missense mutation located in exons coding for transmembrane region in five patients [R334W (1); I336K (2); R117H (1); H1054D (1)]; (ii) a splice mutation in two patients [2789 + 5G-->A], (iii) an uncharacterised mutations in one patient. These results strongly suggest less severe CF phenotype to be associated with these mutations and strengthen the hypothesis that less severe phenotype are genetically determined.

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4 All these patients are compound heterozygote, seven carrying the AF508 and one the G542X on one allele. Login to comment
57 '*'* < GENOTYPE * • • ;i^>"; ' ' FUJ34W/G542X Exon 7/ Exon 11 Arginine •-> Tryptophan AF508/I336K Exon 10/Exon 7 Isoleucine ••> Lysine AF5O8/H1054O Exon 10/Exon 17b Histidine --> Aspartc Acid AF508 Unknown AF508 / 2789+5 G->A Exon 10/ Exon 14b Splice mutation AF508/2789+5 G->A Exon 10/ Exon 14b Splice mutation AF5O8/R117H Exon 10/Exon 4 Arginine -->Histidine AF508/O36K Exon 10/Exon 7 Isoteucine ••> Lysine SEX 4 M F M M F F F M DIAGNOSiS 13 13 27 17 39 32 30 17 PROFESSIONAL; . Login to comment

[hide] Direct sequencing of the complete CFTR gene: the m... Hum Mol Genet. 1993 Oct;2(10):1551-6. Cheadle JP, Goodchild MC, Meredith AL
Direct sequencing of the complete CFTR gene: the molecular characterisation of 99.5% of CF chromosomes in Wales.
Hum Mol Genet. 1993 Oct;2(10):1551-6., [PMID:7505689]

Abstract [show]
We have performed an extensive mutation analysis on 184 CF families in Wales. In our previous study, mutations on 329/369 CF chromosomes were identified after screening for delta F508 and sixteen other mutations. To identify the mutations on the remaining 40 uncharacterized CF chromosomes, we have carried out direct DNA sequencing over the complete coding region, intron splice sites, and part of the promoter region of the CFTR gene. During this study we have designed a set of internal sequencing primers which allow clear sequencing through the aforementioned regions. Sequence analysis revealed 15 further mutations (4 of which are novel), and 10 previously described polymorphisms. In total, we have identified 29 mutations, the distribution of which provides further insight into the functional domains of the CFTR protein. We have characterised 99.5% of the CF chromosomes (365/367, one sample degraded). In order to ascertain accurate frequency data for the Welsh population, CF families with at least 3 'Welsh' grandparents were strictly regarded as 'Welsh'. Of these 91 families, delta F508 accounts for 71.6%, 621 + 1G-->T 6.6% and 1898 + 1G-->A 5.5%. The implications for CF population screening in Wales are discussed.

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115 Further ARMS tests are in development and in principle it should be possible to use a three tube ARMS test to assay rapidly for 13 mutations (delta F508, 621 + 1G-T, G551D, G542X, R1283M, W1282X, R553X, N13O3K, delta 1507, 1898+1G-A, R117H, R560T, and 1717-1G-A) (S.Little, personal comm.). Login to comment

[hide] CFTR transcripts are undetectable in lymphocytes a... J Med Genet. 1993 Oct;30(10):833-7. Will K, Reiss J, Dean M, Schlosser M, Slomski R, Schmidtke J, Stuhrmann M
CFTR transcripts are undetectable in lymphocytes and respiratory epithelial cells of a CF patient homozygous for the nonsense mutation R553X.
J Med Genet. 1993 Oct;30(10):833-7., [PMID:7693946]

Abstract [show]
In order to analyse the influence of the nonsense mutation R553X on CFTR gene expression, transcripts from epithelial cells and lymphocytes were examined from nine subjects (one CF patient homozygous for R553X, one CF patient compound heterozygous for R553X/delta F508, four CF carriers heterozygous for R553X, one CF carrier with the genotype delta F508/N, and two uncharacterized normal adults). After reverse transcription of the region from exons 10 to 13 to cDNA, fragments of the expected size were amplified from all heterozygous and normal subjects. In three subjects an additional alternatively spliced product was observed, which was found to contain a termination codon. In repeated experiments it was not possible to detect any CFTR mRNA in cells derived from the R553X homozygous patient. Furthermore, in subjects heterozygous for R553X we could not detect by hybridisation with a specific oligonucleotide probe and direct sequencing any CFTR mRNA derived from the R553X allele. However, the wild type product was present in all of these subjects. Our results support the view that nonsense mutations in the CFTR gene can lead to a reduction or absence of cytoplasmic CFTR mRNA.

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11 The most frequent mutation, AF508,6 located in the first nucleotide binding fold, is usually associated with severe CF,7 but in exceptional cases AF508 homozygotes are only mildly affected.8 Three nonsense mutations, G542X,9 R553X,'0 and W1282X," represent the second, fourth, and fifth most frequent CF mutations worldwide (Cystic Fibrosis Genetics Analysis Consortium, unpublished data). Login to comment
13 Two CF patients, homozygous for the G542X mutation, have been described as mildly affected."213 Cutting et al14 found two patients heterozygous for W1316X/R553X and S1255X/G542X, respectively, to exhibit severe pancreatic but only mild pulmonary disease. Login to comment

[hide] Analysis of the 27 exons and flanking regions of t... Hum Mol Genet. 1993 Aug;2(8):1209-13. Claustres M, Laussel M, Desgeorges M, Giansily M, Culard JF, Razakatsara G, Demaille J
Analysis of the 27 exons and flanking regions of the cystic fibrosis gene: 40 different mutations account for 91.2% of the mutant alleles in southern France.
Hum Mol Genet. 1993 Aug;2(8):1209-13., [PMID:7691344]

Abstract [show]
In order to characterize the non-delta F508 mutations that account for 36% of cystic fibrosis (CF) chromosomes in Southern France in a sample of 137 patients, we have systematically screened the entire coding region and adjacent sequences of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by the single strand conformation polymorphism (SSCP) technique followed by direct sequencing of the mutant DNAs. We identified 13 novel mutations (9 reported in this paper) and 4 novel rare nucleotide sequence variations. Forty different mutations including delta F508, located in 15 exons, account for only 91.2% of mutants in a population originating from Southern France, in contrast with a recent report on the Celtic population of Brittany demonstrating that 90% of mutations can be detected with only three mutations. We present a very large spectrum of different CF mutations identified in a small geographical area.

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26 Mutations identified in a Southern french population mutation AF5O8 M1K 300delA P67L R74W G85E 394detTT 406-6 (T-C) Y122X I148T 621 + 1G-T 62/+2T-G L206W 1078deIT R334W R347H R347P AI507 1717-1G-A G542X R553X S549N G551D E585X 2184delA K710X R792X S945L Y1092X 3272-26A-G R1158X R1162X 3737delA 3659delC 11234V D1270N W1282X N13O3H N13O3K 4382delA Exon 10 1 3 3 3 3 3 intron 3 4 4 intron 4 intron 4 6a 7 7 7 7 10 intron 10 11 11 11 11 , 12 13 13 13 15 17b intron 17a 19 19 19 19 19 20 20 21 21 24 Amino acid change 3 bp deletion start-Lys at 1 frameshift Pro-Leu at67 Arg-Trp at 74 Gly-Glu at 85 frameshift splice mutation? Login to comment

[hide] Cystic fibrosis mutations impair the fertilization... Hum Reprod. 1993 Aug;8(8):1259-63. Patrizio P, Ord T, Silber SJ, Asch RH
Cystic fibrosis mutations impair the fertilization rate of epididymal sperm from men with congenital absence of the vas deferens.
Hum Reprod. 1993 Aug;8(8):1259-63., [PMID:7691870]

Abstract [show]
One of the limiting factors for the successful treatment of male sterility due to congenital absence of the vas deferens (CAVD) is the low (< 20%) and extremely unpredictable rate of in-vitro fertilization of their epididymal spermatozoa. The recent demonstration that CAVD is a mild form of cystic fibrosis (CF) disease, almost exclusively involving the genital tract, has prompted us to investigate the hypothesis of whether there could be an association between particular CF genotype mutations and the in-vitro performance of epididymal sperm. In this study 63 patients with surgically confirmed diagnosis of CAVD undergoing microsurgical epididymal sperm aspiration (MESA) and in-vitro fertilization (IVF) were evaluated. The genetic screening was carried out on DNA extracted from peripheral lymphocytes and amplified by the polymerase chain reaction. A total of 12 mutations in the cystic fibrosis transmembrane regulator gene (CFTR), representing approximately 90% of the total known CF mutations, were tested. The presence or absence of mutations, as well as the type of mutation found, was correlated with the IVF and pregnancy rates. Of the 63 patients examined, 40 (64%) were positive to the CF screening and 23 were negative. In three cases no spermatozoa were found because of rete testis blockage. The difference in fertilization rates between patients testing positive and negative was highly significant (P = 0.000003), 13 versus 23%, respectively. In order to pinpoint which mutation could account for the most deleterious effect on the IVF results, we analysed these by keeping the patients separated by CF mutations.(ABSTRACT TRUNCATED AT 250 WORDS)

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38 Briefly, genomic DNA extracted from peripheral lymphocytes was amplified by the polymerase chain reaction (PCR) and analysed for 12 mutations in the CFTR gene: Delta F508, G542X, G551D, R553X, W1282X, N1303K, Delta 1507, 1717G-A, R560T, S549N, R117H and 621 + 1. Login to comment
56 DF508/ DF508/R117H R117H/R553X R117H/R117H W1282X/ R553X/ N1303K/ G542X/ 1717G-A 21 4 Negative Table H. Login to comment
69 Four patients had other, rarer mutations (1717G-A, 1303K, G542X, R553X). Login to comment

[hide] CF2603/4delT, a new frameshift mutation in exon 13... Hum Genet. 1993 Jul;91(6):614-5. Ezquieta B, Molano J
CF2603/4delT, a new frameshift mutation in exon 13 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.
Hum Genet. 1993 Jul;91(6):614-5., [PMID:7687986]

Abstract [show]
By using temperature gradient gel electrophoresis to screen for mutations in cystic fibrosis (CF) patients, we have found a new mutation in the CF transmembrane conductance regulator gene. It is a frameshift mutation named CF2603/4delT located at the 3'-end of exon 13. A thymidine at position 2603 or 2604 is lost. The mutation eliminates an MseI site and, therefore, can be screened by restriction enzyme analysis.

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8 The most frequent of these mutations account for as much as 20% of the non-AF508 mutations in the Spanish CF population (e.g. G542X, RII62X, N1303K, W1282X, G551D; Nunes et al. 1991). Login to comment

[hide] Microsatellite haplotypes for cystic fibrosis: mut... Hum Mol Genet. 1993 Jul;2(7):1015-22. Morral N, Nunes V, Casals T, Chillon M, Gimenez J, Bertranpetit J, Estivill X
Microsatellite haplotypes for cystic fibrosis: mutation frameworks and evolutionary tracers.
Hum Mol Genet. 1993 Jul;2(7):1015-22., [PMID:7689896]

Abstract [show]
Highly informative intragenic microsatellite markers within the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene allow the analysis of associations between specific mutations and haplotypes. We have analysed 440 Spanish CF families carrying 22 different CF mutations and have established haplotypes in 1,036 chromosomes for microsatellites IVS8CA, IVS17BTA and IVS17BCA. No new alleles were detected at the three CFTR microsatellites, in more than 3,000 meiosis analysed (estimated mutation rate of less than 3.3 x 10(-4)). The evolution of 16 haplotypes associated with the most common CF mutation, delta F508, and the low mutation rate at these microsatellite loci suggest that delta F508 originated within the 23-31-13 haplotype at least 53,000 years ago, very early in the history of the European population. The number of haplotype changes seen for two other common mutations, G542X (haplotype 23-33-13) and N1303K (23-31-13), suggests that they originated at least 35,000 years ago. Microsatellite allele variability associated with delta F508, G542X and N1303K demonstrates that slippage and mispairing is the main mechanism generating microsatellite alleles. In spite of the haplotype variability detected for these 3 common mutations, the association between haplotype and mutations is very strong. Mutations 1609delCA, 3667del4, delta I507 and G551D are all associated with haplotype 16-7-17, which has a frequency of 14.5% in normal chromosomes. 5 haplotypes bearing specific CF mutations were not found in normal chromosomes. Haplotype 16-46-13 is strongly associated with CF mutations E92K and 3601-111G-->C. About 23% of CF chromosomes with unknown mutations show significant linkage disequilibrium for microsatellite haplotypes.(ABSTRACT TRUNCATED AT 250 WORDS)

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6 The number of haplotype changes seen for two other common mutations, G542X (haplotype 23-33-13) and N1303K (23 - 31 -13), suggests that they originated at least 35,000 years ago. Login to comment
7 Microsatellite allele variability associated with AF508, G542X and N1303K demonstrates that slippage and mispairing is the main mechanism generating microsatellite alleles. Login to comment
19 Only 4 non-AF508 mutations (G542X, G551D, W1282X and N13O3K) are present in most geographical and ethnic subgroups with frequencies higher than 1% each, but with considerable variation (5-8). Login to comment
32 Only 5 mutations have frequencies of more than 1 % in the sample of chromosomes analysed (AF508, G542X, N1303K, 3601-111G-C, and R1162X), accounting for a total of 63.8% of CF chromosomes; 17 mutations have frequencies of less than 1% each, accounting for 5.4% of CF chromosomes; and 271 chromosomes (30.8%) have as yet uncharacterized mutations (Table 1 and Figure 1). Login to comment
58 CF mutations identified in the Spanish population Mutation AF5O8 G542X N13O3K 36O1-111G-C R1162X 1609delCA 2869insG W1282X AI507 G551D 1949del84 CF50KBdel tt 1 K710X 621 + 1G-T R334W 1078delT E92K 3667deM R1158X A120T I148T 386Oins31 Unknown Total N 437 73 18 18 14 8 6 6 5 4 3 3 3 2 2 1 1 1 1 1 1 1 271 880 % 49.7 8.3 2.1 2.1 1.6 0.9 0.7 0.7 0.6 0.5 0.3 0.3 0.3 0.2 0.2 0. Login to comment
79 CFTR microsateDite haplotypes for me three most common CF mutations (AF508, G542X and N13O3K) 16 16 16 16 16 16 16 16 16 17 16 16 16 16 17 23 23 23 4: 3: 30 31 32 29 33 25 35 28 31 31 46 44 45 7 7 33 30 31 18 35 13; 17 49 11: 13 13 13 13 13 13 13 13 14 13 13 13 13 17 17 13 13 13 A A B C 17 52 11: 16 34 83 52 37 26 12 12 11 7 7 6 16 10 6 75 15 12 6 6 16.0 10.0 7.1 5.0 2.3 2.3 2.1 1.3 1.3 1.1 3.0 1.9 1.1 14.5 2.8 2.3 1.1 1.1 15 7 17; 23 7 17; 23 22 17. Login to comment
83 Haplotypes 8CA 17BTA 17BCA Mutation NCF Chromosomes %CF %Normal 23 23 23 22 24 23 17 17 17 17 18 16 16 16 16 21 23 23 23 23 23 17 16 23 22 24 24 23 31 32 30 31 31 31 32 31 33 41 32 31 30 31 7 7 33 32 34 31 37 33 32 31 31 31 32 30 13 13 13 13 13 14 13 13 13 13 13 13 13 14 17 17 13 13 13 13 13 13 13 13 13 13 13 13 AF5O8 (49.7) Total G542X (8.396) Total N13O3K (2.1%) Total 106 2 2 2 2 1 34 29 2 3 1 1 1 1 1 2 190 36 18 4 1 1 1 1 62 10 3 2 1 1 17 55.7 1.1 1.1 1.1 1.1 0.5 17.9 15.3 1.1 1.5 0.5 0.5 0.5 0.5 0.5 1.1 100.0 58.0 29.0 6.5 1.6 1.6 1.6 1.6 100.0 58.8 17.6 11.8 5.9 5.9 100.0 1.1 0.4 1.1 - 0.2 - - 1.1 0.2 - - 10.0 16.0 1.3 14.5 0.2 31.4 2.3 0.4 0.6 1.1 - 0.2 7.1 11.7 1.1 - 0.2 0.2 1.1 2.6 N CF, number of CF chromosomes; CF, cystic fibrosis; (), frequency of CF mutations. Login to comment
103 The three most common CF mutations are associated with the same haplotype subgroup About 90% of G542X chromosomes are associated with haplotypes 23-33-13 or 23-32-13, which account for 2.3% and 0.4% of normal chromosomes, respectively. Login to comment
104 Haplotype distribution for each allelic system and the haplotype frequency G542X N1303K Figure 3. Login to comment
105 Parsimonious tree of evolution for haplotypes carrying mutations AF5O8, G542X and N13O3K. Login to comment
112 The most parsimonious tree for G542X would suggest a hypothetical haplotype 17-32-13 or 16-33-13, which would explain haplotype 17-33-13. Login to comment
113 However, these haplotypes probably each derive independently from the most common G542X haplotypes 23-33-13 and 23-32 - 13, respectively. Login to comment
115 show that G542X originally arose with allele 23 for the IVS8CA locus, and 13 for the IVS17BCA. Login to comment
116 IVS17BTA alleles 32 and 33 are found in two haplotypes each; the highfrequencyof haplotype 23-33-13 makes it most likely that this haplotype is the original wherein G542X arose. Login to comment
117 Haplotypes 16-32-13 or 17-33-13 could be derived independently from the two most common G542X haplotypes by a deletion of 6 and 7 dinucleotides respectively at the IVS8 locus (Table 3 and Figure 3). Login to comment
120 The only difference between haplotype 23-31 -13 (AF508 and N1303K) and 23-33-13 (G542X) is two TA repeats at the IVS17BTA locus. Login to comment
124 Age of mutations AF508, G542X and N1303K The current knowledge on mutations generating new alleles at microsatellite loci suggests a mutation rate of about 1 x 10~4 (ref. 26). Login to comment
131 For the G542X chromosomes the mean number of mutations of the three microsatellites, according to the reconstructed tree (Figure 3), is 0.451, representing about 1,350 generations or 34,000 years as the minimum age of G542X. Login to comment
132 For N13O3K there are 17 chromosomes that would give a result very similar to that of G542X (at least 35,000 years), which, nonetheless, must be considered with caution because of the low sample size. Login to comment
180 The variability observed for G542X and N1303K haplotypes also suggests an ancient origin of, more than 35,000 years ago, for these two mutations. Login to comment
188 Therefore, mutation analysis can be performed, firstly by screening only for the most common mutations (i.e. AF508 and G542X, in the case of the Spanish population); then, having the microsatellite haplotypes allows us to conduct a specific search for known mutations pertaining to each framework (i.e. 17-7-17 with mutations W1282X or Rl 158X, 14-31 -13 with 1949del84, etc); finally, the negative cases are analysed by searching for the unknown molecular defects. Login to comment
201 G542X was analysed by restriction site-generation PCR and digestion with ScrFl (36). Login to comment
213 As at the departure point the mutations AF508, G542X and N1303K occurred each in a single haplotype, all the mutations at the microsatellite loci have accumulated in the genealogy. Login to comment

[hide] Severe cystic fibrosis in a child homozygous for t... J Med Genet. 1993 Jul;30(7):621-2. Bienvenu T, Beldjord C, Fonknechten N, Kaplan JC, Lenoir G
Severe cystic fibrosis in a child homozygous for the G542 nonsense mutation in the CFTR gene.
J Med Genet. 1993 Jul;30(7):621-2., [PMID:7692049]

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48 Severe cystic fibrosis in a child homozygous for the G542 nonsense mutation in the CFTR gene Several homozygous nonsense mutations in the CFTR gene (S1255X/G542X, W1316X/ R553X, G542X/G542X, R553X/R553X, and RI 162X/R1 162X) have been reported. Login to comment
52 We present the clinical and molecular findings in a child homozygous for G542X with severe pancreatic and pulmonary disease. This Turkish boy was born at term (birth weight 3200 g) and presented with meconium ileus, successfully treated with enemas. Login to comment
59 A homozygous G542X mutation was found by DGGE and identified by exon 11 group.bmj.comon October 25, 2012 - Published byjmg.bmj.comDownloaded from Letters to the Editor amplification using a modified primer on the 5' side of codon 542 and subsequent digestion with BstNI.5 The mutation (G-'T at 1756) was characterised by direct sequencing using the Sequenase USB kit by standard methods. Login to comment
60 G542 is a nonsense mutation in which a glycine at codon 542 is replaced with a stop codon G542X in the cystic fibrosis transmembrane conductance regulator gene.' This boy has a severe lung involvement, meconium ileus, and pancreatic insufficiency, as indicated by the high degree of steatorrhoea. Login to comment

[hide] A comprehensive CFTR mutation analysis of German c... Hum Mol Genet. 1993 Jun;2(6):809-11. Reiss J, Ellermeyer U, Rininsland F, Ballhausen P, Lenz U, Wagner S, Schlosser M
A comprehensive CFTR mutation analysis of German cystic fibrosis patients.
Hum Mol Genet. 1993 Jun;2(6):809-11., [PMID:7689013]

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10 Of these patients, 8 were heterozygous for AF508, one was heterozygous for G542X and for one, no mutation was known. Login to comment
13 R117P (CGC-CCQ was found in the patient with the G542X allele and confirmed by family analysis. This mutation is the only novel one we have found in the 5' part of the CFTR gene and replaces the same nucleotide as the former described mutation R117H (10). Login to comment
56 Frequency of CFTR mutations checked routinely in German CF patients Mutation AF508 AI507 R553X G542X G551D R347P N1303K 1717 -lg-a S549R others in exon total Exon 10 10 11 11 11 7 21 llfb) 11 ll(c) Frequency 375/500 1/500 17/124(a) 13/124 7/124 6/124 6/124 5/124 2/124 0/124 % of total CF chromosomes 75.1 0.2 3.4 2.6 1.4 1.2 1.2 1.0 0.4 0.0 86.5 Reference (3) (17) (18) (17) (18) (10) (19) (17) (17) (20) (a) Non-AF508 chromosomes. Login to comment

[hide] Psoralen-modified oligonucleotide primers improve ... Hum Mol Genet. 1993 Apr;2(4):393-7. Costes B, Girodon E, Ghanem N, Chassignol M, Thuong NT, Dupret D, Goossens M
Psoralen-modified oligonucleotide primers improve detection of mutations by denaturing gradient gel electrophoresis and provide an alternative to GC-clamping.
Hum Mol Genet. 1993 Apr;2(4):393-7., [PMID:7684943]

Abstract [show]
Denaturing gradient gel electrophoresis (DGGE), a mutation-scanning procedure separating DNA fragments differing by as little as a single base change, is widely used in studies of genomic nucleotide sequence variability. The efficiency of the technique is greatly enhanced by attaching, through polymerase chain reaction (PCR) incorporation, a long GC-tail to the test DNA sequence which, as a result, becomes analysable throughout. As synthesis of GC-rich specific PCR primers is costly and time-consuming, we attempted to clamp the DNA fragment using a psoralen derivative (ChemiClamp) that promotes photo-induced cross-linking at one end. We found that this procedure provides an attractive alternative to GC-clamp in DGGE (and temperature gradient gel electrophoresis) and should prove useful in both research and diagnostic laboratories.

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62 DGGE analysis of the CF mutation G542X. Login to comment
64 Lanes 1 and 3: G542X heterozygote; Lanes 2 and 4: Normal controls, h: heteroduplexes. Login to comment
67 Figure 4 shows, as an example, the results obtained with a G542X (exon 11) heterozygote. Login to comment

[hide] Two cystic fibrosis patients with the genotype G54... Hum Genet. 1993 Mar;91(1):78-9. Reiss J, Ellermeyer U, Schloesser M, Fuhrmann W, Drews D, Posselt HG
Two cystic fibrosis patients with the genotype G542X/G551D.
Hum Genet. 1993 Mar;91(1):78-9., [PMID:7681035]

Abstract [show]
Two adult sisters affected by cystic fibrosis were both shown to carry two different alterations within exon 11 of the CFTR gene, the nonsense mutation G542X and the missense mutation G551D. Both patients exhibit a relatively benign clinical course. In the described patients, G542X functions as a "mild" allele and is, in this respect, dominant to the "severe" G551D.

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1 Two adult sisters affected by cystic fibrosis were both shown to carry two different alterations within exon 11 of the CFTR gene, the nonsense mutation G542X and the missense mutation G551D. Login to comment
3 In the described patients, G542X functions as a "mild" allele and is, in this respect, dominant to the "severe" G551D. Login to comment
9 Among these are the nonsense mutation G542X and the missense mutation G551D, which account for 2.6% and 1.4%, respectively, of CF alleles in the German population. Login to comment
11 The phenotypic effects of Stop codon mutations are controversial, especially with regard to patients homozygous for G542X (Cuppens et al. 1990; Kalaydjieva 1991). Login to comment
12 We report here the phenotype of two adult patients with the genotype G551 D/G542X. Login to comment
34 The heterozygous signals reflecting the mutations G542X (left) and G551D (right) can be recognized by small peaks and are designated by N tion G551D. Login to comment
35 The exon 11 amplification product was then subjected to automated direct sequencing, which both confirmed the presence of a G551D mutation and detected heterozygosity for the Stop codon mutation G542X (Fig. Login to comment
42 (1991) have both reported patients homozygous for the mutation G542X. Login to comment
44 However, in the former study, two patients are described; the one assigned as having milder symptoms, was homozygous for G542X and was diagnosed earlier than her heterozygous nephew. Login to comment
46 The relatively mild cystic fibrosis in patients homozygous for G542X has been confirmed by the report of Bonduelle et al. Login to comment

[hide] Two new mutations detected by single-strand confor... Hum Genet. 1993 Mar;91(1):63-5. Ivaschenko TE, Baranov VS, Dean M
Two new mutations detected by single-strand conformation polymorphism analysis in cystic fibrosis from Russia.
Hum Genet. 1993 Mar;91(1):63-5., [PMID:7681034]

Abstract [show]
Single-strand conformation polymorphism (SSCP) analysis followed by direct sequencing of exons containing ATP-binding domains of the cystic fibrosis transmembrane conductance regulator (CFTR) gene was performed on 80 Russian DNA samples. Two new alterations--S1196X (exon 19) and W1282R (exon 20)--and two novel polymorphisms--1525-61 (intron 9) and 1716+12 T-C (intron 10)--were identified. Mutation S1196X changes a TCA codon to TGA and destroys an EcoRI site. Alteration W1282R results from a T-to-C change at position 3976. It was found in one Russian patient and creates an AciI site; however, it is unclear whether this is a disease-causing mutation or a polymorphism. Polymorphism 1525-61 results from an A-to-G change. Alteration 1716+12 T-C was found in a Moldovian patient and creates a new MaeII site. It is not known whether this alteration affects the splicing of the mRNA. The previously described A4002G polymorphism was encountered in approximately 9% of Russian CF chromosomes. In addition, we have found the previously described 3732delA mutation in 7 CF chromosomes, making it the second (after delta F508) most frequent mutation in the Russian population.

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14 The AF508 mutation accounts for 30% - 80% of all CF chromosomes, and several others (G551D, R553X, G542X, N1303K, and W1282X) consitute as high as 5% of the CF chromosomes in some populations (Cutting et al. 1990; Kerem et al. 1990; Os- Correspondenceto: M. Dean borne et al. 1991; Vidaud et al. 1990). Login to comment

[hide] Multiplex PCR amplification from the CFTR gene usi... Hum Mol Genet. 1993 Feb;2(2):159-63. Richards B, Skoletsky J, Shuber AP, Balfour R, Stern RC, Dorkin HL, Parad RB, Witt D, Klinger KW
Multiplex PCR amplification from the CFTR gene using DNA prepared from buccal brushes/swabs.
Hum Mol Genet. 1993 Feb;2(2):159-63., [PMID:7684637]

Abstract [show]
Traditionally, DNA used for PCR-based diagnostic analysis has originated from white cells fractionated from whole blood. Although this method yields substantial quantities of DNA, there are some drawbacks to the procedure, including the inconvenience of drawing blood, risk of exposure to blood-borne pathogens, liquid sample handling, and the somewhat involved extraction procedure. Alternatively, DNA for genetic diagnosis has been derived from finger stick blood samples, hair roots, cheek scrapings, and urine samples. Oral saline rinses have also been used extensively as a means of collecting buccal epithelial cells as a DNA source. However, this method still requires liquid sample handling. Herein, we present our results involving the rapid extraction of DNA from buccal cells collected on cytology brushes and swabs for use in PCR reactions, specifically the multiplex amplification of 5 exons within the CFTR gene. The quality of DNA isolated from buccal cells, collected in this manner, has been sufficient to reproducibly support multiplex amplification. Cheek cell samples and the DNA prepared from them as described here are highly stable. The success rate of PCR amplification on DNA prepared from buccal cells is 99%. In a blind study comparing the analysis of 12 mutations responsible for cystic fibrosis in multiplex products amplified with DNA from both blood and buccal cell samples from 464 individuals, there was 100% correlation of results for blood and cheek cell DNA, validating the use of DNA extracted from cheek cells collected on cytology brushes for use in genetic testing.

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108 observed AF5O8 G542X G551D W1282X R553X R560T 1717-1 N1303K 621 + 1 R117H S549N 1507 280 7 16 2 4 2 2 5 6 11 1 •Includes affected individuals and known carriers. Login to comment

[hide] Efficient 12-mutation testing in the CFTR gene: a ... Hum Mol Genet. 1993 Feb;2(2):153-8. Shuber AP, Skoletsky J, Stern R, Handelin BL
Efficient 12-mutation testing in the CFTR gene: a general model for complex mutation analysis.
Hum Mol Genet. 1993 Feb;2(2):153-8., [PMID:7684636]

Abstract [show]
The identification of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has led to the identification of more than 225 presumed disease-causing mutations at the locus. The diagnosis of cystic fibrosis or the carrier state by direct DNA analysis is hindered by this large number. A practical assay must be able to detect enough mutations to achieve clinically significant sensitivity. The use of allele-specific oligonucleotide probes is the most promising of the available methods. However, to date this has generally involved tedious probe-by-probe hybridizations, due to variations in the oligonucleotides' denaturation temperatures caused by differences in their G-C base-pair content. We have developed a rapid, cost-effective assay that simultaneously detects 12 CFTR mutations after multiplex polymerase-chain-reaction amplification of genomic DNA. The test may be readily extended to detect additional mutations at minimal increase in the cost per test or the turnaround time. We improve specificity and avoid the need for individual hybridizations by the use of tetramethylammonium chloride to virtually eliminate the effects of G-C differences. Coupled with non-invasive sample-collection methods, this is an immediately practical assay for cystic fibrosis. More generally, it will serve as a model for the development of diagnostic tests in other genetic disorders involving complex mutation analysis.

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48 A diird was hybridized with a pool of ASOs for the five most frequent CFTR mutations after AF508: G551D, G542X, W1282X, N1303K, and R553X. Login to comment
50 Percent GC content of allele-speciik oligonucleotides GC Content/ASO Mutation G542X G551D R553X W1282X N1303K DI507 1717-1 G - A R560T S549N R117H 621 + 1 G - T 40 53 53 47 41 30 41 53 53 47 30 HS«3X W12»2X N1J0JK £507 H117M • 21 SS4tN RSOOT Table 2. Login to comment
53 1) Number of ASOs (N = 12) 2) Mutation Frequencies # ofCF Mutation Chromosomes Frequency A508 G551D G542X W1282X N1303K R553X 621 + 1 G - T R117H 1717-1 G - A R560T AI507 S549N unknown Total 548 15 13 13 12. Login to comment
54 9 9 '3 3 2 1 1 133 764 • Minimal Number of Independent Conclusion (4 hybridizations) Filter # 1 N(A5O8) 2 A5O8 72.0% 2.0% 1.7% 2.0% 1.5% 1.1% 1.1% 0.4% 0.4% 0.3% 0.1% 0.1% 17.0% Hybridizations 3 4 G551D 621 + 1 G - T G542X R117H W1282X 1717-1 G - A N13O3K R560T R553X A507 S549N with the remaining six mutations: 621 + 1 G^T, R117H, 1717-1 G - A , R560T, AI507, and S549N. Login to comment
72 Example of results obtained from hybridizing four identical filters with ASOs specific for N (the normal sequence at position 508), AF508 (a 3 bp deletion at position 508), pool 1 (G542X, G551D, R553X, W1282X, and N13O3K), and pool 2 (AI507, R117H, 621 + 1 G - T , S549N, R560T, 1717-1 G-A). Login to comment
73 Row A contains positive control samples with the following genotypes: 1A (AF508/AF508); 2A (AF508/G542X); 3A (AF5O8/G551D); 4A (N/R553X); 5A (NAV1282X); 6A (AF5O8/N13O3K); 7A (N/AI5O7); 8A (N/R117H); 9A (N/621 + 1 G-T); 10A (AF508/S549N); HA (AF508/R560T); and 12A (N/1717-1 G-A). Login to comment
79 Pool 1: lane 1 (G542X); lane 2 (G551D); lane 3 (R553X); lane 4 (W1282X); lane 5 (N1303K). Login to comment
86 Row A contains positive control samples: lane 2 (G542X); lane 3 (G551D); lane 4 (R553X); lane 5 (W1282X); lane 6 (N13O3K); lane 7 (AI507); lane 8 (R117H); lane 9 (621 +1 G-T)); lane 10 (S549N); lane 11 (R560T); lane 12 (1717-1 G-A)). Login to comment

[hide] Aetiology of congenital absence of vas deferens: g... Hum Reprod. 1993 Feb;8(2):215-20. Patrizio P, Asch RH, Handelin B, Silber SJ
Aetiology of congenital absence of vas deferens: genetic study of three generations.
Hum Reprod. 1993 Feb;8(2):215-20., [PMID:8473422]

Abstract [show]
Bilateral congenital absence of the vas deferens (CAVD) is a form of male sterility (found in otherwise normal men) of unknown aetiology. Because males with cystic fibrosis (CF) almost invariably have CAVD as well, we investigated the hypothesis that men with isolated CAVD might share a common genetic background with males with CF. Genetic testing for CF was carried out in three generations of subjects: 44 patients with CAVD and their wives, 24 of their parents, and 13 of their offspring generated by microsurgical epididymal sperm aspiration (MESA) and in-vitro fertilization (IVF). DNA extracted from peripheral lymphocytes was amplified by the polymerase chain reaction (PCR) and then analysed for 12 mutations in the cystic fibrosis transmembrane conductance regulatory (CFTR) gene. Among 44 patients tested with CAVD, 26 (59%) were positive for at least one CF mutation, while the carrier frequency for CF mutations in the general population is only 4%. Four patients were found to be compound heterozygotes, three with genotypes Delta F-508/R117H, one with R553X/R117H. Among 24 parents tested, 15 (seven fathers, eight mothers) had sons with CAVD who were positive for CF mutations. Of these, nine (four fathers and five mothers) were found to be carriers for CF mutations. These four fathers, although carriers of CF mutations, were obviously fertile. Of the 13 offspring tested, six (three boys and three girls) had CF positive fathers. Of these, three (two girls and one boy) were found to be carriers for CF mutations. These MESA/IVF children are the first offspring to whom men with CAVD have been able to transmit CF mutations.(ABSTRACT TRUNCATED AT 250 WORDS)

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45 In six patients the mutation identified was W1282X, a nonsense mutation, localized on exon 20 and predicting the production of truncated polypeptides of CFTR; one patient was found to have the R553X mutation, a nonsense mutation of the CG to TG rule localized on exon 11; one patient had the mutation 1717G-A, localized on intron 10, which would cause defective RNA splicing; one patient was found to have the G542X mutation, a nonsense mutation in exon 11. Login to comment
51 Exon 4,10,11,20 and 21 were amplified in a multiplex reaction followed by allele specific oligonucleotide (ASO) probe analysis for the mutations Delta F508, G542X, G551D, R553X, W1282X, N13O3K, Delta 1507, 1717G-A, R560T, S549N, R117H and 621 + 1. Login to comment
85 DF5O8/ DF5O8/ DF5O8/ DF508/ DF508/ DF508/ DF508/ DF5O8/ DF5O8/ DF5O8/ DF508/ DF5O8/ DF508/ W1282X/ W1282X/ W1282X/ W1282X/ W1282X/ DF508/R117H DF508/R117H DF508/R117H R553X/R117H R553X/ 1717G-A/ G542X/ Neg. NA NA NA NA DF508 NA NA NA Neg. NA NA NA NA Neg. NA NA NA DF508 NA R117H NA NA 1717G-A NA DF508 Neg. NA NA NA Neg. NA NA NA DF508 NA NA NA NA W1282X NA NA NA R117H NA DF508 NA NA Neg. NA Neg. Neg. NA NA NA Neg. NA Neg. Neg. Neg. Neg. Neg. Neg. NA Neg. NA Neg. Neg. Neg. NA R117H NA NA NA Neg. (g) DF508 - ... - - (g) Neg. - ... _. - - - - (b) Neg. - ... - - (g) DF5O8 (b) DF508 - - - (b) Neg. - b = boy; g = girl; NA = data not available; -, no offspring; DF5O8 = Delta F508. Login to comment
127 However in the present study, we extended the genetic screening to the parents and the offspring of men with CAVD and moreover, by testing for 12 CF mutations, we demonstrated other rare CF mutations (R117H, G542X and 1717G-A) in patients with CAVD. Login to comment
144 The six CFTR mutations identified in this study occur on four exons plus one intron and represent deletion (Delta F508), nonsense (W1282X, 1717G-A and G542X) and amino acid substitution (R553X and R117H) mutations. Login to comment

[hide] Absence of cystic fibrosis mutations in a large As... J Med Genet. 1993 Feb;30(2):164-6. Curtis A, Richardson RJ, Boohene J, Jackson A, Nelson R, Bhattacharya SS
Absence of cystic fibrosis mutations in a large Asian population sample and occurrence of a homozygous S549N mutation in an inbred Pakistani family.
J Med Genet. 1993 Feb;30(2):164-6., [PMID:7680378]

Abstract [show]
The occurrence of cystic fibrosis is very rare in the Asian population. Carriers of the mutations most commonly found in Caucasians were not detected in a large Asian population sample of almost 900 chromosomes. However, an affected Pakistani child born to consanguineous parents was further investigated and shown to be homozygous for the mutation S549N (G-->A). Molecular and clinical data are presented which may improve our understanding of the phenotypic effects of the S549N mutation in the CFTR gene.

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81 A CFTR product cystic fibrosis patient with the nonsense mutation G542X zygous R553X and the splice site mutation 1717-1 J Med Genet*1991;28:878-80. in a substitution io Chalkley G, Harris A. Login to comment
84 A CFTR product cystic fibrosis patient with the nonsense mutation G542X zygous R553X and the splice site mutation 1717-1 J Med Genet *1991;28:878-80. in a substitution io Chalkley G, Harris A. Login to comment

[hide] Genetic determinants of airways' colonisation with... Lancet. 1993 Jan 23;341(8839):189-93. Kubesch P, Dork T, Wulbrand U, Kalin N, Neumann T, Wulf B, Geerlings H, Weissbrodt H, von der Hardt H, Tummler B
Genetic determinants of airways' colonisation with Pseudomonas aeruginosa in cystic fibrosis.
Lancet. 1993 Jan 23;341(8839):189-93., [PMID:7678316]

Abstract [show]
Exocrine pancreatic insufficiency and lung infection with Pseudomonas aeruginosa are major features of cystic fibrosis (CF). This monogenic disease is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. 267 children and adolescents with CF who were regularly seen at the same centre were assessed for an association of the CFTR mutation genotype with exocrine pancreatic function and the age of onset of chronic colonisation with P aeruginosa. The major mutation delta F508 accounted for 74% of CF alleles; 33 further CFTR mutations had been detected on the CF chromosomes of the study population by June, 1992. With the exception of delta F508/R347P compound heterozygotes, patients of the same mutation genotype were either pancreas insufficient (PI) or pancreas sufficient (PS). The age-specific colonisation rates with P aeruginosa were significantly lower in PS than in PI patients. The missense and splice site mutations that are "mild" CF alleles with respect to exocrine pancreatic function were also "low risk" alleles for the acquisition of P aeruginosa. On the other hand, the proportion of P aeruginosa-positive patients increased most rapidly in the PI delta F508 compound heterozygotes who were carrying a termination mutation in the nucleotide binding fold-encoding exons. Pancreatic status and the risk of chronic airways' colonisation with P aeruginosa are predisposed by the CFTR mutation genotype and can be differentiated by the type and location of the mutations in the CFTR gene.

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62 most rapidly in AF508 compound heterozygotes with a stop mutation (G542X, R553X, R1162X, W1282X) or another mutation in the NBF-encoding exons on the second disease allele: Risk factor* Compound heterozygote (95% Cl) p AF508/nonsense 2-47 (1-42-4-29) < O-(Xn AF508/missense 0-78 (0-43-1-41) NS AF508/frameshift 0-52(021-132) NS AF508/splice site 0.40 (0-19--()-S6) < 0-05 OF508/NBFl orNBF2 1-99 (1-22-3-25) <0-01 AF508/TM1 or TM2 0-62 (0-30-1-30) NS AF508/R domain 0-46 (0-17-1-29) NS *Relative to AF508 homozygous group. Login to comment

[hide] The molecular biology of cystic fibrosis. Annu Rev Med. 1993;44:133-44. Sferra TJ, Collins FS
The molecular biology of cystic fibrosis.
Annu Rev Med. 1993;44:133-44., [PMID:7682803]

Abstract [show]
Cystic fibrosis afflicts many children and, as the life expectancy for patients with this disease steadily increases, many adults as well. The identification and characterization of the gene responsible for this disorder provide a basis for understanding the pathophysiology of cystic fibrosis. Basic science advances are rapidly being integrated into the diagnostic and therapeutic regimens used by physicians in the care of patients with this disease.

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75 AF508,G542X, G551D, R533X, W1282X, and Nl303K account for 84.5% ofCF chromosomes in the non-Askenazic, North AmericanCaucasian population (43). Login to comment

[hide] Identification of 12 novel mutations in the CFTR g... Hum Mol Genet. 1993 Jan;2(1):51-4. Audrezet MP, Mercier B, Guillermit H, Quere I, Verlingue C, Rault G, Ferec C
Identification of 12 novel mutations in the CFTR gene.
Hum Mol Genet. 1993 Jan;2(1):51-4., [PMID:7683952]

Abstract [show]
Over 200 mutations, besides the deletion delta F508, have been identified in the CFTR gene and are known to cause CF. In order to characterize the molecular defects of non delta F508 CF chromosomes of various French origin, we have combined the techniques of denaturing gradient gel electrophoresis (DGGE) and direct sequencing to screen for mutations in the whole coding sequence of the CFTR gene corresponding to the 27 exons and their exon-intron boundaries. This approach enabled us to identify 12 novel mutations which are described here. We have systematically tested a large number of other nucleotide changes distributed in the 27 exons, each of them was clearly detected. These data support the notion that the DGGE conditions we have defined for screening coding sequence of the CFTR gene allows the identification of most of, if not all, the CFTR gene mutations.

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62 His genotype was G542X/Q1238X. Login to comment

[hide] Ethnic heterogeneity and cystic fibrosis transmemb... Am J Hum Genet. 1992 Dec;51(6):1344-8. Ober C, Lester LA, Mott C, Billstrand C, Lemke A, van der Ven K, Marcus S, Kraut J, Lloyd-Still J, Booth C
Ethnic heterogeneity and cystic fibrosis transmembrane regulator (CFTR) mutation frequencies in Chicago-area CF families.
Am J Hum Genet. 1992 Dec;51(6):1344-8., [PMID:1281385]

Abstract [show]
The identification of a common mutation, delta F508, in the CFTR gene allowed, for the first time, the detection of cystic fibrosis (CF) carriers in the general population. Further genetic studies revealed > 100 additional disease-causing mutations in this gene, few of which occur on > 1% of CF chromosomes in any ethnic group. Prior to establishing counseling guidelines and carrier risk assessments, we sought to establish the frequencies of the CFTR mutations that are present in CF families living in the Chicago area, a region notable for its ethnic heterogeneity. Our sample included 283 unrelated CF carriers, with the following ethnic composition: 78% non-Ashkenazi Caucasians, 5% Ashkenazi, 9% African-American, 3% Mexican, 0.3% Native American, and 5% mixed ancestry. When a panel of 10 mutations (delta F508, delta I507, G542X, G551D, R553X, S549N, R1162X, W1282X, N1303K, and 1717-1G-->A) was used, detection rates ranged from 75% in non-Ashkenazi Caucasians to 40% in African-Americans. These data suggest that the goal of screening for 90%-95% of CF mutations may be unrealistic in this and other, similar U.S. populations.

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7 When a panel of 10 mutations (AF508, AI507, G542X, G551D, R553X, S549N, R1162X, W1282X, N1303K, and 1717-1G--A) was used, detection rates ranged from 75% in non-Ashkenazi Caucasians to 40% in African-Americans. Login to comment
36 Mutation Analysis DNA from each subject was screened for the following mutations: AF508 (Kerem et al. 1989a), G542X (Kerem et al. 1990), GS51D (Cutting et al. 1990), R553X (Cutting et al. 1990), S549N (Cutting et al. 1990), R1162X (Gasparini et al. 1991), W1282X (Vidaud et al. 1990), N1303K (Osborne et al. 1991), and 1717-1G- A (Guillermit et al. 1990; Kerem et al. 1990). Login to comment
44 G542X. Login to comment
60 The fact that only approximately 60% of our Caucasian sample was of northern European ancestry may account for the relatively low frequency (.60) of AF508 in Chicago-area non-Ashkenazi Caucasians. The frequencies of the next two most common mutations, G542X and G551D, are also consistent with the ethnic composition of our sample. Login to comment
61 The G542X mutation is fairly common throughout Europe, particularly in southern Europe (Nunes et al. 1991). Login to comment
80 Among 20 Pueblo and Navajo CF chromosomes, only one mutation (G542X) was detected by a panel of six mutations (AF508, G542X, GSS1D, R553X, G542X, and N1303K). Login to comment

[hide] Non-radioactive detection of the most common mutat... Hum Genet. 1992 Dec;90(4):375-8. Fortina P, Conant R, Monokian G, Dotti G, Parrella T, Hitchcock W, Kant J, Scanlin T, Rappaport E, Schwartz E, et al.
Non-radioactive detection of the most common mutations in the cystic fibrosis transmembrane conductance regulator gene by multiplex allele-specific polymerase chain reaction.
Hum Genet. 1992 Dec;90(4):375-8., [PMID:1282898]

Abstract [show]
A rapid, simple, nonradioactive method for detection of four common mutations causing cystic fibrosis (CF) has been developed combining multiplexing with allele-specific polymerase chain reaction amplification. This approach (MASPCR) provides an easy assay for direct genotyping of normal and mutant CF alleles in homozygotes and heterozygotes. The strategy involves multiplex PCR of exons 10, 11, and 21 within the cystic fibrosis transmembrane conductance regulator (CFTR) gene in a single reaction containing three common oligoprimers and either the four normal or four mutant oligos corresponding to the delta F508, G551D, G542X, and N1303K mutations. Primers are chosen so that the size of the four PCR products differ, thereby facilitating detection on agarose gels following amplification in the same reaction. Patient samples are primed with either four normal or four mutant oligo mixtures, and PCR products run in parallel on gels to detect band presence or absence. This approach provides a simple and potentially automated method for cost-effective population screening.

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3 The strategy involves multiplex PCR of exons 10, 11, and 21 within the cystic fibrosis transmembrane conductance regulator (CFTR) gene in a single reaction containing three common oligoprimers and either the four normal or four mutant oligos corresponding to the AF508, G551D, G542X, and N1303K mutations. Login to comment
11 Five mutations have been identified (AF508, G551D, R553X, G542X, and N1303K), which account for 84% of all Caucasian CF chromosomes, and a rapid, reliable and cost-effective screening method is required that includes detection of these prevalent mutations. Login to comment
18 Oligonucleotide primers used for mutation detection by multiplex allele-specific polymerase chain reaction (MASPCR) Primers ~ Sequence b pMoV CF-W1468-N CF-508RP AF508 5' IVS-11 R553X-N R553X-M G551D-N G551D-M G542X-N G542X-M 5' IVS-2I N1303K-N N1303K-M 5'-GGCACCATTAAAGAAAATATCATCTT-3' l5 5'-TAGTGTGAAGGGTTCATATGCATAAT-3' 15 5'-GGCACCATTAAAGAAAATATCATTGG-3' 15 5'-CAACTGTGGTTAAAGCAATAGTGT-3' 20 5'-CTAAAGAAATTCTTGCTCG-3' 10 5'-CTAAAGAAATTCTTGCTCA-3' 10 5'-GAAATTCTTGCTCGTTGA C-3' 5 5'-GAAATTCTTGCTCGTTGAT-3 ' 5 5'-GTGTGATTCCACCTTCTCC-3' 2(1 5'-GTGTGATTCCACCTTCTCA-3' 20 5'-AAGAATGATACAAAGCAGACATG-3' 40 5'-CACTGTTCATAGGGATCCAAG-3' 40 5'-CACTGTTCATAGGGATCCAAC-3' 4(1 a Normal (N) and mutant (M) primers b Bold letters indicate single-base mutations c Amount (pMol) of each primer used in multiplex reactions single lane of a gel. Login to comment
22 DNA samples were obtained from CF patients with the AF508, G551D, R553X, and G542X mutations. Login to comment
30 Each reaction contained the three common primers (CF-508RP, 5' IVS-11, and 5' IVS-21) and either four normal or four mutant primer sets for the AF508, G551D, G542X, and NI303K sequences (Table 1). Login to comment
33 5' IVS-11 5' IVS-21 f,f A AA CF-W1468-N.--1~(139 bp) d'---- CF-508RP AFS08 .~1P.(136bp) <1~ CF-508RP ._~ (174 bp) ~ G542X (N or M) 5' IVS-11 (202 bp) ~ G551D (N or M) 5' IVS-21 ~ (240 bp) ~I~N1303K (N or M) Fig. 1. Login to comment
36 Multiplex amplification is accomplished using common primers (CF-508RP, 5' IVS-I1, and 5' IVS-21) and a mixture of either the four normal (CF-W1468-N, G551D-N, G542X-N, and N 1303K-N) or four mutant primer (AF508, G55 l DM, G542X-M, and NI303K-M) sets (Table 1) in each PeR reaction. Login to comment
47 All amplifications were done with either normal (N) or mutant (M) primers corresponding to the AF508, G551D, G542X, and N1303K regions of the human CFTR gene. Login to comment
48 Amplifications were done with DNA from normal controls using normal primers (lane 2 N1303K-N and 5' IVS-21; lane 4 G551D-N and 5' IVS-11; lane 6 G542X-N and 5' IVS-11; and lane 8 CF-W1468-Nand CF-508RP) or mutant primers for the same regions (lane 3 N1303K-Mand 5' IVS-21; lane 5 G551D-M and 5' IVS-11;lane 7 G542X-M and 5' IVS-11; and lane 9 AF508and CF-508RP). Login to comment
52 Multiplex amplification of four regions in normal DNA (lanes 2-5) encompassing AF508, G551D, G542X, and N1303K mutations with normal primer (N; lanes 2 and 4) or mutant (M) primer sets (lanes 3 and 5). Login to comment
53 Multiplex amplification was done using separate reactions containing a mixture of either the normal or corresponding mutant primer sets with DNA from a heterozygote for the AF508mutation (lanes 6 and 7), DNA from a heterozygote for the G542X mutation (lanes 8 and 9), and DNA from a compound heterozygote for the AF508and G542X mutations (lanes 10 and 11), respectively. Login to comment
61 We have successfully detected the G542X, G551D, and AF508 mutations using this multiplex approach. Login to comment
63 Data are shown confirming the following genotypes: a AF508 heterozygote (lanes 6 and 7), a G542X heterozygote (lanes 8 and 9), and a AF508/G542X compound heterozygote (lanes 10 and 11). Login to comment
73 For example, in one sample, the four normal primers produce normal amplification products, while the mutant primer set yields only a 174-bp fragment, consistent with heterozygosity for the G542X mu- tation. Login to comment

[hide] Screening for five mutations detects 97% of cystic... Am J Hum Genet. 1992 Nov;51(5):951-6. Abeliovich D, Lavon IP, Lerer I, Cohen T, Springer C, Avital A, Cutting GR
Screening for five mutations detects 97% of cystic fibrosis (CF) chromosomes and predicts a carrier frequency of 1:29 in the Jewish Ashkenazi population.
Am J Hum Genet. 1992 Nov;51(5):951-6., [PMID:1384328]

Abstract [show]
To determine the distribution and frequency of cystic fibrosis (CF) mutations in the Israeli population, we have screened 96 patients for 11 relatively common mutations. Five mutations--delta F508, G542X, W1282X, N1303K, and 3849 + 10kb C-->T--were found to account for 97% of the CF alleles in the Ashkenazi Jews. In contrast, of the 11 mutations tested, only delta F508 was detected in Jewish patients of Sephardic or Oriental origin, accounting for 43% of the CF alleles. Four mutations--delta F508, G542X, W1282X, and N1303K--accounted for 55% of the CF alleles in Arab patients. In a pilot screening study, a random sample of 424 Ashkenazi individuals was analyzed for three mutations--delta F508, W1282X, and G542X. Thirteen individuals were detected as heterozygotes (six for delta F508 and seven for W1282X), predicting a heterozygote frequency of 1:29. This is similar to the frequency of carriers in the Caucasian population of northern European ancestry. On the basis of these data, the Ashkenazi population is considered to be a candidate for CF heterozygote screening.

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33 The regions encompassing the mutations of interest were simultaneously amplified by PCR using DNA primers: C16B and C16D for AF508 (Kerem et al. 1989), lli5 and 11i3 for G542X, 20i5 and 20i3 for W1282X, and 21i5 and 21i3 for N1303K (Kerem et al. 1990; Vidaud et al. 1990; Osborne et al. 1991; Zielenski et al. 1991 b). Login to comment
37 Mutation Detection Mutations AF508 and G542X were detected by using oligonucleotides oligo-N, oligo AF, 542NL, and 542X according to a method described elsewhere (Kerem et al. 1989 and 1990). Login to comment
42 The hybridization and wash conditions were as follows: W1282X-hybridization at 370C for 60 min and three washes in 2 x SSC, 0.1% SDS (twice at room temperature for 10 min and then at 590C for 10 min); AF508-hybridization 370C for 60 min and then three washes in 2 x SSC, 0.1% SDS (twice at 370C for 10 min and then at 420C for 10 min); G542X-hybridization at 420C for 2 h and then two washes in 2 x SSC, 0.1% SDS (once at 420C for 10 min and once at 450C for 10 min). Login to comment
46 The same approach was used as an alternative method to detect AF508 and G542X. Login to comment
54 Results Five mutations-W1282X, AF508, G542X, N1303K, and 3849 + lOkb C-'lT-were found in our patients. Login to comment
57 Amongthe Ashkenazi, three mutations-W1282X (48%), AF508 (30%), and G542X (12%)-accounted for 90% of the CF mutations, while mutations N1303K and 3849 + 1Okb C--T were found on an additional 7% of CF chromosomes. Login to comment
60 In the Arab patients, four mutations-AF508, N1303K, W1282X, and G542X-were found, accounting for 55% ofthe CF mutations. Login to comment
62 To evaluate the gene frequency in the Ashkenazi population, a random sample of 424 Ashkenazi individuals (848 chromosomes) was screened for the presence of the three mutations W1282X, AF508, and G542X. Thirteen individuals were identified as heterozygotes- six with the AF508 mutation and seven with the W1282X mutation. Login to comment
63 In this sample we did not identify any carrier having the G542X mutation. Login to comment
64 Discussion The predominant mutation in Caucasians, AF508, was found in all ethnic communities in Israel but at a significantly lower frequency (31%) than in northern Table 2 Distribution of CF Mutations, by Ethnic Group FREQUENCY (no.) IN Jews Ashkenazi Non-Ashkenazi Turkish Arabs MUTATION (n = 94) (n = 37) (n = 7) (n = 31) F508 ........................... .30 (28) .43 (16) .29 (2) .26 (8) G542X .................. .12 (11) .43 (3) .03 (1) W1282X ................... .48 (45) .03 (1) .14 (1) .10 (3) N1303K .................. .03 (3) .16 (5) 3849 + 10kb C-T .......... .04 (4) .14 (1) Subtotal .................. .97 .46 1.00 .55 Unidentified mutations .... .03 (3) .54 (20) 0 .45 (14) NOTE. Login to comment
82 We screened for three mutations-AF508, W1282X, and G542X-which are expected to detect 90% of the carriers. Login to comment

[hide] Cystic fibrosis genotypes and views on screening a... Am J Hum Genet. 1992 Nov;51(5):943-50. Scriver CR, Fujiwara TM
Cystic fibrosis genotypes and views on screening are both heterogeneous and population related.
Am J Hum Genet. 1992 Nov;51(5):943-50., [PMID:1384327]

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76 of CF chromosomes]) Distribution and Mutation (%) Askhenazi from Israel (Abeliovich et al. 1992 [94]; Shoshani et al. 1992 [95]): W1282X .......................................... AFS08 ............................................. G542X ............................................ 3849 + 10 kb, CT ............................ N1303K ........................................... Total ............................................ Celtic Bretons from France (Ferec et al. 1992 [365]): AF508 ............................................. 1078delT ......................................... G5S1D ............................................ 1717-1 G-A ..................................... W846X ............................................ G91R .............................................. Login to comment
78 Ten alleles at 0.27% each .................... Total ............................................ French Canadians from northeastern Quebec (Rozen et al. 1992 [181]): 48 30 12 4 3 97 60 22 4 0 4 92a 82 4 1 1 1 2 3 98a AFS08 ......... ..................... 58 621+1G-T .............................. 23 A455E .......... .................... 8 G85E ......... ..................... 1 711+1 G-T ............................... 1 G542X .......... .................... 1 Y1092X .............................. 1 N1303K ........... ................... 1 Total .......... .................... 93a a Because of rounding, individual values do not sum to the total. Login to comment

[hide] A termination mutation (2143delT) in the CFTR gene... Hum Genet. 1992 Nov;90(3):279-84. Dork T, Kalin N, Stuhrmann M, Schmidtke J, Tummler B
A termination mutation (2143delT) in the CFTR gene of German cystic fibrosis patients.
Hum Genet. 1992 Nov;90(3):279-84., [PMID:1283149]

Abstract [show]
German patients with cystic fibrosis (CF) were screened for molecular lesions in exon 13 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by single strand conformation polymorphism (SSCP) and chemical cleavage of mismatch analyses. Direct sequencing of four samples that displayed the same SSCP pattern and that were susceptible to cleavage of hetero-duplexes by osmium tetroxide revealed, in all cases, a deletion of a single T residue at nucleotide position 2143 within codon 671 of the CFTR gene. As a result, leucine codon 671 is changed into a termination codon. In total, the 2143delT mutation was confirmed in 6 out of 271 German non-delta F508 CF chromosomes by artificial restriction fragment length polymorphism analysis, indicating that this frameshift mutation accounts for about 2% of German non-delta F508 mutations. The 6 pancreas insufficient patients who are compound heterozygous for 2143-delT suffer from the typical features of pulmonary and gastrointestinal CF disease. The 2143delT mutation completes the panel of the more frequent CFTR mutations that reside on the "delta F508 haplotype" and that contribute to its overpresentation among German non-delta F508 alleles that are associated with severe forms of disease.

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86 All six identified alleles carry the typical "risk haplotype" for CF that is also associated with other common severe mutations, such as zXF508, N1303K, G542X or 621 + 1G-T (Kerem et al. 1989, 1990: Osborne et al. 1991: Zielenski et al. 1991b). Login to comment

[hide] The spectrum of CFTR mutations in south-west Germa... Hum Genet. 1992 Nov;90(3):267-9. Lindner M, Wolf A, Moh B, Steinbach P, Kleihauer E, Bartram CR, Kulozik AE
The spectrum of CFTR mutations in south-west German cystic fibrosis patients.
Hum Genet. 1992 Nov;90(3):267-9., [PMID:1283148]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) gene of 110 cystic fibrosis (CF) patients from the south-west of Germany was screened for 12 different mutations. This analysis resulted in an identification of 79% of all CF mutations and a complete genotype in 66% of the families. The most common mutation found was delta F508 (67%). Another 5 mutations accounted for a further 12.5% (4% G542X; 3% R553X; 3% N1303K; 2% 1717-1 G-->A; 0.5% G551D) whereas 6 mutations (R117H, A455E, delta I507, S549I, S549N, and R1162X) were not found. Fifty-four (49%) patients were delta F508 homozygotes and 18 (16.5%) were compound heterozygotes for delta F508 and one of the rarer mutations. These frequencies differ slightly from those found in the north of Germany and considerably from those reported from the south of Europe, which seems to be consistent with a north to south decline of the relative abundance of delta F508. Two patients, age 6 and 25 years, were compound heterozygotes for G542X and N1303K. The clinical features of the 6 year old were characterised by severe gastrointestinal and as yet only mild pulmonary complications whereas the 25 year old manifested severe pulmonary and gastrointestinal symptoms indicating that the N1303K mutation of the C-terminal CFTR nucleotide binding fold significantly impairs protein function in both the pancreas and the lungs.

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4 Another 5 mutations accounted for a further 12.5% (4% G542X; 3% R553X; 3% N1303K; 2% 1717-1 G--~A; 0.5% G551D) whereas 6 mutations (R117H, A455E, AI507, $549I, $549N, and R1162X) were not found. Login to comment
7 Two patients, age 6 and 25 years, were compound heterozygotes for G542X and N1303K. Login to comment
25 Sequences derived from Dean et al. (1990); Kerem et al. (1990); Riordan et al. (1989); Guillermit et al. (1990); Cutting et al. (1990) Exon PCR primers Mutation Allele specific probes 4 5'-AGTCACCAAAGCAGTACAGC-3' R117H N: 5'-GCTATTCTCATCTGCATTCC-3' M: 9 5'-TAATGGATCATGGGCCATGT-3' A455 E N: 5'-ACAGTG~ITGAATGTGGTGCA-3' M: 5'-GTTTTCCTGGATTATGCCTGGCAC-3' N: 5'-GTTGGCATGCTTTGATGACGCTTC-3' M: t0 11 5'-CAACTGTGGTTAAAGCAATAGTGT-3' 5'-GCACAGATTCTGAGTAACCATAAT-3' 5'-GAGGAACGCTCTATC-3' 5'-GAGGAACACTCTATC-3' 5'-GTTGTTGGCG GTI'GCT-3' 5'-GTTGTTGGAG GTTGCT-3' zXF508 5'-CACCAAAGATGATATTTTC-3' 5'-AACACCAATGATATTTTCTT-3' AI507 1717-1G--+A N: 5'-TTGGTAATAGGACATCTCCA-3' M: 5'-TTGGTAATAAGACATCTCCA-3' G542X N: 5'-CCACCTTCTCCAAGAACTAT-3' M: 5'-CCACCTTCTCAAAGAACTAT-3 G551D N: 5'-CTCGTTGACCTCCACTCAGT-3' M: 5'-CTCGTTGATCTCCACTCAGT-3' 19 5'-GCCCGACAAATAACCAAGTGA-3' R 1162X 5'-GCTAACACATTGCTFCAGGCT-3' 21 5'-AATGTTCACAAGGGACTCCA-3' N1303K 5'-CAAAAGTACCTGTTGCTCCA-3' N: 5'-AGGGATCCAAGTTTTTTCTA-3 ' M: 5'-AGGGATCCAACTTTTTTCTA-3' Materials and methods Determination of the CFTR genotype All mutation analyses were performed on Taq-PCR (polymerase chain reaction) amplified DNA fragments (30 cycles at 92~176 72~ for 30/60/90 s) using primers as shown in Table 1 (Sambrook et al. 1989). Login to comment
26 The R117H, A455E, 1717-1G~A, AF508, G542X, G551D, and NI303K mutations were identified by allele specific oligonucleotide hybridisation (ASO, Sambrook et al. 1989) using 32p-labelled probes specific for the normal or the mutated sequence (Table 1). Login to comment
33 The other five mutations were found on a further 12% of the CF chromosomes, the most numerous of these being G542X, R553X, and N1303K (Table 2).This distribution is similar to that found in a group of patients from the north of Germany, although in contrast to our Table 2. Login to comment
34 Mutations observed in 220 defective cystic fibrosis transmembrane conductance regulator (CFTR) alleles Mutation n (%) Rll7H 0 A455E 0 AI507 0 AF508 147 (67) 1717-1G--*A 4 (2) G542X 9 (4) $549I 0 $549N 0 G551D 1 (<0.5) R553X 7 (3) R1162X 0 N1303K 6 (3) Unknown 46 (21) Total 220 (100) patients in that group R553X was considerably more common than G542X (Plieth et al. 1992). Login to comment
36 Fifty-four patients (49%) were homozygous for A508, 18 (16.5%) were compound heterozygotes for AF508 and one of the rarer mutations, and 2 were compound heterozygotes for G542X and N1303K. Login to comment
40 Genotype of 110 CF patients Genotype n (%) F508/F508a 54 (49) F508/1717-1G---~A 4 (3.5) F508/G542X 3 (3) F508/G551D 1 (1) F508/R553Xb 5 (4.5) F508/N1303K 4 (3.5) F508/unknown c 22 (20) G542X/N1303K 2 (2) G542X/unknown 4 (3.5) R553X/unknown 2 (2) Unknown/unknown d 9 (8) Total 110 (100) a Includes one Italian patient b Includes one patient with a French father and a German mother c Includes two Italian and one French patient Includes one Greek and one Turkish patient sorption is also indicated by repeated low stool chymotrypsin levels (<0.3 U/g) and a reduced excretion (< 10%) of para-amino benzoic acid (PABA) following a standardised oral challenge. Login to comment

[hide] The spectrum of cystic fibrosis mutations. Trends Genet. 1992 Nov;8(11):392-8. Tsui LC
The spectrum of cystic fibrosis mutations.
Trends Genet. 1992 Nov;8(11):392-8., [PMID:1279852]

Abstract [show]
Although the major mutation causing cystic fibrosis accounts for almost 70% of mutant chromosomes screened, almost 300 sequence alterations have been identified in the gene during the past two and a half years. At least 230 of these mutations are probably associated with disease. This rapid accumulation of data is in part due to the highly coordinated effort by members of the Cystic Fibrosis Genetic Analysis Consortium. The information is not only essential to genetic diagnosis, but also will aid in understanding the structure and function of the protein, and possibly in correlating genotype with phenotype.

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61 In a study based in Milan, the data show that AF508 accounts for 55% of the CF chromosomes, whereas G542X, N1303K and 1717-1G+A are relatively more frequent than in other populations (Ref. 13 and M. Ferrari, pers. Login to comment
64 Frequent cystic fibrosis mutations Name Relative freqeenc~ Mutation Con~,~'~luence Ref. Z~508 67.2 G542X 3.4 G551D 2.4 W1282X 2.1 3905insT 2.1 N1303K 1.8 3849+10kbC-+T 1.4 1717-1G-+A 1078delT 2789+5G--+A Deletion of 3 bp between nt 1652 and t655 in exon 10 G-+T at nt 1756 in exon 11 G-+A at nt 1784 in exon 1I G-+A at nt 3978 in exon 20 Insertion of T after nt 3905 in exon 20 C-+G at nt 4041 in exon 21 C-->T in a 6.2 kb EcoRI fragment 10 kb from 5' junction of intron 19 3849+4A-+G 1.0 7tt÷IG--+T 0.9 Rl162X 0.9 1898+lG-+A 0.9 Rll7H 0.8 3659delc 0.8 G85E 0.7 2184delA 0.7 AI5W 0.5 R347P 0.5 R~ 0.4 1,3 C-+T at nt 1"789in exon 11 1.3 G-+T at nt 1 from 5' junction of intron 4 1.1 G--+A at nt 1 from 3' junction of intron 10 1.1 Deletion of T at nt 1078 in exon 7 1.1 G-cA at 5 nt from 5' end of intron 14b A-->G at 4 nt from 5' end of intron 19 G-+T at nt 1 from 5' junction of intron 5 C-+T at nt 3616 in exon 19 G-+A at nt 1 from 5' junction of intron 12 G--)A at nt 482 in exon Deletion of C at nt 3659 in exon 19 G-+A at nt 386 in exon 3 A-->G at nt 2183 and deletion of A at nt 2184 in exon 13 Deletion of 3 bp between nt 1648 and 1653 in exon 10 G-+C at nt 1172 in exon 7 G-~C at nt 1811 in exon 11 A455E 0.4 R334W 0.4 Y122X 0.3 S549R(T-+G) 0.3 Q493X 0.3 V520F 0.2 S549N 0.2 C-+A at nt 1496 in exon 9 C-+T at nt 1132 in exon 7 T-cA at nt ~i98 in exon 4 T--+G at nt 1779 in exon 11 C-+T at nt 1609 in exon 10 G-+T at nt 1690 in exon 10 G-->A at nt I778 in exon !1 Deletion of Phe at codon 508 Gly-+Stop at codon 542 12 Gly-~Asp at codon 551 10 l"rp-->Stop at codon t282 35 Frameshift -~ Asn-+Lys at codon 1303 36 Aberrant splicing -~ Arg~Stop ~ codon 553 Splice mutation 10 37 Splice mutation 12 Frameshift 38 Splice mutation _c Splice mutation? Login to comment

[hide] Mutation analysis of the cystic fibrosis transmemb... Am J Hum Genet. 1992 Oct;51(4):736-40. Grebe TA, Doane WW, Richter SF, Clericuzio C, Norman RA, Seltzer WK, Rhodes SN, Goldberg BE, Hernried LS, McClure M, et al.
Mutation analysis of the cystic fibrosis transmembrane regulator gene in Native American populations of the southwest.
Am J Hum Genet. 1992 Oct;51(4):736-40., [PMID:1384321]

Abstract [show]
We report DNA and clinical analyses of cystic fibrosis (CF) in two previously unstudied, genetically isolated populations: Pueblo and Navajo Native Americans. Direct mutation analysis of six mutations of the CFTR gene--namely, delta F508, G542X, G551D, R553X, N1303K, and W1282X--was performed on PCR-amplified genomic DNA extracted from blood samples. Haplotype analyses with marker/enzyme pairs XV2c/TaqI and KM19/PstI were performed as well. Of the 12 affected individuals studied, no delta F508 mutation was detected; only one G542X mutation was found. None of the other mutations was detected. All affected individuals have either an AA, AC, or CC haplotype, except for the one carrying the G542X mutation, who has the haplotype AB. Clinically, six of the affected individuals examined exhibit growth deficiency, and five (all from the Zuni Pueblo) have a severe CF phenotype. Four of the six Zunis with CF are also microcephalic, a finding not previously noted in CF patients. Our DNA data have serious implications for risk assessment of CF carrier status for these people.

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5 Direct mutation analysis of six mutations of the CFTR gene-namely, AF508, G542X, G551D, R553X, N1303K, and W1282X-was performed on PCR-amplified genomic DNA extracted from blood samples. Login to comment
7 Of the 12 affected individuals studied, no AF508 mutation was detected; only one G542X mutation was found. Login to comment
9 All affected individuals have either an AA, AC, or CC haplotype, except for the one carrying the G542X mutation, who has the haplotype AB. Login to comment
24 These mutations are G551D (Cutting et al. 1990), G542X (Kerem et al. 1990), and R553X (Cutting et al. 1990), which are all in exon 11, and N1303K (Osborne et al. 1991), in exon 21. Login to comment
62 After hybridization, the filters were washed for 5 min at room temperature in 5 x SSC and then for 30 min in 2 x SSC at the following temperatures: 49°C for the AF508 probe and 43°C for its normal homologue, 430C for G542X and its normal sequence, and 51 'C for N1303K and its normal sequence. Login to comment
79 One patient (008-1), however, carries a single copy of G542X. Login to comment

[hide] Cystic fibrosis gene. Br Med Bull. 1992 Oct;48(4):738-53. Harris A
Cystic fibrosis gene.
Br Med Bull. 1992 Oct;48(4):738-53., [PMID:1281033]

Abstract [show]
The cystic fibrosis gene, located at 7q31, spans about 230 kb of genomic DNA and contains 27 exons. The cDNA of 6.2kb would predict an 1480 amino acid protein, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR has a high degree of homology with members of the ABC-transporter super family. The predicted protein structure consists of two membrane-spanning domains, each of 6 sub-units, anchoring CFTR in the apical membrane of specialized epithelial cells, 2 nucleotide binding folds (NBF) and a regulatory (R) domain. Disease-associated mutations in the CF gene are mainly clustered in the nucleotide-binding folds. The most common mutation, occurring in 70% of CF genes in Northern Europe and North America, is the deletion of amino acid phenylalanine at position 508 in the first NBF (ie delta F508).

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98 It has been reported that the G542X mutation when present in a homozygous form may lead to a milder form of CF than AF508 homozygotes or heterozygotes AF508/G542X.57 Similarly other patients with predicted 'stop' mutations in both CF genes may be less severely affected than AF508 homozygotes.58 In other words if the 'stop' mutation actually abolishes production of CFTR this may be less deleterious than having a malfunctioning protein such as the A508 protein. Login to comment

[hide] Milestones in cystic fibrosis. Br Med Bull. 1992 Oct;48(4):717-37. Super M
Milestones in cystic fibrosis.
Br Med Bull. 1992 Oct;48(4):717-37., [PMID:1281032]

Abstract [show]
The study of cystic fibrosis (CF) provides a fascinating insight into developments in medicine in the 20th century. Milestones include the first clear clinical descriptions in the 1930s, discovery of a sweat electrolyte abnormality, establishing the autosomal recessive mode of inheritance and improvements in treatment. Microdissection experiments on sweat glands allowed the main defect to be delineated as one of chloride transport. Location of the gene to chromosome 7 made prenatal diagnosis feasible and carrier detection in siblings. The CF gene--its product being the cystic fibrosis transmembrane conductance regulator (CFTR), and its major mutation Delta F508 was discovered in 1989. World-wide collaboration has resulted in discovery of more than 150 further mutations. Incorporation of CFTR into non-chloride transporting insect cells by conferring chloride transport, proved it a chloride channel. CFTR incorporated into adenovirus results in correction of the chloride transport defect in airway cells, bringing gene therapy closer.

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159 Homozygotes for G542X have mild disease.67 It is postulated that alternate routes of chloride transport may open up when CFTR does not function at all. Login to comment
160 Thus a protein conformational change in CFTR resulting in a signif- FIBROSIS Table 4 CF Mutations encountered in United Kingdom Mutation Delta F508 G551D R553 G542X R56OT N1303K DI507 R117H 621+1G-T G85E W1282X E60X R75Q V520F 1717-1 G-A CF chromosomes screened 1 Mutations encountered 1062 199 (non Delta F508) 199 199 199 199 199 199 199 199 199 30 15 199 199 CF chromosomes with mutation in North-West England 863 37 8 11 6 6 4 5 10 4 2 2 1 3 3 Percentage 81.2 3.48 0 75 1.03 0.58 0 58 038 0.47 0.98 0.38 0 19 ? Login to comment
171 The upper track shows 621+1 G->T, the next G551D, then G542X and the lowest track shows Delta F5O8. Login to comment
172 Two contiguous tracks belong to each specimen tested, for the absence and presence of the allele Thus the patterns shown are: negative, negative, heterozygous for Delta F508, homozygous for Delta F508, compound heterozygote for 621+1 and Delta F5O8, heterozygous for G551D, and compound heterozygote for G542X and Delta F508, then the size ladder ARMS is a registerd trademark of ICI Diagnostics Table 5 Applications of earner testing to families with a known history of CF. Login to comment
175 A positive correlation of the rare mutation G542X/Delta F508 is awaiting confirmation, but our own figure of 5 out of 7 with meconium ileus seems likely to be confirmed. Login to comment

[hide] CFTR nonsense mutations G542X and W1282X associate... Hum Mol Genet. 1992 Oct;1(7):542-4. Hamosh A, Rosenstein BJ, Cutting GR
CFTR nonsense mutations G542X and W1282X associated with severe reduction of CFTR mRNA in nasal epithelial cells.
Hum Mol Genet. 1992 Oct;1(7):542-4., [PMID:1284888]

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1 1, No. 7 542-544 CFTR nonsense mutations G542X and W1282X associated with severe reduction of CFTR mRNA in nasal epithelial cells Ada Hamosh12 *, Beryl J.Rosenstein2 and Garry R.Cutting1 '2 -3 1 Center for Medical Genetics, departments of Pediatrics and 3 Medicine, the Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA Received July 8, 1992; Revised and Accepted August 28, 1992 Cystic fibrosis (CF) is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene (1). Login to comment
4 The nonsense mutations G542X and W1282X (5) are also among the most common CFTR mutations (CF Genetic Analysis Consortium). Login to comment
6 To study the effect of these nonsense mutations upon mRNA processing, nasal epithelial cells were obtained from two carriers of the G542X mutation, three compound heterozygotes for G542X and AF5O8, two carriers of the W1282X mutation, one homozygote for the W1282X mutation and one W1282X/AF5O8 nasal polyp cell culture. Login to comment
28 For the G542X mutation, cDNA was amplified using CFTR primers Exon 7-5' and Exon 11-3' (3). Login to comment
32 Figure la shows the results of ASO hybridization to amplified genomic DNA and cDNA from one compound heterozygote for the G542X mutation. Login to comment
42 Quantitation by densitometry (The Discovery Series™ Scanner, PDI, Huntingdon Station, NY) demonstrated that the level of transcripts derived from the G542X nonsense mutation was consistently less than 10% of the normal or AF5O8 allele, while that of the W1282X mutation was consistently less than 5% of the normal or AF508 allele. In this study, we demonstrate that the CFTR nonsense mutations G542X and W1282X are associated with severely reduced levels of CFTR mRNA in nasal epithelial cells. Login to comment
43 Decreased levels of mRNA carrying the G542X mutation have been observed in both respiratory and rectal epithelia (10). Login to comment
44 These findings suggest that the G542X and W1282X mutations should result in severely decreased or undetectable CFTR protein and add to the growing evidence that nonsense mutations usually result in reduced transcript levels, as has been observed in many human disease genes (11, 12). Login to comment
47 No large scale analysis of patients carrying the G542X mutation has been reported. Login to comment
49 Approximately 3% of CF patients will be homozygous or compound heterozygous for the G542X, R553X, and W1282X mutations. Login to comment
107 Joanna Rommens for the sample of CFTR plasmid DNA, Dr Arthur Beaudet for genomic DNA from a G542X homozygote, Dr Bat-Sheva Kerem for the 542X and 542NL ASOs, and Dr Iain Mclntosh for advice. Login to comment

[hide] G27X: a novel mutation in exon 2 of the CF gene. Hum Mol Genet. 1992 Sep;1(6):445. Shackleton S, Harris A
G27X: a novel mutation in exon 2 of the CF gene.
Hum Mol Genet. 1992 Sep;1(6):445., [PMID:1284531]

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11 To date more than 27 predicted 'stop' mutations have been defined in the CF gene (4), several of these including G542X (5), R553X (6) and W1282X (7) being sufficently common to encounter homozygotes for the mutation, thus enabling potentially more informative predictions of genotype/phenotype associations. Login to comment

[hide] Mutation analysis of 184 cystic fibrosis families ... J Med Genet. 1992 Sep;29(9):642-6. Cheadle J, Myring J, al-Jader L, Meredith L
Mutation analysis of 184 cystic fibrosis families in Wales.
J Med Genet. 1992 Sep;29(9):642-6., [PMID:1357180]

Abstract [show]
We describe a molecular analysis of 184 cystic fibrosis (CF) families in Wales. To determine accurate frequency data for the CF mutations in the Welsh population, families with at least three Welsh grandparents were strictly regarded as Welsh. Of these 74 families, we have identified approximately 90% of mutations causing CF, with delta F508 accounting for 71.8% and 621 + 1G greater than T 6.7%. We observed a significant difference between the Welsh and Scottish frequencies of 621 + 1G greater than T. To allow the rapid and efficient screening for the more common mutations we modified a multiplex used by Watson et al enabling the detection of delta F508, G551D, and R553X simultaneously with 621 + 1G greater than T. In parallel to this system we ran the Cellmark Diagnostics ARMS multiplex kit, which detects delta F508, 621 + 1G greater than T, G551D, and G542X. RFLP analysis of the 184 families shows that the delta F508 chromosomes are almost exclusively found on the B haplotype (XV2c 1, KM19 2); the other CF mutations have more heterogeneous backgrounds. Strong haplotype correlations exist between the markers XV2c, KM19, D9, and G2 and the other CF mutations. Haplotype data suggest that there are at least seven mutations that remain to be identified in these families.

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13 '`3 Marked variations in the frequency of AF508 have been reported from different populations.4 For example, in different regions of Europe, Figure 1 The ARMS multiplex analysed on a 3% NuSieve gel. Samples I to 3 are normal, 4 is a AF508 heterozygote, 5 a G542X/AF508, 6 a G551D/AF508, 7 a 621 + IG > T/AF508, and 8 a GSSID homozygote. Login to comment
25 A multiplex test based on the Amplification Refractory Mutation System (ARMS), developed by Cellmark Diagnostics,'6 allows the simultaneous detection of AF508, G551D, G542X, and 621 + 1G > T. Login to comment
27 The A reaction identifies normal 621 + 1G> T/ AF508 sequences and mutant G551D/G542X sequences, whereas the B reaction identifies mutant 621 + G> T/AF508 sequences and normal G551D/G542X sequences (fig 1). Login to comment
41 We have identified a total of 89-8% of CF mutations in the Welsh population, with AF508 accounting for 718%, 621+1G>T 6 7%, G542X 2-7%, and R1283M 2-0%. Login to comment
61 Welsh Mixed Undefined Total Mutation No % No % No % No % AF508 107/149 71-8 92/126 73 0 69/94 73 4 268/369 72-6 621 + 1G>T 10/42* 6-7 5/34* 4-0 4/25* 4-3 19/101* 51 G551D 2/42* 1-3 6/34* 4-8 3/25* 3-2 11/101* 3 0 G542X 4/42* 2-7 4/34* 3-2 1/25* 1.1 9/101* 2-4 G85E 0/41* 0-0 2/34* 1 6 3/24* 3*4 5/99* 1-4 R553X 2/42* 1-3 2/34* 16 0/25* 00 4/101* 1-1 R1283M 3/42* 2.0 0/34* 0.0 0/25* 0.0 3/101* 0-8 N1303K 1/42* 0 7 1/34* 0-8 0/24* 0.0 2/100* 0-6 AI507 2/149 1-3 0/126 0.0 0/94 0.0 2/369 0-5 R117H 1/42* 0 7 1/34* 0-8 0/25* 0.0 2/101* 0-5 1717- 1G>A 2/42* 1-3 0/34* 0 0 0/25* 0 0 2/101* 0-5 R560T 0/42* 00 0/34* 00 1/25* 1 1 1/101* 03 1154InsTC 0/40* 0 0 1/33* 0 9 0/24* 0.0 1/97* 0-3 V520F 0/42* 0 0 0/34* 0 0 0/25* 0.0 0/101* 0 0 W1282X 0/42* 0 0 0/34* 0.0 0/25* 0.0 0/101* 0 0 R347P 0/42* 0 0 0/34* 0 0 0/24* 0.0 0/100* 0 0 Q493X 0/42* 0 0 0/34* 0 0 0/24* 0 0 0/100* 00 Total (%) 89-8 90 7 86-5 891 * Non-AF508 chromosomes. Login to comment
76 In parallel to this we ran the ARMS multiplex which detects AF508, G551D, G542X, and 621 + 1G > T, thus accounting for 83-1 % of our total CF chromosomes. Login to comment
97 We wish to thank Professor P S Harper, Dr D Shaw, and Dr A Clarke for their useful comments on this manuscript, Dr L Sandkuijl for his help with the statistical analyses, Dr M 0 621 + 1G> T Total G551D Total G542X Total G85E Total R553X Total R1283M Total N1303K Total AI507 Total RI 17H Total 1717-1 Total R560T Total 1154Ins TC Total 0 1 1 o o o 0 o 0 0 2 0 o o 0 1 0 0 I 1 1 1 I I I Al-Jader, Meredith Table S Distribution of haplotypes in the uncharacterised CF chromosomes. Login to comment

[hide] Development, multiplexing, and application of ARMS... Am J Hum Genet. 1992 Aug;51(2):251-62. Ferrie RM, Schwarz MJ, Robertson NH, Vaudin S, Super M, Malone G, Little S
Development, multiplexing, and application of ARMS tests for common mutations in the CFTR gene.
Am J Hum Genet. 1992 Aug;51(2):251-62., [PMID:1379414]

Abstract [show]
The amplification refractory mutation system (ARMS) is a simple, rapid and reliable method for the detection of any mutation involving single base changes or small deletions. We have applied ARMS methodology to the detection of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Single ARMS tests have been developed for 11 CFTR mutations found in the northwest of England. ARMS reactions for the most common mutations have been multiplexed to give a test which will detect the presence of the delta F508, G551D, G542X, and 621 + 1G----T mutations in a DNA sample. The multiplex test has been validated by the analysis of over 500 previously genotyped samples and has been found to be completely accurate. The rapid detection of the most common mutations has enabled early molecular confirmation of suspected cystic fibrosis in neonates, rapid typing of cystic fibrosis patients and their relatives, and testing of sperm and egg donors.

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6 ARMS reactions for the most common mutations have been multiplexed to give a test which will detect the presence of the AF508, G551D, G542X, and 621 + 1G--T mutations in a DNA sample. Login to comment
57 The mutations detected, their frequencies, and the methods used to Table I CF Mutations in 1,030 Chromosomes from the Northwest of England Observed Frequency Detection Mutation (%) Methoda Reference R117H ......... .4 A Dean et al. 1990 621 + 1G>T ... 1.0 R Zielenski et al. 1991 AI507 ......... .5 S Schwartz et al. 1991 AF508 ......... 80.7 S and A Kerem et al. 1989 1717-1G>A ... .3 A Guillermit et al. 1990 G542X ......... 1.1 A Kerem et al. 1990 GSS1D ......... 3.4 R Cutting et al. 1990 RS53X ......... .8 R Cutting et al. 1990 RS60T ......... .6 R Kerem et al. 1990 W1282X ....... .2 A Vidaud et al. 1990 N1303K ........ .6 A Osborne et al. 1991 a A = ASO hybridization; R = RFLP; and S = size difference. Login to comment
65 ARMS tests were developed for the following mutations: AF508 (Riordan et al. 1989), A1507 (Schwarz et al. 1991), R117H (Dean et al. 1990), 621 + 1G-T (Zielenski et al. 1991), 1717G--oA (Guillermit et al. 1990), W1282X (Vidaud et al. 1990), GSS1D and R553X (Cutting et al. 1990), G542X and R560T (Kerem et al. 1990), and N1303K (Osborne et al. 1991). Login to comment
115 The mutations for which ARMS tests were developed were R560T, R553X, GS5iD, G542X, 1717-1G--A, 621 + 1G--T, N1303K, and W1282X. Login to comment
118 The Development of a Multiplex ARMS Test for the Four Most Common CF Mutations in the Northwest of England In order to obtain a multiplex ARMS test for these four mutations (AF508, GSSiD, G542X, and 621 + 1G-T; table 1), it was necessary to develop a method for the simultaneous detection of the two closely linked exon XI mutations, GSSiD and G542X, which are separated by only 28 bp. Login to comment
138 R553X: CACCTTGCTAAAGAAATTCTTGCTAG ................. CACCTTGCTAAAGAAATTCTTGCTAA ................. GS51D: GCTAAAGAAATTCTTGCTCGTTGCC ................. AGCTAAAGAAATTCTTGCTCGTTGCT ................. G542X: ACTCAGTGTGATTCCACCTTCTAC ...................... CACTCAGTGTGATTCCACCTTCTCA .................... 1717-1G>A: GTCTTTCTCTGCAAACTTGGAGATGTGC ............ GTCTTTCTCTGCAAACTTGGAGATGTGT ............ R117H: CACATATGGTATGACCCTCTATATAAACTC. Login to comment
152 In the multiplex, the normal ARMS reactions for AF508 and 621 + 1G--T are combined with the mutant overARMS reactions for G551D and G542X in the "A" Figure 2 Development of a single ARMS test for R117H. Login to comment
155 Shown are the results a n m n m R560T 317bp c n m n m 0551 D 286bp obtained using primers RH-d-N and RH-d-M at 0.86 pM (a), at reduced primer concentration 0.10 ItM (b), at 0.86 FM with the inclusion of AAT1 /2 control primers (c), and using primers RHh-N and RH-h-M (which are identical to RH-d-N/M, except for C/C mismatch at position - 2). Login to comment
156 b n m n m R553X 291bp * control - 220bp d n m n m G542X 256bp4- control 220bp e n m n m 621 +1G>T 380bp gn m n m f n m n m 360bp 4----- control 220bp h n m n m 1717G>A 220bp -- control - * 360bp W1282X I 178bp Figure 3 Single ARMS tests. Login to comment
161 The ARMS tests are as follows: a, R56OT; b, R553X; c, GSS1D; d, G542X; e, 621 + 1G T; f, N1303K; g, 1717G-A; and h, W1282X. Login to comment
191 In addition to the validation and application data reported here, theARMS multiplex is also being tested Table 4 Sequences and Concentration of ARMS Primers Used in the Multiplex ARMS Test Size Concentration Mutation and Sequence (5' to 3') Code (bp) Tube (x standard) AFS08: GACTTCACTTCTAATGATGATTATGGGAGA ...... DF-C A/B 1.0 GTATCTATATTCATCATAGGAAACACCACA ....... DF-j-N 160 A 1.0 GTATCTATATTCATCATAGGAAACACCATT ....... DF-w-M 157 B 2.0 Exon 11: TAAAATTTCAGCAATGTTGTTTTTGACC ......... 11-C A/B 2.0 GS51D: GCTAAAGAAATTCTTGCTCGTTACC .................. GD-j2d3-N 285 B 2.0 AGCTAAAGAAATTCTTGCTCGTTGCT ................ GD-j-M 286 A 1.0 G542X: ACTCAGTGTGATTCCACCTTCTAC..................... GX-e-N 256 B 1.0 CACTCAGTGTGATTCCACCTTCTCA ................... GX-M 257 A 1.0 621 + 1G>T: TCACATATGGTATGACCCTCTATATAAACT ....... 621-C A/B .5 TGCCATGGGGCCTGTGCAAGGAAGTATTCC ..... 621-j-N 380 A .5 TGCCATGGGGCCTGTGCAAGGAAGTATTGA .... 621-r-M 380 B .5 4 5 6 7 8 9 10 11 Figure 4 Results obtained using the standard ARMS multiplex test. Login to comment
193 The first contains the products of the normal 621 + 1G-T and AF508 primers and ofthe mutant GSS1D and G542X primers. Login to comment
194 The corresponding products for the 621 + 1G-T and AFS08 mutant primers and for the GSS1D and G542X normal primers are in the second track of the pair. Login to comment
196 The genotypes of the 11 samples are as follows: 1, normal; 2, normal; 3, normal; 4, 621 + 1G-T heterozygote; 5, G551D heterozygote; 6, G542X heterozygote; 7, AFS08 heterozygote; 8, 621 + 1G-T, AFS08; 9, GSS1D,AFS08; 10, G542X,AFS08; and 11, AFS08 homozygote. as part ofa large multicenter study. Login to comment
216 1 2 3 blank 621 +lG>T G551D - ~ G542X F508 621 + 1 G>T G551 D G542X F508 621 +lG>T G551D \_-_ G542X F508 The use of long primers (typically 30 mers) ensured that false priming events were minimized and that primer template interactions were stabilized. Login to comment
229 This approach did not work, because the yields of several of the reactions were too low (GSSlD-normal, G542X-mutant, and AFS08 mutant) or too high (621 + 1G-T-normal and mutant) when compared with the yield of the other reactions (data not shown). Login to comment
238 An additional problem in the development ofa multiplex for the four most common CF mutations is the close proximity of GSS1D and G542X; these mutations are within 28 bp of each other in exon XI of the CFTR gene. Login to comment
241 A potential problem with this method is the priming ofthe GSS1D product with the G542X primers or product. Login to comment
243 In our hands the approach worked well and allowed simultaneous detection of GS51D and G542X, although the relative yield ofthese two products was sensitive to changes in mismatch strength and primer concentration. Login to comment
58 The mutations detected, their frequencies, and the methods used to Table I CF Mutations in 1,030 Chromosomes from the Northwest of England Observed Frequency Detection Mutation (%) Methoda Reference R117H ......... .4 A Dean et al. 1990 621 + 1G>T ... 1.0 R Zielenski et al. 1991 AI507 ......... .5 S Schwartz et al. 1991 AF508 ......... 80.7 S and A Kerem et al. 1989 1717-1G>A ... .3 A Guillermit et al. 1990 G542X ......... 1.1 A Kerem et al. 1990 GSS1D ......... 3.4 R Cutting et al. 1990 RS53X ......... .8 R Cutting et al. 1990 RS60T ......... .6 R Kerem et al. 1990 W1282X ....... .2 A Vidaud et al. 1990 N1303K ........ .6 A Osborne et al. 1991 a A = ASO hybridization; R = RFLP; and S = size difference. Login to comment
66 ARMS tests were developed for the following mutations: AF508 (Riordan et al. 1989), A1507 (Schwarz et al. 1991), R117H (Dean et al. 1990), 621 + 1G-T (Zielenski et al. 1991), 1717G--oA (Guillermit et al. 1990), W1282X (Vidaud et al. 1990), GSS1D and R553X (Cutting et al. 1990), G542X and R560T (Kerem et al. 1990), and N1303K (Osborne et al. 1991). Login to comment
116 The mutations for which ARMS tests were developed were R560T, R553X, GS5iD, G542X, 1717-1G--A, 621 + 1G--T, N1303K, and W1282X. Login to comment
119 The Development of a Multiplex ARMS Test for the Four Most Common CF Mutations in the Northwest of England In order to obtain a multiplex ARMS test for these four mutations (AF508, GSSiD, G542X, and 621 + 1G-T; table 1), it was necessary to develop a method for the simultaneous detection of the two closely linked exon XI mutations, GSSiD and G542X, which are separated by only 28 bp. Login to comment
141 R553X: CACCTTGCTAAAGAAATTCTTGCTAG ................. CACCTTGCTAAAGAAATTCTTGCTAA ................. GS51D: GCTAAAGAAATTCTTGCTCGTTGCC ................. AGCTAAAGAAATTCTTGCTCGTTGCT ................. G542X: ACTCAGTGTGATTCCACCTTCTAC ...................... CACTCAGTGTGATTCCACCTTCTCA .................... 1717-1G>A: GTCTTTCTCTGCAAACTTGGAGATGTGC ............ GTCTTTCTCTGCAAACTTGGAGATGTGT ............ R117H: CACATATGGTATGACCCTCTATATAAACTC. Login to comment
159 b n m n m R553X 291bp * control - 220bp d n m n m G542X 256bp 4- control 220bp e n m n m 621 +1G>T 380bp gn m n m f n m n m 360bp 4----- control 220bp h n m n m 1717G>A 220bp -- control - * 360bp W1282X I 178bp Figure 3 Single ARMS tests. Login to comment
164 The ARMS tests are as follows: a, R56OT; b, R553X; c, GSS1D; d, G542X; e, 621 + 1G T; f, N1303K; g, 1717G-A; and h, W1282X. Login to comment
197 The corresponding products for the 621 + 1G-T and AFS08 mutant primers and for the GSS1D and G542X normal primers are in the second track of the pair. Login to comment
199 The genotypes of the 11 samples are as follows: 1, normal; 2, normal; 3, normal; 4, 621 + 1G-T heterozygote; 5, G551D heterozygote; 6, G542X heterozygote; 7, AFS08 heterozygote; 8, 621 + 1G-T, AFS08; 9, GSS1D,AFS08; 10, G542X,AFS08; and 11, AFS08 homozygote. Login to comment
220 1 2 3 blank 621 +lG>T G551D - ~ G542X F508 621 + 1 G>T G551 D G542X F508 621 +lG>T G551D \_-_ G542X F508 The use of long primers (typically 30 mers) ensured that false priming events were minimized and that primer template interactions were stabilized. Login to comment
233 This approach did not work, because the yields of several of the reactions were too low (GSSlD-normal, G542X-mutant, and AFS08 mutant) or too high (621 + 1G-T-normal and mutant) when compared with the yield of the other reactions (data not shown). Login to comment
242 An additional problem in the development ofa multiplex for the four most common CF mutations is the close proximity of GSS1D and G542X; these mutations are within 28 bp of each other in exon XI of the CFTR gene. Login to comment
245 A potential problem with this method is the priming ofthe GSS1D product with the G542X primers or product. Login to comment
247 In our hands the approach worked well and allowed simultaneous detection of GS51D and G542X, although the relative yield ofthese two products was sensitive to changes in mismatch strength and primer concentration. Login to comment

[hide] Incidence and expression of the N1303K mutation of... Hum Genet. 1992 Aug;89(6):653-8. Osborne L, Santis G, Schwarz M, Klinger K, Dork T, McIntosh I, Schwartz M, Nunes V, Macek M Jr, Reiss J, et al.
Incidence and expression of the N1303K mutation of the cystic fibrosis (CFTR) gene.
Hum Genet. 1992 Aug;89(6):653-8., [PMID:1380943]

Abstract [show]
The N1303K mutation was identified in the second nucleotide binding fold of the cystic fibrosis (CF) gene last year. We have gathered data from laboratories throughout Europe and the United States of America in order to estimate its frequency and to attempt to characterise the clinical manifestations of this mutation. N1303K, identified on 216 of nearly 15,000 CF chromosomes tested, accounts for 1.5% of all CF chromosomes. The frequency of the N1303K allele varies significantly between countries and ethnic groups, being more common in Southern than in Northern Europe. This variation is independent of the delta F508 allele. It was not found on UK Asian, American Black or Australian chromosomes. N1303K is associated with four different linked marker haplotypes for the polymorphic markers XV-2c, KM.19 and pMP6d-9. Ten patients are homozygous for this mutation, whereas 106 of the remainder carry one of 12 known CF mutations in the other CF allele. We classify N1303K as a "severe" mutation with respect to the pancreas, but can find no correlation between this mutation, in either the homozygous or heterozygous state, and the severity of lung disease.

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63 Fifteen patients were born with meconium ileus, 10 with the AF508/N1303K genotype; 4 of the remaining 5 were G542X/N1303K patients for whom data was available, and 2 patients were N1303K/unknown. Login to comment
67 All the G542X/N1303K patients were diagnosed at birth. Login to comment
75 N/A data not available Genotype Number Meana (range) age Male/ Pancreatic status Meconium Sputum female ileusb colonisation ofof patients Current At diagnosis PS PI P.aeruginosa Yes No Mean (range) of FEVj percentage of that predicted AF508 79 10 0.7 • 0.6 16 / 20 0 55 10/58 20 8 N1303K (0.5-36) (0-2.5) N1303K 10 7 1 • 1 3/3 0 6 1/6 3 1 N1303K (3-12) (0.1-1.5) G551D 6 19 9.7 • 2.2 1/ 2 0 4 0/4 3 1 N1303K (17-22) (8-12) G542X 8 7 0 • 0 2/3 0 4 4/5 0 2 N1303K (0.1-12) 621+lG---~T 1 22 18 1/0 0 1 0/1 1 0 N1303K W1282X 4 13.5 0.7 • 0.6 3 / 1 0 4 0/4 0 1 N1303K (5-23) (0.25-1.7) R560T 1 5 41 1 / 0 0 1 0/1 0 1 N1303K R553X 1 N/A N/A N/A 0 1 0/1 N/A N/A N1303K R334W 1 19 N/A 1/0 0 1 0/1 N/A N/A N1303K R1162X 1 N/A N/A N/A N/A N/A N/A N/A N/A N1303K 1717-1G---~A 2 7 N/A 0/1 N/A N/A N/A N/A N/A N1303K 3659delC 1 21 74 0/1 0 1 0/1 1 0 N1303K 1078delT 1 N/A N/A N/A N/A N/A N/A N/A N/A N1303K Other 90 12 4.3 • 5.6 9/9 5 19 2/17 4 10 N1303K (2-24) (0.1-15) 63 (32-101) 65 (46-84) 80 (66-93) N/A 55 70 N/A N/A N/A N/A N/A 26 N/A 66 a Mean +_SD b Shown as a fraction of the patients for whom data was available the percentage of FEV1 between age-matched N1303K homozygotes and AF508/N1303K heterozygotes (65% vs 75%, P> 0.1). Login to comment
94 Of the 15 patients who presented with meconium ileus, 4 were found to have the G542X/N1303K genotype. Login to comment
96 Because none of the other genotypes reported in this study showed any association with meconium ileus, it seems likely that this observation relates to the G542X, and not the N1303K, allele. Login to comment

[hide] Abnormal localization of cystic fibrosis transmemb... J Cell Biol. 1992 Aug;118(3):551-9. Denning GM, Ostedgaard LS, Welsh MJ
Abnormal localization of cystic fibrosis transmembrane conductance regulator in primary cultures of cystic fibrosis airway epithelia.
J Cell Biol. 1992 Aug;118(3):551-9., [PMID:1379244]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a membrane glycoprotein that forms Cl- channels. Previous work has shown that when some CF-associated mutants of CFTR are expressed in heterologous cells, their glycosylation is incomplete. That observation led to the hypothesis that such mutants are not delivered to the plasma membrane where they can mediate Cl- transport. Testing this hypothesis requires localization of CFTR in nonrecombinant cells and a specific determination of whether CFTR is in the apical membrane of normal and CF epithelia. To test the hypothesis, we used primary cultures of airway epithelia grown on permeable supports because they polarize and express the CF defect in apical Cl- permeability. Moreover, their dysfunction contributes to disease. We developed a semiquantitative assay, using nonpermeabilized epithelia, an antibody directed against an extracellular epitope of CFTR, and large (1 microns) fluorescent beads which bound to secondary antibodies. We observed specific binding to airway epithelia from non-CF subjects, indicating that CFTR is located in the apical membrane. In contrast, there was no specific binding to the apical membrane of CF airway epithelia. These data were supported by qualitative studies using confocal microscopy: the most prominent immunostaining was in the apical region of non-CF cells and in cytoplasmic regions of CF cells. The results indicate that CFTR is either missing from the apical membrane of these CF cells or it is present at a much reduced level. The data support the proposed defective delivery of some CF-associated mutants to the plasma membrane and explain the lack of apical Cl- permeability in most CF airway epithelia.

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154 The genotype of the CF cells was AF508/N1303K for CF1 and CF4, AF508/G542X for CF2, AF508/other for CF3, and other/other for CF5. Login to comment
156 The N1303K and G542X mutations are two of the more common non-AF508 mutations (see ref. Login to comment
158 As with CFTRAF508, CFTR-N1303K is not appropriately glycosylated in a recombinant system (9, 19). Login to comment
159 We expect that CFTR-G542X is a truncated protein or, if the levels of mRNA are decreased (20), that there is no G542X protein at all. Login to comment
163 Thus, we conclude that some mutant CFTR proteins, including CFTRAF508, CFTR-N1303K, CFTR-G542X, and at least one other mutant CFTR, are not present or are present at greatly reduced amounts in the apical membrane of these CF epithelia. Login to comment
211 This conclusion is valid for CFTRAF508, CFTR- Denning et al. Abnormal Localization of CFTRin CFAirway Epithelia N1303K, CFTR-G542X, and at least one other undetermined mutation. Login to comment
162 Thus, we conclude that some mutant CFTR proteins, including CFTRAF508, CFTR-N1303K, CFTR-G542X, and at least one other mutant CFTR, are not present or are present at greatly reduced amounts in the apical membrane of these CF epithelia. Login to comment
208 This conclusion is valid for CFTRAF508, CFTR- Denning et al. Abnormal Localization of CFTRin CFAirway Epithelia N1303K, CFTR-G542X, and at least one other undetermined mutation. Login to comment

[hide] Nine cystic fibrosis patients homozygous for the C... J Med Genet. 1992 Aug;29(8):558-62. Gasparini P, Borgo G, Mastella G, Bonizzato A, Dognini M, Pignatti PF
Nine cystic fibrosis patients homozygous for the CFTR nonsense mutation R1162X have mild or moderate lung disease.
J Med Genet. 1992 Aug;29(8):558-62., [PMID:1381442]

Abstract [show]
The clinical course of nine cystic fibrosis patients homozygous for the CF gene nonsense mutation R1162X was investigated. Since this mutation should lead to an interruption in the synthesis of the cystic fibrosis transmembrane regulator (CFTR) protein, a severe clinical course was expected. All patients showed pancreatic insufficiency, while the course of the lung disease was mild to moderate. These results suggest that this form of truncated CFTR protein, still containing the regulatory region, the first ATP binding domain, and both transmembrane domains, could be partially working in the lung tissues.

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113 We investigated both the approaches pressed so that a reduced quantity of CFTR mentioned above, but for want of genotype could be produced, as recently suggested from characterised cohorts, we think that the com- studies on nasal epithelial cells of two patients parison of the R1162X homozygotes with a homozygous for the G542X nonsense muta- 'mixed genotype' cohort (fig 1), in which chest tion. Login to comment
121 Cutting et al'6 reported two compound heterozygotes for stop mutations S1255X/G542X and W1316X/R553X (see above), respectively. Login to comment
124 Beaudet et aP7 described a patient homozygous for mutation G542X with pancreatic insufficiency and pulmonary disease milder than in the average CF patient of comparable age. Login to comment
207 Possible suppression of the termination codon in CF patients with the G542X mutation. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... FASEB J. 1992 Jul;6(10):2775-82. McIntosh I, Cutting GR
Cystic fibrosis transmembrane conductance regulator and the etiology and pathogenesis of cystic fibrosis.
FASEB J. 1992 Jul;6(10):2775-82., [PMID:1378801]

Abstract [show]
Cystic fibrosis (CF) is an inherited disorder causing pancreatic, pulmonary, and sinus disease in children and young adults. Abnormal viscosity of mucous secretions is a hallmark of the disease, and is believed to be the result of altered electrolyte transport across epithelial cell membranes. The monogenic etiology of this disease has been apparent for more than 40 years, but the defective gene has only recently been identified. This was made possible because of a revolution in genetic technology, called positional cloning, which can pinpoint disease genes without previous knowledge of the abnormal protein product. The protein encoded by the gene defective in CF has been termed the CF transmembrane conductance regulator (CFTR) because of its postulated role in electrolyte transport. Studies investigating the normal function of CFTR and how mutations affect that function, thereby causing CF, have required the combined skills of clinicians, geneticists, molecular biologists, and physiologists. From this collaborative effort a greater understanding of the pathogenesis of this disorder is now emerging. It may soon be possible to introduce novel therapies derived from this new knowledge that will be aimed directly at the basic defect. An ever-increasing number of genes of unknown function will be identified by continuing advances in molecular genetic technology and the advent of the genome sequencing project. The experience in cystic fibrosis research may prove to be a paradigm for investigation of the function of genes isolated by positional cloning methods.

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69 Apart from the iF508 mutation, five other mutations (G542X,3 G551D, R553X, W1282X, and N1303K) occur at frequencies greater than 1% in the majority of populations (4, 16). Login to comment
109 CF mutations that occur at a frequency of 5% or greater in certain population groups Region Mutation Population group Frequency Reference Transmembrane 1-6 621 + 1GT 711+1GT French Canadian; Saguenay-Lac St. Jean French Canadian; Urban Quebec 0.23 0.09 72 72 NBF 1 A455E SF508 G542X G551D French Canadian; Saguenay-Lac St. Jean Worldwide Ashkenazi Jewish Spanish Scottish 0.08 0.30-0.88 0.12 0.05 0.05 72 14 73 74 75 Transmembrane 7-12 R1162X N.E. Italian 0.05 74 NBF 2 W1282X Ashkenazi Jewish 0.48 56, 73 press CFTR and in those used for transient expression studies (33). Login to comment

[hide] Molecular characterization of cystic fibrosis: 16 ... Genomics. 1992 Jul;13(3):770-6. Fanen P, Ghanem N, Vidaud M, Besmond C, Martin J, Costes B, Plassa F, Goossens M
Molecular characterization of cystic fibrosis: 16 novel mutations identified by analysis of the whole cystic fibrosis conductance transmembrane regulator (CFTR) coding regions and splice site junctions.
Genomics. 1992 Jul;13(3):770-6., [PMID:1379210]

Abstract [show]
The spectrum of cystic fibrosis (CF) mutations was determined in 105 patients by using denaturing gradient gel electrophoresis to screen the entire coding regions and adjacent cystic fibrosis transmembrane conductance regulator (CFTR) gene sequences. The nucleotide substitutions detected included 16 novel mutations, 11 previously described defects, and 11 nucleotide sequence polymorphisms. Among the novel mutations, 6 were of the missense type, 4 were nonsense mutations, 4 were frameshift defects, and 2 affected mRNA splicing. The mutations involved all the CFTR domains, including the R domain. Of the 61 non-delta F508 CF chromosomes studied, mutations were found on 36 (59%), raising the proportion of CF alleles characterized in our patient cohort to 88%. Given the efficacy of the screening method used, the remaining uncharacterized mutations probably lie in DNA sequences outside the regions studied, e.g., upstream-promoter sequences, the large introns, or putative regulatory regions. Our results further document the highly heterogeneous nature of CF mutations and provide the information required for DNA-based genetic testing.

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55 Six of these mutations, G542X, G551D, 1717-1 G-A (Fig. l), 3659delC, R1162X, and N1303K, have been reported previously (Cutting et al., 1990; Gasparini et al., 1991b; Guillermit et al., 1990; Kerem et al., 1990; Osborne et al., 1991). Login to comment
67 T C225R R334W G542X G551D 1717-l G -+ A K710X Lys -b Stop at 710 A-+Tat2260 G628R Gly + Arg at 628 G+Aat2014 2043 delG Frameshift 1 -bp deletion W846X Trp --, Stop at 846 G-+Aat2670 2789 + 5 G - A Splice mutation G + A at 2789 + 5 Y913C Tyr --) Cys at 913 A-,Gat2870 3272-26 A -+ G Splice mutation A + G at 3272-26 W1063X Trp -+ Stop at 1063 G+Aat3321 R1066C Arg + Cys at 1066 C+Tat3328 Y1092X Tyr + Stop at 1092 C + A at 3408 3659delC Frameshift l-bp deletion 19 3732deIA Frameshift 1-bp deletion 19 K1200E Lys --, Glu at 1200 A+Gat3730 19 R1162X Arg - Stop at 1162 C + T at 3616 19 W1282X Trp + Stop at 1282 G+Aat3978 20 N1303K Asn -+ Lys at 1303 C -+ G at 4041 21 4374 + 1 G + A Splice mutation G+Aat4374+ 1 Intron 23 Asp + Gly at 44 Frameshift Frameshift Gly + Arg at 178 Splice mutation Cys + Arg at 225 Arg + Trp at 334 Gly + Stop at 542 Gly + Asp at 551 Splice mutation A+Gat263 2 2bp deletion 2 1-bp deletion 4 G --, A at 664 5 G + Tat 711 + 1 Intron 5 T+Cat805 6a C + Tat 1132 7 G + T at 1756 11 G+Aat1784 11 G + A at 1717-l Intron 10 Haplotype Restriction (XV-2c, KM-19) site change Reference A B A A or C A D A B, D B B Hinfl(-) - - - - SecI (+) MspI (6) - Mb01 (+) - 13 13 13 14a Intron 14 b 15 Intron 17a 17b 17b 17b C A B A D A A C B C XmnI (-) - - - MnlI (-) - - This study This study This study Zielenski et al. (1991) Zielenski et al. (1991) This study Gasparini et al. (1991b) Kerem et al. (1990) Cutting et al. (1990) Kerem et al. (1990); Guillermit et al. (1990) This study This study This study Vidaud et al. (1990a) Highsmith et al. (1990) Vidaud et al. (1990a) This study This study This study Bozon (personal communication) Kerem et al. (1990) This study Together with 3732delA Gasparini et al. (1991b) Vidaud et al. (1990a) Osborne et al. (1991) This study Note. Previously undescribed mutations are shown in bold type. Login to comment
108 Two mutations in exon 11-G551D and G542X-are more frequent than the other non-AF508 mutations: G542X represents 4-6% in all parts of the country except Brittany, while G551D is present at a rate of 4% in the population of Celtic origin (Ferec, personal communication). Login to comment

[hide] Analysis of four diverse population groups indicat... Am J Hum Genet. 1992 Jun;50(6):1185-94. Cutting GR, Curristin SM, Nash E, Rosenstein BJ, Lerer I, Abeliovich D, Hill A, Graham C
Analysis of four diverse population groups indicates that a subset of cystic fibrosis mutations occur in common among Caucasians.
Am J Hum Genet. 1992 Jun;50(6):1185-94., [PMID:1376017]

Abstract [show]
To determine the nature and frequency of non-delta F508 cystic fibrosis (CF) mutations among diverse populations, we have sequenced exons 9-12 and 19-23 of the CF transmembrane conductance regulator (CFTR) gene from 128 CF chromosomes (39 U.S. Caucasian, 27 African-American, 42 Northern Irish, and 20 Israeli chromosomes). These regions were chosen because they encode the two putative ATP-binding folds of CFTR, domains which appear to have functional significance. In addition, CFTR exons 1 and 2 were analyzed in the American patients. Mutations were found on 49 of the 128 CF chromosomes. Nineteen different mutations were observed; six were novel, while the remaining 13 had been reported previously by our group or by other investigators. Six of nine different mutations found in African-American patients were unique to that population. However, the vast majority of the mutations found in U.S. Caucasians (eight of nine), Northern Irish (four of five), and Israelis (three of three) also occurred in other Caucasian groups. The preponderance of previously reported mutations in these three groups suggested that a subset of the non-delta F508 mutations occur in common among Caucasians. A survey of mutation frequencies in other Caucasian groups confirmed this observation. Unfortunately, this subset accounts for less than half of non-delta F508 CF mutations in most groups. These data suggest that screening for delta F508 and this select group of mutations will efficiently and economically maximize the number of CF mutations identified in Caucasian groups. However, it will be difficult to detect more than 90% of mutant CFTR alleles except in ethnically and geographically discrete populations where CF is the result of founder effect.

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95 One mutation, G542X, was observed in all four groups. Login to comment
125 However, any explanation should take into account both the high frequency of the null allele G542X in almost all populations studied and the predominance of the nonsense mutation W1282X in Ashkenazi patients. Login to comment
212 Lerer I, Sagi M, Cutting GR, Abeliovich D (1992) Cystic fibrosis mutations delta F508 and G542X in Jewish patients. Login to comment

[hide] Genetic determination of exocrine pancreatic funct... Am J Hum Genet. 1992 Jun;50(6):1178-84. Kristidis P, Bozon D, Corey M, Markiewicz D, Rommens J, Tsui LC, Durie P
Genetic determination of exocrine pancreatic function in cystic fibrosis.
Am J Hum Genet. 1992 Jun;50(6):1178-84., [PMID:1376016]

Abstract [show]
We showed elsewhere that the pancreatic function status of cystic fibrosis (CF) patients could be correlated to mutations in the CF transmembrane conductance regulator (CFTR) gene. Although the majority of CF mutations--including the most common, delta F508--strongly correlated with pancreatic insufficiency (PI), approximately 10% of the mutant alleles may confer pancreatic sufficiency (PS). To extend this observation, genomic DNA of 538 CF patients with well-documented pancreatic function status were analyzed for a series of known mutations in their CFTR genes. Only 20 of the 25 mutations tested were found in this population. They accounted for 84% of the CF chromosomes, with delta F508 being the most frequent (71%), and the other mutations accounted for less than 5% each. A total of 30 different, complete genotypes could be determined in 394 (73%) of the patients. The data showed that each genotype was associated only with PI or only with PS, but not with both. This result is thus consistent with the hypothesis that PI and PS in CF are predisposed by the genotype at the CFTR locus; the PS phenotype occurs in patients who have one or two mild CFTR mutations, such as R117H, R334W, R347P, A455E, and P574H, whereas the PI phenotype occurs in patients with two severe alleles, such as delta F508, delta I507, Q493X, G542X, R553X, W1282X, 621 + 1G----T, 1717-1G----A, 556delA, 3659delC, I148T, G480C, V520F, G551D, and R560T.

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10 This result is thus consistent with the hypothesis that PI and PS in CF are predisposed by the genotype at the CFTR locus; the PS phenotype occurs in patients who have one or two mild CFTR mutations, such as R117H, R334W, R347P, A455E, and P574H, whereas the PI phenotype occurs in patients with two severe alleles, such as AF508, A1507, Q493X, G542X, R553X, W1282X, 621 + 1G-PT, 1717-1G--'A, 556delA, 3659delC, I148T, G480C, V520F, G551D, and R560T. Login to comment
50 The next most common were GSS1D (3.1%), G542X (2.2%), and 621 + 1G--T (1.3%). Login to comment
58 Intron 10: 1717-1G-'A Exon 11: G542X .......... S549R ........... G551D .......... R553X .......... R560T .......... Exon 12: Y563N .......... P574H .......... Exon 19: 3659delC ....... Exon 20: W1282X ....... Exon 21: N1303K ..... G460-C A deletion G482-'A A deletion T575-C 621 + 1G-T C1132-T C1172- G C1496-A G1505-'T G1570-T C1609-T 3-bp deletion 3-bp deletion G1690-T G1717-1-A G1756-T T1779-G G1784- A C1789-T G1811-C T1819- A C1853- A C deletion G3978-A C4041-G Asp 110-His Frameshift Arg 117-His Frameshift Ile 148-Thr Splice mutation Arg 334-Trp Arg 347-Pro Ala 455- Glu Gly 458-'Val Gly480-Cys Gln 493- stop del of Ile 507 del of Phe 508 Val 520-Phe Splice mutation Gly 542- stop Ser 549-'Arg Gly 551-WAsp Arg 553- stop Arg 560- Thr Tyr 563- Asn Pro 574-His Frameshift Trp 1282-stop Asn 1303-Lys Dean et al. 1990 White et al. 1991 Dean et al. 1990 Zielenski et al. 1991a F. Rininsland, D. Bozon, and L.-C. Tsui, unpublished data Zielenski et al. 1991a Gasparini et al. 1991 Dean et al. 1990 Kerem et al. 1990b Cuppens et al. 1990 Strong et al. 1991 Kerem et al. 1990b Kerem et al. 1990b Kerem et al. 1989b Jones et al. 1991 Kerem et al. 1990b Kerem et al. 1990b Kerem et al. 1990b Cutting et al. 1990 Cutting et al. 1990 Kerem et al. 1990b Kerem et al. 1990b Kerem et al. 1990b Kerem et al. 1990b Vidaud et al. 1990 Osborne et al. 1990 PI or PS, but not with both. Login to comment
66 As shown in table 3, meconium ileus Table 2 1181 Table 3 Frequency of 25 CF Mutations in Chromosomes of the Toronto Study Population Mutation AF508 ...... G551D...... G542X...... 621 +1G-'T N1303K..... W1282X..... R1 17H...... 1717-1G-~A R560T...... A1507 ...... R553X...... V52OF ...... R334W ..... A455E...... I148T ...... Q493X...... P574H...... R347P ...... SS6delA ..... 3659delC .... G480C...... 444delA ..... D110H...... G458V...... S549R ...... Y563N...... Login to comment
68 Although meconium ileus was found in patients who had diverse genotypes, it appeared to be overrepresented (9/18) in patients who had the AF508/G542X genotype. Login to comment
73 Complete CF Genotypes for 394 Patients No. OF PATIENTS GENOTYPE WITH Allele 1 Allele 2 pla P AF508 ...... AF508 277 (49) 2 G551D 21 (1) 0 G542X 18 (9)c 0 621+1G-~T 11 (1) 0 AI507 7 (1) 0 N1303K 6 (1) 0 R560T 5 0 1717-lG-A 5 (1) 0 556delA 3 0 Q493X 3 0 R553X 3 (1) 0 W1282X 3 0 3659delC 2 0 1148T 1 0 R117H 0 9 A445E 0 2 P574H 0 2 R347P 0 1 G551D ..... 1717-lG-~A 2 0 621+1G-~T 1 0 G480C 1 0 G551D 1 0 V520F 1 (1) 0 G542X ..... V520F 1 0 1148T ...... W1282X 1 (1) 0 W1282X .... W1282X 1 0 N1303K .... R553X 1 (1) 0 R117H ..... R117H 0 1 G542X 0 1 R334W ..... R334W 0 1 a1 Numbers in parentheses are number of patients with neonatal meconium ileus. Login to comment
81 Table 4 Classification of CF Gene Mutations as Severe or Mild with Respect to Pancreatic Function Type of Mutation Severe (location) Mild (location) Missense (point mutation) ...... 1148T (exon 4) R117H (exon 4) G480C (exon 9) R334W (exon 7) VS2OF (exon 10) GSS1D (exon 11) R347P (exon 7) RS60T (exon 11) A455E (exon 9) N1303K (exon 21) P574H (exon 12) Single amino acid deletion ........ AFS08 (exon 10) A1507 (exon 10) Stop codon (nonsense) ..... Q493X (exon 10) G542X (exon 11) R553X (exon 11) W1282X (exon 20) Splice junction ... 621 + 1G-T (intron 4) 1717-1G-T (intron 10) Frameshift ........ 556delA (exon 4) 3659delC (exon 19) with any of the mild mutations was associated with PS. Login to comment
85 Accordingly, the mutations R117H, R334W, R347P, A455E, and P574H may be regarded as mild, whereas AF508, AI507, Q493X, G542X, R553X, W1282X, 621 + 1G-T, 1717-1G--A, 556delA, 3659delC, 1148T, G480C, V520F, GSS1D, and R560T are severe. Login to comment
99 Forexample, nine of 18 patients with the AF508/ G542X genotype have been found to have neonatal meconium ileus; this frequency is much above the overall proportion observed in our clinic (17%). Login to comment

[hide] A frameshift mutation (2869insG) in the second tra... Am J Hum Genet. 1992 May;50(5):1140-2. Nunes V, Bonizzato A, Gaona A, Dognini M, Chillon M, Casals T, Pignatti PF, Novelli G, Estivill X, Gasparini P
A frameshift mutation (2869insG) in the second transmembrane domain of the CFTR gene: identification, regional distribution, and clinical presentation.
Am J Hum Genet. 1992 May;50(5):1140-2., [PMID:1373935]

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28 Three other mutations are relatively common in the Spanish population-G542X (Kerem et al. 1990), R1162X (Gasparini et al. 1991), and N1303K (Osborne et al. 1991), which have frequen- Letters to the Editor A C G T 4-G M N Figure I Identification of the CF mutation 2869insG by direct sequencing: comparison ofthemutated (left) and normal (right) sequences of the CFTR region containing exon 15. Login to comment
39 Of the five patients carrying this mutation, one is homozygous, andfourarecompound heterozygotes with other mutations-AF508 in two cases, G542X in one case, and an unknown mutation in the other. Login to comment
54 The four compound heterozygotes show a severe clinical course, which is probably due to the combined effect of 2869insG and either mutation AF508 or mutation G542X. Login to comment

[hide] The occurrence of various non-delta F508 CFTR gene... Hum Genet. 1992 May;89(2):245-6. Nemeti M, Johnson JP, Papp Z, Louie E
The occurrence of various non-delta F508 CFTR gene mutations among Hungarian cystic fibrosis patients.
Hum Genet. 1992 May;89(2):245-6., [PMID:1375186]

Abstract [show]
Cystic fibrosis (CF) is an autosomal recessive disease caused by different mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The frequency of the major mutation (delta F508) in the Hungarian population is 64%. To identify other common mutations in CF families from Hungary, 30 non-delta F508 CF chromosomes were analyzed for selected mutations in exon 11 (G551D, R553X, G542X), intron 4 (621 + 1G----T), intron 10 (1717-1G----A), exon 20 (W1282X), and in exon 21 (N1303K) of the CFTR gene. In 6 of the 30 non-delta F508 CF chromosomes the following mutations were detected: R553X, G542X, 1717-1G----A, W1282X, and N1303K. After analysis of the above eight mutations, 30% of CF chromosomes are as yet undefined and further analysis is planned.

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3 To identify other common mutations in CF families from Hungary, 30 non-AF508 CF chromosomes were analyzed for selected mutations in exon 11 (G551D, R553X, G542X), intron 4 (621+1G--~T), intron 10 (1717-1G---~A), exon 20 (W1282X), and in exon 21 (N1303K) of the CFTR gene. Login to comment
4 In 6 of the 30 non-AF508 CF chromosomes the following mutations were detected: R553X, G542X, 1717-1G---~A, W1282X, and N1303K. Login to comment
16 As 13 different mutations have been reported within exon 11 alone (Cutting et al. 1990; Devoto et al. 1991; DOrk et al. 1991; Kerem et al. 1990; Sangiuolo et al. 1991; Strong et al. 1991), our first aim was to check the frequency of three of the more frequent mutations in this exon, namely G551D, R553X, and G542X. Login to comment
22 The G542X mutation was detected using the modified primer and BstNI digestion as described by Ng et al. (1991). Login to comment
25 The primer modification strategy was the same as mentioned above for detection of the G542X mutation and 1717-1G--~A splice mutation (Haliassos et al. 1989). Login to comment
32 Screening of 84 Hungarian CF chromosomes for eight CFTR gene mutations Name of mutation No. of No. of chromosomes mutant screeneda chromosomes AF508 (Exon 10) 84 54 G551D (Exon 11) 30 0 R553X (Exon 11) 30 2 G542X (Exon 11) 28 1 621+lG--*T (Intron 4) 27 0 1717-1G--~A (Intron 10) 27 1 W1282X (Exon 20) 26 1 N1303K (Exon 21) 25 1 a Chromosomes with an identified mutation were excluded for examination of other mutations Results and discussion The results of screening 84 parental CF chromosomes for these 8 mutations are summarized in Table 1. Login to comment
34 In the 6 non-AF508 chromosomes the following mutations were found: R553X (2 chromosomes), G542X, 1717-1G---,A, W1282X, and N1303K. Login to comment

[hide] A new missense mutation (R1283M) in exon 20 of the... Hum Mol Genet. 1992 May;1(2):123-5. Cheadle JP, Meredith AL, al-Jader LN
A new missense mutation (R1283M) in exon 20 of the cystic fibrosis transmembrane conductance regulator gene.
Hum Mol Genet. 1992 May;1(2):123-5., [PMID:1284468]

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5 From a sample of 373 CF chromosomes, 318 proved to have one of the following mutations: delta F508, delta 1507, G551D, R553X, G542X, R117H, 1717-1G>A, R560T and 621 + 1G>T. Login to comment

[hide] Intra- and extragenic marker haplotypes of CFTR mu... Hum Genet. 1992 Feb;88(4):417-25. Dork T, Neumann T, Wulbrand U, Wulf B, Kalin N, Maass G, Krawczak M, Guillermit H, Ferec C, Horn G, et al.
Intra- and extragenic marker haplotypes of CFTR mutations in cystic fibrosis families.
Hum Genet. 1992 Feb;88(4):417-25., [PMID:1371263]

Abstract [show]
In order to facilitate the screening for the less common mutations in the cystic fibrosis (CF) gene viz., the CF transmembrane conductance regulator gene (CFTR), marker haplotypes were determined for German non-CF (N) and CF chromosomes by polymerase chain reaction analysis of four polymorphisms upstream of the CF gene (XV-2c, KM.19, MP6-D9, J44) and six intragenic polymorphisms (GATT, TUB9, M470V, T854T, TUB18, TUB20) that span the CFTR gene from exon 6 through exon 21. Novel informative sequence variants of CFTR were detected in front of exons 10 (1525-61 A or G), 19 (3601-65 C or A), and 21 (4006-200 A or G). The CF locus exhibits strong long-range marker-marker linkage disequilibrium with breakpoints of recombination between XV-2c and KM.19, and between exons 10 and 19 of CFTR. Marker alleles of GATT-TUB9 and TUB18-TUB20 were found to be in absolute linkage disequilibrium. Four major haplotypes encompass more than 90% of German N and CF chromosomes. Fifteen CFTR mutations detected on 421 out of 500 CF chromosomes were each identified on one of these four predominant 7-marker haplotypes. Whereas all analysed delta F508 chromosomes carried the same KM.19-D9-J44-GATT-TUB9-M470V-T854T haplotype, another frequent mutation in Germany, R553X, was identified on two different major haplotypes. Hence, a priori haplotyping cannot exclude a particular CF mutation, but in combination with population genetic data, enables mutations to be ranked by decreasing probability.

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26 A few other mutations were analysed either by allele-specific oligonucleotide hybridization (Rl17H, G542X, 1717-1 G---~A) (Dean et al. 1990b; Guillermit et al. 1990; Kerem et al. 1990), single strand conformation polymorphism (Orita et al. 1989) (R347P, 3659delC) (Dean et al. 1990b; Kerem et al. 1990), temperature gradient gel electrophoresis (Rosenbaum and Riesner 1987) (1717-1 G--~A) (Guillermitet al. 1990; Kerem et al. 1990) or by allele-specific PCR (G542X, NI303K) (Kerem et al. 1990; Osborne et al. 1991). Login to comment
92 The more frequent mutations G542X, G551D, R553X are located in sequence motifs that are evolutionarily conserved amongst the members of ABC transport proteins (Riordan et al. 1989; Hyde et al. 1990). Login to comment
98 ASO, Allele-specificoligonucleotide hybridization; TGGE, temperature gradient gel electrophoresis; SSCP, single strand conformation polymorphism Mutation Localization No. % Method of detectiona Reference R117H Exon 4 2 0.4 ASO Dean et al. (1990b) R334W Exon 7 2 0.4 RFLP MspI Gasparini et al. (1991b) R347P Exon 7 5 1.0 RFLP NcoI Dean et al. (1990b) A455E Exon 9 1 0.2 RFLP AciI Kerem et al. (1990) F508C2 Exon 10 1 0.2 Nondenaturing PAGE Kobayashi et al. (1990) AF508 Exon 10 370 74.0 Nondenaturing PAGE Kerem et al. (1989) 1717-1 G---~A Intron 10 2 0.4 TGGE Kerem et al. (1990) G542X Exon 11 5 1.0 Allele-specificPCR Kerem et al. (1990) G551D Exon 11 5 1.0 RFLP DpnII Cutting et al. (1990) R553X Exon 11 12 2.4 RFLP HincII Cutting et al. (1990) 2789 + 5 G---~A Intron 14B 3 0.6 RFLP SspI Highsmith et al. (1990) Rl162X Exon 19 1 0.2 RFLP DdeI Gasparini et al. (1991b) 3659delC Exon 19 3 0.6 SSCP Kerem et al. (1990) W1282X Exon 20 2 0.4 RFLP MnlI Vidaud et al. (1990) N1303K Exon 21 7 1.4 Allele-specificPCR Osborne et al. (1991) Unpublished 13 2.6 Unknown 66 13.2 Total 500 a All non-AF508 mutations were subsequently verified by direct genomic sequencing of the respective PCR product b F508C was first detected on an N chromosome (Kobayashi et al. 1990) and hence is suspected to represent a benign missense mutation Table 5. Login to comment
100 The four major haplotypes are indicated in bold type KM.19 D9 J44 GATT TUB9 M470V T854T TUB18 TUB20 Mutation 1 l 2 1 2 2 1 1 2 2 2 1 1 2 1 2 2 1 2 2 1 1 2 1 2 1 2 2 2 1 2 1 1 1 1 2 2 2 1 2 1 1 1 2 i 1 2 1 2 1 1 2 1 2 R347P, F508C, R1162X, 3659delC 1717-1 G--~A, G551D, R553X (n = 2), 2789 + 5 G---~A,W1282X R117H R334W, A455E, G542X, N1303K, AF508 (96%) ~F508 (4%) R553X (n = 10) a Haplotypes were assigned from the individual pedigrees mutation was located on a single KM. 19-D9-J44-GATT-TUB9-M470V-T854T haplotype. Login to comment
106 Several more frequent disease-causing sequence variations, such as R334W, A455E, G542X, and N1303K, reside on the typical AF508 haplotype. Login to comment

[hide] Association of a nonsense mutation (W1282X), the m... Am J Hum Genet. 1992 Jan;50(1):222-8. Shoshani T, Augarten A, Gazit E, Bashan N, Yahav Y, Rivlin Y, Tal A, Seret H, Yaar L, Kerem E, et al.
Association of a nonsense mutation (W1282X), the most common mutation in the Ashkenazi Jewish cystic fibrosis patients in Israel, with presentation of severe disease.
Am J Hum Genet. 1992 Jan;50(1):222-8., [PMID:1370365]

Abstract [show]
Only about 30% of the cystic fibrosis chromosomes in the Israeli cystic fibrosis patient populations carry the major CF mutation (delta F508). Since different Jewish ethnic groups tended to live as closed isolates until recent times, high frequencies of specific mutations are expected among the remainder cystic fibrosis chromosomes of these ethnic groups. Genetic factors appear to influence the severity of the disease. It is therefore expected that different mutations will be associated with either severe or mild phenotype. Direct genomic sequencing of exons included in the two nucleotide-binding folds of the putative CFTR protein was performed on 119 Israeli cystic fibrosis patients from 97 families. One sequence alteration which is expected to create a termination at residue 1282 (W1282X) was found in 63 chromosomes. Of 95 chromosomes, 57 (60%) are of Ashkenazi origin. Together with the delta F508 (23% in this group), G542X, N1303K, and 1717-1G----A mutations, the identification of 92% of cystic fibrosis chromosomes of Ashkenazi origin becomes possible. Patients homozygous for the W1282X mutation (n = 16) and patients heterozygous for the delta F508 and W1282X mutations (n = 22) had similarly severe disease, reflected by pancreatic insufficiency, high incidence of meconium ileus (37% and 27%, respectively), early age at diagnosis, poor nutritional status, and variable pulmonary function. In conclusion, the W1282X mutation is the most common cystic fibrosis mutation in the Ashkenazi Jewish patient population in Israel. This nonsense mutation is associated with presentation of severe disease.

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10 Together with the AF508 (23% in this group), G542X, N1303K, and 1717-1G-*A mutations, the identification of 92% of cystic fibrosis chromosomes of Ashkenazi origin becomes possible. Login to comment
29 In a previous study of a small group of Israeli CF patients, four additional rare mutations (1717- 1G--A, G542X, S5491, and S549R) were found (Kerem et al. 1990b). Login to comment
56 The G542X, S5491, S549R, N1303K, and 1717- 1G--A mutations were detected by PCR and subse- quent allele specific oligo nucleotide hybridization as described elsewhere (Kerem et al. 1990b; Osborne et al. 1991). Login to comment
72 patients screened, 16 (15 Ashkenazi and 1 Arab) were homozygous for W1282X, 22 (20 Ashkenazi, 1 Sephardic, and 1 from an unclassified origin) were heterozygous for W1282X and AF508, 2 (Ashkenazi) patients were heterozygous for W1282X and G542X, and 11 patients were heterozygous for W1282X and an as yet unidentified mutation. Login to comment
77 Hence, together with AF508, G542X, N1303K, and 1717-1G--A, 92% of the CF chromosomes could be identified in Jewish patients of Ashkenazi origin. Login to comment
79 Therefore, for assessment of the severity of the W1282X mutation, it would have been appropriate to compare patients homozygous for the W1282X mutation with patients homozygous for the AF508 mutation and with patients heterozygous for Table I Analysis of Mutation Frequencies Identified in 97 Israeli CF Patients JEWISH PATIENTS ARAB Ashkenazim Sepharadim Unclassified PATIENTS No. of chromosomes sampled.... 95 51 8 40 W1282X (no. [%]) .................. 57 (60) 1 (2) 3 (38) 2 (5) AF508 (no. [%])..................... 21 (23) 18 (35) 2 (25) 10 (25) G542X (no. [%]).................... 4 (4) 2 (4)0 2 (5) N1303K (no. [%]) .................. 4 (4) 0 0 2 (5) 1717-1G-A (no. [%]) ............. 1 (1) 0 0 1 (3) S5491 (no. [%])...................... 0 0 0 2 (5) S549R (no. [%])..................... 0 1 (2) 2 (25) 2 (5) Total mutations (no. [%])...... 87 (92) 22 (43) 7 (88) 21 (53) both mutations. Login to comment
91 Two patients were heterozygous for two different termination mutations, W1282X and G542X. Login to comment

[hide] Single-strand conformation polymorphism (SSCP) ana... Hum Genet. 1992 Jan;88(3):283-7. Plieth J, Rininsland F, Schlosser M, Cooper DN, Reiss J
Single-strand conformation polymorphism (SSCP) analysis of exon 11 of the CFTR gene reliably detects more than one third of non-delta F508 mutations in German cystic fibrosis patients.
Hum Genet. 1992 Jan;88(3):283-7., [PMID:1370807]

Abstract [show]
In Central Europe, the delta F508 deletion accounts for approximately 75% of mutations in the cystic fibrosis transmembrane conductance regulator gene causing cystic fibrosis. The remainder comprise a large number of individually infrequent mutations whose detection requires a disproportionately large effort. However, a sizeable proportion of non-delta F508 mutations have been found to cluster within exon 11. We have taken advantage of this clustering to detect a total of five previously described point mutations present on 26/72 (36%) non-delta F508 chromosomes by polymerase chain reaction/direct sequencing of exon 11. These exon 11 mutations were then subjected to single-strand conformation polymorphism (SSCP) analysis, which was shown (i) to discriminate reliably between mutant and wildtype alleles and (ii) to generate reproducible mutation-specific band patterns. This analysis thus represents the first attempt to assess SSCP analysis retrospectively, and serves to illustrate the potential of this screening technique in diagnostic medicine.

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16 Indeed, two mutational "hot spots" have been identified, one of which, located within the 95-bp exon 11 (Cutting et al. 1990), contains a number of mutations including G551D, R553X, 1717-1G--~A (splice-site mutation), G542X and $549R. Login to comment
18 A combination of restriction enzyme digestion (for R553X and G551D) and/or allele-specific oligonucleotide hybridization (for G542X and 1717-1G--~A) is now routinely performed to detect known mutations in polymerase chain reaction (PCR) amplified DNA samples. Login to comment
47 Nearly half of these chromosomes contained R553X but the remainder contained G551D, G542X, 1717-1G--~A and $549R. Login to comment
54 Distribution of non-AF508mutations in 59 German patients Non-AF508mutations Chromo- % (n = 72) somes In exon 11 R553X 12 16.6 G542X 5 6.9 G551D 4 5.5 1717-1G---~A(5' splicesite) 3 4.1 $549R 2 2.8 Others 9 12.5 Undetermined 37 51.4 Fig. la-c. Login to comment
59 W Wildtype control, lane 1 $549R/ $549R, lane 2 G551D, lane 3 1717-1G---~A,lane 4 G542X, lane 5 R553X, lane 6 R553X/R553X, lane 7 G542X/1717-1 SSCP analysis was then performed upon the same set of DNA samples to test the mutation-detection efficiency of this method. Login to comment
63 Mutation-specific bands can thus be noted in patient samples bearing the heterozygous mutations G551D, 1717-1G--+A, G542X and R553X. Login to comment
64 Furthermore, individuals homozygous for the mutations $549R and R553X, and compound heterozygous for G542X/1717-1G-->A were readily distinguishable from patients heterozygous for these mutations because of the absence of wildtype bands in the former (Fig. 1). Login to comment
72 SSCP analysis performed on DNAs of six individuals with the following genotypes (nucleotide change in brackets): lanes W wildtype/wildtype, lanes 1 G551D/wildtype (G-*A), lanes 2 1717- 1G-~A/wildtype (G--~A), lanes 3 G542X/wildtype (G--~T), lanes 4 R553X/wildtype (C----~T),lanes 5 S549R/S549R(T-*G). Login to comment
75 In the case of the mutations G551D, G542X and $549, the coding strands of mutant and wild-type allele are separated, whereas the non-coding strands co-migrate. Login to comment

[hide] The gene defect in cystic fibrosis and clinical ap... J R Soc Med. 1992;85 Suppl 19:6-8. Super M
The gene defect in cystic fibrosis and clinical applications of the knowledge.
J R Soc Med. 1992;85 Suppl 19:6-8., [PMID:1375961]

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27 One of the stop mutations, G542X with the 49% ofpatients demonstrating homozygosity occurring in compound heterozygote form with AF5N for AF5N. Login to comment
45 83 a stop codon or G542X or W1282X), insertions at Al? Login to comment
51 Factors outside the gene seem to be associated with differing 1008 818 81.15% levels ofseverity and, ofcourse, one must remember G551D 215* 34 2.986 differing environmental influences such as time of G542X 203* 11 1.02% diagnosis, vigour oftreatment and social class which R560T 113* 6 1.00% may play a role. Login to comment

[hide] Screening for non-delta F508 mutations in five exo... Am J Hum Genet. 1991 Jun;48(6):1127-32. Devoto M, Ronchetto P, Fanen P, Orriols JJ, Romeo G, Goossens M, Ferrari M, Magnani C, Seia M, Cremonesi L
Screening for non-delta F508 mutations in five exons of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in Italy.
Am J Hum Genet. 1991 Jun;48(6):1127-32., [PMID:1709778]

Abstract [show]
Analysis of exons 10, 11, 14a, 15, and 20 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by denaturing-gradient-gel electrophoresis (DGGE) allowed the identification of mutations causing cystic fibrosis (CF) in 25 of 109 non-delta F508 chromosomes, as well as identification of a number of polymorphisms and sequence variations. Direct sequencing of the PCR fragments which showed an altered electrophoretic behavior not attributable to known mutations has led to the characterization of four new mutations, two in exon 11, and one each in exons 15 and 20. Screening for the different mutations thus far identified in our patients by the DGGE analysis and other independent methods should allow detection of about 70% of the molecular defects causing CF in Italy. Mutations located in exons 11 and 20 account for at least 30% of the non-delta F508 mutations present in Italian CF patients.

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27 In addition, a variable number of the same chromosomes had been previously tested, with other methods (ASO and/or digestion with restriction enzymes as described below), for the presence of other known mutations-in particular, 91 for G542X (Kerem et al. 1990), 56 for W1282X (Vidaud et al. 1990), and 96 for R553X (Cutting et al. 1990b). Login to comment
28 For DGGE screening, a first group of 61 chromosomes originated from a corresponding number of com- poundheterozygous patients who carried an identified mutation (57 deltaF508, two W1282X, one G542X, andone R553X) on one oftheirtwo CFchromosomes. Login to comment
32 Screening for CFTR gene mutations already published or known through the CF Genetic Analysis Consortium was carried out on random samples of CF chromosomes by PCR amplification ofspecific exons that was followed by digestion with restriction enzymes indicated in table 2 which recognize mutation sites inside the amplification product and, in one case (G542X), by hybridization with the allele-specific oligonucleotides (ASO). Login to comment
34 Table 2 Results of Screening for Known Mutations Total No. of No. of Chromosomes Chromosomes Overall Mutation Exon Method With the Mutationa Screenedb Frequencyc Reference R334W......... 7 MspI 2 198 1.01 X. Estivill, personal communication R347P......... 7 HhaI or NcoI 4 183 2.19 Dean et al. 1990 G542X ......... 11 ASO (DGGE) 15 (4) 176 (18) 9.79 Kerem et al. 1990 S549N......... 11 DdeI 1 159 .63 Cutting et al. 1990b G5S1D ......... 11 HincII or MboI 0 186 Cutting et al. 1990b R553X ......... 11 HincIl (DGGE) 5 (1) 186 (13) 3.02 Cutting et al. 1990b 1717-1G-A .... 11 (DGGE) (12) (109) 11.01 Guillermit et al. 1990 S1255X ......... 20 HindIII 0 130 Cutting et al. 1990a W1282X......... 20 MnlI (DGGE) 7 (3) 124 (53) 5.65 Vidaud et al. 1990 a Numbers in parentheses are number of mutations found through DGGE. Login to comment
57 In this way, a total of four G542X, one R553X, and 12 1717-1G--oA in exon 11 and of three W1282X in exon 20 could be identified (see table 2). Login to comment

[hide] Effects of cystic fibrosis and congenital bilatera... Am J Hum Genet. 2000 May;66(5):1485-95. Epub 2000 Apr 4. Mickle JE, Milewski MI, Macek M Jr, Cutting GR
Effects of cystic fibrosis and congenital bilateral absence of the vas deferens-associated mutations on cystic fibrosis transmembrane conductance regulator-mediated regulation of separate channels.
Am J Hum Genet. 2000 May;66(5):1485-95. Epub 2000 Apr 4., [PMID:10762539]

Abstract [show]
The protein defective in cystic fibrosis (CF), the CF transmembrane-conductance regulator (CFTR), functions as an epithelial chloride channel and as a regulator of separate ion channels. Although the consequences that disease-causing mutations have on the chloride-channel function have been studied extensively, little is known about the effects that mutations have on the regulatory function. To address this issue, we transiently expressed CFTR-bearing mutations associated with CF or its milder phenotype, congenital bilateral absence of the vas deferens, and determined whether mutant CFTR could regulate outwardly rectifying chloride channels (ORCCs). CFTR bearing a CF-associated mutation in the first nucleotide-binding domain (NBD1), DeltaF508, functioned as a chloride channel but did not regulate ORCCs. However, CFTR bearing disease-associated mutations in other domains retained both functions, regardless of the associated phenotype. Thus, a relationship between loss of CFTR regulatory function and disease severity is evident for NBD1, a region of CFTR that appears important for regulation of separate channels.

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47 DNA was assayed for 16 common CFTR mutations (R117H, 62111GrT, R334W, R349P, A455E, DI507, DF508, 1717-1GrA, G542X, S549N, G551D, R553X, R560T, 3849110 Kb CrT, W1282X, and N1303K), by reverse dot-blot hybridization (Mickle et al. 1998). Login to comment

[hide] [Acute recurrent pancreatitis and heterozygous mut... Arch Pediatr. 2000 Apr;7(4):388-90. Vodoff MV, Gilbert B, Piguet C, Sautereau D, de Lumley L
[Acute recurrent pancreatitis and heterozygous mutation of the cystic fibrosis gene: a case report].
Arch Pediatr. 2000 Apr;7(4):388-90., [PMID:10793926]

Abstract [show]
Recurrent pancreatitis is seldom associated with CFTR (Cystic Fibrosis Transmembrane Regulator) gene mutation. CASE REPORT: A 17-year-old boy presented with isolated idiopathic pancreatitis. CFTR gene mutations study revealed delta F508 heterozygous mutation. CONCLUSION: Mutations for the CFTR gene should be explored in case of recurrent pancreatitis.

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33 La recherche des principales autres mutations (d1.507, Wl282X, G542X, 1717-lG-A. Login to comment

[hide] High frequency of cystic fibrosis transmembrane re... Am J Hum Genet. 2000 Jun;66(6):2013-4. Gomez Lira M, Benetazzo MG, Marzari MG, Bombieri C, Belpinati F, Castellani C, Cavallini GC, Mastella G, Pignatti PF
High frequency of cystic fibrosis transmembrane regulator mutation L997F in patients with recurrent idiopathic pancreatitis and in newborns with hypertrypsinemia.
Am J Hum Genet. 2000 Jun;66(6):2013-4., [PMID:10801389]

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12 Among these rare mutations, L997F was identified in 4 (12.5%) of 32 patients with idiopathic pancreatitis (genotypes L997F/DF508, L997F/5T, and twice L997F/no mutation identified, respectively), and in 4 (8%) of 49 newborns with hypertrypsinemia (genotypes L997F/G542X, L997F/R553X, L997F/DF508, and L997F-F1052V phase unknown, respectively). Login to comment
17 This classification implies that the L997F heterozygotes compounded with common CF mutations found among the patients with idiopathic pancreatitis (DF508) and that the hypertrypsinemic newborns with negative sweat chloride (DF508, G542X, and R553X) should be diagnosed as affected by an atypical form of CF; a close follow-up is indicated for these patients. Login to comment

[hide] Prenatal diagnosis of cystic fibrosis: a case of t... Clin Chim Acta. 2000 Aug;298(1-2):121-33. Castaldo G, Martinelli P, Massa C, Fuccio A, Grosso M, Rippa E, Paladini D, Salvatore F
Prenatal diagnosis of cystic fibrosis: a case of twin pregnancy diagnosis and a review of 5 years' experience.
Clin Chim Acta. 2000 Aug;298(1-2):121-33., [PMID:10876009]

Abstract [show]
We performed prenatal diagnoses for cystic fibrosis in 32 high risk (1:4) couples (including a dizygotic pregnancy). Chorionic villi sampling did not cause abortion or fetal malformation in any case. The preliminary analysis of 9 short tandem repeats always excluded maternal contamination of the DNA extracted from chorionic villi and confirmed paternity. Twenty-two prenatal diagnoses were made by direct analysis of the mutations. In seven cases diagnosis was made by the analysis of intragenic polymorphisms; in three cases, we analyzed two extragenic polymorphisms. The prenatal diagnosis (including genetic counselling) was completed within 24 h from the sampling. Seven prenatal diagnoses revealed an affected fetus; all couples opted for therapeutic abortion. In 17 cases the fetus was heterozygote, and in seven cases it was non carrier of mutated alleles. In the twin pregnancy, mutations were DeltaF508/N1303K. Direct analysis of the DNA extracted from the two independent samples of chorionic villi revealed one fetus non carrier of mutated alleles and the other a carrier of the N1303K mutation. Analysis of the HPRT locus predicted both the fetuses as males. Furthermore, the genotype of each fetus was defined after birth. The prenatal diagnosis with chorionic villi sampling plays a key role in the prevention of cystic fibrosis. The laboratories must be equipped for both the direct analysis of mutations and for the analysis of a large number of polymorphisms. The preliminary analysis of short tandem repeats is recommended both to exclude maternal contamination and to confirm parentage.

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65 Mutation and polymorphism analyses of the CFTR gene A panel of 13 CF mutations (i.e. DF508, N1303K, G542X, 1148T, R553X, W1282X, 1717-1 G . Login to comment

[hide] Pancreatic function and extended mutation analysis... J Pediatr. 2000 Aug;137(2):214-20. Massie RJ, Wilcken B, Van Asperen P, Dorney S, Gruca M, Wiley V, Gaskin K
Pancreatic function and extended mutation analysis in DeltaF508 heterozygous infants with an elevated immunoreactive trypsinogen but normal sweat electrolyte levels.
J Pediatr. 2000 Aug;137(2):214-20., [PMID:10931414]

Abstract [show]
BACKGROUND: Newborn screening for cystic fibrosis (CF) with immunoreactive trypsinogen (IRT) and DeltaF508 analysis followed by sweat testing misses some infants with CF and detects more DeltaF508 carriers than expected. Some of the apparent DeltaF508 carriers may be DeltaF508 compound heterozygotes with normal sweat electrolyte levels. METHODS: Infants identified by newborn screening with an elevated IRT level, one DeltaF508 allele, and a sweat chloride level <60 mmol/L underwent CF mutation analysis, pancreatic stimulation testing, and repeat IRT analysis followed by clinical review and repeat sweat test at 12 months. RESULTS: Over a 24-month period we identified 122 DeltaF508 heterozygotes and recruited 57; 4 had borderline sweat chloride levels (40 to 60 mmol/L), 5 (8.8%, 95% CI 1.4, 16.2) had a second CF mutation (R117H), and 11 (20%, 95% CI 10, 30) had the intron 8 5T allele. Three had clinical CF at 12 months (initial sweat chloride levels: 53, 51, and 32 mmol/L). Pancreatic electrolyte secretion in the subjects with a borderline sweat chloride level was similar to that in patients with known CF. CONCLUSION: The excess of DeltaF508 heterozygotes detected by IRT/DNA screening is associated with the presence of a second mutation or the 5T allele in some infants. Screened infants with borderline sweat chloride levels almost certainly have CF, but long-term follow-up of the infants with the genotype DeltaF508/R117H and DeltaF508/5T is required to determine their outcome. In the meantime, newborn screening should be confined to severe mutations associated with classic CF.

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26 Measurement of Cl levels was done by colorimetry, and measurement of sodium levels was done by flame cytometry.16 Gene Mutation Analysis Blood was taken and DNA extract- ed17 for an extended cystic fibrosis transmembrane conductance regulator protein gene mutation analysis as described previously.18 The following mutations were included: ࢞F508, ࢞I507, R117H, G551D, A455E, G542X, N1303K, W1282X, 1717-1G࢐A, R560T, R347P, R334W, R553X, R1162X, S549N, 3849+10C࢐T, and 621+1G࢐T. Login to comment

[hide] A novel CFTR frame-shift mutation, 935delA, in two... Mol Genet Metab. 2000 Aug;70(4):316-21. Wang J, Bowman CM, Wong LJ
A novel CFTR frame-shift mutation, 935delA, in two Hispanic cystic fibrosis patients.
Mol Genet Metab. 2000 Aug;70(4):316-21., [PMID:10993719]

Abstract [show]
The currently available mutation analysis panel detects about 50-60% of CFTR mutations in Hispanic patients. In order to search for Hispanic CF mutations, we developed a temporal temperature gradient gel electrophoresis (TTGE) method to screen for unknown mutations. Using TTGE to study the CFTR gene has lead to the discovery of many novel mutations in Hispanic patients. A novel frame-shift mutation, 935delA, was found in two unrelated patients. One was heterozygous for two novel frame-shift mutations, 663delT and 935delA, and the other was heterozygous for DeltaF508 and 935delA. Both patients showed severe phenotype with meconium ileus, pancreatic insufficiency, and early pulmonary microbial colonization with Pseudomonas aeruginosa. Patient 1 died at 4 years of age. Patient 2 had an upper lobectomy. The 935delA mutation produces a truncated polypeptide with only 21% of the full-length protein. The severe course of clinical manifestation is consistent with two oppressively truncated mutant polypeptides encoded by both mutant alleles in patient 1 and the compound heterozygosity truncation and DeltaF508 mutations in patient 2.

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91 The two most common types of truncated CFTR proteins were present in these patients; one was missing the portion beyond the first ATP binding domain (for example, the R553X and the G542X) and the other was truncated beyond the second ATP binding domain (for example the W1282X). Login to comment

[hide] Worldwide genetic analysis of the CFTR region. Am J Hum Genet. 2001 Jan;68(1):103-17. Epub 2000 Dec 4. Mateu E, Calafell F, Lao O, Bonne-Tamir B, Kidd JR, Pakstis A, Kidd KK, Bertranpetit J
Worldwide genetic analysis of the CFTR region.
Am J Hum Genet. 2001 Jan;68(1):103-17. Epub 2000 Dec 4., [PMID:11104661]

Abstract [show]
Mutations at the cystic fibrosis transmembrane conductance regulator gene (CFTR) cause cystic fibrosis, the most prevalent severe genetic disorder in individuals of European descent. We have analyzed normal allele and haplotype variation at four short tandem repeat polymorphisms (STRPs) and two single-nucleotide polymorphisms (SNPs) in CFTR in 18 worldwide population samples, comprising a total of 1,944 chromosomes. The rooted phylogeny of the SNP haplotypes was established by typing ape samples. STRP variation within SNP haplotype backgrounds was highest in most ancestral haplotypes-although, when STRP allele sizes were taken into account, differences among haplotypes became smaller. Haplotype background determines STRP diversity to a greater extent than populations do, which indicates that haplotype backgrounds are older than populations. Heterogeneity among STRPs can be understood as the outcome of differences in mutation rate and pattern. STRP sites had higher heterozygosities in Africans, although, when whole haplotypes were considered, no significant differences remained. Linkage disequilibrium (LD) shows a complex pattern not easily related to physical distance. The analysis of the fraction of possible different haplotypes not found may circumvent some of the methodological difficulties of LD measure. LD analysis showed a positive correlation with locus polymorphism, which could partly explain the unusual pattern of similar LD between Africans and non-Africans. The low values found in non-Africans may imply that the size of the modern human population that emerged "Out of Africa" may be larger than what previous LD studies suggested.

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23 Only four other mutations (G542X, N1303K, G551D, and W1282X) have overall allele frequencies among CF chromosomes 11% (Estivill et al. 1997). Login to comment
122 Haplotype backgrounds for the major CF mutations are: 1-2, for DF508, G542X, and N1303K mutations, and 2-1, for G551D and W1282X mutations (Morral et al. 1996). Login to comment

[hide] A simplified cyclic adenosine monophosphate-mediat... J Pediatr. 2000 Dec;137(6):849-55. Callen A, Diener-West M, Zeitlin PL, Rubenstein RC
A simplified cyclic adenosine monophosphate-mediated sweat rate test for quantitative measure of cystic fibrosis transmembrane regulator (CFTR) function.
J Pediatr. 2000 Dec;137(6):849-55., [PMID:11113843]

Abstract [show]
OBJECTIVE: Sweat production is stimulated by both cholinergic and beta-adrenergic pathways in the sweat gland secretory coil. beta-Adrenergic pathway-mediated sweating is absent in cystic fibrosis (CF) because cyclic adenosine monophosphate (cAMP)-mediated chloride transport through the cystic fibrosis transmembrane regulator (CFTR) is disrupted. We report the development of a rapid, reproducible, macroscopic, and quantitative methodology to test the hypothesis that beta-adrenergic sweat rate discriminates among 3 different CFTR phenotypes-CF, heterozygote CF carriers, and non-CF. STUDY DESIGN: Intradermal injection of a mixture of 50 micromol/L isoproterenol, 5 mmol/L aminophylline (to potentiate the beta-adrenergic stimulation), and 140 micromol/L atropine (to block potential cholinergic stimulation) in lactated Ringer's solution was performed in duplicate on one forearm. A single injection of 0.5 mmol/L methacholine to stimulate sweat production by the cholinergic pathway was performed on the other forearm. Sweat rate was determined as the amount of sweat collected on filter paper over 20 minutes. RESULTS AND CONCLUSIONS: Median cAMP-mediated sweat rates were 1.45 mg/20 min (CF, n = 29), 2.55 mg/20 min (CF heterozygote carriers, n = 30), and 3.65 mg/20 min (non-CF, n = 30) and were significantly different in all 3 groups (P =.0001, Kruskal-Wallis test). Methacholine-stimulated sweat rates were similar for all 3 groups. The cAMP-mediated sweat rate test may be a useful endpoint for studies of new agents to increase the function of CFTR.

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60 Among the females with CF, 7 had homozygous ࢞F508 genotypes; 2 had ࢞F508/unknown genotypes; and one each had a genotype of ࢞F508/G551D, ࢞F508/R117H, ࢞F508/3489 + 10 kb C to T, ࢞F508/G542X, ࢞F508/R347P, and R117H/unknown. Login to comment
61 Of the males with CF, 11 had homozygous ࢞F508 genotypes, one had a ࢞F508/G542X genotype, and 2 had unknown genotypes. Login to comment

[hide] [CFTR gene analyis in 207 patients with cystic fib... Arch Pediatr. 2001 Feb;8(2):150-7. Federici S, Iron A, Reboul MP, Desgeorges M, Claustres M, Bremont F, Bieth E
[CFTR gene analyis in 207 patients with cystic fibrosis in southwest France: high frequency of N1303K and 1811+1.6bA>G mutations].
Arch Pediatr. 2001 Feb;8(2):150-7., [PMID:11232455]

Abstract [show]
The large molecular heterogeneity in cystic fibrosis (CF) represents the main difficulty for the genotype characterization. Moreover, numerous studies have reported considerable variations in frequencies of cystic fibrosis transmembrane conductance regulator (CFTR) mutations in different populations. MATERIAL AND METHODS: We analyzed the genotype of 207 CF children living in southwest France. RESULTS: Among 50 identified mutations, we report for some of them a widely modified incidence compared with those observed in other regions of France. These differences were more significant in the subset of the CF chromosomes originating in southwest France. Thus, the 1811 + 1.6 kbA > G mutation, rarely observed in the other French regions (< 0.5%), proved to be, with a frequency of 8.8%, the most frequent mutation after the F508 deletion (57%). The frequencies of N1303K, 1811 + 1.6 kbA > G and R334W mutations were also clearly increased: 7.9 and 2.6%, respectively. CONCLUSION: We show that the southwest of France is characterized by a specific mutational spectrum. We consider that these regional data on the spectrum of CF mutations are crucial to develop more accurate and less expensive molecular screening strategies for cystic fibrosis in France.

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47 Le criblage des mutations a &#e9;t&#e9; effectu&#e9; par &#e9;tapes avec des m&#e9;thodes incluant toutes une amplification pr&#e9;alable par PCR (polymerase chain reaction) suivie d`une d&#e9;tection des h&#e9;t&#e9;roduplexes pour la mutation ࢞F508 ou d`une analyse par hybridation en dot blot inverse avec la trousse INNO-Lipa CF8 (Innogenetics, Zwiljnaarde, Belgique) pour les mutations ࢞F508, G542X, N1303K, G551D, R553X, W1282X, 1717G>A et ࢞I507. Login to comment
58 Cinq autres mutations ont une fr&#e9;quence sup&#e9;rieure &#e0; 1 % : G542X (5,3 %), N1303K (3,9 %), 1811+1,6kbA>G (3,4 %), 1717-1G>A (1,4 %) et R334W (1,2 %). Login to comment
61 Origine g&#e9;ographique (% all&#e8;les) Sud-Ouest (nos donn&#e9;es) France [6] Italie/Espagne [12, 16] a b c d/e (n = 414) (n = 114) (n = 7 190) (n = 3 492/1 944) ࢞F508 64,2 57 67,21 51/50,6 G542X 5,3 5,3 2,89 5/8 N1303K 3,9 7,9 2,05 5/ ? Login to comment
76 La mutation G542X, d&#e9;j&#e0; connue pour avoir une fr&#e9;quence &#e9;lev&#e9;e dans le sud de l`Europe et les pays m&#e9;diterran&#e9;ens [2], repr&#e9;sente 5,3 % des chromosomes CF de notre s&#e9;rie alors que la fr&#e9;quence nationale est de 2,9 %. Login to comment
77 L`analyse des chromosomes CF originaires du seul Sud-Ouest depuis au moins deux g&#e9;n&#e9;rations montre une fr&#e9;quence identique de 5,3 %, sugg&#e9;rant que l`augmentation de fr&#e9;quence de G542X par rapport &#e0; la moyenne nationale n`est pas li&#e9;e &#e0; un effet fondateur. Login to comment
78 Pour ces quatre mutations, ࢞F508, N1303K, 1811+1,6kbA>G et G542X, les deux distributions (population fran&#e7;aise et population du Sud-Ouest) sont significativement diff&#e9;rentes au seuil de 5 % (p < 0,05). Login to comment
133 Ces r&#e9;sultats sont en accord avec d`autres donn&#e9;es sugg&#e9;rant que la mutation N1303K fait partie avec ࢞F508 et G542X des trois mutations apparues il y a plus de 30 000 ans [9, 10]. Login to comment
143 Enfin, si les quatre mutations les plus fr&#e9;quentes du Sud-Ouest (࢞F508, G542X, N1303K et 1811+1,6kbA>G) ne sont pas retrouv&#e9;es, le risque r&#e9;siduel n`est que de 1/130, montrant l`int&#e9;r&#ea;t d`un d&#e9;pistage cibl&#e9; des mutations, avec en particulier la recherche de la mutation 1811+1,6kbA>G chez les patients originaires de cette r&#e9;gion. Login to comment

[hide] Activity of fucosyltransferases and altered glycos... Biochimie. 2001 Aug;83(8):743-7. Glick MC, Kothari VA, Liu A, Stoykova LI, Scanlin TF
Activity of fucosyltransferases and altered glycosylation in cystic fibrosis airway epithelial cells.
Biochimie. 2001 Aug;83(8):743-7., [PMID:11530206]

Abstract [show]
Cystic fibrosis (CF) glycoconjugates have a glycosylation phenotype of increased fucosylation and/or decreased sialylation when compared with non-CF. A major increase in fucosyl residues linked alpha 1,3 to antennary GlcNAc was observed when surface membrane glycoproteins of CF airway epithelial cells were compared to those of non-CF airway cells. Importantly, the increase in the fucosyl residues was reversed with transfection of CF cells with wild type CFTR cDNA under conditions which brought about a functional correction of the Cl(-) channel defect in the CF cells. In contrast, examination of fucosyl residues in alpha 1,2 linkage by a specific alpha 1,2 fucosidase showed that cell surface glycoproteins of the non-CF cells had a higher percentage of fucose in alpha 1,2 linkage than the CF cells. Airway epithelial cells in primary culture had a similar reciprocal relationship of alpha 1,2- and alpha 1,3-fucosylation when CF and non-CF surface membrane glycoconjugates were compared. In striking contrast, the enzyme activity and the mRNA of alpha 1,2 fucosyltransferase did not reflect the difference in glycoconjugates observed between the CF and non-CF cells. We hypothesize that mutated CFTR may cause faulty compartmentalization in the Golgi so that the nascent glycoproteins encounter alpha 1,3FucT before either the sialyl- or alpha 1,2 fucosyltransferases. In subsequent compartments, little or no terminal glycosylation can take place since the sialyl- or alpha 1,2 fucosyltransferases are unable to utilize a substrate, which is fucosylated in alpha 1,3 position on antennary GlcNAc. This hypothesis, if proven correct, could account for the CF glycophenotype.

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103 Some mutations (e.g., G542X) produce a truncated transcript and no protein, while other mutations (e.g., R117H) produce a protein that has impaired conduction properties. Login to comment

[hide] Analysis of 31 CFTR mutations in 55 families from ... Early Hum Dev. 2001 Nov;65 Suppl:S161-4. Gomez-Llorente MA, Suarez A, Gomez-Llorente C, Munoz A, Arauzo M, Antunez A, Navarro M, Gil A, Gomez-Capilla JA
Analysis of 31 CFTR mutations in 55 families from the South of Spain.
Early Hum Dev. 2001 Nov;65 Suppl:S161-4., [PMID:11755047]

Abstract [show]
We carried out a molecular analysis of 350 chromosomes from 55 families originating from the South of Spain (Andalucia) who were diagnosed with cystic fibrosis (CF). We used polymerase chain reaction, followed by an oligonucleotide ligation assay (OLA) and sequence-coded separation using capillary electrophoresis. A frequency of 43.5% for DeltaF508 was found, making it the most common CF mutation in our sample. Seven more mutations (G542X, R334W, R1162X, 2789+5G-->A, R117H, DeltaI507 and W1282X) were detected and accounted for 24.7% of the total. The remaining mutations (31.8%) were undetectable with the methodology used in this study.

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5 Seven more mutations (G542X, R334W, R1162X, 2789 + 5G ! Login to comment
29 A I.10 R117H E.4 G542X E.11 Y122X E.4 G551D E.11 711 + 1G ! Login to comment
48 Interestingly, this frequency is Table 2 CFTR mutations identified in 55 families (350 chromosomes) from the south of Spain Mutations Frequency (%) Location Exon/Intron DF508 43.5 E.10 G542X 11.4 E.11 R334W 5.0 E.7 R1162X 3.0 E.19 2789 + 5G ! Login to comment
53 They were G542X, R334W, R1162X, 2789 + 5G ! Login to comment
55 G542X was the second most common mutation found in the present study, at a similar frequency to that reported for the rest of the Spanish population [2]. Login to comment
56 G542X and DF508 together represented 80% of all mutations detected in the present study. Login to comment

[hide] Gentamicin in pharmacogenetic approach to treatmen... Lancet. 2001 Dec 15;358(9298):2014-6. Hamilton JW
Gentamicin in pharmacogenetic approach to treatment of cystic fibrosis.
Lancet. 2001 Dec 15;358(9298):2014-6., [PMID:11755605]

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75 THE LANCET ߦ Vol 358 ߦ December 15, 2001 2015 COMMENTARY Effect of CFTR mutations in epithelial cells Lumen Cytoplasm Nucleus Class 4 (eg, R117H) Class 3 (eg, G551D) Class 2 (eg, èc;F508) Class 1 (eg, G542X) Class 5 (eg, A455E) Apical surface Basolateral surface ATP CFTR MSD NBD NBD RD Golgi ER CFTR gene Collectively, the five mutant classes result in little or no functional CFTR expression at the apical surface of epithelial cells, but through different mechanisms. Login to comment
76 Class 1 mutations are nonsense, frameshift, and splice-junction mutations (such as the nonsense mutation G542X) that cause little or no translation of CFTR mRNA to protein. Login to comment

[hide] Therapeutic strategies to correct malfunction of C... Paediatr Respir Rev. 2001 Jun;2(2):159-64. Lim M, Zeitlin PL
Therapeutic strategies to correct malfunction of CFTR.
Paediatr Respir Rev. 2001 Jun;2(2):159-64., [PMID:12531063]

Abstract [show]
Cystic fibrosis (CF) is a systemic autosomal recessive inherited disorder that results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although the gene was cloned 11 years ago, there still is no definitive treatment to correct the functional deficit. Current treatment strategies focus on pancreatic enzyme replacement and control of pulmonary inflammation and infection. This review examines novel strategies still in preclinical development or phase 1 clinical trials. Gene therapy is an evolving area of study that offers the potential for a cure for cystic fibrosis. CF lung disease is a significant barrier to effective gene delivery and transfer, but new vectors show promise in overcoming these limitations. There are also new pharmacological therapies aimed at correcting defects in CFTR processing and function. These are tailored to the specific class of mutation but may offer therapeutic benefit to many patients. They include phenylbutyrate, flavonoids, aminoglycosides and xanthines.

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60 Type Genotype Phenotypea Defect Potential therapeutics Class I G542X PI No CFTR synthesis, aminoglycosides 621 + 1 G ࢐T No cell surface Cl- 3905insT transport W1282X R553X 1717-1 G ࢐ A Class II F508b PI Defective CFTR 4-PBA, flavonoids, N1303K trafficking and chemical chaperones, P574Hb processing xanthines A455Eb Class III G551D PI Defective channel flavonoids, milrinone G551S regulation, reduced or absent Cl-transport Class IV R117H PS Reduced Cl-transport 4-PBA, xanthines, R334W flavonoids G314E R347P F508b P574Hb ClassV 3849 + 10 kb C࢐T PS Reduced number of flavonoids, milrinone, 2789 + 5 G ࢐A normal CFTR proteins 4-PBA 3272 - 26 A ࢐ G Reduced Cl-transport A455Eb 3120+1 G࢐A 1811 + 1.6 kb A ࢐ G a PI indicates pancreatic insufficiency; PS indicates pancreatic sufficiency. Login to comment

[hide] Cystic fibrosis: basic science. Paediatr Respir Rev. 2000 Jun;1(2):93-100. McAuley DF, Elborn JS
Cystic fibrosis: basic science.
Paediatr Respir Rev. 2000 Jun;1(2):93-100., [PMID:12531100]

Abstract [show]
Advances in the elucidation of cystic fibrosis transmembrane regulator (CFTR) function have resulted in a greater understanding of the relationship between the CF gene defect and clinical disease. The clinical phenotype is influenced by the class of mutation and possibly by other modifier genes. CFTR regulates the volume and composition of airways surface liquid, primarily by controlling chloride ion transport. However, CFTR also regulates other membrane channels and transports other molecules which may be important in mucocilary clearance and innate defence mechanisms. CFTR may also modulate the inflammatory response in respiratory epithelial cells and other inflammatory cells through a range of mechanisms. It is apparent that CFTR dysfunction results in a range of effects which may contribute to the clinical phenotype. These may contribute to the development of clinical disease at different stages of the natural history of cystic fibrosis. A greater understanding of the basic defect and its implications is likely to result in novel therapeutic approaches.

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23 After the ࢞F508 mutation, substitution mutations such as G542X (2.4%) and G551D (1.8%) are the next most common mutations.2 Patients who are homozygous for the ࢞F508 gene generally have lung disease, pancreatic insufficiency, obstructive azoospermia, and universally have elevated sweat chlorides. Login to comment

[hide] CFTR gene: molecular analysis in patients from Sou... Mol Genet Metab. 2003 Apr;78(4):259-64. Streit C, Burlamaque-Neto AC, de Abreu e Silva F, Giugliani R, Saraiva Pereira ML
CFTR gene: molecular analysis in patients from South Brazil.
Mol Genet Metab. 2003 Apr;78(4):259-64., [PMID:12706377]

Abstract [show]
Cystic fibrosis (CF) is the most common genetic disease among Caucasians. The CF gene, named cystic fibrosis transmembrane conductance regulator (CFTR), codifies a protein that acts as a channel through the epithelial membrane. The present work aimed (1) to detect sequence alterations in the nucleotide binding regions and at the membrane spanning domain of the CFTR gene and (2) to detect the following frequent mutations R347P, R347H, R334W, and Q359K (located in exon 7), DeltaF508 (located in exon 10), G542X, G551D, R553X, and S549N (located in exon 11), W1282X (located in exon 20), and N1303K (located in exon 21). Seventy-seven unrelated CF patients were analyzed, who were previously diagnosed and currently under treatment at the Pneumology Service of our hospital. Regions of interest were amplified by PCR using specific primers. Each sample was analyzed by a non-radioactive single-stranded conformational polymorphism (SSCP) analysis technique and restriction enzyme digestion. The DeltaF508 mutation was found in 48.7% of the alleles. Frequencies of G542X, R334W, R553X, and W1282X mutations in our population were 3.25, 1.3, 0.65, and 0.65%, respectively. No alleles were found to carry mutations G551D, R334W, R347P, R347H, Q359K, S549N, and N1303K, which were included in the screening protocol. This study allowed the characterization of 84 out of 154 CF mutant alleles (54.5%). The incidence of main CF mutations analyzed was similar to that of the south European population. Mutation data presented here will be useful for designing new DNA testing strategies for CF in South Brazil.

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2 The present work aimed (1) to detect sequence alterations in the nucleotide binding regions and at the membrane spanning domain of the CFTR gene and (2) to detect the following frequent mutations R347P, R347H, R334W, and Q359K (located in exon 7), DF508 (located in exon 10), G542X, G551D, R553X, and S549N (located in exon 11), W1282X (located in exon 20), and N1303K (located in exon 21). Login to comment
7 Frequencies of G542X, R334W, R553X, and W1282X mutations in our population were 3.25, 1.3, 0.65, and 0.65%, respectively. Login to comment
34 The main aims of the present work were (1) to establish the frequency of the DF508 mutation this studied population, (2) to identify alterations in the nucleotide sequence of the exons 3, 5, and 7 which are located in the first membrane spanning domain (MSD1); of exons 9, 10, 11, and 12 which are located in the first nucleotide binding domain (NBD1); of exons 19, 20, 21, and 22 which are located in the second nucleotide binding domain (NBD2) of the CFTR gene, and finally (3) to identify some specific frequent mutations (R347P, R347H, R334W, Q359K, G542X, G551D, R553X, S54 9N, W1282X, and N1303K) in these patients. Login to comment
59 Restriction fragment length polymorphism Mutations R347P, R347H, R334W, Q359K (located in exon 7), G542X, S549N, G551D, R553X mutations (exon 11), W1282X (exon 20), and N1303K (exon 21) were identified by restriction fragment length polymorphism (RFLP) protocol, using specific restriction endonucleases. Login to comment
61 RFLP using BstNI was used to detect the G542X mutation, because this alteration destroys the recognition site for this enzyme. Login to comment
66 Positive controls were used along with samples to be tested for G542X, G551D, and R553X mutations during the experiments. Login to comment
76 Screening of four additional mutations (G542X, R553X, R334W, and W1282X) together with DF508 Table 2 Mutations detected in 77 CF patients from south region of Brazil Mutation Location Number of alleles Frequency (%) R334W Exon 7 2 1.3 R347P Exon 7 0 0 R347H Exon 7 0 0 Q359K Exon 7 0 0 DF508 Exon 10 75 48.7 S549N Exon 11 0 0 G542X Exon 11 5 3.2 G551D Exon 11 0 0 R553X Exon 11 1 0.7 W1282X Exon 20 1 0.7 N1303K Exon 21 0 0 ? Login to comment
83 Three patients (3.9%) carry the G542X mutation in only one allele and an unknown mutation in the other (G542X/?). Login to comment
84 Two patients (2.6%) were a compound heterozygote for the DF508 and the G542X mutation (DF508/G542X). Login to comment
101 On the other hand, estimated frequencies of G542X, N1303K, and W1282X mutations among alleles of affected patients in our population were 8.35, 1.6, and 0.8%, respectively [26]. Login to comment
102 Frequencies of DF508, G542X, G551D, and R553X mutations in our study were similar to that established for South Europe [27]. Login to comment
111 The G542X and R553X mutations showed frequencies similar to those observed in south European countries (http://www.genet.sickids.on.ca) [25]. Login to comment
113 DNA screening for four common CF mutations (G542X, R553X, R334W, and W1282X), together with DF508, enabled the detection of 84 out of the 154 CF alleles in our sample, that represents 54.5% of studied alleles. Login to comment

[hide] Effect of genotype on phenotype and mortality in c... Lancet. 2003 May 17;361(9370):1671-6. McKone EF, Emerson SS, Edwards KL, Aitken ML
Effect of genotype on phenotype and mortality in cystic fibrosis: a retrospective cohort study.
Lancet. 2003 May 17;361(9370):1671-6., [PMID:12767731]

Abstract [show]
BACKGROUND: Over 1000 mutations of the cystic fibrosis transmembrane conductance regulator gene (CFTR) that cause cystic fibrosis have been identified. We examined the effect of CFTR genotype on mortality and disease phenotype. METHODS: Using the US Cystic Fibrosis Foundation National Registry, we did a retrospective cohort study to compare standardised mortality rates for the 11 most common genotypes heterozygous for DeltaF508 with those homozygous for DeltaF508. Of the 28455 patients enrolled in the registry at the time of our analysis, 17853 (63%) were genotyped. We also compared the clinical phenotype, including lung function, age at diagnosis, and nutritional measures, of 22 DeltaF508 heterozygous genotypes. Mortality rates and clinical phenotype were also compared between genotypes classified into six classes on the basis of their functional effect on CFTR production. FINDINGS: Between 1991 and 1999, genetic and clinical data were available for 17853 patients with cystic fibrosis, which was 63% of the total cohort. There were 1547 deaths during the 9 years of follow-up. In the analysis of the 11 most common genotypes, DeltaF508/R117H, DeltaF508/DeltaI507, DeltaF508/3849+10kbC-->T, and DeltaF508/2789+5G-->A had a significantly lower mortality rate (4.7, 8.0, 11.9, and 4.4, respectively) than the genotype homozygous for DeltaF508 (21.8, p=0.0060). DeltaF508/R117H, DeltaF508/DeltaI507, DeltaF508/ 3849+10 kbC-->T, DeltaF508/2789+5G-->A, and DeltaF508/A455E have a milder clinical phenotype. Outcomes for all functional classes were compared with that of class II (containing DeltaF508 homozygotes) and classes IV and V had a significantly lower mortality rate and milder clinical phenotype. INTERPRETATION: Patients with cystic fibrosis have distinct genetic subgroups that are associated with mild clinical manifestations and low mortality. These differences in phenotype are also related to the functional classification of CFTR genotype.

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47 ARTICLES 1672 THE LANCET ߦ Vol 361 ߦ May 17, 2003 ߦ www.thelancet.com Panel 1: Frequencies of CFTR mutations* CFTR Allele CFTR Allele mutation frequency (%) mutation frequency èc;F508 69&#b7;4% 2789+5G࢐A 0&#b7;3% Unknown 15&#b7;7% R1162X 0&#b7;3% G542X 2&#b7;3% G85E 0&#b7;3% G551D 2&#b7;2% R560T 0&#b7;2% èc;I507 1&#b7;6% R334W 0&#b7;2% W1282X 1&#b7;4% 3659èc;C 0&#b7;2% N1303K 1&#b7;2% A455E 0&#b7;1% R553X 0&#b7;9% 711+1G࢐T 0&#b7;1% 621+1G࢐T 0&#b7;8% 1898+1G࢐A 0&#b7;1% R117H 0&#b7;7% 2184èc;A 0&#b7;1% 3849+10 kbC࢐T 0&#b7;7% S549N 0&#b7;1% 1717-IG࢐A 0&#b7;5% 1078èc;T 0&#b7;03% R347P 0&#b7;3% *n=17 853. Login to comment
48 Panel 2: Functional classification of CFTR alleles Class Functional effect of Allele mutation I Defective protein G542X, R553X, W1282X, production R1162X, 621-1G࢐T, 1717-1G࢐A, 1078èc;T, 3659èc;C II Defective protein èc;F508, èc;I507, N1303K, processing S549N III Defective protein G551D, R560T regulation IV Defective protein R117H, R334W, G85E, conductance R347P V Reduced amounts of 3849+10KbC࢐T, functioning CFTR protein 2789+5G࢐A, A455E Unknown 711+1G࢐T, 2184DA, 1898+1G࢐A Total cohort Genotyped cohort (n=28 455) (n=17 853) Person-years at risk 152 011 96 870 Sex (% male) 53% 52% Race (% white) 96% 96% Age (years) 11&#b7;9 (11&#b7;1) 10&#b7;9 (11&#b7;2) Age at diagnosis (years) 3&#b7;5 (7&#b7;1) 3&#b7;6 (7&#b7;5) Sweat test (mmol/L) 101 (19) 100 (20) FEV1 (L) 1&#b7;72 (0&#b7;91) 1&#b7;80 (0&#b7;92) FEV1 (% predicted) 69 (29) 72 (28) FVC (L) 2&#b7;41 (1&#b7;18) 2&#b7;50 (1&#b7;21) FVC (% predicted) 81% (28) 84% (24) Height (cm) 121% (41) 117% (41) Weight (kg) 30&#b7;0 (21&#b7;3) 28&#b7;6 (21&#b7;8) Pancreatic insufficiency (%) 90% 87% P aeruginosa colonisation (%) 49% 46% Number of deaths (%) 3548 (12%) 1547 (9%) Data are mean (SD) unless otherwise stated. Login to comment
61 Standardised mortality rates did not differ between patients homozygous for èc;F508 and the èc;F508 heterozygotes with the G551D, G542X, N1303K, W1282X, R553X, 621+1G࢐T, and 1717-1G࢐A alleles. Login to comment
64 ARTICLES THE LANCET ߦ Vol 361 ߦ May 17, 2003 ߦ www.thelancet.com 1673 Patients Person-years Deaths Crude Standardised p valueߤ (n) mortality mortality rate* rate* (95% CI) Genotype èc;F508/èc;F508 9144 51 164 1019 19&#b7;9 21&#b7;8 (20&#b7;5-23&#b7;1) &#b7;&#b7; èc;F508/G551D 593 3247 60 18&#b7;5 16&#b7;6 (12&#b7;4-20&#b7;8) 0&#b7;019 èc;F508/G542X 574 3239 57 17&#b7;6 18&#b7;9 (14&#b7;1-23&#b7;7) 0&#b7;257 èc;F508/N1303K 303 1778 30 16&#b7;9 16&#b7;2 (10&#b7;3-22&#b7;0) 0&#b7;063 èc;F508/W1282X 278 1618 36 22&#b7;3 21&#b7;6 (14&#b7;5-28&#b7;6) 0&#b7;950 èc;F508/R553X 230 1335 21 15&#b7;7 25&#b7;0 (11&#b7;8-38&#b7;1) 0&#b7;641 èc;F508/621-1G࢐T 213 1268 27 21&#b7;0 19&#b7;2 (11&#b7;6-26&#b7;7) 0&#b7;503 èc;F508/1717-1G࢐A 120 619 13 21&#b7;0 20&#b7;6 (9&#b7;9-31&#b7;4) 0&#b7;833 èc;F508/èc;I507 318 897 8 8&#b7;9 8&#b7;0 (2&#b7;7-13&#b7;3) <0&#b7;0001 èc;F508/R117H 177 844 8 9&#b7;5 4&#b7;7 (0&#b7;8-8&#b7;5) <0&#b7;0001 èc;F508/3849+10 kbC࢐T 151 700 13 18&#b7;6 11&#b7;9 (5&#b7;0-18&#b7;9) 0&#b7;006 èc;F508/2789+5G࢐A 86 444 4 9&#b7;0 4&#b7;4 (0&#b7;0-8&#b7;9) <0&#b7;0001 èc;F508/other 3434 19 170 372 19&#b7;4 17&#b7;6 (15&#b7;8-19&#b7;4) 0&#b7;0002 Other/other 2232 10 494 233 22&#b7;2 20&#b7;5 (17&#b7;9-23&#b7;1) 0&#b7;380 *Per 1000 person-years. Login to comment
67 Table 2: Standardised and crude mortality rates (including organ transplantation) by genotype Genotype No of Age at Sweat FEV1 FVC Height Weight Pancreatic P&#b7; aeruginosa Subjects Diagnosis Chloride (% predicted)* (% predicted)* (cms)* (kg)* Insufficiency Colonization (yrs) (mmol) (%)ߤ (%)ߤ èc;F508/èc;F508 6 213 2&#b7;5 &#b1; 0&#b7;1 104 &#b1; 0&#b7;2 77 &#b1; 0&#b7;3 89 &#b1; 0&#b7;3 141 &#b1; 0&#b7;2 37&#b7;0 &#b1; 0&#b7;1 92 (91-92) 60 (59-61) èc;F508/G551D 411 3&#b7;7 &#b1; 0&#b7;3ߥ 108 &#b1; 0&#b7;9ߥ 76 &#b1; 1&#b7;2 89 &#b1; 1&#b7;2 142 &#b1; 0&#b7;7&#a7; 38&#b7;2 &#b1; 0&#b7;6&#a7; 92 (89-94) 59 (54-64) èc;F508/G542X 389 1&#b7;9 &#b1; 0&#b7;2 104 &#b1; 0&#b7;8 79 &#b1; 1&#b7;2 91 &#b1; 1&#b7;2 141 &#b1; 0&#b7;7 37&#b7;3 &#b1; 0&#b7;5 93 (89-95) 57 (52-62) èc;F508/N1303K 213 2&#b7;1 &#b1; 0&#b7;3 106 &#b1; 1&#b7;2 80 &#b1; 1&#b7;8 91 &#b1; 1&#b7;7 141 &#b1; 1&#b7;0 37&#b7;1 &#b1; 0&#b7;6 92 (87-95) 61 (55--68) èc;F508/W1282X 205 1&#b7;6 &#b1; 0&#b7;2 103 &#b1; 1&#b7;2 80 &#b1; 1&#b7;7 92 &#b1; 1&#b7;6 141 &#b1; 0&#b7;9 37&#b7;4 &#b1; 0&#b7;7 94 (90-97) 59 (52-65) èc;F508/R553X 164 2&#b7;5 &#b1; 0&#b7;4 106 &#b1; 1&#b7;4 76 &#b1; 1&#b7;8 89 &#b1; 1&#b7;6 139 &#b1; 0&#b7;9 35&#b7;4 &#b1; 0&#b7;7&#a7; 90 (85-94) 60 (53-67) èc;F508/621-1G 162 2&#b7;5 &#b1; 0&#b7;4 107 &#b1; 1&#b7;3 78 &#b1; 1&#b7;8 89 &#b1; 1&#b7;5 143 &#b1; 1&#b7;0&#a7; 38&#b7;8 &#b1; 0&#b7;8&#a7; 87 (80-91)&#a7; 57 (49-64) èc;F508/èc;I507 149 8&#b7;5 &#b1; 1&#b7;1ߥ 95 &#b1; 1&#b7;9ߥ 86 &#b1; 2&#b7;1ߥ 93 &#b1; 1&#b7;8&#a7; 137 &#b1; 1&#b7;4&#a7; 37&#b7;4 &#b1; 1&#b7;25 84 (78-89)ߥ 39 (31-48)ߥ èc;F508/R117H 123 13&#b7;7 &#b1; 1&#b7;2ߥ 80 &#b1; 1&#b7;9ߥ 91 &#b1; 2&#b7;1ߥ 97 &#b1; 1&#b7;7ߥ 143 &#b1; 1&#b7;8 42&#b7;9 &#b1; 1&#b7;7ߥ 65 (55-73)ߥ 22 (16-29)ߥ èc;F508/3849+10 kB 114 11&#b7;3 &#b1; 0&#b7;9ߥ 72 &#b1; 2&#b7;5ߥ 77 &#b1; 2&#b7;1 87 &#b1; 1&#b7;9 144 &#b1; 1&#b7;4&#a7; 41&#b7;2 &#b1; 1&#b7;2ߥ 66 (57-74)ߥ 69 (59-77) èc;F508/2789+5G 63 13&#b7;4 &#b1; 1&#b7;6ߥ 102 &#b1; 2&#b7;1 88 &#b1; 2&#b7;8ߥ 97 &#b1; 2&#b7;3ߥ 140 &#b1; 2&#b7;5 41&#b7;8 &#b1; 2&#b7;2&#a7; 71 (59-81)ߥ 32 (22-44)ߥ èc;F508/1717-1G 74 1&#b7;3 &#b1; 0&#b7;3 103 &#b1; 2&#b7;0 75 &#b1; 2&#b7;7 86 &#b1; 2&#b7;4 139 &#b1; 1&#b7;5 35&#b7;7 &#b1; 0&#b7;9 96 (88-99) 59 (48-69) èc;F508/R560T 46 1&#b7;7 &#b1; 0&#b7;5 104 &#b1; 2&#b7;0 84 &#b1; 3&#b7;3ߥ 96&#b1; 2&#b7;8&#a7; 142 &#b1; 1&#b7;9 38&#b7;4 &#b1; 1&#b7;4 91 (79-97) 63 (48-75) èc;F508/R347P 44 5&#b7;9 &#b1; 1&#b7;1&#a7; 105 &#b1; 2&#b7;6 76 &#b1; 3&#b7;0 90 &#b1; 2&#b7;9 142 &#b1; 2&#b7;4 38&#b7;7 &#b1; 1&#b7;8 67 (52-79)ߥ 53 (38-68) èc;F508/G85E 43 9&#b7;2 &#b1; 1&#b7;8ߥ 99 &#b1; 2&#b7;3&#a7; 76 &#b1; 2&#b7;5 90 &#b1; 2&#b7;5 142 &#b1; 2&#b7;9 38&#b7;3 &#b1; 2&#b7;2 88 (75-95) 52 (35-68) èc;F508/3659DC 40 1&#b7;1 &#b1; 0&#b7;4 105 &#b1; 2&#b7;1 76 &#b1; 3&#b7;9 88 &#b1; 4&#b7;1 139 &#b1; 1&#b7;9 36&#b7;6 &#b1; 1&#b7;2 92 (77-97) 55 (39-69) èc;F508/A455E 29 14&#b7;3 &#b1; 2&#b7;0ߥ 89 &#b1; 3&#b7;1ߥ 98 &#b1; 4&#b7;0ߥ 104 &#b1; 3&#b7;4ߥ 138 &#b1; 3&#b7;4 42&#b7;1 &#b1; 2&#b7;5&#a7; 60 (41--76)ߥ 17 (8-32)ߥ èc;F508/R334W 28 13&#b7;2 &#b1; 3&#b7;0ߥ 104 &#b1; 3&#b7;2 86 &#b1; 3&#b7;4&#a7; 94 &#b1; 3&#b7;3 138 &#b1; 3&#b7;2 42&#b7;3 &#b1; 3&#b7;5 67 (46-82)ߥ 51 (32--70) èc;F508/R1162X 26 1&#b7;9 &#b1; 1&#b7;1 101 &#b1; 2&#b7;3 77 &#b1; 4&#b7;2 92 &#b1; 4&#b7;6 138 &#b1; 1&#b7;8 36&#b7;5 &#b1; 1&#b7;4 92 (75-98) 65 (47-80) èc;F508/1898+1G 20 1&#b7;2 &#b1; 0&#b7;3 99 &#b1; 2&#b7;8 83 &#b1; 4&#b7;1 94 &#b1; 4&#b7;4 138 &#b1; 3&#b7;3 35&#b7;1 &#b1; 2&#b7;1 85 (61--95) 63 (39-82) èc;F508/2184DA 20 2&#b7;3 &#b1; 0&#b7;9 106 &#b1; 5&#b7;3 82 &#b1; 4&#b7;3 92 &#b1; 4&#b7;4 141 &#b1; 3&#b7;0 36&#b7;5 &#b1; 1&#b7;5 94 (69-99) 60 (38-79) èc;F508/711+1G 17 1&#b7;3 &#b1; 0&#b7;5 108 &#b1; 4&#b7;6 83 &#b1; 4&#b7;2 94 &#b1; 4&#b7;4 137 &#b1; 3&#b7;4 36&#b7;7 &#b1; 2&#b7;9 100 73 (50-88) èc;F508/S549N 11 6&#b7;4 &#b1; 1&#b7;9&#a7; 109 &#b1; 5&#b7;7 67 &#b1; 6&#b7;1 77 &#b1; 7&#b7;2 140 &#b1; 3&#b7;2 36&#b7;7 &#b1; 2&#b7;6 92 (62-99) 71 (40--90) èc;F508/Other 2 262 5&#b7;8 &#b1; 0&#b7;2ߥ 99 &#b1; 0&#b7;4ߥ 80 &#b1; 0&#b7;5ߥ 91 &#b1; 0&#b7;5ߥ 141 &#b1; 0&#b7;3 38&#b7;1 &#b1; 0&#b7;3ߥ 86 (84-87)ߥ 50 (48-52)ߥ Other/Other 1 551 7&#b7;5 &#b1; 0&#b7;3ߥ 93 &#b1; 0&#b7;6ߥ 82 &#b1; 0&#b7;6ߥ 90 &#b1; 0&#b7;6&#a7; 141 &#b1; 0&#b7;4 38&#b7;3 &#b1; 0&#b7;3ߥ 81 (80-84)ߥ 40 (38-43)ߥ Data are mean (SE) unless otherwise indicated. Login to comment

[hide] Simultaneous screening for 11 mutations in the cys... Mol Cell Probes. 1992 Feb;6(1):33-9. Cuppens H, Buyse I, Baens M, Marynen P, Cassiman JJ
Simultaneous screening for 11 mutations in the cystic fibrosis transmembrane conductance regulator gene by multiplex amplification and reverse dot-blot.
Mol Cell Probes. 1992 Feb;6(1):33-9., [PMID:1372093]

Abstract [show]
An assay is described in which 11 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene can be screened simultaneously. Six different exons of the CFTR gene are amplified in a single multiplex amplification. Biotinylated dUTP is incorporated into the different fragments during the amplification process. A sample of this mixture is then hybridized to 21 different poly-dT tailed oligonucleotide probes which are bound to a nylon membrane. In order to screen the different mutations in a single step hybridization, the length of the different oligonucleotides and the amount used in the assay were optimized. The detection is performed by binding avidin-alkaline phosphatase to the biotin, followed by a chemiluminescent reaction. By means of this fast and sensitive assay, about 85% of all the cystic fibrosis mutations in the Belgian population can be detected.

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35 Mutation Number of CF chromosomes with the mutation Reference AF508 138(71-1%) 3 G542X 11 (5 .7%) 9 N1303K 6(3-1%) 15 1717-1G--*A 5 (2.6%) 8, 9 A455E 2 (1 .0%) 9 W1282X 2 (1 .0%) 10 G458V 1 (0.5%) 4 A1507 1 (0.5%) 9 Unidentified 28 (14.4%) cation was carried out on a DNA Thermal Cycler (Perkin Elmer-Cetus Instruments) . Login to comment
63 Cystic fibrosis patients homozygous for the AF508, G542X and N1303K mutations were available in our Belgian population. Login to comment
75 PCR fragments obtained by multiplex amplification of normal genomic DNA, genomic DNA of a carrier for the A1507 mutation, and genomic DNA of patients homozygous for the AF508, G542X or N1303K mutations were hybridized . Login to comment
82 The G542X, N1303K and 1717-1G->A mutations turned out to be the most frequent in non-AF508 CF chromosomes in our population . Login to comment
97 621 + IG-T A455E G458V A1507 AF508 1717-IG - A G542X G551D R553X WI282X N 1303 K 621+IG- .T A455E G458V A1507 AF508 1717-I G-> A G542X G551D R553X W1282X N1303K Fig. 2. Login to comment
101 A E M W Non-radioactive CF reverse dot-blot 37 B F M W C M W D M W G H 621 + IGT A455E G458V A1507 A F508 1717-IGA G542X G551D R553 X W1282X N1303K 621+IG-ߦT A455E G458V L 1507 AF508 1717-IG- A G542 X G551D R553X W1282X N 1303 K A F M W G B M W C H M W D M W E J M W Fig. 3. Login to comment
103 Hybridization of PCR fragments obtained by multiplex amplification of G458V/G542X (A), AF508/AF508 (B), AF508/? Login to comment
105 denotes a yet unidentified CF mutation) (C), AF508/N1303K (D), AF508/G542X (E), C458V/normal (F), AF508/1717-1G-+A (G), G542X/? Login to comment

[hide] Cystic fibrosis mutations delta F508 and G542X in ... J Med Genet. 1992 Feb;29(2):131-3. Lerer I, Sagi M, Cutting GR, Abeliovich D
Cystic fibrosis mutations delta F508 and G542X in Jewish patients.
J Med Genet. 1992 Feb;29(2):131-3., [PMID:1377276]

Abstract [show]
We have screened our CF patients for mutations in exons 10 and 11 of the CFTR gene. Two mutations, delta F508 and G542X, have been found in 66 Jewish CF patients. The average frequency of the delta F508 mutation in the Jewish population is 33.8%. The G542X mutation accounts for 13% of the Ashkenazi CF mutations and has been found in three out of seven chromosomes of Jewish patients from Turkey (probably descended from Ashkenazi immigrants). The G542X mutation was not found in any of the other non-Ashkenazi patients. All the G542X bearing chromosomes have the same haplotype. Based on these observations it is concluded that the G542X mutation was introduced into the Jewish people after the split into Ashkenazi and non-Ashkenazi.

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0 I Med Genet 1992 29: 131-133 Cystic fibrosis mutations AF508 and G542X in Jewish patients I Lerer, M Sagi, G R Cutting, D Abeliovich Abstract We have screened our CF patients for mutations in exons 10 and 11 of the CFTR gene. Login to comment
1 Two mutations, AF508 and G542X, have been found in 66 Jewish CF patients. Login to comment
3 The G542X mutation accounts for 13% of the Ashkenazi CF mutations and has been found in three out of seven chromosomes of Jewish patients from Turkey (probably descended from Ashkenazi immigrants). Login to comment
4 The G542X mutation was not found in any of the other non-Ashkenazi patients. Login to comment
5 All the G542X bearing chromosomes have the same haplotype. Login to comment
6 Based on these observations it is concluded that the G542X mutation was introduced into the Jewish people after the split into Ashkenazi and non-Ashkenazi. Login to comment
8 The majority of these are rare mutations observed in single families but some mutations are relatively frequent.3 We have screened CF patients for the AF508 mutation and for G542X, G551D, R553X, and S549N or I, all within exon 11, and for the splice mutation 1717-1G-.A. Login to comment
9 Two mutations have been found among the Jewish patients, AF508 and G542X. Login to comment
18 Exon 11 was amplified by PCR using 1 li-5 and 1 i-3 as primers.3 The mutations G542X and 1717-1G--A were analysed by ASO hybridisation. Login to comment
32 The G542X mutation was found in 14 CF chromosomes, in 11 (13%) chromosomes of Ashkenazi origin and in three out of a total of seven (43%) chromosomes of Turkish origin. Login to comment
33 None ofthe other Sephardic CF chromosomes had the G542X mutation. Login to comment
34 We did not find any patients homozygous for the G542X mutation. Login to comment
35 The haplotype of the G542X bearing chromosomes was allele 1 of T6/20-MspI, haplotype B at the D7S23 locus, allele 1 of J3.11/ MspI, allele 2 of both MetH/TaqI and MetD/ BanI, and allele 1 of MetD/TaqI. Login to comment
38 Locus Met D7S23 CFTR D7S8 DNA probe MetH MetD MetD KM19 - XV2c T6/20 J3.11 Enzyme TaqI TaqI BanI PstI TaqI MspI MspI Allele haplotype 1 2 1 2 1 2 1-1 2-1 1-2 2-2 1 2 1 2 A B C D Normal chromosomes Ashkenazi (n=52) 25 24 34 4 17 33 15 8 27 2 19 3 26 25 Non-Ashkenazi (n=30) 14 9 15 7 12 17 6 4 16 4 12 3 14 16 AF508 chromosomes (n = 43) Ashkenazi (n=25) 13 4 13 0 13 5 0 22 0 3 18 0 2 21 Non-Ashkenazi (n= 18) 8 3 8 3 7 7 1 17 0 0 12 0 2 13 G542X chromosomes (n = 14) Auhrkiesho (gi (=3) 1 6 7 0 0 9 0 14 0 0 12 0 11 0 CF chromosomes, unknown mutations (n = 70) Ashkenazi (n=48) 28 2 22 1 24 6 0 43 4 1 617 3 40 Non-Ashkenazi (n=22) 11 7 15 1 12 8 1 8 9 1 7 2 7 11 Table 2 Allelic association coefficient (A)* of closely linked RFLPs and the AF508 mutation. Login to comment
47 G542X is a termination mutation in codon 542 of the putative CFTR protein gene. Login to comment
51 This argument is strengthened by the fact that one of the families from Turkey with G542X has the surname Ashkenazi, which was given to the Ashkenazi immigrants. Login to comment
52 The high proportion of G542X in the patients from Turkey indicates a possible founder effect. Login to comment
53 The haplotype ofthe G542X chromosome is unique, even when one examines the distant RFLPs at the Met and J3.11 loci. Login to comment
55 It would be interesting to compare the haplotypes of those G542X chromosomes to those from other sources in Europe. Login to comment
56 The mutations AF508 and G542X together account for less than 50% of the CF mutations in the Jewish patients. Login to comment
70 132 Cystic fibrosis mutations AF508 and G542X in 3rewish patients 4 Cutting GR, Kasch LM, Rosenstein BJ, et al. A cluster of cystic fibrosis mutations in the first nucleotide-binding fold of the CFTR protein. Login to comment
106 U U KJ _// 1 6 133 #P's -d- ,Ilk, -44 I 44 .4w 0 Aw. of -40 VI* A&- Case 2 .3 16M... ........A 4 to 11%, "W... ow A Case 3 S. group.bmj.com on December 5, 2015 - Published by http://jmg.bmj.com/ Downloaded from G542X in Jewish patients. Login to comment

[hide] [Clinical aspects and genetic specificities of cys... Arch Pediatr. 2003 Nov;10(11):955-9. Flodrops H, Renouil M, Lesure F, Marechal D, Piyaraly S, Arvin-Berod C, Robillard PY, Fourmaintraux A, Cartault F
[Clinical aspects and genetic specificities of cystic fibrosis in Reunion Island].
Arch Pediatr. 2003 Nov;10(11):955-9., [PMID:14613688]

Abstract [show]
OBJECTIVES: Evaluation of the phenotype-genotype correlation of a specific mucoviscidosis mutation, "Y122X", in Reunion Island. This mutation represents 25% of our cases. PATIENTS AND METHODS: Retrospective study of a cohort of 84 children presenting cystic fibrosis (CF) during a 5-year period (1994-1998). Diagnosis was based on one or two identified genetic mutations and/or minimum two abnormal chloride sweat tests (Cl > 70 mmol/l). Follow-up of this cohort was performed in the two referral centers of the Island following the French national guidelines (INSERM U 155). RESULTS: In our population, we identified 10 mutations, of which three of them represented more than 80% of the cases: Delta F508 (51.8%), Y122X (24.4%) and 3120 + 1G --> A (4.8%). The authors report clinical significant differences in children with the homozygote mutation Y122X as compared with children presenting the Delta F508 CF-mutation: failure to thrive affecting mainly the height with, paradoxically, a relatively normal weight development, and a better pulmonary function. CONCLUSION: The frequent Y122X CF-mutation reported in "la Reunion" seems to affect mainly height in children with a relatively good nutritional outcome. This failure to thrive does not seem to be of digestive origin. These results suggest that growth gene(s) located nearby the cystic fibrosis transmembrane conductance regulator (CFTR) may have suffered the same segregation than the Y122X mutation or that clusters of this specific Caucasian population known as "petits blancs" in la Reunion are smallest for ethnic reasons.

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82 = mutation non identifi&#e9;e. Tableau 3 Distribution des mutations &#e0; la R&#e9;union Mutation Nombre de chromosomes Total % Total cumul&#e9; nord (1) sud (2) D F508 53 34 87 51,8 87 Y122X 17 24 41 24,4 128 3120+1G࢐A 6 2 8 4,8 136 A455E 2 1 3 1,8 139 G551D - 1 1 0,6 140 2789+5G࢐A 1 - 1 0,6 141 1161 del C - 1 1 0,6 142 1078 del T - 1 1 0,6 143 1717-1G࢐A 1 - 1 0,6 144 G542X - 1 1 0,6 145 ??? Login to comment

[hide] Unusually common cystic fibrosis mutation in Portu... Biochem Biophys Res Commun. 2003 Nov 21;311(3):665-71. Mendes F, Roxo Rosa M, Dragomir A, Farinha CM, Roomans GM, Amaral MD, Penque D
Unusually common cystic fibrosis mutation in Portugal encodes a misprocessed protein.
Biochem Biophys Res Commun. 2003 Nov 21;311(3):665-71., [PMID:14623323]

Abstract [show]
A561E, a novel cystic fibrosis (CF) associated mutation in the first nucleotide binding domain of CFTR, is the second most common CF mutation in Portugal. Properties of the A561E-CFTR protein were studied by immunoblotting, pulse-chase, immunocytochemistry, and MQAE halide-efflux assay in stably transfected BHK cells. Altogether, results presented here suggest that A561E causes protein mislocalization in the endoplasmic reticulum where the mutant protein must be trapped by the quality control mechanism. We conclude that A561E originates a protein trafficking defect, thus belonging to class II of CFTR mutations. As it is the case for F508del-CFTR (the most common CF mutant), low temperature treatment partially rescues a functional A561E-CFTR channel, suggesting that substitution of glutamic acid for alanine at position 561 does not completely abolish CFTR function. Pharmacological strategies previously reported for treatment of CF patients with the F508del mutation could thus be also effective in CF patients bearing the A561E mutation.

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147 To date, 14 patients carrying A561E were identified in Portugal; nine are compound heterozygotes with F508del, one with G542X and four are homozygous for A561E mutation (Pacheco et al., personal communication and manuscript in preparation). Login to comment

[hide] Variation in a repeat sequence determines whether ... Am J Hum Genet. 2004 Jan;74(1):176-9. Epub 2003 Dec 18. Groman JD, Hefferon TW, Casals T, Bassas L, Estivill X, Des Georges M, Guittard C, Koudova M, Fallin MD, Nemeth K, Fekete G, Kadasi L, Friedman K, Schwarz M, Bombieri C, Pignatti PF, Kanavakis E, Tzetis M, Schwartz M, Novelli G, D'Apice MR, Sobczynska-Tomaszewska A, Bal J, Stuhrmann M, Macek M Jr, Claustres M, Cutting GR
Variation in a repeat sequence determines whether a common variant of the cystic fibrosis transmembrane conductance regulator gene is pathogenic or benign.
Am J Hum Genet. 2004 Jan;74(1):176-9. Epub 2003 Dec 18., [PMID:14685937]

Abstract [show]
An abbreviated tract of five thymidines (5T) in intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is found in approximately 10% of individuals in the general population. When found in trans with a severe CFTR mutation, 5T can result in male infertility, nonclassic cystic fibrosis, or a normal phenotype. To test whether the number of TG repeats adjacent to 5T influences disease penetrance, we determined TG repeat number in 98 patients with male infertility due to congenital absence of the vas deferens, 9 patients with nonclassic CF, and 27 unaffected individuals (fertile men). Each of the individuals in this study had a severe CFTR mutation on one CFTR gene and 5T on the other. Of the unaffected individuals, 78% (21 of 27) had 5T adjacent to 11 TG repeats, compared with 9% (10 of 107) of affected individuals. Conversely, 91% (97 of 107) of affected individuals had 12 or 13 TG repeats, versus only 22% (6 of 27) of unaffected individuals (P<.00001). Those individuals with 5T adjacent to either 12 or 13 TG repeats were substantially more likely to exhibit an abnormal phenotype than those with 5T adjacent to 11 TG repeats (odds ratio 34.0, 95% CI 11.1-103.7, P<.00001). Thus, determination of TG repeat number will allow for more accurate prediction of benign versus pathogenic 5T alleles.

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32 Each of the 27 fathers with 5T had one of the following mutations: DF508 (18), G542X (2), 1812-1GrA (2), DI507 (1), 936delTA (1), N1303K (1), 3600af9;2insT (1), or 1717-1 GrA (1). Login to comment
37 Each of the 98 patients with CBAVD had 5T with one of the following mutations: DF508 (78), G542X (6), N1303K (3), 711af9;1GrT (2), R1066C (2), R1162X (2), R764X (1), Y563X (1), H609R (1), L206W (1), or R334W (1). Login to comment

[hide] Cystic fibrosis. Am J Clin Pathol. 2003 Dec;120 Suppl:S3-13. Lewis MJ, Lewis EH 3rd, Amos JA, Tsongalis GJ
Cystic fibrosis.
Am J Clin Pathol. 2003 Dec;120 Suppl:S3-13., [PMID:15298139]

Abstract [show]
On a daily basis, pathologists examine the fundamental basis of human diseases using morphologic, immunologic, and molecular techniques. Cystic fibrosis (CF), as a clinically heterogeneous disease, exemplifies the complex challenges of genetic diseases for the pathologist who attempts to explain the mechanisms of disease and provide rationale for clinical management. This review includes an overview of CF and a discussion of pathophysiologic features and practical components of clinical and anatomic pathology, and concludes with a review of molecular diagnostics.

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9 G542X is the most frequent mutation in Caucasians after ࢞F508, occurring at a frequency of 2%. Login to comment
95 ēa; ēa;Table 3ēa; ēa; Recommended Mutation Panel for Cystic Fibrosis Carrier Screening ࢞F508 ࢞I507 G542X G551D W1282X N1303K R553X 621+1G>T R117H 1717-1G>A A455E R560T R1162X G85E R334W R347P 711+1G>T 1898+1G>A 2184delA 1078delT 3849+10kbC>T 2789+5G>A 3659delC I148T 3120+1G>A I506V* I507V* F508C* 5T/7T/9T* * Reflex tests. Login to comment

[hide] Diagnosis of cystic fibrosis in adults with diffus... J Cyst Fibros. 2004 Mar;3(1):15-22. Hubert D, Fajac I, Bienvenu T, Desmazes-Dufeu N, Ellaffi M, Dall'ava-Santucci J, Dusser D
Diagnosis of cystic fibrosis in adults with diffuse bronchiectasis.
J Cyst Fibros. 2004 Mar;3(1):15-22., [PMID:15463882]

Abstract [show]
We assessed the contribution of the sweat test, genotyping and nasal potential difference (NPD) in the diagnosis of cystic fibrosis (CF) in adults with diffuse bronchiectasis (DB). Among 601 adults referred for DB from 1992 to 2001, 46 were diagnosed with CF. The sweat test was positive in 37 patients and normal or intermediate in nine patients. Two CF mutations were identified in 18 patients (39%) by screening for 31 mutations and in 36 patients (78%) after complete genetic analysis. NPD was suggestive of CF in 71% of the patients. The combination of the sweat test and genetic analysis led to the diagnosis of CF in 45 patients. In the nine patients with normal or intermediate sweat test, the diagnosis was confirmed by screening for 31 mutations in five, by complete genetic screening in three, and by NPD in the remaining patient. Searching for CF should start with sweat test. If the sweat test is normal or intermediate, screening for 31 mutations may help to diagnose CF. A complete genetic analysis is indicated when only one mutation is detected and/or when other clinical features, such as obstructive azoospermia or pancreatic insufficiency, are suggestive of CF. NPD measurement is indicated in controversial cases.

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47 We used an oligonucleotide ligation assay using a commercially available kit (Cystic Fibrosis Assay, Applied Biosystems, Foster City, CA, USA) to seek 31 mutations in the CFTR gene (F508del, I507del, Q943X, V520F, 1717y1GࡊA, G542X, G551D, R553X, R560T, S549R, S549 N, 3849q10kbCࡊT, 3849q4AࡊG, R1162X, 3659delC, W1282X, 3905insT, 621q1GࡊT, R117H, Y122X, 711q1GࡊT, 1078delT, R347P, R347H, R334 W, A455E, N1303K, G85E, 1898q1GࡊA, 2183AAࡊG, 2789q5GࡊA) which allowed to detect 82% of the CF alleles in France. Login to comment
128 The consensus opinion is Table 3 Sensitivity of the sweat test and genotyping for the diagnosis of cystic fibrosis Positive diagnosis of CF Number of patients (percentage) Sweat test (sweat chloride)60 mmol l ) y1 37 (80%) Molecular analysis: -screening for 31 mutations 18 (39%) -complete screening 36 (78%) Combination of sweat test and molecular analysis: -sweat testqscreening for 31 mutations 42 (91%) -sweat testqcomplete screening 45 (98%) Table 4 Efficacy of the CFTR genetic analysis according to the screening method used Identified mutations Two mutations At least one mutation No mutations Screening for F508del Number of patients (%) 3 (7%) 31 (67%) 15 (33%) Screening for nine mutations* Number of patients (%) 6 (13%) 36 (78%) 10 (22%) Screening for 31 mutations** Number of patients (%) 18 (39%) 43 (93%) 3 (7%) Complete screening Number of patients (%) 36 (78%) 46 (100%) 0 Nine mutations: F508del, I507del, G542X, G551D, R553X, 621q1GࡊT, G85E, R117H, N1303K. Login to comment
129 * 31 mutations: F508del, I507del, Q493X, V520F, 1717y1GࡊA, G542X, G551D, R553X, R560T, S549R, S549 N, 3849q10kbCࡊT, 3849q ** 4AࡊG, R1162X, 3659delC, W1282X, 3905insT, 621q1GࡊT, R117H, Y122X, 711q1GࡊT, 1078delT, R347P, R347H, R334 W, A455E, N1303K, G85E, 1898q1GࡊA, 2183AAࡊG, 2789q5GࡊA. that the laboratory criteria for the diagnosis of CF should be expanded to include identification of CFTR mutations and abnormal bioelectrical properties of the nasal epithelium, in addition to the sweat test w7x. Login to comment
148 The F508del mutation was found in 31 patients and the G542X mutation was found in five patients. Login to comment

[hide] Prevalence of deltaF508, G551D, G542X, and R553X m... Braz J Med Biol Res. 2005 Jan;38(1):11-5. Epub 2005 Jan 18. Araujo FG, Novaes FC, Santos NP, Martins VC, Souza SM, Santos SE, Ribeiro-dos-Santos AK
Prevalence of deltaF508, G551D, G542X, and R553X mutations among cystic fibrosis patients in the North of Brazil.
Braz J Med Biol Res. 2005 Jan;38(1):11-5. Epub 2005 Jan 18., [PMID:15665983]

Abstract [show]
Cystic fibrosis (CF) is the most common genetic disease among Caucasians and is rare among sub-Saharan Africans. The Brazilian population is not ethnically homogeneous but it is the result of three-way ethnic admixture of Europeans, Africans and Amerindians in varying proportions, depending on the region. In the present study, we investigated 33 patients who had been diagnosed and are currently under treatment for CF at the University Hospital Joao de Barros Barreto, Belem, Para State. The molecular analysis for G542X, G551D and R553X mutations was performed by PCR followed by RFLP using BstNI, HincII and MboI, respectively, in polyacrylamide gel eletrophoresis and stained with AgNO3. ThedeltaF508 mutation (a deletion of 3 bp) was only analyzed by polyacrylamide gel electrophoresis and stained with AgNO3. Each sample was analyzed for regions of interest in the CFTR gene using amplified by PCR and specific primers. The deltaF508 and G551D mutations presented frequencies of 22.7 and 3%, respectively. In 74.3% of the remaining patients, none of the mutations investigated was found. The present study characterized in a sample of patients with an established clinical diagnosis of CF (asthma, repeated bronchopneumonia, disorders of nutritional status, etc.) the most frequent mutation (deltaF508) in the North region of Brazil and is also the first report of the G551D mutation. In spite of the wide spectrum of CF mutations and the heterogeneous ethnic origin of the Amazon population, the molecular diagnosis is a helpful additional tool for the diagnosis and treatment of CF patients.

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14 ࢞F508 is the most common CF mutation (66%) in the worldwide populations studied to date, although other mutations such as G542X (2.4%), G551D (1.6%), N1303K Brazilian Journal of Medical and Biological Research (2005) 38: 11-15 ISSN 0100-879X Short Communication (1.3%), and W1282X (1.2%) (5-13) may be relatively frequent depending on the ethnic origin of the population. Login to comment
24 (8)about CF in S&#e3;o Paulo State demonstrated the presence of the G542X, N1303K and W1282X mutations, with frequencies of 8.35, 1.6 and 0.8%, respectively. Login to comment
26 (7) first reported two patients from the city of Rio de Janeiro carrying the G542X mutation and five other patients carrying splicing mutations 3120+1G࢐A. ThewidespectrumofCFmutations(more than 900 already described) and the heterogeneous ethnic origin of the Brazilian population are important factors that must be considered to draw a broader map of this disease in the Brazilian territory. Login to comment
27 Thus, the main objective of the present study was to investigate the four most frequent CF mutations (࢞F508, G542X, G551D, and R553X) in a sample of patients with an established clinical diagnosis of CF in the North region of Brazil. Login to comment
33 Region Number of alleles Mutations (%) Reference ࢞F508 G542X G551D R553X South Rio Grande do Sul 106 49.1 2.8 0 0 5 Rio Grande do Sul 154 48.7 3.2 0 0.7 12 Santa Catarina 58 55.2 3.5 0 0 5 Santa Catarina 48 27 0 0 0 9 Paran&#e1; 100 39 9 0 2 5 Southeast S&#e3;o Paulo 110 52.7 10 0.9 2.7 5 Rio de Janeiro 88 30.7 2.3 1.1 0 6 Rio de Janeiro 148 25.7 0 0 0 7 Rio de Janeiro 34 35.3 - - - 9 Minas Gerais 62 32.6 4.2 0 0 5 North Bel&#e9;m 66 22.7 0 3 0 Present study approved this research. Login to comment
36 The four mutations (࢞F508, G542X, G551D, and R553X) were amplified using primers and conditions described below. Login to comment
38 The volume 50 &#b5;l was used to observe the G542X, G551D and R553X mutations (exon 11). Login to comment
40 A region of 114 bp was amplified and then three mutations were investigated: G542X, G551D and R553X. Login to comment
44 The mutation G542X made use of the endonuclease BstN I. Login to comment
45 For the individuals who presented the pattern (+/+), which indicates absence of the mutation G542X, the investigation of a second restriction site was made for the endonuclease HincII (G551D). Login to comment
50 The analysis of the G542X, G551D and R553X mutations were observed by 9% polyacrylamide gel electrophoresis. Login to comment

[hide] A 96-well formatted method for exon and exon/intro... Anal Biochem. 2006 Jun 15;353(2):226-35. Epub 2006 Apr 5. Lucarelli M, Narzi L, Piergentili R, Ferraguti G, Grandoni F, Quattrucci S, Strom R
A 96-well formatted method for exon and exon/intron boundary full sequencing of the CFTR gene.
Anal Biochem. 2006 Jun 15;353(2):226-35. Epub 2006 Apr 5., [PMID:16635477]

Abstract [show]
Full genotypic characterization of subjects affected by cystic fibrosis (CF) is essential for the definition of the genotype-phenotype correlation as well as for the enhancement of the diagnostic and prognostic value of the genetic investigation. High-sensitivity diagnostic methods, capable of full scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, are needed to enhance the significance of these genetic assays. A method for extensive sequencing of the CFTR gene was optimized. This method was applied to subjects clinically positive for CF and to controls from the general population of central Italy as well as to a single subject heterozygous for a mild mutation and with an uncertain diagnosis. Some points that are crucial for the optimization of the method emerged: a 96-well format, primer project and purification, and amplicon purification. The optimized method displayed a high degree of diagnostic sensitivity; we identified a subset of 13 CFTR mutations that greatly enhanced the diagnostic sensitivity of common methods of mutational analysis. A novel G1244R disease causing mutation, leading to a CF phenotype with pancreatic sufficiency but early onset of pulmonary involvement, was detected in the subject with an uncertain diagnosis. Some discrepancies between our results and previously published CFTR sequence were found.

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26 None of these subjects showed any clinical manifestations of CF, nor were any positive for CFTR mutations when analyzed by means of the PCR/OLA/SCS method (Celera Diagnostics) [21], which searches for the most common worldwide 31 CFTR mutations (G85E, R117H, Y122X, 621+1G->T, 711+1G->T, 1078delT, R347P, R347H, R334W, A455E, DF508, DI507, Q493X, V520F, 1717-1G->A, G542X, G551D, R553X, R560T, S549R(T->G), S549N, 1898+1G->A, 2183AA->G, 2789+5G->A, R1162X, 3659delC, 3849+10kbC->T, 3849+4A->G, W1282X, 3905insT, N1303K), including the 12 most common in Italy [1,22]. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... J Cyst Fibros. 2006 Aug;5(3):159-64. Epub 2006 Mar 6. Ngiam NS, Chong SS, Shek LP, Goh DL, Ong KC, Chng SY, Yeo GH, Goh DY
Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in Asians with chronic pulmonary disease: a pilot study.
J Cyst Fibros. 2006 Aug;5(3):159-64. Epub 2006 Mar 6., [PMID:16678503]

Abstract [show]
BACKGROUND: Little is known about the relationship between cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in Asian patients and severe asthma or idiopathic bronchiectasis. We investigated this potential relationship in the Singaporean Chinese. METHODS: Twenty patients with chronic pulmonary disease, 14 with severe asthma and 6 with idiopathic bronchiectasis, were screened for CFTR mutations by direct gene sequencing. The frequencies of identified putative mutations were compared against 40 unaffected controls and 96 unselected population samples. RESULTS: Three missense mutations (I125T, I556V, and Q1352H) and 1 splice site variant (intron 8 12TG5T) were identified in a total of 10 patients, representing a combined mutant/variant allele frequency of 0.25. These alleles were also observed in the controls, but at a significantly lower allele frequency of 0.09 (P<0.01). Furthermore, the I125T mutation was significantly associated with the idiopathic bronchiectasis sub-group (P<0.05). CONCLUSIONS: The significantly higher frequency of CFTR mutations among patients with chronic pulmonary disease compared with unaffected controls suggests that these mutations may increase risk for disease. The association of I125T with idiopathic bronchiectasis alone suggests that different mutations predispose to different disease.

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102 These are R117H (exon 4), 621UVG>T (intron 4), F508del (exon 10), 1717-1 G>A (intron 10), G542X (exon 11), G551D (exon 11), R553X (exon 11), R1162X (exon 19), W1282X (exon 20) and N1303K (exon 21). Login to comment

[hide] The genetic background of osteoporosis in cystic f... J Cyst Fibros. 2006 Dec;5(4):229-35. Epub 2006 May 18. Castellani C, Malerba G, Sangalli A, Delmarco A, Petrelli E, Rossini M, Assael BM, Mottes M
The genetic background of osteoporosis in cystic fibrosis: association analysis with polymorphic markers in four candidate genes.
J Cyst Fibros. 2006 Dec;5(4):229-35. Epub 2006 May 18., [PMID:16713399]

Abstract [show]
BACKGROUND: Reduced Bone Mass Density (BMD) is frequent in Cystic Fibrosis (CF). Potentially, other genes than the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene may contribute to the bone phenotype variability in CF patients. METHODS: Four candidate genes likely associated with BMD variability were studied: the vitamin D receptor (VDR) gene, the estrogen receptor alpha (ESR1), the calcitonin receptor (CALCR) and the type I alpha 1 collagen (COL1A1) gene. A complete bone and CF evaluation was obtained for 82 subjects (39 m, 43 f): 15 had normal BMD (group 1), 46 were osteopenic (group 2), and 21 were osteoporotic (group 3). RESULTS: No statistical difference was found among the three groups for age, sex, pancreatic status, and vertebral fractures, nor for any of the biochemical markers. Weight, Body Mass Index (BMI), and FEV1, scored significantly worse in the two groups with the lowest T score. The CFTR mutations R1162X and F508del were more frequent in patients with lower BMD (p=0.044 and p=0.071). There was no significant difference in the distribution of the five marker genotypes among the 3 groups defined according to the unadjusted or adjusted (BMI and FEV1) BMD T score. No significant correlation was found between the VDR, CALCR, or COL1A1 gene polymorphisms and reduced BMD values. The individual ESR1 PvuII-XbaI haplotype C-A is associated to elevated u-calcium levels whereas the haplotype T-A is associated to lower values (p=0.00251). CONCLUSIONS: There was no evidence that the genes under study, with the possible exception of ESR1 gene variants, may modulate bone phenotype in CF.

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74 CFTR analysis Patients selected for the study had been characterized previously for CFTR mutations with a reverse dot blot (RDB) Table 1 Anthropometric and CF-associated variables in normal, osteopenic and osteoporotic patients Group 1 normal bone density Group 2 osteopenia Group 3 osteoporosis Statistical evaluation Numerosity 15/82 46/82 21/82 - Mean age (years) 27.73T4.19 26.71T5.93 28.1T9.51 NS Males/females 9/6 19/27 11/10 NS CFTR genotype F508del/UK: 3 UK/ UK: 3 F508del/ F508del: 2 F508del/ G542X: 2 F508del/ R553X: 2 1717-1G> AvW1282X: 1 F508del/ N1303K: 1 G542X/UK: 1 F508del/F508del: 6 UK/UK: 5 F508del/ UK: 3 2183AA>G/UK: 3 2789+5G> A /UK: 3 F508del/N1303K: 3 F508del / R1162X: 3 F508del/2183AA>G: 2 N1303K/ N1303K: 2 R1162X/R1162X: 2 R1162X/ 2183AA>G: 2 2183AA>G/G542X: 1 F508del/ 1898+3A>G: 1 F508del/2789+5G> A: 1 F508del/711+5G>A: 1 F508del/ Q353X: 1 I507del/R1162X: 1 Q552X/ UK: 1 N1303K/G542X: 1 R1162X/3849+ 10KbC>T: 1 R1162X/711+5G>A: 1 T338I/ UK: 1 R553X/UK: 1 F508del/F508del: 4 F508del/ UK: 3 F508del/N1303K: 2 UK/ UK: 2 2789+5G>A/2789+5G> A: 1 F508del/1898+3A>G: 1 F508del/ 2183AA>G: 1 F508del/3849+10KbC> T: 1 F508del/G542X: 1 F508del/ R1066H: 1 F508del/R1162X: 1 Q552X/: 1 R352Q/: 1 R553X/UK: 1 Weight (kg) 61.7T5.89 56.5T8.25 48.9T9.40 p <0.0001 BMI 22.6T1.3 20.3T2.7 18T2.9 p <0.0001 PS/PI 3/12 10/36 7/14 NS FEV1% predicted 52.33T16.73 49.82T21.44 37.1T21.07 p =0.0205 NS = not significant. Login to comment
80 assay which allows the simultaneous analysis of the commonest CFTR mutations in North-eastern Italy (F508del, I507del, R117H, R1162X, 2183AA>G, N1303K, 3849+10KbC>T, G542X, 1717-1G>A, R553X, Q552X, G85E, 711+5G>A, W1282X, 3132delTG and 2789+5G>A) [25]. Login to comment

[hide] CFTR gene analysis in Latin American CF patients: ... J Cyst Fibros. 2007 May;6(3):194-208. Epub 2006 Sep 11. Perez MM, Luna MC, Pivetta OH, Keyeux G
CFTR gene analysis in Latin American CF patients: heterogeneous origin and distribution of mutations across the continent.
J Cyst Fibros. 2007 May;6(3):194-208. Epub 2006 Sep 11., [PMID:16963320]

Abstract [show]
BACKGROUND: Cystic Fibrosis (CF) is the most prevalent Mendelian disorder in European populations. Despite the fact that many Latin American countries have a predominant population of European-descent, CF has remained an unknown entity until recently. Argentina and Brazil have detected the first patients around three decades ago, but in most countries this disease has remained poorly documented. Recently, other countries started publishing their results. METHODS: We present a compilation and statistical analysis of the data obtained in 10 countries (Argentina, Brazil, Chile, Colombia, Costa Rica, Cuba, Ecuador, Mexico, Uruguay and Venezuela), with a total of 4354 unrelated CF chromosomes studied. RESULTS: The results show a wide distribution of 89 different mutations, with a maximum coverage of 62.8% of CF chromosomes/alleles in the patient's sample. Most of these mutations are frequent in Spain, Italy, and Portugal, consistent with the origin of the European settlers. A few African mutations are also present in those countries which were part of the slave trade. New mutations were also found, possibly originating in America. CONCLUSION: The profile of mutations in the CFTR gene, which reflects the heterogeneity of its inhabitants, shows the complexity of the molecular diagnosis of CF mutations in most of the Latin American countries.

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42 Some have concentrated in the search of specific mutations that are Table 1 Mutations found in the Latin American CF patients Exon 1 p.L6VÌe; Exon 3 p.W57X, p.R75X, p.G85E Exon 4 p.R117H Exon 6a p.H199Y, p.V201M, p.L206W, p.Q220X, p.V232D, c.846delTÌe; Exon 6b p.Y275XÌe;, c.935delA Exon 7 p.R334W, p.R347P, p.Y362XÌe;, c.1078delT, c.1215delG Exon 8 c.1323_1324insAÌe; Exon 9 c.1460_1461delATÌe;, c.1353_1354insTÌe;,# Exon 10 p.I506T, p.I507del, p.F508del Exon 11 p.G542X, p.S549N, p.S549R, p.G551D, p.G551S, p.R553X, p.L558S, p.A559T, c.1782delA Exon 12 p.S589I Exon 13 p.H609RÌe;, p.P750L, p.V754M, c.1924_1930del, c.2055_2063del, c.2183AA NG;c.2184delA, c.2184delA, c.2185_2186insC, c.2347delG, c.2566_2567insTÌe;, c.2594_2595delGTÌe; Exon 14a p.R851L, c.2686_2687insTÌe; Exon 15 c.2869_2870insG Exon 16 c.3120+1GNA Exon 17a p.I1027T, c.3171delC, c.3199_3204del Exon 17b p.G1061R, p.R1066C, p.W1069X#, p.W1089X, p.Y1092X, p.W1098CÌe; Exon 19 p.R1162X, p.W1204X, p.Q1238X, c.3617_3618delGAÌe;#, c.3659delC Exon 20 p.W1282X, p.R1283M Exon 21 p.N1303K, c.4016_4017insT Exon 22 c.4160_4161insGGGGÌe; 5' flanking c.-834GNT Intron 2 c.297-1GNAÌe;, c.297-2ANG Intron 3 c.406-1GNA Intron 4 c.621+1GNT Intron 5 c.711+1GNT Intron 8 c.IVS8-5T Intron 10 c.1716GNA, c.1717-1GNA Intron 11 c.1811+1.6KbANG, c.1812-1GNA Intron 12 c.1898+1GNA, c.1898+3ANG Intron 14 c.2789+2_2789+3insA, c.2789+5GNA Intron 17a c.3272-26ANG Intron 17b c.3500-2ANGÌe; Intron 19 c.3849+1GNA, c.3849+10KbCNT Intron 20 c.4005+1GNA, c.4005-1GNA# Mutations are listed according to their position in the gene. Login to comment
46 of chromosomes analysed p.F508del p.G542X p.W1282X p.N1303K p.R1162X p.L6VÌe; p.W57X p.R75X p.G85E p.R117H p.H199Y p.V201M p.L206W p.Q220X p.V232D p.Y275XÌe; p.R334W p.R347P p.Y362XÌe; p.I506T Argentina 98 61 440 258 18 12 12 2 1 1 3 1 5 1 310 181 20 7 5 5 7 0 5 0 222 135 15 7 5 1 26 14 2 1 1 150 88 6 6 1 2 3 Subtotal and frequency (%) 1246 100 737 59.15 61 4.90 27 2.17 28 2.25 9 0.72 1 0.08 1 0.08 13 1.04 1 0.08 13 1.04 1 0.08 Brazil 468 221 26 11 74 38 2 1 320 155 28 3 8 8 4 1 2 1 1 8 122 62 120 38 10 3 148 38 4 0 0 48 15 154 75 5 1 0 2 0 386 154 24 6 10 17 9 0 10 1 18 4 0 0 2 0 0 0 0 Subtotal and frequency (%) 1858 100 800 43.06 99 5.33 11 0.59 34 1.83 25 1.35 13 0.70 1 0.05 2 0.11 1 0.05 1 0.05 20 1.07 1 0.05 Chile 72 21 36 11 3 0 44 22 4 3 1 1 100 45 7 5 0 2 0 2 0 Subtotal and frequency (%) 252 100 99 41.28 14 5.55 8 3.17 3 1.19 3 1.19 Colombia 184 77 7 2 1 2 1 34 13 2 1 1 Subtotal and frequency (%) 218 100 90 41.28 9 4.13 3 1.38 2 0.92 2 0.92 1 0.46 Costa Rica Frequency (%) 48 100 11 22.91 12 25.00 0 0 0 0 0 Cuba Frequency (%) 144 100 49 34.03 Ecuador 32 11 1 50 16 2 2 20 5 0 0 0 Subtotal and frequency (%) 102 100 32 31.37 2 1.96 1 0.98 2 1.96 Mexico 194 79 12 4 3 1 1 1 2 80 36 4 1 Subtotal and frequency (%) 274 100 115 41.97 16 5.84 5 1.82 3 1.09 1 0.36 1 0.36 1 0.36 2 0.73 Uruguay Frequency (%) 76 100 43 56.58 6 7.89 2 2.63 3 3.95 3 3.95 2 2.63 Venezuela 54 16 2 82 41 Subtotal and frequency (%) 136 100 57 41.91 2 1.47 Total 4354 2033 221 49 72 42 1 1 3 32 1 1 1 2 1 1 1 39 1 1 2 Frequency (%) 100 46.69 5.08 1.13 1.65 0.96 0.02 0.02 0.07 0.73 0.02 0.02 0.02 0.05 0.02 0.02 0.02 0.90 0.02 0.02 0.05 The five most frequent mutations are shown on the left-hand side, followed by the rest of the mutations in 5'-3' and exon-intron order. Login to comment
62 The p.G542X mutation, with a total frequency of 5.07% (25% in Costa Rica to 1.47% in Venezuela), is the second most frequent mutation in Latin America, with the exception of Colombia and Costa Rica. Login to comment
64 Costa Rica is a unique case, where p.G542X (12/48 chromosomes) and p.F508del (11/48 chromosomes) have almost the same frequencies. Login to comment
85 Costa Rica sits on an isolated branch, apparently due to the weight given by the exceptionally high frequency of the p.G542X mutation in these patients (25%). Login to comment
89 Table 3 Most frequent mutations (N1%) in Latin American patients Country Chromosomes analysed p.F508del p.G542X p.N1303K p.W1282X p.R1162X Unknown n % n % n % n % n % n % Argentina 1246 737 59.15 61 4.90 28 2.25 27 2.17 9 0.72 271 21.75 Brazil 1858 800 43.06 99 5.33 34 1.83 11 0.59 25 1.35 789 42.46 Chile 252 99 39.28 14 5.55 0 0.00 8 3.17 3 1.19 115 45.63 Colombia 218 90 41.28 9 4.13 2 0.92 3 1.38 2 0.92 84 38.53 Costa Rica 48 11 22.92 12 25.00 - - - - - - 25 52.08 Cuba 144 49 34.03 - - - - - - - - 95 65.97 Ecuador 102 32 31.37 2 1.96 1 0.98 - - - - 65 63.72 Mexico 274 115 41.97 16 5.84 5 1.82 - - - - 88 32.11 Uruguay 76 43 56.58 6 7.89 2 2.63 - - 3 3.95 11 14.47 Venezuela 136 57 41.91 2 1.47 - - - - - - 77 56.62 Total 4354 2033 46.69 221 5.08 72 1.65 49 1.13 42 0.96 1620 37.21 A - sign indicates that these mutations were not tested in the sample of patients, therefore their real frequency remains unknown. Login to comment
101 Furthermore, if we take three of the most frequent mutations in Europeans (p.F508del, p.G542X and p. N1303K), along with a very frequent mutation in Italians, p.R1162X [9], the percentage of these in Argentinean and Uruguayan CF patients adds up 67% and 71%, respectively, of the mutations (Table 3). Login to comment
111 As discussed, another way to disclose similarities or differences in the distribution of mutations in the CF patients from Latin Table 6 Screening panel of CFTR mutations Country Total number of mutations Minimum panel Detection power Uruguay 12 6 mutations: p.F508del, p.G542X, p.R1162X, p.N1303K (p.R334W, p.G85E) 78% Argentina 52 7 mutations: p.F508del, p.G542X, p.R1162X, p.W1282X, p.N1303K (p.R334W, p.G85E) 71% M&#e9;xico 35 8 mutations: p.F508del, p.G542X, p.N1303K (p.R75X, p.I507del, p.S549N,c.406-1GNA, c.3849+10kbGNA) 58% Colombia 19 7 mutations: p.F508del, p.G542X, p.R1162X, p.W1282X, p.N1303K (p.S549R, c.1811+1.6kbANG) 56% Brazil 41 6 mutations: p.F508del, p.G542X, p.R1162X, p.W1282X, p.N1303K (p.R334W) 53% The total number of mutations found in each country is indicated in the second column from left. Login to comment
126 In this country, p. G542X (25%) is the first mutation, followed by p.F508del (22.92%) (Table 3). Login to comment
127 As argued by the authors, this unusual high frequency of p.G542X, which is almost four times that observed overall in Spain (7.66%) and Andalusia (8.6%) [9], could be a consequence of a strong founding effect due to the small number of settling families from this region established in Costa Rica in the XVIth-XVIIth century. Login to comment

[hide] Molecular targeting of CFTR as a therapeutic appro... Trends Pharmacol Sci. 2007 Jul;28(7):334-41. Epub 2007 Jun 18. Amaral MD, Kunzelmann K
Molecular targeting of CFTR as a therapeutic approach to cystic fibrosis.
Trends Pharmacol Sci. 2007 Jul;28(7):334-41. Epub 2007 Jun 18., [PMID:17573123]

Abstract [show]
One of the major challenges facing the pharmaceutical field is the identification of novel, 'druggable' targets common to distinct diseases that, despite their clinical diversity, share the same basic molecular defect(s) - thus, being termed 'horizontal diseases'. Membrane proteins constitute one of the largest families in the human genome and, given their major roles in cells and organisms, they are relevant to common human disorders such as cardiovascular disease and cancer, but also to rare genetic conditions such as cystic fibrosis (CF). Here, we review therapeutic approaches to correcting the basic defect in CF, which is caused mainly by the intracellular retention of a misfolded protein, and focus on various recent drug-discovery strategies for this important and paradigmatic disease. These strategies have possible applications in many membrane protein disorders, including other channelopathies. The mechanisms of action of potent and specific compounds, representing promising drug leads for CF pharmacotherapy, are explained and discussed.

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46 Class I mutations, which abrogate protein production, often include mutations that generate premature stop codons (e.g. G542X) that lead to mRNA degradation by nonsense-mediated decay. Login to comment

[hide] XV-2c/KM19 haplotypes analysis of cystic fibrosis ... Acta Physiol Hung. 2008 Sep;95(3):313-25. doi: 10.1556/APhysiol.95.2008.3.7. Flores-Martinez SE, Martinez JF, Machorro-Lazo MV, Garcia-Zapien AG, Salgado-Goytia L, Cruz-Quevedo EG, Moran-Moguel MC, Sanchez-Corona J
XV-2c/KM19 haplotypes analysis of cystic fibrosis patients from western Mexico.
Acta Physiol Hung. 2008 Sep;95(3):313-25. doi: 10.1556/APhysiol.95.2008.3.7., [PMID:18788470]

Abstract [show]
The analysis of polymorphic markers within or closely linked to the cystic fibrosis transmembrane regulator (CFTR) gene is useful as a molecular tool for carrier detection of known and unknown mutations. To establish the association between mutations in the CFTR gene in western Mexican cystic fibrosis (CF) patients, the distribution of XV2c/KM19 haplotypes was analyzed by PCR and restriction enzyme digestion in 384 chromosomes from 74 CF patients, their unaffected parents, and normal subjects. The haplotype analysis revealed that haplotype B was present in 71.9% of CF chromosomes compared to 0% of non-CF chromosomes. The F508del and G542X mutations were strongly associated with haplotype B (96.7% and 100% of chromosomes, respectively). The haplotype distribution of the CF chromosomes carrying other CFTR mutations had a more heterogeneous background. Our results show that haplotype B is associated with CFTR mutations. Therefore, haplotype analysis is a suitable alternate strategy for screening CF patients with a heterogeneous clinical picture from populations with a high molecular heterogeneity where carrier detection programs are not available. In addition, it may be a helpful diagnostic tool for genetic counseling and carrier detection in the relatives of CF patients and in couples who are planning to have children.

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3 The F508del and G542X mutations were strongly associated with haplotype B (96.7% and 100% of chromosomes, respectively). Login to comment
71 Of the 64 CF chromosomes, 30 carried the F508del mutation, 7 the G542X mutation, and the remaining 27 chromosomes carried the 3849+10 Kb C>T, 621+1 G>T, G551D, R553X, F311de1, R352Q, R74W mutations, as well as the new mutation 2053-2060del (unpublished data) or unidentified mutations; all of these were grouped as "other" (Table II). Login to comment
72 The majority of the chromosomes with the F508del mutation (29/30) were found on chromosomes with haplotype B (96.7%), whereas all 7 chromosomes carrying the G542X mutation were on chromosomes with haplotype B (100%). Login to comment
78 (%) of XV-2c/KM19 haplotypes Chromosomes A B C D CF (n=64) 10 (15.6%) 46 (71.9%) 5 (7.8%) 3 (4.7%) F508del (n=30) - 29 (96.7%) - 1 (3.3%) G542X (n=7) - 7 (100%) - - Other* (n=27) 10 (37.0%) 10 (37.0%) 5 (18.5%) 2 (7.4%) Non-CF (n=30) 18 (60%) 0 (0%) 9 (30%) 3 (10%) *Chromosomes bearing detected mutations (3849+10 Kb C>T, 621+1 G>T, G551D, R553X, F311del, R352Q, R74W, 2053-2060del), or unidentified mutations. Login to comment
99 The difference between the studies is more evident when the frequencies of haplotype B and the G542X mutation are compared. Login to comment
101 (20) reported that 56.7% of CF chromosomes were mainly associated with haplotype B, and that the G542X mutation was associated with haplotype B in 72.7% of the chromosomes. Login to comment
102 In our CF chromosomes, haplotype B was present in 71.9% of CF chromosomes and in 100% of G542X chromosomes (Table III). Login to comment
124 Herein, we have identified a very high association of the F508del mutation and an even higher association of G542X mutation with haplotype B. Login to comment
128 Therefore, a haplotype analysis using markers KM19 and XV-2c might be useful as a first screening strategy to offer a molecular diagnosis for the western Mexican families, emphasizing the fact that, in our study, haplotype B was absent in all chromosomes of individuals free of the clinical features of CF, and because the association of the F508del and G542X mutations with haplotype B in CF patients from western Mexico was higher than reported in Latin American and some Caucasian populations. Login to comment
129 The use of XV-2c/KM19 haplotype analysis as a first screening strategy could offers several advantages, such as: the assessment of the carrier risk of patients, particularly with F508del and G542X mutations; the screening of individuals bearing mutations in the CFTR gene (heterozygotes detection); and allowing the implementation of detection programs for carrier detection of known and unknown mutations in first-degree relatives of CF patients who wish to know their carrier status. Login to comment

[hide] p.F508del in a heterogeneous cystic fibrosis popul... Braz J Med Biol Res. 2008 Aug;41(8):643-7. Vidigal PV, Reis FJ, Boson WL, De Marco LA, Brasileiro-Filho G
p.F508del in a heterogeneous cystic fibrosis population from Minas Gerais, Brazil.
Braz J Med Biol Res. 2008 Aug;41(8):643-7., [PMID:18797695]

Abstract [show]
Cystic fibrosis (CF) is the most common autosomal recessive disease of the Caucasian population. Among the various CF mutations, p.F508del is the most frequent, accounting for two-thirds of the global CF chromosomes, although showing great variability among populations. We have studied 115 unrelated CF patients from a mixed population of Minas Gerais (Brazil). To evaluate part of the DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, blood DNA was obtained and PCR was performed using two pairs of primers that anneal to exons 10 and 24 of the CFTR gene. The PCR product was then submitted to automatic sequencing using the ABI PRISM 310 Genetic Analyzer. The p.F508del mutation was found in 50 (21.7%) of 230 unrelated CF alleles. Fifteen (13.0%) patients were homozygous for this mutation, while 20 (17.4%) were heterozygous; the remaining 80 (69.6%) patients did not carry the p.F508del mutation. Exon 24 sequence had no change in 75 (65.2%) patients, 21 (18.3%) had the sequence variation 4521G/A, 11 (9.6%) had a not yet described sequence variation 4407T/A and 8 (7.0%) patients had both sequence variations (4521G/A and 4407T/A). The polymorphism 4407T/A results in an amino acid modification from aspartic acid to glutamic acid, which will probably have no function effect in CFTR. This low p.F508del prevalence can be due to the variable ethnic origin of this population from Minas Gerais, which may have a high diversity of CF rare mutations.

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19 Among the various CF mutations, a deletion of 3 bp at codon 508 (p.F508del) is the most frequent accounting for two-thirds of the global CFchromosomes.Only4othermutations(G542X,N1303K, G551D, and W1282X) have overall frequencies above 1% among CF chromosomes. Login to comment

[hide] [Why and how to assess hypospermia?]. Gynecol Obstet Fertil. 2008 Oct;36(10):1035-42. doi: 10.1016/j.gyobfe.2008.04.021. Epub 2008 Sep 17. Robin G, Marcelli F, Mitchell V, Marchetti C, Lemaitre L, Dewailly D, Leroy-Billiard M, Rigot JM
[Why and how to assess hypospermia?].
Gynecol Obstet Fertil. 2008 Oct;36(10):1035-42. doi: 10.1016/j.gyobfe.2008.04.021. Epub 2008 Sep 17., [PMID:18801689]

Abstract [show]
Hypospermia is a semen volume lower than 2 mL on at least two semen analyses. The etiologies of hypospermia are many and may be divided into two pathophysiologic sub-groups: disturbances of ejaculation reflex leading to partial retrograde ejaculation and seminal glands and ducts anatomic and functional anomalies. In this last pathologic mechanism, the mutations of CFTR gene, involved in many different forms of cystic fibrosis, represent a possible cause of hypospermia. The molecular anomaly of CFTR gene's screening is very important for the potential descendents and for the patient himself. It must be considered any time clinic and/or paraclinic context is evocative.

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153 C`est pourquoi, le recours aux kits de recherche de mutations du g&#e8;ne CFTR qui ne permettent de d&#e9;pister qu`une trentaine de mutations impliqu&#e9;es dans les formes s&#e9;v&#e8;res compl&#e8;tes de la mucoviscidose (comme les mutations DF508 (70 %), G542X (3 %), N1303K (2 %). Login to comment

[hide] [The French nationwide cystic fibrosis newborn scr... Arch Pediatr. 2008 Jun;15 Suppl 1:S1-6. doi: 10.1016/S0929-693X(08)73940-X. Munck A, Roussey M
[The French nationwide cystic fibrosis newborn screening program: strategy and results].
Arch Pediatr. 2008 Jun;15 Suppl 1:S1-6. doi: 10.1016/S0929-693X(08)73940-X., [PMID:18822253]

Abstract [show]
In 2002 France implemented a nationwide newborn screening program for cystic fibrosis (CF). The strategy combined immunoreactive trypsinogen and, in case of a value over the cut-off level, DNA analysis in dried blood samples at day 3. Data were centralized and periodically analyzed thus maintaining the percentage of samples requiring mutation analysis (0.6%), limiting the number of false-positive cases (0.1%) without increasing the number of false-negative cases (3.2%). 3.527.353 infants were screened between 2002 and 2006. The overall cystic fibrosis incidence was 1/ 4136 with a wide range of regional variations. Dilemma case presentation occurred for 14 % of the patients; an European working group is actively working on this topic, attempting to establish a consensus on the adequate procedures. Cystic fibrosis newborn screening is feasible all over a nation but needs a strong organization from maternity wards to CF care centers.

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50 L`organigramme du DNN (fig. 1) pr&#e9;voit une valeur seuil de TIR &#e0; J3 d&#e9;termin&#e9;e sur les donn&#e9;es des r&#e9;gions fran&#e7;aises ayant d&#e9;but&#e9; ce d&#e9;pistage il y a plus de 10 ans afin de s&#e9;lec- Mutations recherch&#e9;es par le Kit Elucigen dans le cadre du d&#e9;pistage n&#e9;onatal de la mucoviscidose (Kit CF30) : F508del ; I 507del ; 1078delT, 1717-1 G>A ; 2183AA>G ; 3659delC ; 3849+10kbC>T ; 621+1G>T ; A455E ; E60X ; G542X ; G551D ; N1303K ; R1162X ; R117H ; R334W ; R347P ; R553X ; S1251N ;W1282X ; 1811+1.6kbA>G ; 2789+5G>A ; 3120+1G>A ; 3272-26A>G ; 394delT ; 711+1G>T ; G85E ; Y1092X ; Y122X ;W846X. Login to comment

[hide] Cystic fibrosis in a boy with meconium ileus and m... Turk J Pediatr. 2008 Jul-Aug;50(4):383-5. Yalcin E, Ozcelik U, Yilmaz E, Dogru D, Kiper N, Ferec C
Cystic fibrosis in a boy with meconium ileus and mild clinical phenotype associated with 2183AA-G/D1152H genotype.
Turk J Pediatr. 2008 Jul-Aug;50(4):383-5., [PMID:19014055]

Abstract [show]
We report a 16-year-old boy with cystic fibrosis presenting with meconium ileus in the neonatal period who showed mild clinical phenotype later. He had sufficient pancreatic function, mild lung involvement and borderline sweat chloride levels. Analysis of the cystic fibrosis transmembrane regulator protein gene revealed the rare mutation: 2183AA-G/D1152H. To our knowledge, this is the first report concerning such a mutation combination in cystic fibrosis.

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31 These nine patients carried delF508, G542X, G1244E and 2789+5G-A on the other CF allele. Login to comment

[hide] Pharmaceuticals targeting nonsense mutations in ge... BioDrugs. 2009;23(3):165-74. doi: 10.2165/00063030-200923030-00003. Rowe SM, Clancy JP
Pharmaceuticals targeting nonsense mutations in genetic diseases: progress in development.
BioDrugs. 2009;23(3):165-74. doi: 10.2165/00063030-200923030-00003., [PMID:19627168]

Abstract [show]
Premature termination codons (PTCs) are a cause of numerous genetic disorders spanning diseases that affect children and adults, and are produced by base pair substitutions that create abnormal stop codons within the open reading frame. Several ribosome-binding drugs, including select aminoglycosides and synthetic novel small molecules, induce 'translational readthrough' of PTCs, restoring full-length functional protein in a number of preclinical and clinical settings. In this review, we examine the mechanistic underpinnings of PTC suppression, including the nature of the interactions between agents that suppress PTCs and the eukaryotic ribosome regulation of transcript levels in eukaryotic cells, and the importance of the mRNA context in suppression of PTCs. We also examine results from proof-of-concept studies in preclinical model systems and clinical trials (with a focus on PTC124). Several of the published studies in cystic fibrosis have reported improvements in cystic fibrosis transmembrane conductance regulator (CFTR) biomarkers during short-term evaluation, including topical and systemic aminoglycoside treatment, and oral dosing with PTC124. These results, coupled with our improved understanding of how translation termination is regulated at PTCs, will help guide future directions of research involving this innovative treatment strategy for genetic diseases.

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484 [26] They extended this work to the four most common disease-causing mutations in CFTR (G542X, R553X, R1162X, and W1282X), including studies in an immortalized lower airway cell line (IB3-1) isolated from a CF patient heterozygous for the W1282X CFTR mutation. Login to comment
518 Suppression of PTCs in Animal Models Two mouse models have been developed that possess PTCs in disease-causing genes, including the mdx mouse (a model of muscular dystrophy) and the transgenic G542X-hCFTR mouse (a model of CF). Login to comment
522 Complementary studies in the G542X-hCFTR mouse (expressing the human G542X CFTR cDNA under regulatory control of the FABP promoter on a Cftr knockout background) confirmed that gentamicin treatment increased the detection of full-length CFTR protein in the gut (the site of CF-related mortality in this genetic model) and was associated with the appearance of cAMP-stimulated Cl-conductance in isolated gut tissue studied ex vivo. Login to comment
524 Follow-up studies confirmed these effects and extended the findings to clinically relevant doses of both gentamicin and amikacin; amikacin suppressed the G542X-hCFTR premature stop mutation more effectively than gentamicin at clinically relevant doses. Login to comment
530 [24] In the G542X-hCFTR mouse, oral and intraperitonealadministration led to detectable full-length CFTR localization at the apical cell membrane of gut glandular cells by immunofluorescent staining, and improved Cl-conductance as assayed by transepithelial ion transport. Login to comment
571 A rank order of Y122X, W1282X, G542X, and R1162X was seen among the most frequent stop mutations after gentamicin treatment, with relative suppression varying by as much as 7.2-fold, post-therapy. Login to comment
585 [60] The detection of rescued CFTR activity in several patients with the G542X CFTR mutation (and others) in the studies conducted in Israel[55] and France/ Belgium,[64] suggests that the treatment effect is not limited to individuals with the W1282X mutation and that the agent can potentially exhibit a broad spectrum of activity across multiple PTCs in vivo. Login to comment

[hide] Frequency of 8 CFTR gene mutations in cystic fibro... Braz J Med Biol Res. 2010 Feb;43(2):134-8. Epub 2010 Jan 15. Perone C, Medeiros GS, del Castillo DM, de Aguiar MJ, Januario JN
Frequency of 8 CFTR gene mutations in cystic fibrosis patients in Minas Gerais, Brazil, diagnosed by neonatal screening.
Braz J Med Biol Res. 2010 Feb;43(2):134-8. Epub 2010 Jan 15., [PMID:20098842]

Abstract [show]
The nature and frequency of cystic fibrosis mutations in Brazil is not uniform due to the highly varied ethnic composition of the population. The average frequency of the F508del mutation has been reported to be 48.6%. Other common mutations in Brazil are G542X, R1162X, and N1303K. The aim of this study was to analyze the frequency of 8 mutations (F508del, G542X, R1162X, N1303K, W1282X, G85E, 3120+1G>A, and 711+1G>T) in a sample of 111 newborn patients with cystic fibrosis diagnosed by the Cystic Fibrosis Neonatal Screening Program of Minas Gerais State. The mutations were tested by allele-specific oligonucleotide PCR with specially designed primers. An allele frequency of 48.2% was observed for the F508del mutation, and allele frequencies of 5.41, 4.50, 4.05, and 3.60% were found for the R1162X, G542X, 3120+1G>A, and G85E mutations, respectively. The genotypes obtained were in Hardy-Weinberg equilibrium. These data demonstrate that the 8-mutation panel studied here has extensive coverage (68%) for the cystic fibrosis mutations in Minas Gerais. These data improve our knowledge of cystic fibrosis in Brazil, particularly in this region. In addition, this investigation contributed to the establishment of a sensitive and population-specific mutation panel, which can be helpful for molecular diagnosis of cystic fibrosis.

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2 Other common mutations in Brazil are G542X, R1162X, and N1303K. Login to comment
3 The aim of this study was to analyze the frequency of 8 mutations (F508del, G542X, R1162X, N1303K, W1282X, G85E, 3120+1G>A, and 711+1G>T) in a sample of 111 newborn patients with cystic fibrosis diagnosed by the Cystic Fibrosis Neonatal Screening Program of Minas Gerais State. Login to comment
5 An allele frequency of 48.2% was observed for the F508del mutation, and allele frequencies of 5.41, 4.50, 4.05, and 3.60% were found for the R1162X, G542X, 3120+1G>A, and G85E mutations, respectively. Login to comment
16 Other mutations such as G542X, G551D and N1303K are commonly found throughout the world, depending on geographical and ethnic features (5). Login to comment
18 Other common mutations in this region are G542X, N1303K, W1282X, and R1162X (6). Login to comment
26 Despite the large variations in ethnicbackgroundamongdifferentregions,G542X,N1303K and R1162X have been considered to be the most frequent non-F508del mutations in Brazil (6,11,13). Login to comment
29 The objective of the present investigation was to determine the frequency of 8 CFTR mutations (G85E, 711+1G>T, F508del, G542X, 3120+1G>A, R1162X, W1282X, and N1303K) in 111 sweat test-positive newborns screened by the CFNS program in the State of Minas Gerais, Brazil. Login to comment
43 The mutations are: G85E, 711+1G>T, F508del, G542X, 3120+1G>A, R1162X, W1282X, and N1303K. Login to comment
47 Mutation Primer sequence (5` ࢐ 3`) Amplicon size (bp) Annealing temperature (&#b0;C) G85E-S GGA GAT TTA TGT TCT ATG G 245 52 G85E-M GGA GAT TTA TGT TCT ATG A G85E-R GTA AAT TGC CAC CCG TGT TCC AGG 711+1G>T-S CCA ACA ACC TGA ACA AAT TTG ATG AAG 340 64 711+1G>T-M CCA ACA ACC TGA ACA AAT TTG ATG AAT 711+1G>T-R TTG CTC AGG TAT CAT ATC TGG CC F508del-S ACC ATT AAA GAA AAT ATC ATC TT 262 54 F508del-M ACC ATT AAA GAA AAT ATC ATT GG F508del-R TGC AAG CTT CTT AAA GCA TA G542X-S GCA GAG AAA GAC AAT ATA GTT CTT G 213/217 58 G542X-M GTT TGC AGA GAA AGA CAA TAT AGT TCT TTT G542X-R CCA CTA GCC ATA AAA CCC CAG G 3120+1G>A-S CTT ACC ATA TTT GAC TTC ATC CAG G 191 62 3120+1G>A-M CTT ACC ATA TTT GAC TTC ATC CAG A 3120+1G>A-R TTA CTA AAC TTA TGT CTA TTT TGA AGG C R1162X-S TTA TTT CAG ATG CGA TCT GTG AGC C 117 63 R1162X-M TTA TTT CAG ATG CGA TCT GTG AGC TT R1162X-R AAT CAT AAC TTT CGA GAG TTG GCC W1282X-S GGG ATT CAA TAA CTT TGC AAC AGT GG 203 67 W1282X-M GGG ATT CAA TAA CTT TGC AAC AGT GA W1282X-R TCT GCC TAT GAG AAA ACT GCA CTG GAG N1303K-S TTT TTT CTG GAA CAT TTA GAA AAA AC 137 58 N1303K-M TTT TTT CTG GAA CAT TTA GAA AAA AG N1303K-R GCC ATT TGT GTT GGT ATG AGT TAC CCC The -S suffix indicates a wild allele specific primer, the -M suffix a mutant allele primer, and the -R suffix the primer used in both wild and mutant allele amplification. Login to comment
63 R1162X, G542X, 3120+1G>A, and G85E were the most frequent non-F508del mutations observed, comprising 12 (5.4%), 10 (4.5%), 9 (4.1%), and 8 (3.6%) of the chromosomes studied. Login to comment
69 28 (25.23%) 34.38 (30.97%) F508del / G542X 5 (4.50%) 4.84 (4.36%) F508del / 3120+1G>A 4 (3.60%) 4.36 (3.93%) F508del / R1162X 4 (3.60%) 5.81 (5.23%) F508del / G85E 4 (3.60%) 3.87 (3.49%) F508del / 711+1G>T 2 (1.80%) 0.97 (0.87%) F508del / W1282X 1 (0.90%) 0.48 (0.43%) F508del / N1303K 1 (0.90%) 0.97 (0.87%) G542X / ? Login to comment
84 Mutation N Frequency (%) Cumulative frequency (%) G85E 8 3.60 3.60 711+1G>T 2 0.90 4.50 F508del 107 48.20 52.70 G542X 10 4.50 57.20 3120+1G>A 9 4.05 61.25 R1162X 12 5.41 66.66 W1282X 1 0.45 67.11 N1303K 2 0.90 68.01 Unknown alleles 71 31.99 Total 222 100.00 100.00 N = number of observed alleles. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Drugs. 2010 Feb 12;70(3):241-59. doi: 10.2165/11316160-000000000-00000. Becq F
Cystic fibrosis transmembrane conductance regulator modulators for personalized drug treatment of cystic fibrosis: progress to date.
Drugs. 2010 Feb 12;70(3):241-59. doi: 10.2165/11316160-000000000-00000., [PMID:20166764]

Abstract [show]
This article considers the issue of personalized drug discovery for the orphan disease cystic fibrosis (CF) to deliver a candidate for therapeutic development. CF is a very complicated disease due to numerous anomalies of the gene leading to progressive severity and morbidity. Despite extensive research efforts, 20 years after the cloning of the CF gene, CF patients are still waiting for a curative treatment as prescribed medications still target the secondary manifestations of the disease rather than the gene or the CF transmembrane conductance regulator (CFTR) protein. New therapeutics aimed at improving mutant CFTR functions, also known as 'protein repair therapy' are nevertheless hoped and predicted to replace some of the currently used therapy, while improving the quality of life as well as life expectancy of CF patients. Although there is substantial variability in the cost of treating CF between countries, a protein repair therapy should also alleviate the financial burden of medical costs for CF patients and their families. Finding new drugs or rediscovering old ones for CF is critically dependent on the delivery of molecular and structural information on the CFTR protein, on its mutated version and on the network of CFTR-interacting proteins. The expertise needed to turn compounds into marketable drugs for CF will depend on our ability to provide biological information obtained from pertinent models of the disease and on our success in transferring safe molecules to clinical trials. Predicting a drug-induced response is also an attractive challenge that could be rapidly applied to patients.

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67 [25,26] Besides F508del, other frequent mutations are found in North African CF patients, in particular W1282X, G542X, R1162X and N1303K. Login to comment
97 Class I includes nonsense, frame shift and splice site mutations (e.g. stop mutations: G542X, R553X, W1282X) leading to unstable transcripts and failure of CFTR translation. Login to comment
133 [47] In a prospective phase II clinical trial on CF patients selected for expressing CFTR variants with a class I mutation (G542X, W1282X), oral administration of ataluren reduced the epithelial electrophysiological abnormalities caused by CFTR channel dysfunction. Login to comment

[hide] [Cystic fibrosis in a woman aged seventy]. Ned Tijdschr Geneeskd. 2010;154:A1342. Ras JE, van Velzen E, van Berkhout FT, van den Brand JJ
[Cystic fibrosis in a woman aged seventy].
Ned Tijdschr Geneeskd. 2010;154:A1342., [PMID:20619026]

Abstract [show]
A seventy-year-old woman was admitted to hospital with a Staphylococcus aureus respiratory tract infection. She had a history of extensive bronchiectasis and allergic bronchopulmonary aspergillosis (ABPA). Cystic fibrosis (CF) was suspected and cystic fibrosis transmembrane conductance regulator (CFTR) gene analysis showed F508del and R117H-7T mutations. In these mutations there is residual activity in the chloride channel in the cell membrane coded by the CFTR gene. This results in a much milder disease pattern varying from no disease at all to isolated organ disease. This type of disease is known as non-classical cystic fibrosis. In our patient the diagnosis of cystic fibrosis was made exceptionally late in life.

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63 TABEL 1 Classificatie van mutaties in het 'cystic fibrosis transmembrane conductance regulator`(CFTR)-gen op chromosoom 7 klasse mechanisme enkele bekende mutaties I geen synthese van het CFTR-eiwit G542X R553X W1282X R1162X 621-1G࢐T 1717-1G࢐A 1078࢞T 3659࢞C II defect in eiwitrijping met voortijdig afbraak ࢞F508 ࢞I507 N1303K S549N III verstoorde regulatie van de CFTR-functie G551D R56OT IV verstoorde conductie van chloride of verstoorde kanaalopening R117H R334W G85E R347P V minder synthese van het CFTR-eiwit 3849+10KbC࢐T 2789+5G࢐A A455E TABEL 2 Diagnostiek van cystische fibrose test testuitslag klassieke CF* niet-klassieke CFߤ zweettest chlorideconcentratie > 60 mmol/l chlorideconcentratie ࣘ 60 mmol/l neuspotentiaalmeting afwijkend niet-afwijkend CFTR-mutatie-analyse 2 mutaties 2 mutaties CF = cystische fibrose; CFTR = 'cystic fibrosis transporter regulator`-gen. Login to comment

[hide] Newborn screening for cystic fibrosis in Alberta: ... Paediatr Child Health. 2010 Nov;15(9):590-4. Lilley M, Christian S, Hume S, Scott P, Montgomery M, Semple L, Zuberbuhler P, Tabak J, Bamforth F, Somerville MJ
Newborn screening for cystic fibrosis in Alberta: Two years of experience.
Paediatr Child Health. 2010 Nov;15(9):590-4., [PMID:22043142]

Abstract [show]
On April 1, 2007, Alberta became the first province in Canada to introduce cystic fibrosis (CF) to its newborn screening program. The Alberta protocol involves a two-tier algorithm involving an immunoreactive trypsinogen measurement followed by molecular analysis using a CF panel for 39 mutations. Positive screens are followed up with sweat chloride testing and an assessment by a CF specialist. Of the 99,408 newborns screened in Alberta during the first two years of the program, 221 had a positive CF newborn screen. The program subsequently identified and initiated treatment in 31 newborns with CF. A relatively high frequency of the R117H mutation and the M1101K mutation was noted. The M1101K mutation is common in the Hutterite population. The presence of the R117H mutation has created both counselling and management dilemmas. The ability to offer CF transmembrane regulator full sequencing may help resolve diagnostic dilemmas. Counselling and management challenges are created when mutations are mild or of unknown clinical significance.

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46 These include the following mutations: delF508, I507del, G542X, G85E, R117H, 621+1G࢐T, 711+1G࢐T, G551D, R334W, R347P, A455E, 1717-1G࢐A, R560T, R553X, N1303K, 1898+1G࢐A, 2184delA, 2789+5G࢐A, 3120+1G࢐A, R1162X, 3659delC, 3849+10kbC࢐T, W1282X, 1078delT, 394delTT, Y122X, R347H, V520F, A559T, S549N, S549R, 1898+5G࢐T, 2183AA࢐G, 2307insA, Y1092X, M1101K, S1255X, 3876delA and 3905insT. Login to comment

[hide] [Emergencies in adult mucoviscidosis patients]. Internist (Berl). 2012 May;53(5):575-84. doi: 10.1007/s00108-012-3019-4. Smaczny C, Born T, Wagner TO
[Emergencies in adult mucoviscidosis patients].
Internist (Berl). 2012 May;53(5):575-84. doi: 10.1007/s00108-012-3019-4., [PMID:22527665]

Abstract [show]
Cystic fibrosis is an inherited autosomal recessive metabolic disease caused by mutations on the CFTR gene. This leads to defective chloride channels on epithelial cell membranes and causes various disorders of the respiratory, gastrointestinal, and urogenital tracts.As a result, all exocrine glands produce a viscous secretion, leading to pulmonary symptoms such as chronic cough, secretion retention, recurring infections as well as bronchiectasis and obstructive lung emphysema. Gastrointestinal effects include exocrine and often also endocrine pancreatic insufficiency with chronic diarrhea and maldigestion syndrome as well as pancreoprivic diabetes mellitus; biliary cirrhosis occurs in 10% of cases. Additional effects include reduced fertility in women and infertility in men.Life-threatening complications include bleeding from the bronchial arteries, pneumothorax, and distal intestinal obstruction syndrome (DIOS), previously known as meconium ileus equivalent. Treatment requires rapid diagnosis and should be carried out in experienced centres, since the mortality rate can otherwise be up to 50%.

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19 Bei Neugeborenen kommt es typischerweise zu einem Tab. 1ߓ Typische Mutationen bei der Mukoviszidose Mutationsklasse Defektbeschreibung Beispiele (alte Nomenklatur) I Vollst&#e4;ndigerVerlust der CFTR-Protein- synthese R553X, G542X, N1303K, Êf; 1717-1 G &#e1;ߙ A II St&#f6;rung der Reifung und des intrazellul&#e4;- renTransports des CFTR-Proteins DF508 III Regulationsst&#f6;rung des Ionenkanals G551D IV St&#f6;rung der Ionenleitf&#e4;higkeit des CFTR-Kanals R347H, R117H V Verminderte CFTR-Konzentration in der Zelle 3849+10kBÊf;C &#e1;ߙ T VI Beschleunigter CFTR-Abbau ߕ CFTRÉe;Cystic fibrosis transmembrane conductance regulator". Login to comment

[hide] Suppression of premature termination codons as a t... Crit Rev Biochem Mol Biol. 2012 Sep;47(5):444-63. doi: 10.3109/10409238.2012.694846. Epub 2012 Jun 7. Keeling KM, Wang D, Conard SE, Bedwell DM
Suppression of premature termination codons as a therapeutic approach.
Crit Rev Biochem Mol Biol. 2012 Sep;47(5):444-63. doi: 10.3109/10409238.2012.694846. Epub 2012 Jun 7., [PMID:22672057]

Abstract [show]
In this review, we describe our current understanding of translation termination and pharmacological agents that influence the accuracy of this process. A number of drugs have been identified that induce suppression of translation termination at in-frame premature termination codons (PTCs; also known as nonsense mutations) in mammalian cells. We discuss efforts to utilize these drugs to suppress disease-causing PTCs that result in the loss of protein expression and function. In-frame PTCs represent a genotypic subset of mutations that make up ~11% of all known mutations that cause genetic diseases, and millions of patients have diseases attributable to PTCs. Current approaches aimed at reducing the efficiency of translation termination at PTCs (referred to as PTC suppression therapy) have the goal of alleviating the phenotypic consequences of a wide range of genetic diseases. Suppression therapy is currently in clinical trials for treatment of several genetic diseases caused by PTCs, and preliminary results suggest that some patients have shown clinical improvements. While current progress is promising, we discuss various approaches that may further enhance the efficiency of this novel therapeutic approach.

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218 The G542X mutation, found in 2% of Caucasian CF patients, is the most common CFTR nonsense mutation. Login to comment
234 Studies to examine suppression of a CFTR nonsense mutation utilized a transgenic mouse that carried a human CFTR cDNA containing the G542X nonsense mutation. Login to comment
236 The CFTR-G542X transgene was then crossed into a Cftrtm1Cam knockout mouse, resulting in a mouse that was suitable to evaluate the ability of suppression therapy to restore CFTR protein function (Du et al., 2002). Login to comment
244 In summary, a number of different drugs were found to suppress the common G542X mutation in a transgenic CF mouse model. Login to comment
518 Aminoglycoside suppression of a premature stop mutation in a Cftr-/- mouse carrying a human CFTR-G542X transgene. Login to comment
521 Clinical doses of amikacin provide more effective suppression of the human CFTR-G542X stop mutation than gentamicin in a transgenic CF mouse model. Login to comment
524 PTC124 is an orally bioavailable compound that promotes suppression of the human CFTR-G542X nonsense allele in a CF mouse model. Login to comment
531 Poly-l-aspartic acid enhances and prolongs gentamicin-mediated suppression of the CFTR-G542X mutation in a cystic fibrosis mouse model. Login to comment

[hide] [Genetics and modifier genes, atypical and rare fo... Arch Pediatr. 2012 May;19 Suppl 1:S3-7. doi: 10.1016/S0929-693X(12)71099-0. Ferec C, Scotet V, Beucher J, Corvol H
[Genetics and modifier genes, atypical and rare forms].
Arch Pediatr. 2012 May;19 Suppl 1:S3-7. doi: 10.1016/S0929-693X(12)71099-0., [PMID:22682487]

Abstract [show]
Cystic fibrosis (CF) is defined as the most common life shortening genetic disorder in the Caucasian populations. The cloning of the gene responsible for the disease - the CFTR (Cystic Fibrosis Transmembrane conductance Regulator) gene - twenty years ago has greatly improved our knowledge of the pathophysiology of CF. That disease is characterized by a highly phenotypic variability and the CFTR mutations cannot explain all the variability observed in the disease severity. The possible influence of the environment and modifier genes has therefore been evocated. Several genetic variants coding for genes involved in the physiopathology of the disease have been studied, like genes involve in the immunity and the inflammatory response. Some of these genes have indeed been shown to influence the disease severity. A new approach has also been developed, analyzing the whole genome. This review summarizes the genetic basis of CF in its classical and atypical forms, as well as the work performed in the field of modifier genes.

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39 La mutation G542X est fr&#e9;quente dans les populations du pourtour m&#e9;diterran&#e9;en. Login to comment

[hide] Gastrointestinal complications of cystic fibrosis. Clin Gastroenterol Hepatol. 2013 Apr;11(4):333-42; quiz e30-1. doi: 10.1016/j.cgh.2012.11.006. Epub 2012 Nov 8. Gelfond D, Borowitz D
Gastrointestinal complications of cystic fibrosis.
Clin Gastroenterol Hepatol. 2013 Apr;11(4):333-42; quiz e30-1. doi: 10.1016/j.cgh.2012.11.006. Epub 2012 Nov 8., [PMID:23142604]

Abstract [show]
The cystic fibrosis transmembrane regulator protein (CFTR) is an ion channel in the apical surface of epithelial membranes that regulates other ion channels. Dysfunction of CFTR leads to the clinical entity of CF when mutations in CFTR are inherited in an autosomal recessive fashion. Although airway obstruction, inflammation, and infection are usually the most serious consequences of CFTR dysfunction because they lead to respiratory failure, CFTR dysfunction affects the intestinal tract and the pancreatic and hepatobiliary ducts in a similar fashion, leading to significant morbidity. This review outlines pathophysiology and common gastrointestinal ailments in the CF population along with current medical and surgical management.

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20 The most common genotypes in infants with MI are severe mutations (F508del, G542X, W1282X, R553X, G551D), although most patients with these mutations do not present with MI.9 Recent genome-wide association studies have been able to account for approximately 17% of the phenotypic variability,14 implying that other non-CFTR genetic factors also contribute to this clinical presentation. Login to comment

[hide] [Mucoviscidosis: CFTR mutation-specific therapy: a... Arch Pediatr. 2013 Jan;20(1):63-73. doi: 10.1016/j.arcped.2012.10.018. Epub 2012 Nov 27. Leonard A, Leal T, Lebecque P
[Mucoviscidosis: CFTR mutation-specific therapy: a ray of sunshine in a cloudy sky].
Arch Pediatr. 2013 Jan;20(1):63-73. doi: 10.1016/j.arcped.2012.10.018. Epub 2012 Nov 27., [PMID:23199563]

Abstract [show]
There is a need to find a cure for pulmonary disease in cystic fibrosis (CF), though full benefit of this approach will be restricted to those patients with well-preserved lungs. The most promising route is currently that of a pharmacological mutation-specific approach aiming at correcting the mechanism by which mutations lead to impairment of chloride conductance across respiratory epithelial cells. In the past 14years, 7 candidate drugs (CPX, 4PBA, gentamicin, PTC124, VX-770 or Ivacaftor, VX-809 or Lumacaftor, and Miglustat) have been investigated in CF patients. A postulate of 14 out of the 15 published studies has been that an effective agent had to improve total chloride secretion as assessed in vivo by nasal potential difference measurements. The present review casts a critical look at these studies. Apparent inconsistencies are discussed as well as possible limitations of nasal potential difference measurements as outcome parameters in these trials. Primarily targeting a mutation carried by less than 2% of French CF patients, the 2 Ivacaftor studies could well be a milestone on the long road toward a cure for CF. However, further data on safety and long-term efficacy are obviously needed and the current price of this medication in the US would make it unaffordable for European patients.

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125 Gentamicine par voir intraveineuse Une premie `re e &#b4;tude a e &#b4;value &#b4; les taux de chlorure dans la sueur et le PN - a ` 6 reprises - chez 5 patients porteurs d`une seule mutation de classe I (dont 3 G542X) et 5 patients homozygotes F508del [37]. Login to comment
130 Dans une partie distincte du travail, les auteurs de &#b4;montraient in vitro une translecture en pre &#b4;sence de gentamicine 4 a ` 7 fois plus efficace pour la mutation Y122X que pour les mutations W1282X, G542X et R1162X. Login to comment
317 [21] Du M, Liu X, Welch EM, et al. PTC124 is an orally bioavailable compound that promotes suppression of the human CFTR-G542X nonsense allele in a CF mouse model. Login to comment

[hide] Genetics of pancreatitis with a focus on the pancr... Minerva Gastroenterol Dietol. 2012 Dec;58(4):299-308. Larusch J, Whitcomb DC
Genetics of pancreatitis with a focus on the pancreatic ducts.
Minerva Gastroenterol Dietol. 2012 Dec;58(4):299-308., [PMID:23207607]

Abstract [show]
Genetic risk for acute pancreatitis (AP), recurrent acute pancreatitis (RAP) and chronic pancreatitis (CP) are increasingly recognized. The exocrine pancreas is composed of both acinar cells and duct cells, with genetic factors associated with AP, RAP and CP linked to one cell type or the other. Increased susceptibility to pancreatitis occurs when the normal physiological mechanisms that allow the pancreas to respond to common stresses or injury are altered. Currently, most our knowledge about genetics focuses on three genes that play critical roles in pancreatic function (PRSS1, CFTR, SPINK1) such that isolated defects lead to disease. However, recent data suggest that more complex combination of genetic and environmental factors are also as important, or more important than Mendelian genetic risk. Understanding of complex interactions requires modeling of these factors so that the response to stresses or injury can be simulated and critical interactions understood. A simple duct cell model is given to illustration the relationship between CFTR, CASR, aquaporins, claudins, and SPINK1, and how they interact. The role of CFTR variants in pancreatic diseases is then discussed.

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119 These drugs have been reported and reviewed in a number of articles in detail, both in ongoing clinical trials and approved by FDA F508del and G551D (65), G542X, W1282X and all other premature termination mutations (66, 67), leading us to speculate on the impact or utility of these drugs on pancreatitis. Login to comment

[hide] Assessing the Disease-Liability of Mutations in CF... Cold Spring Harb Perspect Med. 2012 Dec 1;2(12):a009480. doi: 10.1101/cshperspect.a009480. Ferec C, Cutting GR
Assessing the Disease-Liability of Mutations in CFTR.
Cold Spring Harb Perspect Med. 2012 Dec 1;2(12):a009480. doi: 10.1101/cshperspect.a009480., [PMID:23209179]

Abstract [show]
Over 1900 mutations have been reported in the cystic fibrosis transmembrane conductance regulator (CFTR), the gene defective in patients with cystic fibrosis. These mutations have been discovered primarily in individuals who have features consistent with the diagnosis of CF. In some cases, it has been recognized that the mutations are not causative of cystic fibrosis but are responsible for disorders with features similar to CF, and these conditions have been termed CFTR-related disorders or CFTR-RD. There are also mutations in CFTR that do not contribute to any known disease state. Distinguishing CFTR mutations according to their penetrance for an abnormal phenotype is important for clinical management, structure/function analysis of CFTR, and understanding the molecular and cellular mechanisms underlying CF.

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37 Worldwide, only a limited number of mutant alleles display a frequency higher than 1%, as for example the G542X (p.Gly542X), which is common in the Mediterranean area of Europe and Africa, which may be associated with the Phoenician migration around the Mediterranean Sea (Loirat et al. 1997). Login to comment
41 In North America, the distribution of CFTR mutations reflects European descent (the five more common mutations in the U.S. with afrequencyover 1% are F508del, G542X, G551D, W1282X, and N1303K) (Bobadilla et al. 2002). Login to comment
55 Mutations in CFTR including G542X, R553X (p.Arg553X), and W1282X have been shown to lead to NMRD in primary airway cells (Hamosh et al. 1991, 1992b; Will et al. 1995). Login to comment
93 On identification of the CFTR gene, it was shown that F508del and several other loss-of-function mutations including G551D and G542X are highly associated with pancreatic insufficiency. Login to comment
114 PI-CF patients bearing nonsense mutation G542X have shown the highest rate of MI, whereasthosewith F508del have an intermediate rate, and patients with the G551D mutation have a relatively low rate of MI (Hamosh et al. 1992a; Kristidis et al. 1992; Feingold and Guilloud-Bataille 1999). Login to comment

[hide] Distribution of CFTR mutations in the Czech popula... J Cyst Fibros. 2013 Sep;12(5):532-7. doi: 10.1016/j.jcf.2012.12.002. Epub 2012 Dec 29. Krenkova P, Piskackova T, Holubova A, Balascakova M, Krulisova V, Camajova J, Turnovec M, Libik M, Norambuena P, Stambergova A, Dvorakova L, Skalicka V, Bartosova J, Kucerova T, Fila L, Zemkova D, Vavrova V, Koudova M, Macek M, Krebsova A, Macek M Jr
Distribution of CFTR mutations in the Czech population: positive impact of integrated clinical and laboratory expertise, detection of novel/de novo alleles and relevance for related/derived populations.
J Cyst Fibros. 2013 Sep;12(5):532-7. doi: 10.1016/j.jcf.2012.12.002. Epub 2012 Dec 29., [PMID:23276700]

Abstract [show]
BACKGROUND: This two decade long study presents a comprehensive overview of the CFTR mutation distribution in a representative cohort of 600 Czech CF patients derived from all regions of the Czech Republic. METHODS: We examined the most common CF-causing mutations using the Elucigene CF-EU2v1 assay, followed by MLPA, mutation scanning and/or sequencing of the entire CFTR coding region and splice site junctions. RESULTS: We identified 99.5% of all mutations (1194/1200 CFTR alleles) in the Czech CF population. Altogether 91 different CFTR mutations, of which 20 were novel, were detected. One case of de novo mutation and a novel polymorphism was revealed. CONCLUSION: The commercial assay achieved 90.7%, the MLPA added 1.0% and sequencing increased the detection rate by 7.8%. These comprehensive data provide a basis for the improvement of CF DNA diagnostics and/or newborn screening in our country. In addition, they are relevant to related Central European populations with lower mutation detection rates, as well as to the sizeable North American "Bohemian diaspora".

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49 Results Altogether we found 91 different CFTR mutations (Table 1), with only seven being present at a frequency N1%: F508del (67.42%), CFTRdele2,3(21kb) (5.75%), G551D (2.92%), N1303K (2.42%), G542X (2.0%), 3849+10kbCNT (1.67%) and 1898+1GNA (1.42%) (using the legacy/traditional nomenclature). Login to comment
73 Similarly, we presume that the "Mediterranean" mutation G542X [8] is, according to historical patterns of CE colonization [17], the most prevalent in the Danube basin, i.e. in SK, HU, AT and DE. Login to comment
89 Mutations/HGVS nomenclature/ Mutations/traditional nomenclature, legacy name/ Czech Republic 2012 (this study) (N=1200) Slovakia 2010 (N=856) Eastern Hungary 2011 (N=80) Germany Bavaria 2002 (N=250) Austria Tyrol 1997 (N=126) Austria NorthEast, North- North 2002 (N=118) Poland (N=1726) c.1521_1523delCTT F508del 67.42 66.80 70.00 74.00 74,60 70.30 57.0 c.54-5940_273+10250del21 kb CFTRdele2,3/21kb 5.75 2.26 5.00 1.2* 2.6# NA 1.80 c.1652GNA G551D 2.91 b0.50 0.00 6.40 1.60 2.50 0.50 c.3909CNG N1303K 2.42 2.03 5.00 2.40 0.00 NA 1.80 c.1624GNT G542X 2.00 4.06 3.75 3.20 2.40 5.10 2.60 c.3718-2477CNT 3849+10kbCNT 1.67 4.28 0.00 NA 0.00 3.40 2.70 c.1766+1GNA 1898+1GNA 1.42 b0.50 0.00 NA 0.00 NA NA c.1040GNC R347P 0.92 1.10 1.25 0.80 1.60 2.50 NA c.2012delT 2143delT 0.92 1.10 0.00 NA 0.00 NA NA c.3140-26ANG 3272-26ANG 0.67 b0.50 0.00 NA 0.00 NA NA c.3846GNA W1282X 0.58 b0.50 0.00 NA 0.00 NA 0.70 c.1007TNA I336K 0.58 0.00 0.00 NA 0.00 NA NA c.1657CNT R553X 0.50 0.90 0.00 1.20 0.00 NA 1.90 c.2657+5GNA 2789+5GNA 0.50 0.00 0.00 NA 2.40 NA NA c.2834CNT S945L 0.50 0.00 0.00 NA 0.00 NA NA c.2052_2053insA 2184insA 0.42 1.58 5.00 NA 0.00 NA NA Legend: data for Slovakia [12], Eastern Hungary [14], Germany-Bavaria [13], Austria-Tyrol [18], Austria North East and North West [13], Poland and *[8], and # [16]. Login to comment

[hide] CFTR-mutation specific applications of CFTR-direct... J Cyst Fibros. 2013 Sep;12(5):487-96. doi: 10.1016/j.jcf.2012.12.005. Epub 2013 Jan 11. van Meegen MA, Terheggen SW, Koymans KJ, Vijftigschild LA, Dekkers JF, van der Ent CK, Beekman JM
CFTR-mutation specific applications of CFTR-directed monoclonal antibodies.
J Cyst Fibros. 2013 Sep;12(5):487-96. doi: 10.1016/j.jcf.2012.12.005. Epub 2013 Jan 11., [PMID:23317763]

Abstract [show]
BACKGROUND: Over the last decade novel monoclonal CFTR-specific antibodies have been developed. We here present a paired analysis to detect wild-type and mutant CFTR using Western blot analysis, flow cytometry and confocal microscopy in several cellular expression systems. METHODS: The following CFTR-specific antibodies were used; 217, 432, 450, 570, 769, 596, 660, L12B4 and 24.1. Mutant CFTR was detected in HEK293 cells transiently expressing the mutations; G542X, R1162X, F508del, N1303K, G551D, R117H, A455E. RESULTS: The majority of these antibodies are suitable for most applications tested. Using immunofluorescence, some antibodies can better detect mutant forms of CFTR (F508del and N1303K by mAbs 596 and 769), or display lower aspecific detection by Western blot analysis (mAbs 432, 450, 769 and 596) or immunofluorescence (mAbs 432, 450, 570 and 769). CONCLUSION: Optimal detection of CFTR by monoclonal antibodies depends on CFTR mutation and the specific research application.

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3 Mutant CFTR was detected in HEK293 cells transiently expressing the mutations; G542X, R1162X, F508del, N1303K, G551D, R117H, A455E. Login to comment
14 Class I mutations such as G542X and R1162X encode for premature stop codons and affect CFTR protein translation [6]. Login to comment
76 We selected wt-CFTR, F508del (class II), G542X (I), R1162X (I), G551D (III), R117H (IV), A455E (V), and N1303K (II) that were ectopically expressed in HEK293 cells. Login to comment
79 G542X could only be detected by L12B4 (50 kDa) and R1162X was detected around 110 kDa but not using NBD2 and C-terminal-specific mAbs. Login to comment
111 G551D was expressed at levels comparable to wt-CFTR, expression of R117H, N1303K, A455E and R1162X was intermediate, and low levels were observed for F508del and G542X. Login to comment
114 As expected, expression levels for R1162X were similar to empty vector, however, some expression was consistently observed for G542X, possibly due to production of an alternative transcript from this cDNA construct downstream of position 542. Login to comment
145 Western blots containing 25 bc;g of total protein of HEK293 cells transiently transfected with pcDNA3 containing; CFTR-wt, F508del, G542X, R1162X, G551D, R117H, A455E, N1303K or empty vector. Login to comment
153 Mutant CFTR G542X and F508del could not be detected by mAb 450. Login to comment
155 Detection of CFTR was limited for F508del and A455E and CFTR mutants G542X and R1162X were not detectable. Login to comment

[hide] Impaired CFTR function in mild cystic fibrosis ass... J Cyst Fibros. 2013 Dec;12(6):821-5. doi: 10.1016/j.jcf.2012.12.014. Epub 2013 Jan 27. Sorio C, Angiari C, Johansson J, Verze G, Ettorre M, Buffelli M, Castellani C, Assael BM, Melotti P
Impaired CFTR function in mild cystic fibrosis associated with the S977F/T5TG12complex allele in trans with F508del mutation.
J Cyst Fibros. 2013 Dec;12(6):821-5. doi: 10.1016/j.jcf.2012.12.014. Epub 2013 Jan 27., [PMID:23361109]

Abstract [show]
BACKGROUND: The S977F mutation (c.2930C>T) in the CFTR gene (CFTR/ABCC7) is extremely rare. We describe the case of an adult patient carrying the complex allele S977F/T5TG12 in trans with the F508del mutation. Mild respiratory manifestations arose in adulthood associated with azoospermia, acute pancreatitis, minor hemoptysis and Cl(-) levels ranging from 40 to 42 mEq/L. METHOD: Diagnosis was confirmed by repeated NPD measurements, genetic DHPLC analysis and a recently described functional assay measuring cAMP-dependent cell depolarization in peripheral blood monocytes. RESULTS: NPD measurements, DHPLC and monocyte functional assay (CF index=-18). Results were consistent with a CF phenotype. CONCLUSIONS: The combined application of DHPLC and NPD analysis in the algorithm for CF diagnosis appears useful for the management of similar cases. In addition, the novel monocyte functional assay might contribute to improve our diagnostic capability, counseling and better treatment of these challenging clinical cases.

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39 This prediction is consistent with the case reported in the literature, where a subject carrying the S977F mutation, in addition to a "severe" classic CF-causing mutation on the other allele (CFTR genotype G542X/S977F), presented with clinical evidence of CF [9]. Login to comment

[hide] CFTR p.Arg117His associated with CBAVD and other C... J Med Genet. 2013 Apr;50(4):220-7. doi: 10.1136/jmedgenet-2012-101427. Epub 2013 Feb 1. Thauvin-Robinet C, Munck A, Huet F, de Becdelievre A, Jimenez C, Lalau G, Gautier E, Rollet J, Flori J, Nove-Josserand R, Soufir JC, Haloun A, Hubert D, Houssin E, Bellis G, Rault G, David A, Janny L, Chiron R, Rives N, Hairion D, Collignon P, Valeri A, Karsenty G, Rossi A, Audrezet MP, Ferec C, Leclerc J, Georges Md, Claustres M, Bienvenu T, Gerard B, Boisseau P, Cabet-Bey F, Cheillan D, Feldmann D, Clavel C, Bieth E, Iron A, Simon-Bouy B, Izard V, Steffann J, Viville S, Costa C, Drouineaud V, Fauque P, Bi
CFTR p.Arg117His associated with CBAVD and other CFTR-related disorders.
J Med Genet. 2013 Apr;50(4):220-7. doi: 10.1136/jmedgenet-2012-101427. Epub 2013 Feb 1., [PMID:23378603]

Abstract [show]
BACKGROUND: The high frequency of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene mutation p.Arg117His in patients with congenital bilateral absence of the vas deferens (CBAVD) and in newborns screened for CF has created a dilemma. METHODS: Phenotypic and genotypic data were retrospectively collected in 179 non-newborn French individuals carrying p.Arg117His and a second CFTR mutation referred for symptoms or family history, by all French molecular genetics laboratories, referring physicians, CF care centres and infertility clinics. RESULTS: 97% of the patients had the intronic T7 normal variant in cis with p.Arg117His. 89% patients were male, with CBAVD being the reason for referral in 76%. In 166/179 patients with available detailed clinical features, final diagnoses were: four late-onset marked pulmonary disease, 83 isolated CBAVD, 67 other CFTR-related phenotypes, including 44 CBAVD with pulmonary and/or pancreatic symptoms and 12 asymptomatic cases. Respiratory symptoms were observed in 30% of the patients, but the overall phenotype was mild. No correlation was observed between sweat chloride concentrations and disease severity. Five couples at risk of CF offspring were identified and four benefited from prenatal or preimplantation genetic diagnoses (PND or PGD). Eight children were born, including four who were compound heterozygous for p.Arg117His and one with a severe CF mutation. CONCLUSIONS: Patients with CBAVD carrying p.Arg117His and a severe CF mutation should benefit from a clinical evaluation and follow-up. Depending on the CBAVD patients' genotype, a CFTR analysis should be considered in their partners in order to identify CF carrier couples and offer PND or PGD.

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44 The other mutations consisted of c.3846G>A (p.Trp1282*, W1282X) (2%), c.1624G>T (p.Gly542*, G542X) (2%), c.262_263del (394delTT) (2%), c.1585-1G>A (1717-1G>A) (2%) and other mutations (25%). Login to comment

[hide] Recent developments in targeting protein misfoldin... Bioorg Med Chem Lett. 2013 Apr 1;23(7):1935-44. doi: 10.1016/j.bmcl.2013.01.089. Epub 2013 Feb 4. Denny RA, Gavrin LK, Saiah E
Recent developments in targeting protein misfolding diseases.
Bioorg Med Chem Lett. 2013 Apr 1;23(7):1935-44. doi: 10.1016/j.bmcl.2013.01.089. Epub 2013 Feb 4., [PMID:23454013]

Abstract [show]
Protein misfolding is an emerging field that crosses multiple therapeutic areas and causes many serious diseases. As the biological pathways of protein misfolding become more clearly elucidated, small molecule approaches in this arena are gaining increased attention. This manuscript will survey current small molecules from the literature that are known to modulate misfolding, stabilization or proteostasis. Specifically, the following targets and approaches will be discussed: CFTR, glucocerebrosidase, modulation of toxic oligomers, serum amyloid P (SAP) sections and HSF1 activators.

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55 Class I mutations contribute to the formation of proteins with incomplete length and provide protein with complete loss of activity (i.e., mutation: G542X). Login to comment

[hide] Targeting a genetic defect: cystic fibrosis transm... Eur Respir Rev. 2013 Mar 1;22(127):58-65. doi: 10.1183/09059180.00008412. Derichs N
Targeting a genetic defect: cystic fibrosis transmembrane conductance regulator modulators in cystic fibrosis.
Eur Respir Rev. 2013 Mar 1;22(127):58-65. doi: 10.1183/09059180.00008412., [PMID:23457166]

Abstract [show]
Cystic fibrosis (CF) is caused by genetic mutations that affect the cystic fibrosis transmembrane conductance regulator (CFTR) protein. These mutations can impact the synthesis and transfer of the CFTR protein to the apical membrane of epithelial cells, as well as influencing the gating or conductance of chloride and bicarbonate ions through the channel. CFTR dysfunction results in ionic imbalance of epithelial secretions in several organ systems, such as the pancreas, gastrointestinal tract, liver and the respiratory system. Since discovery of the CFTR gene in 1989, research has focussed on targeting the underlying genetic defect to identify a disease-modifying treatment for CF. Investigated management strategies have included gene therapy and the development of small molecules that target CFTR mutations, known as CFTR modulators. CFTR modulators are typically identified by high-throughput screening assays, followed by preclinical validation using cell culture systems. Recently, one such modulator, the CFTR potentiator ivacaftor, was approved as an oral therapy for CF patients with the G551D-CFTR mutation. The clinical development of ivacaftor not only represents a breakthrough in CF care but also serves as a noteworthy example of personalised medicine.

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24 In fact, notwithstanding the common F508del variant, only four CFTR mutations have a frequency .0.1%: G551D, W1282X, G542X and N1303K, having a worldwide prevalence of around 1-3% each [9, 11]. Login to comment
28 For example, G542X is a nonsense or stop mutation, where introduction of a premature termination codon (or stop codon) results in premature cessation of translation and production of truncated CFTR protein. Login to comment

[hide] The impact of personalised therapies on respirator... Eur Respir Rev. 2013 Mar 1;22(127):72-4. doi: 10.1183/09059180.00008212. Elborn JS
The impact of personalised therapies on respiratory medicine.
Eur Respir Rev. 2013 Mar 1;22(127):72-4. doi: 10.1183/09059180.00008212., [PMID:23457168]

Abstract [show]
Stratified approaches to treating disease are very attractive, as efficacy is maximised by identifying responders using a companion diagnostic or by careful phenotyping. This approach will spare non-responders form potential side-effects. This has been pioneered in oncology where single genes or gene signatures indicate tumours that will respond to specific chemotherapies. Stratified approaches to the treatment of asthma with biological therapies are currently being extensively studied. In cystic fibrosis (CF), therapies have been developed that are targeted at specific functional classes of mutations. Ivacaftor, the first of such therapies, potentiates dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) protein Class III mutations and is now available in the USA and some European countries. Pivotal studies in patients with a G551D mutation, the most common Class III mutation, have demonstrated significant improvements in clinically important outcomes such as spirometry and exacerbations. Sweat chloride was significantly reduced demonstrating a functional effect on the dysfunctional CFTR protein produced by the G551D mutation. Symptom scores are also greatly improved to a level that indicates that this is a transformational treatment for many patients. This stratified approach to the development of therapies based on the functional class of the mutations in CF is likely to lead to new drugs or combinations that will correct the basic defect in many patients with CF.

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52 This optimism must TABLE 1 Mechanism of action and targets of emerging cystic fibrosis transmembrane conductance regulator (CFTR) modulators Mutation class Nature of defect Example mutations Pancreatic function# Modulator strategy Agents in development I Biosynthesis G542X Insufficient Suppressor Suppressor and potentiator" Ataluren II Trafficking F508del Insufficient Corrector Corrector plus potentiator VX-809 plus ivacaftor+ VX-661 plus ivacaftor+ III Channel gating G551D Insufficient Potentiator Ivacaftor (FDA approved, G551D) Other gating mutations?" Login to comment

[hide] Molecular and functional analysis of the large 5' ... J Mol Diagn. 2013 May;15(3):331-40. doi: 10.1016/j.jmoldx.2013.01.001. Epub 2013 Mar 5. Giordano S, Amato F, Elce A, Monti M, Iannone C, Pucci P, Seia M, Angioni A, Zarrilli F, Castaldo G, Tomaiuolo R
Molecular and functional analysis of the large 5' promoter region of CFTR gene revealed pathogenic mutations in CF and CFTR-related disorders.
J Mol Diagn. 2013 May;15(3):331-40. doi: 10.1016/j.jmoldx.2013.01.001. Epub 2013 Mar 5., [PMID:23470247]

Abstract [show]
Patients with cystic fibrosis (CF) manifest a multisystemic disease due to mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR); despite extensive testing of coding regions, a proportion of CF alleles remains unidentified. We studied 118 patients with CF and CFTR-related disorders, most with one or both unknown mutations after the scanning of CFTR coding regions, and a non-CF control group (n = 75) by sequencing the 6000-bp region at the 5' of the CFTR gene. We identified 23 mutations, of which 9 were novel. We expressed such mutations in vitro using four cell systems to explore their functional effect, relating the data to the clinical expression of each patient. Some mutations reduced expression of the gene reporter firefly luciferase in various cell lines and may act as disease-causing mutations. Other mutations caused an increase in luciferase expression in some cell lines. One mutation had a different effect in different cells. For other mutations, the expression assay excluded a functional role. Gene variants in the large 5' region may cause altered regulation of CFTR gene expression, acting as disease-causing mutations or modifiers of its clinical phenotype. Studies of in vitro expression in different cell systems may help reveal the effect of such mutations.

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115 Case 7 had c.-1308A>G in trans with G542X; he has PI and mild pulmonary expression. Login to comment
151 Diagnosis Clinical expression Sweat chloride (mEq/L) CFTR genotype FEV 1% Current age (years) 1 CBAVD CBAVD alone 38 [N1303K;c.-674T>C]/c.-674T>C 90 32 2 CBAVD CBAVD alone 36 F508del/c.-674T>C 106 28 3 CBAVD CBAVD alone 36 F508del/c.-674T>C 104 30 4 CF PI, mild P, no L 70 F508del/c.-869T[8_9] 78 41 5 CF PI, mild P, nasal polyposis, chronic sinusitis, no L 76 F508del/[TG12-T5-470V; c.-1308A>G] 80 30 6 CF PI, mild P, colonization by P. aeruginosa, no L 68 N1303K/c.-1308A>G 84 32 7 CF PI, mild P, no L 71 G542X/c.-1308A>G 80 17 8 CF PI, severe P, severe L 115 [2789&#fe;5G>A;c.-1773_ -1772delAT]Y849X* 44 20 9 CF PI, mild P, no L 90 F508del/c.-1773_-1772delAT 76 28 10 CBAVD CBAVD alone 40 F508del/c.-812T>G 96 31 11 CBAVD CBAVD alone 23 F508del/c.-812T>G 102 41 12 CF PS, mild P, no L 20 F508del/[1525-1delG;TG12-T5-470V;c.-2200G>A]y 84 20 13 CF PS, mild P, no L 70 F508del/c.-3632G>T 88 26 14 CBAVD CBAVD alone 22 c.-5914A>G/U 98 *The patient originally had the genotype 2789&#fe;5G>A/unknown; during the present study, we revealed the second mutation (ie, Y849X). Login to comment

[hide] PGD for cystic fibrosis patients and couples at ri... Reprod Biomed Online. 2013 May;26(5):420-30. doi: 10.1016/j.rbmo.2013.01.006. Epub 2013 Jan 29. Rechitsky S, Verlinsky O, Kuliev A
PGD for cystic fibrosis patients and couples at risk of an additional genetic disorder combined with 24-chromosome aneuploidy testing.
Reprod Biomed Online. 2013 May;26(5):420-30. doi: 10.1016/j.rbmo.2013.01.006. Epub 2013 Jan 29., [PMID:23523379]

Abstract [show]
Preimplantation genetic diagnosis (PGD) for inherited disorders is presently applied for more than 300 different conditions. The most frequent PGD indication is cystic fibrosis (CF), the largest series of which is reviewed here, totalling 404 PGD cycles. This involved testing for 52 different CFTR mutations with almost half of the cases (195/404 cycles) performed for DeltaF508 mutation, one-quarter (103/404 cycles) for six other frequent mutations and only a few for the remaining 45 CFTR mutations. There were 44 PGD cycles performed for 25 CF-affected homozygous or double-heterozygous CF patients (18 male and seven female partners), which involved testing simultaneously for three mutations, resulting in birth of 13 healthy CF-free children and no misdiagnosis. PGD was also performed for six couples at a combined risk of producing offspring with CF and another genetic disorder. Concomitant testing for CFTR and other mutations resulted in birth of six healthy children, free of both CF and another genetic disorder in all but one cycle. A total of 96 PGD cycles for CF were performed with simultaneous aneuploidy testing, including microarray-based 24-chromosome analysis, as a comprehensive PGD for two or more conditions in the same biopsy material.

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42 [1075C>A; 1079C>A] p.[Gln359Lys; Thr360Lys] Exon 8 1 1 1 4 1 1 R297Q c.890G>A p.Arg297Gln Exon 8 1 1 1 2 0 0 R347P c.1040G>C p.Arg347Pro Exon 8 3 5 2 4 1 1 T338I c.1013C>T p.Thr338Ile Exon 8 1 1 1 2 1 1 DF508 c.1521_1523delCTT p.Phe508del Exon 11 130 195 172 345 88 (4) 92 DI507 c.1519_1521delATC p.Ile507del Exon 11 1 5 5 11 2 1 Q493R c.1478A>G p.Gln493Arg Exon 11 5 5 2 2 2 2 1717-1G-A c.1585-1G>A - Intron 11 6 10 9 18 6 8 G542X c.1624G>T p.Gly542X Exon 12 14 17 15 34 10 10 G551S c.1651G>A p.Gly551Ser Exon 12 1 1 1 2 1 1 G551D c.1652G>A p.Gly551Asp Exon 12 12 22 19 33 7 8 I556V c.1666A>G p.Ile556Val Exon 12 1 2 2 4 1 1 R553X c.1657C>T p.Arg553X Exon 12 3 4 2 4 0 0 R560T c.1679G>C p.Arg560Thr Exon 12 1 1 1 2 1 2 1898+1G-A c.1766 &#b1; 1G>A - Intron 13 1 1 1 2 1 1 2184delA c.2052delA p.Lys684AsnfsX38 Exon 14 1 1 0 0 0 0 G622D c.1865G>A p.Gly622Asp Exon 14 1 1 1 3 0 0 N703S c.2108A>G p.Asn703Ser Exon 14 1 2 2 3 2 2 S737F c.2210C>T p.Ser737Phe Exon 14 1 1 0 0 0 0 2622+1G-A c.2490 &#b1; 1G>A - Intron 14 1 5 5 13 1 1 2752-26A-G c.2620-26A>G - Intron 15 1 2 2 4 0 0 2789+5G-A c.2657 &#b1; 5G>A - Intron 16 3 5 4 8 0 0 3120G-A c.2988G>A - Exon 18 2 2 1 2 1 0 3067-72del c.3067_3072del p.Ile1023_Val1024del Exon 19 1 1 1 1 0 0 I1027T c.3080T>C p.Ile1027Thr Exon 19 1 1 1 1 0 0 L997F c.2991G>C p.Leu997Phe Exon 19 1 2 2 4 1 (1) 0 M1028R c.3083T>G p.Met1028Arg Exon 19 1 1 1 2 1 2 F1052V c.3154T>G p.Phe1052Val Exon 20 1 1 0 0 0 0 Y1092X c.3276C>A p.Tyr1092X Exon 20 1 2 1 2 1 1 A1136T c.3406G>A p.Ala1136Thr Exon 21 1 2 1 2 1 0 D1152H c.3454G>C p.Asp1152His Exon 21 3 7 7 15 1 1 3659 del C c.3528delC p.Lys1177SerfsX15 Exon 22 2 4 3 7 3 3 R1162X c.3484C>T p.Arg1162X Exon 22 1 3 2 5 2 2 S1235R c.3705T>G p.Ser1235Arg Exon 22 2 3 3 5 2 1 3849+10kbC>T c.3717 &#b1; 12191C>T - Intron 22 2 4 4 5 0 0 W1282X c.3846G>A p.Trp1282X Exon 23 15 20 20 42 11 11 N1303K c.3909C>G p.Asn1303Lys Exon 24 9 12 11 24 4 5 Q1352H c.4056G>C p.Gln1352His Exon 25 1 1 1 1 1 1 Total 265 404 345 685 172 (6a ) 175 Values are n unless otherwise stated. Login to comment
53 Almost half of these (195/404 cycles) were performed for DF508 mutation, one-quarter (103/404 cycles)forsixotherfrequentmutations(W1282X,R117H,G551D, G542X, N1303K, 1717-1G>A), and only a few for each of the remaining 45 CFTR mutations (Table 2). Login to comment
56 (CA)n EXON 4 (GATT)n Intron 4 Poly T tract Intron 10 R117H G--A R75XH C--T A120T G--A I148T T--C A349V C--T 1259 Ins A 621+1 G--T EXON 3 EXON 7 EXON 8 Delta I 507 EXON 10 Delta F 508 EXON 11 1717-1 G--A G542X G--T G550X G--T G551D G--A R553X C--T R560T G--C EXON 19 EXON 20 EXON 21 R1162X C--T W1282X G--A N1303K C--G IVS 1 Mutations in CFTR gene (PGD PERFORMED FOR 52 MUTATIONS) IVS 6 a IVS 8 (CA)n (CA)n IVS 17b (TA)n (CA)n D7S486 D7S522 D7S633 D7S677 D7S2847 D7S655 115,89 116.07 117.01 117.13 117.19 117.20 118.6 118.81 Mb IVS8-1 IVS8-2 Figure 1 Mutations (above) and linked markers (below) in CFTR that were used in multiplex PCR. Login to comment
81 Because the female partner was double heterozygous, involving two different mutations (R117H and G542X), and the male partner was a carrier of R117H mutation, PGD was based on blastomere biopsy. Login to comment
88 Two unaffected embryos (nos. 6 and 7) were transferred, resulting in an unaffected pregnancy and birth of a healthy baby girl, confirmed to be a carrier of the maternal G542X mutation. Login to comment
109 (Upper panel) Pedigree, showing that the maternal partner is a double-heterozygous CF-affected patient, with two different CFTR mutations, R117H and G542X, while the paternal partner is a carrier of the 1717 mutation. Login to comment

[hide] Tobramycin is a suppressor of premature terminatio... J Cyst Fibros. 2013 Dec;12(6):806-11. doi: 10.1016/j.jcf.2013.02.007. Epub 2013 Mar 27. Altamura N, Castaldo R, Finotti A, Breveglieri G, Salvatori F, Zuccato C, Gambari R, Panin GC, Borgatti M
Tobramycin is a suppressor of premature termination codons.
J Cyst Fibros. 2013 Dec;12(6):806-11. doi: 10.1016/j.jcf.2013.02.007. Epub 2013 Mar 27., [PMID:23540394]

Abstract [show]
Premature translation terminations (PTCs) constitute the molecular basis of many genetic diseases, including cystic fibrosis, as they lead to the synthesis of truncated non-functional or partially functional protein. Suppression of translation terminations at PTCs (read-through) has been developed as a therapeutic strategy to restore full-length protein in several genetic diseases. Phenotypic consequences of PTCs can be exacerbated by the nonsense-mediated mRNA decay (NMD) pathway that detects and degrades mRNA containing PTC. Modulation of NMD, therefore, is also of interest as a potential target for the suppression therapy. Tobramycin is an aminoglycoside antibiotic, normally used to treat Pseudomonas aeruginosa pulmonary infection in CF patients. In the present study, by using yeast as a genetic system, we have examined the ability of Tobramycin to suppress PTCs as a function of the presence or absence of NMD. Results demonstrate that Tobramycin exhibits read-through ability on PTCs and preferentially in absence of NMD.

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73 Discussion In CF at least 5-10% of the CF alleles carry a nonsense mutation (e.g. G542X, R553X, R1162X, W1282X; CF Mutation Analysis Consortium, http://www.genet.sickkids.on.ca/cftr/) that causes a premature arrest of translational termination thus preventing the synthesis of a full-length, often non-functional or partially functional, CFTR [4]. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... PLoS One. 2013 Apr 17;8(4):e61176. doi: 10.1371/journal.pone.0061176. Print 2013. Schippa S, Iebba V, Santangelo F, Gagliardi A, De Biase RV, Stamato A, Bertasi S, Lucarelli M, Conte MP, Quattrucci S
Cystic fibrosis transmembrane conductance regulator (CFTR) allelic variants relate to shifts in faecal microbiota of cystic fibrosis patients.
PLoS One. 2013 Apr 17;8(4):e61176. doi: 10.1371/journal.pone.0061176. Print 2013., [PMID:23613805]

Abstract [show]
INTRODUCTION: In this study we investigated the effects of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene variants on the composition of faecal microbiota, in patients affected by Cystic Fibrosis (CF). CFTR mutations (F508del is the most common) lead to a decreased secretion of chloride/water, and to mucus sticky secretions, in pancreas, respiratory and gastrointestinal tracts. Intestinal manifestations are underestimated in CF, leading to ileum meconium at birth, or small bowel bacterial overgrowth in adult age. METHODS: Thirty-six CF patients, fasting and under no-antibiotic treatment, were CFTR genotyped on both alleles. Faecal samples were subjected to molecular microbial profiling through Temporal Temperature Gradient Electrophoresis and species-specific PCR. Ecological parameters and multivariate algorithms were employed to find out if CFTR variants could be related to the microbiota structure. RESULTS: Patients were classified by two different criteria: 1) presence/absence of F508del mutation; 2) disease severity in heterozygous and homozygous F508del patients. We found that homozygous-F508del and severe CF patients exhibited an enhanced dysbiotic faecal microbiota composition, even within the CF cohort itself, with higher biodiversity and evenness. We also found, by species-specific PCR, that potentially harmful species (Escherichia coli and Eubacterium biforme) were abundant in homozygous-F508del and severe CF patients, while beneficial species (Faecalibacterium prausnitzii, Bifidobacterium spp., and Eubacterium limosum) were reduced. CONCLUSIONS: This is the first report that establishes a link among CFTR variants and shifts in faecal microbiota, opening the way to studies that perceive CF as a 'systemic disease', linking the lung and the gut in a joined axis.

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37 Patient Sex Age (years) CFTR allele, = CFTR allele, R Criterion I(a) Criterion II (1 = severe, 0 = mild)(b) Pancreatic status(d) FEV1% BMI 1 M 17 F508del M1V 2 (1) 1 65 17.91 2 F 23 F508del Y569D 2 (1) 0 97 18.66 3 (s1)(c) F 20 P1013L F508del 2 (0) 0 87 18.67 4 M 11 F508del L997F (without R117L) 2 0 0 110 21.33 5 (s1)(c) M 11 P1013L F508del 2 (0) 0 100 23.14 6 M 8 R553X F508del 2 1 0 80 15.87 7 M 3 F508del unknown 2 (0) 0 nd nd 8 F 33 F508del F508del 1 1 1 73 18.61 9 M 10 F508del L1077P 2 1 0 94 19.79 10 M 9 F508del G542X 2 1 1 100 16.00 11 F 9 4167delCTAAGCC L1065P 3 nd 1 76 14.57 12 F 14 R117C (without (TG)12T5) F508del 2 0 0 94 18.44 13 F 11 F508del 991del5 2 1 1 109 17.80 14 M 42 (TG)12T5 F508del 2 0 0 106 23.78 15 (s2)(c) M 9 F508del F508del 1 1 1 82 15.45 16 M 10 F508del R347P 2 (0) 0 89 15.91 17 (s2)(c) F 6 F508del F508del 1 1 1 110 15.20 18 (s3)(c) M 39 2789+5G.A N1303K 3 nd 0 105 19.33 19 (s3)(c) F 41 2789+5G.A N1303K 3 nd 0 80 19.47 20 F 26 N1303K W1282X 3 nd 1 90 19.57 21 M 7 CFTRdele2,3 (21 kb) N1303K 3 nd 1 107 12.85 22 F 9 F508del L997F (without R117L) 2 0 0 113 25.21 23 M 7 P5L W1282X 3 nd 0 89 22.31 24 M 9 2789+5G.A F508del 2 (1) 1 97 15.60 25 F 2 F508del F508del 1 1 1 nd nd 26 F 32 N1303K N1303K 3 nd 1 107 21.22 27 M 14 L1065R T338I 3 nd 0 116 21.50 28 M 12 711+3A.G S549R(A.C) 3 nd 0 97 20.00 29 M 13 unknown R117H (without (TG)12T5) 3 nd 0 104 19.36 30 M 14 F508del G542X 2 1 1 84 21.87 31 F 13 F508del F508del 1 1 1 85 18.00 32 F 41 2789+5G.A N1303K 3 nd 1 84 21.08 33 F 21 L1065P F508del 2 (0) 0 62 18.29 34 F 50 D1152H F508del 2 (0) 0 63 23.74 35 M 29 F508del 2790-2A.G 2 (1) 0 92 24.46 36 F 45 unknown W1282X 3 nd 0 69 23.42 a (Hm = 1; Ht = 2; N = 3). Login to comment
62 Class I, II or III: G542X, W1282X, F508del, N1303K, L1065P, L1077P, Y569D, S549R(A.C). Login to comment

[hide] Optimal complement-mediated phagocytosis of Pseudo... Am J Respir Cell Mol Biol. 2013 Sep;49(3):463-70. doi: 10.1165/rcmb.2012-0502OC. Van de Weert-van Leeuwen PB, Van Meegen MA, Speirs JJ, Pals DJ, Rooijakkers SH, Van der Ent CK, Terheggen-Lagro SW, Arets HG, Beekman JM
Optimal complement-mediated phagocytosis of Pseudomonas aeruginosa by monocytes is cystic fibrosis transmembrane conductance regulator-dependent.
Am J Respir Cell Mol Biol. 2013 Sep;49(3):463-70. doi: 10.1165/rcmb.2012-0502OC., [PMID:23617438]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and is characterized by chronic pulmonary infections. The mechanisms underlying chronic infection and inflammation remain incompletely understood. Mutant CFTR in nonepithelial tissues such as immune cells has been suggested to contribute to infection, inflammation, and the resultant lung disease. However, much controversy still exists regarding the intrinsic role of CFTR in immune cells, especially phagocytes. Therefore, we investigated CFTR expression and function in neutrophils and monocytes isolated from human peripheral blood. CFTR function was assessed by comparing non-CF and CF cells, before and after the chemical inhibition of CFTR. We found CFTR protein expression in monocytes, but this expression was limited or undetectable in neutrophils. Furthermore, the phagocytosis and intracellular killing of Pseudomonas aeruginosa was reduced in CF monocytes, and impaired phagocyte effector mechanisms were phenocopied in non-CF monocytes upon the pharmacological inhibition of CFTR. Reduced phagocytosis in CF monocytes relied on the complement-dependent opsonization of Pseudomonas aeruginosa, and was also observed in the context of latex particles labeled with purified C3b. In mechanistic terms, we observed that CFTR function in monocytes is required for the optimal expression of CD11b. We observed no role for CFTR in neutrophil-mediated phagocytosis. These data support an intrinsic role for CFTR in monocytes, and suggest that CFTR-dependent alterations in complement-mediated interactions between Pseudomonas aeruginosa and monocytes may contribute to enhanced susceptibility to infection in patients with CF.

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54 Immune cells were isolated from healthy control subjects and patients with CF who carried a G542X/G54X, G542X/F508 del mutation or F508 del/F508 del mutation. Login to comment
64 To confirm CFTR antibody specificity, whole-cell CFTR expression levels were compared with those of isotype control antibodies, and CF cells were derived from a subject with two CFTR nonsense mutations (G542X/G542X), a mutation of which it is known that these cells have no expression of CFTR. Login to comment
86 To confirm CFTR specificity further, whole-blood immune cells were isolated from a healthy control subject and a CF patient with two nonsense mutations (G542X/ G542X), with an expectation of absent expression of CFTR. Login to comment
88 CFTR expression in nasal epithelial cells from a healthy control subject, but not in a patient with CF (G542X/G542X), further supported the contention that our staining procedure could detect CFTR (Figure 1C). Login to comment
93 We confirmed staining specificity in CD141 monocytes from a healthy control subject and a patient with CF and a homozygous G542X/G542X mutation (Figure 2B). Login to comment
118 (B) Whole-cell CFTR (red) expression in peripheral blood immune cells of a healthy control subject, compared with a patient with a G542X/G542X mutation, using L12B4 and 570. Login to comment
119 (C) Whole-cell CFTR (green) expression in nasal epithelial cells of a healthy control subject and a patient with a G542X/G542X mutation, using L12B4. Login to comment
128 (B) Flow cytometric analysis of whole-cell CFTR expression in monocytes of a healthy control subject compared with a patient with CF and carrying a homozygous G542X mutation, using the CFTR mAbs 570 and L12B4. Login to comment

[hide] Newborn screening for cystic fibrosis in Switzerla... J Cyst Fibros. 2013 Dec;12(6):667-74. doi: 10.1016/j.jcf.2013.04.008. Epub 2013 May 24. Torresani T, Fingerhut R, Rueegg CS, Gallati S, Kuehni CE, Baumgartner MR, Barben J
Newborn screening for cystic fibrosis in Switzerland--consequences after analysis of a 4 months pilot study.
J Cyst Fibros. 2013 Dec;12(6):667-74. doi: 10.1016/j.jcf.2013.04.008. Epub 2013 May 24., [PMID:23712087]

Abstract [show]
BACKGROUND: Switzerland introduced newborn screening (NBS) for CF in 2011, using an IRT/DNA/IRT protocol. This paper describes the results of the first year and compares two versions of the protocol with different IRT cut-offs, particularly effects on recall rate, sensitivity and specificity. METHODS: IRT cut-offs were >45 ng/ml (99.0th percentile) in period 1 (months 1-4) and >50 ng/ml (99.2nd percentile) in period 2 (months 5-12). In period 2 we abstained from recalls when none of the 7 most common CF mutations were detected and IRT was <60 ng/ml. RESULTS: In periods 1 and 2, 26,535 and 56,663 tests were performed. Recall rates were 0.94% and 0.48%, respectively (p<0.001), PPV increased from 23% to 47% (p=0.024) and sensitivity was 90% and 100%. CONCLUSIONS: Raising initial IRT cut-off from the 99.0th to the 99.2nd percentile and abstaining from recalls for children with an IRT<60 ng/ml and carrying no major CFTR mutation significantly reduced the recall rate without affecting sensitivity.

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48 CFTR mutation screening (2nd tier) The SNSL determined the seven most common CFTR mutations in Switzerland with an in-house developed kit (SWISS PANEL: F508del, 3905insT, G542X, R553X, W1282X, 1717-1GNA, N1303K) [10,1]. Login to comment

[hide] Clinical and genetic features in patients with cys... Iran J Pediatr. 2013 Apr;23(2):212-5. Farjadian S, Moghtaderi M, Kashef S, Alyasin S, Najib K, Saki F
Clinical and genetic features in patients with cystic fibrosis in southwestern iran.
Iran J Pediatr. 2013 Apr;23(2):212-5., [PMID:23724185]

Abstract [show]
OBJECTIVE: Cystic fibrosis (CF) is a common autosomal recessive genetic disease caused by a mutation in the CF transmembrane conductance regulatory (CFTR) gene. This study attempted to identify the most common CFTR mutations and any correlations between certain mutations and the clinical presentation of the disease in CF patients in southwestern Iran. METHODS: Twenty nine common CFTR gene mutations were examined in 45 CF patients. FINDINGS: Chronic cough, intestinal obstruction, dehydration, heat exhaustion and steatorrhea were the most common early clinical symptoms among our patients. The most common mutation was DeltaF508, with an allele frequency of 21%. The homozygous DeltaF508 mutation was observed in eight patients (18%), and three patients (7%) were DeltaF508 carriers. The 2183AA > G mutation was observed in four patients, one of whom was also a DeltaF508 carrier. The R1162X mutation was detected in two patients. The G542X, R334W and N1303K mutations were detected each in one patient, the first of whom was also a DeltaF508 carrier. CONCLUSION: Out of 45 patients, 27 (60%) had none of the CFTR gene mutations we tested for. The most frequent mutations in southwestern Iranian patients with CF should be identified by sequencing the entire CFTR gene in order to optimize the design of a diagnostic kit for common regional mutations.

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10 The G542X, R334W and N1303K mutations were detected each in one patient, the first of whom was also a ࢞F508 carrier. Login to comment
26 Genomic DNA was extracted from 200 &#b5;L of whole blood with the QiaAmp DNA Mini Kit (Qiagen, Valencia, CA, USA) and 29 common CFTR gene mutations (D1152H, 1717-1G>A, G542X, W1282X, N1303K, ࢞F508, 3849+10kbC>T, 394delTT, 621+1G>T, S1251N, G551D, R117H, R1162X, R334W, A455E, 2183AA>G, 3659delC, 1078delT, ࢞I507, R347P, R553X, E60X, 3120+1G>A, 2789+5G>A, 1898+1G>A, 711+1G>T, G85E, 2184delA and R560T) were analyzed with the ELUCIGENE CF29 v. 2 kit using four multiplex PCR. Login to comment
33 Table 1: Early clinical symptoms in patients from southwestern Iran with cystic fibrosis (n=45) Frequency Clinical symptoms 38 (84%) Chronic cough Respiratory symptoms 37 (82%) Bronchiolitis 35 (78%) Recurrent pneumonia 34 (76%) Purulent sputum 31(69%) Recurrent wheeze 18 (40%) Atelectasias 6 (13%) Bronchiectasis 4 (9%) Recurrent sinusitis 2 (4%) Hemoptysis 1(2%) Cor pulmonale 37 (82%) Steatorrhea Gasterointestinal symptoms 24 (53%) Growth failure 9 (20%) Liver disease 6 (13%) Distal intestinal obstruction 3 (7%) Diarrhea 2 (4%) Constipation 2 (4%) Meconium ileus 2 (4%) Dehydration Other symptoms 2 (4%) Heat exhaustion --- Vasculitis --- Diabetes 214 CFTR Gene Mutations in CF Patients Iran J Pediatr; Vol 23 (No 2), Apr 2013 Published by: Tehran University of Medical Sciences (http://ijp.tums.ac.ir) Table 2: Frequencies of CFTR gene mutations in a sample of patients in southwestern Iran with cystic fibrosis n=45 CFTR gene mutations 8 (18%) ࢞F508 (M)/ ࢞F508 (M) 3 (7%) ࢞F508 (N)/ 2183AA>G 2 (4%) ࢞F508 (N)/ R1162X 1 (2%) ࢞F508 (N)/ R334W 1 (2%) ࢞F508 (N)/ N1303K 1 (2%) ࢞F508 (M)/ G542X 1 (2%) ࢞F508 (M)/ 2183AA>G 1 (2%) ࢞F508 (M)/ ࢞F508 (N) 27 (60%) Undefined Parental consanguinity was found in 40 patients (89%) and a family history of CF was reported by 15 patients (37%) in this group. Login to comment
82 Jalalirad M, Houshmand M, Mirfakhraie R, et al. First study of CF mutations in the CFTR gene of Iranian patients: detection of DeltaF508, G542X, W1282X, A120T, R117H, and R347H mutations. Login to comment

[hide] A functional CFTR assay using primary cystic fibro... Nat Med. 2013 Jul;19(7):939-45. doi: 10.1038/nm.3201. Epub 2013 Jun 2. Dekkers JF, Wiegerinck CL, de Jonge HR, Bronsveld I, Janssens HM, de Winter-de Groot KM, Brandsma AM, de Jong NW, Bijvelds MJ, Scholte BJ, Nieuwenhuis EE, van den Brink S, Clevers H, van der Ent CK, Middendorp S, Beekman JM
A functional CFTR assay using primary cystic fibrosis intestinal organoids.
Nat Med. 2013 Jul;19(7):939-45. doi: 10.1038/nm.3201. Epub 2013 Jun 2., [PMID:23727931]

Abstract [show]
We recently established conditions allowing for long-term expansion of epithelial organoids from intestine, recapitulating essential features of the in vivo tissue architecture. Here we apply this technology to study primary intestinal organoids of people suffering from cystic fibrosis, a disease caused by mutations in CFTR, encoding cystic fibrosis transmembrane conductance regulator. Forskolin induces rapid swelling of organoids derived from healthy controls or wild-type mice, but this effect is strongly reduced in organoids of subjects with cystic fibrosis or in mice carrying the Cftr F508del mutation and is absent in Cftr-deficient organoids. This pattern is phenocopied by CFTR-specific inhibitors. Forskolin-induced swelling of in vitro-expanded human control and cystic fibrosis organoids corresponds quantitatively with forskolin-induced anion currents in freshly excised ex vivo rectal biopsies. Function of the CFTR F508del mutant protein is restored by incubation at low temperature, as well as by CFTR-restoring compounds. This relatively simple and robust assay will facilitate diagnosis, functional studies, drug development and personalized medicine approaches in cystic fibrosis.

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127 (d) Forskolin-induced swelling of rectal organoids derived from three individual healthy controls, two individuals with a mild cystic fibrosis genotype (F508del A455E) and nine individuals with a severe cystic fibrosis genotype (one individual with E60X 4015ATTTdel, one with F508del G542X, one with F508del L927P and six with F508del F508del). Login to comment
134 (Healthy controls, n = 3; mild cystic fibrosis, n = 2; severe cystic fibrosis (F508del F508del), n = 5; severe cystic fibrosis (other; E60X 4015ATTTdel and F508del G542X), n = 2; mean &#b1; s.d.). Login to comment
150 0 500 1,000 1,500 2,000 2,500 0 500 1,000 1,500 2,000 2,500 3,000 0 400 800 1,200 1,600 C8 Corr-4a C8 + Corr-4a VX-809 VX-770 VX-809 + VX-770 VRT-325 Corr-4a VRT-325 + Corr-4a CF1 CF6 CF5 CF4 CF3 CF2 F508del L927P F508del G542X E60X 4015delATTT F508del F508del b d a c 0 20 40 60 80 100 120 V R T - 3 2 5 C o r r - 4 a C 8 V X - 8 0 9 V X - 7 7 0 V R T - 3 2 5 + C o r r - 4 a C 8 + C o r r - 4 a V X - 8 0 9 + V X - 7 7 0 C o n t r o l C o n t r o l F508del F508del HC F508del A455E Organoid swelling (absolute AUC t = 60) Organoid swelling (absolute AUC t = 60) Organoid swelling (absolute AUC t = 60) Organoid swelling (normalized AUC t = 60) Figure 5ߒ Differential FIS between organoids from subjects with cystic fibrosis after chemical CFTR restoration. Login to comment
390 Rectal organoids from healthy controls and subjects with cystic fibrosis were generated from four rectal suction biopsies after ICMs obtained (i) during standard cystic fibrosis care (one individual each harboring E60X 4015ATTTdel, F508del G542X or F508del L927P and five individuals harboring F508del F508del), (ii) for diagnostic purposes (one healthy control) or (iii) during voluntary participation in studies approved by the University Medical Center Utrecht and Erasmus MC ethics committees (two healthy controls and one individual harboring F508del F508del). Login to comment

[hide] Genetic interaction of GSH metabolic pathway genes... BMC Med Genet. 2013 Jun 10;14:60. doi: 10.1186/1471-2350-14-60. de Lima Marson FA, Bertuzzo CS, Secolin R, Ribeiro AF, Ribeiro JD
Genetic interaction of GSH metabolic pathway genes in cystic fibrosis.
BMC Med Genet. 2013 Jun 10;14:60. doi: 10.1186/1471-2350-14-60., [PMID:23758905]

Abstract [show]
BACKGROUND: Cystic fibrosis (CF) is a monogenic disease caused by CFTR gene mutations, with clinical expression similar to complex disease, influenced by genetic and environmental factors. Among the possible modifier genes, those associated to metabolic pathways of glutathione (GSH) have been considered as potential modulators of CF clinical severity. In this way it is of pivotal importance investigate gene polymorphisms at Glutamate-Cysteine Ligase, Catalytic Subunit (GCLC), Glutathione S-transferase Mu 1 (GSTM1), Glutathione S-transferase Theta 1 (GSTT1), and Glutathione S-transferase P1 (GSTP1), which have been associated to the GSH metabolic pathway and CF clinical severity. METHOD: A total of 180 CF's patients were included in this study, which investigated polymorphisms in GCLC and GST genes (GCLC -129C>T and -3506A>G; GSTM1 and GSTT1 genes deletion, and GSTP1*+313A>G) by PCR and PCR-RFLP associating to clinical variables of CF severity, including variables of sex, clinical scores [Shwachman-Kulczycki, Kanga e Bhalla (BS)], body mass index, patient age, age for diagnosis, first clinical symptoms, first colonization by Pseudomonas aeruginosa, sputum's microorganisms, hemoglobin oxygen saturation in the blood, spirometry and comorbidities. The CFTR genotype was investigated in all patients, and the genetic interaction was performed using MDR2.0 and MDRPT0.4.7 software. RESULTS: The analysis of multiple genes in metabolic pathways in diseases with variable clinical expression, as CF disease, enables understanding of phenotypic diversity. Our data show evidence of interaction between the GSTM1 and GSTT1 genes deletion, and GSTP1*+313A>G polymorphism with CFTR gene mutation classes, and BS (Balance testing accuracy=0.6824, p=0.008), which measures the commitment of bronchopulmonary segments by tomography. CONCLUSION: Polymorphisms in genes associated with metabolism of GSH act on the CF's severity.

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54 Data was recorded by the PF BREEZE software version 3.8B for Windows 95/98/NT [32] and the following markers were included: forced Table 2 Genotypic characteristic of gene polymorphisms at GCLC, GSTM1, GSTT1, and GSTP1 genes and CFTR gene mutation among cystic fibrosis patients Gene Chromosome position Location Variation Genotype MAF p* C/C C/T T/T GCLC, rs17883901 6p12 Promoter region C/T 144 (80%) 29 (16.11%) 7 (3.89%) 0.12 <0.005 1 A/A A/G G/G GCLC, rs137852340 6p12 Promoter region A/G 118 (65.56%) 56 (31.11%) 6 (3.33%) 0.19 >0.05 GSTP1, rs1695 11q13 Exon 5 A/G 97 (53.89%) 74 (41.11%) 9 (5%) 0.26 >0.05 Wt/Wt + Wt/del del/del GSTM1 1p13.3 Deletion 108 (60%) 72 (40%) GSTT1 22q11.23 Deletion 117 (65%) 63(35%) CFTR mutation genoytpe N Frequency F508del/F508del 57 31.67% F508del/G542X 12 6.67% F508del/R1162X 5 2.78% F508del/N1303K 4 2.22% F508del/R553X 1 0.56% F508del/S4X 1 0.56% F508del/1717-1G>A 1 0.56% G542X/R1162X 1 0.56% G542X/I618T 1 0.56% G542X/2183A>G 1 0.56% R1162X/R1162X 1 0.56% F508del/- 45 25.00% G542X/- 5 2.78% R1162X/- 1 0.56% -/- 44 24.45% GCLC glutamate-cysteine ligase catalytic subunit, GSTM1 Glutathione S-transferase Mu 1, GSTT1 Glutathione S-transferase theta 1, GSTP1 Glutathione S-transferase P1, CFTR Cystic fibrosis transmembrane conductance regulator, C Cytosine, T Thymine, A Adenine, G Guanine, < minor than, > bigger than, MAF minor allele frequency, % percentage, *p value for Hardy-Weinberg Equilibrium, N number of patients, Wt Wild allele, del deleted allele, (-) CFTR mutation no identified. Login to comment
59 The CFTR gene mutations were investigated by PCR technique (F508del) and the restriction fragment length polymorphism (RFLP) method (G542X, R1162X, R553X, G551D and N1303K). Login to comment

[hide] Quantification of major urinary metabolites of PGE... Prostaglandins Leukot Essent Fatty Acids. 2013 Aug;89(2-3):121-6. doi: 10.1016/j.plefa.2013.06.001. Epub 2013 Jun 20. Jabr S, Gartner S, Milne GL, Roca-Ferrer J, Casas J, Moreno A, Gelpi E, Picado C
Quantification of major urinary metabolites of PGE2 and PGD2 in cystic fibrosis: correlation with disease severity.
Prostaglandins Leukot Essent Fatty Acids. 2013 Aug;89(2-3):121-6. doi: 10.1016/j.plefa.2013.06.001. Epub 2013 Jun 20., [PMID:23791427]

Abstract [show]
Cystic fibrosis transmembrane conductance (CFTR) alterations are involved in the overproduction of prostaglandins (PG) in CF in vitro. We assessed the relationship between PGE-M and PGD-M urinary metabolites of PGE2 and PGD2 and CF severity. Twenty-four controls and 35 CF patients were recruited. PGE-M and PGD-M levels were measured by liquid chromatography/mass spectrometry and results were expressed as median and 25th-75th interquartile of ng/mg creatinine (Cr). PGE-M (15.63; 9.07-43.35ng/mg Cr) and PGD-M (2.16; 1.43-3.53ng/mg Cr) concentrations were higher in CF than in controls: PGE-M, (6.63; 4.35-8.60ng/mg Cr); PGD-M (1.23; 0.96-1.54ng/mg Cr). There was no correlation between metabolite levels and spirometric values. Patients with pancreatic insufficiency (n=29) had higher PGE-M levels (19.09; 9.36-52.69ng/mg Cr) than those with conserved function (n=6) (9.61; 5.78-14.34ng/mg Cr). PGE-M levels were associated with genotype severity: mild (7.14; 5.76-8.76, n=8), moderate (16.67; 13.67-28.62ng/mg Cr, n=5) and severe (22.82; 10.67-84.13ng/mg Cr). Our study confirms the key role of CFTR in the regulation of the cyclooxygenase pathway of arachidonic acid metabolism found in in vitro studies.

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113 Mutations Mutation class Severity Number Pancreatic sufficiency (n) W128X/W128X I/I Severe 1 0 I507/Q890X I/I Severe 1 0 F508del/G542X II/I Severe 2 0 F508del/2188AA4G II/I Severe 1 0 F508del/N1303K II/I Severe 3 0 F508del/1677delTA II/I Severe 1 0 F508del/2188AA4G II/I Severe 1 0 F508del/F508del II/II Severe 10 0 F508del/Q890X II/II Severe 1 0 F508del/E1308X II/II Severe 1 0 F508del/5T-12TG II/III Moderate 2 0 G542X/G85V I/III Moderate 1 0 F508del/124del23kbp II/III Moderate 1 0 G542X/M1137V I/III Moderate 1 1 I507/L206W I/IV Mild 1 0 F508del/L206W I/IV Mild 4 2 711+1G4L206W I/IV Mild 1 1 N1303K/3272-26A4G I/IV Mild 1 1 F508del/F587I II/V Mild 1 1 n&#bc;Number. Login to comment

[hide] Novel CFTR variants identified during the first 3 ... J Mol Diagn. 2013 Sep;15(5):710-22. doi: 10.1016/j.jmoldx.2013.05.006. Epub 2013 Jun 28. Prach L, Koepke R, Kharrazi M, Keiles S, Salinas DB, Reyes MC, Pian M, Opsimos H, Otsuka KN, Hardy KA, Milla CE, Zirbes JM, Chipps B, O'Bra S, Saeed MM, Sudhakar R, Lehto S, Nielson D, Shay GF, Seastrand M, Jhawar S, Nickerson B, Landon C, Thompson A, Nussbaum E, Chin T, Wojtczak H
Novel CFTR variants identified during the first 3 years of cystic fibrosis newborn screening in California.
J Mol Diagn. 2013 Sep;15(5):710-22. doi: 10.1016/j.jmoldx.2013.05.006. Epub 2013 Jun 28., [PMID:23810505]

Abstract [show]
California uses a unique method to screen newborns for cystic fibrosis (CF) that includes gene scanning and DNA sequencing after only one California-40 cystic fibrosis transmembrane conductance regulator (CFTR) panel mutation has been identified in hypertrypsinogenemic specimens. Newborns found by sequencing to have one or more additional mutations or variants (including novel variants) in the CFTR gene are systematically followed, allowing for prospective assessment of the pathogenic potential of these variants. During the first 3 years of screening, 55 novel variants were identified. Six of these novel variants were discovered in five screen-negative participants and three were identified in multiple unrelated participants. Ten novel variants (c.2554_2555insT, p.F1107L, c.-152G>C, p.L323P, p.L32M, c.2883_2886dupGTCA, c.2349_2350insT, p.K114del, c.-602A>T, and c.2822delT) were associated with a CF phenotype (42% of participants were diagnosed at 4 to 25 months of age), whereas 26 were associated with CFTR-related metabolic syndrome to date. Associations with the remaining novel variants were confounded by the presence of other diseases or other mutations in cis or by inadequate follow-up. These findings have implications for how CF newborn screening and follow-up is conducted and will help guide which genotypes should, and which should not, be considered screen positive for CF in California and elsewhere.

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26 Newborns were screened using the California method, which includes i) analysis of serum immunoreactive trypsinogen (IRT) levels using the AutoDELFIA neonatal IRT L kit (PerkinElmer, Waltham, MA) in all newborn blood spot specimens, ii) CFTR mutation panel [29-40 mutations (the mutations on the California panel were selected for the most part according to allelic frequencies found in a comprehensively genotyped group of California CF cases to achieve a 95% race/ethnicity-specific rate of CF case detection in black, white, and Hispanic individuals in California and include c.1585-1G>A, c.1680-1G>A, c.1973-1985del13insAGAAA, c.2175_2176insA, c.164 &#fe; 2T>A (removed on August 12, 2008), c.2988 &#fe; 1G>A, c.3717 &#fe; 12191C>T, c.3744delA, c.274-1G>A, c.489 &#fe; 1G>T, c.579 &#fe; 1G>T, p.A559T, p.F311del, p.F508del, p.I507del, p.G542X, p.G551D, p.G85E, p.H199Y, p.N1303K, p.R1066C, p.R1162X, p.R334W, p.R553X, p.S549N, p.W1089X, p.W1204X (c.3611G>A), p.W1282X, c.1153_1154insAT [added October 4, 2007], c.1923_1931del9insA, c.3140-26A>G, c.531delT, c.803delA, c.54-5940_273 &#fe; 10250del21kb, p.P205S, p.Q98R, p.R75X, p.S492F [added December 12, 2007], c.3659delC, p.G330X, p.W1204X [c.3612G>A] [added August 12, 2008] [Signature CF 2.0 ASR; Asuragen Inc., Austin, TX])] testing of specimens with IRT 62 ng/mL (highest 1.5%), iii) CFTR gene scanning and sequence analysis (Ambry Test: CF; Ambry Genetics, Aliso Viejo, CA) for specimens found to have only one mutation after CFTR mutation panel testing, and iv) referral to 1 of 15 pediatric CF care centers (CFCs) for sweat chloride (SC) testing and follow-up of all newborns with either two CFTR mutations detected during panel testing or one CFTR mutation detected during panel testing and one (or more) additional CFTR mutation and/or variant detected during sequencing. Login to comment
59 of parents receiving CFTR mutation testing Diagnosis/ status Study participants with positive NBS results 1 W, H 83.5 p.F508del* c.2554_2555insTy 7T/9T 2 CF 2 H 527.0 p.F508del c.-877C>T p.F1107L 7T/9T 0 CF 3 W 86.5 p.F508del p.V562Iy c.-837T>Cy 5Tyz /9T 1 CF 4 H 222.3 p.F508del p.I556V c.1278delC NA 0 CF 5 H, O 93.5 p.F508del* c.-152G>Cy 7T/9T 2 CF 6 W, H, B, O 95.4 p.F508del* p.L323Py 5Tyz /9T 2 CF 7 H 70.5 p.F508del p.L32M 7T/9T 0 CF 8 W 209.5 p.F508del c.2883_2886dupGTCA 9T/9T 0 CF 9 H 155.7 p.F508del* c.2349_2350insT 7T/9T 1 CF 10 O 146.8 p.F508del* c.3718-24G>Ay 5Tyx /9T 2 CF 11 B 99.4 p.A559T* p.L206Wy c.-448A>G* 7T/9T 2 CF 12 W, H 90.3 p.P205S p.K114del 7T/7T 0 CF 13 H 69.7 p.P205S p.K114del 7T/7T 0 CF 14 H 82.9 c.274-1G>A* c.-602A>Ty 7T/7T 2 CF 15 W 106.6 p.F508del* c.-461A>Gy c.-983A>T* 7T/9T 2 CRMS 16 W, B 83.9 p.F508del c.4243-5C>T 5T*x /9T 1 CRMS 17 W 81.5 p.F508del* p.I1027T* p.Y325C 7T/9T 2 CRMS 18 H 70.7 p.F508del c.-967T>C 9T/9T 0 CRMS 19 W, H 62.4 p.F508del* c.-635A>G 7T/9T 1 CRMS 20 H 65.4 p.F508dely c.2490 &#fe; 14G>T* 7T/9T 2 CRMS 21 W 69.3 p.F508del* c.744-15T>Cy 7T/9T 2 CRMS 22 W, H, O 66.2 p.F508del p.D249Y 7T/9T 0 CRMS 23 H 94.8 p.F508del p.R811S 7T/9T 0 CRMS 24 W 75.8 p.F508del* p.H1375Ny 7T/9T 2 CRMS 25 H 63.0 p.F508del p.L136P 7T/9T 0 CRMS 26 W, O 63.0 p.F508del* p.M1140L 7T/9T 1 CRMS 27 W, O 91.7 p.F508del p.V1198M 9T/9T 0 CRMS 28 H 69.3 p.F508dely c.1767-13T>G* 7T/9T 2 CRMS 29 H 108.8 p.F508del p.V1322L 7T/9T 0 CRMS 30 H 96.4 p.F508dely p.C76R* 7T/9T 2 CRMS 31 H 69.0 c.3140-26A>G c.-510G>A* 7T/7T 1 CRMS 32 H 100.2 p.G542X c.-684G>A* 7T/9T 1 CRMS 33 H 84.1 c.1153_1154insAT* c.-730A>Gy 7T/7T 2 CRMS 34 H 62.9 c.1973_ 1985del13insAGAAA* p.D112Gy 7T/7T 2 CRMS 35 H 116.7 c.3744delA* p.T887P 7T/7T 1 CRMS 36 B 73.3 c.2988 &#fe; 1G>A c.-288G>C 7T/9T 0 CRMS 37 H 93.5 p.R75X c.3367 &#fe; 3A>C 7T/7T 0 CRMS 38 W, H 81.4 c.3717 &#fe; 12191C>T* c.-769A>Gy 7T/7T 2 CRMS 39 W 79.0 c.3717 &#fe; 12191C>Ty p.R668Cy p.T1396P* 7T/9T 2 CRMS 40 H 87.3 c.274-1G>A p.F315S 7T/7T 0 CRMS 41 H 79.7 p.G542X c.869 &#fe; 8G>T 7T/9T 0 CRMS 42 O 79.8 p.R553X p.T1478R 7T/7T 0 CRMS 43 H 70.5 p.A559T* c.-448A>G* 7T/7T 2 Carrier 44 B 76.2 p.A559T* c.-448A>G* 7T/7T 1 Carrier 45 W, H 69.2 p.G85E* c.744-15T>C* 5Tyz /7T 2 Carrier 46 W 69.1 p.N1303K* c.2490 &#fe; 14G>A* 7T/9T 1 Carrier 47 W, O 111.7 p.F508del c.3963 &#fe; 6G>T 7T/9T 0 ND{ 48 W 80.1 p.F508del p.R1128G 7T/9T 0 ND{ (table continues) sequencing. Login to comment

[hide] Cystic fibrosis transmembrane regulator correctors... Cold Spring Harb Perspect Med. 2013 Jul 1;3(7). pii: a009761. doi: 10.1101/cshperspect.a009761. Rowe SM, Verkman AS
Cystic fibrosis transmembrane regulator correctors and potentiators.
Cold Spring Harb Perspect Med. 2013 Jul 1;3(7). pii: a009761. doi: 10.1101/cshperspect.a009761., [PMID:23818513]

Abstract [show]
Cystic fibrosis (CF) is caused by loss-of-function mutations in the CF transmembrane conductance regulator (CFTR) protein, a cAMP-regulated anion channel expressed primarily at the apical plasma membrane of secretory epithelia. Nearly 2000 mutations in the CFTR gene have been identified that cause disease by impairing its translation, cellular processing, and/or chloride channel gating. The fundamental premise of CFTR corrector and potentiator therapy for CF is that addressing the underlying defects in the cellular processing and chloride channel function of CF-causing mutant CFTR alleles will result in clinical benefit by addressing the basic defect underlying CF. Correctors are principally targeted at F508del cellular misprocessing, whereas potentiators are intended to restore cAMP-dependent chloride channel activity to mutant CFTRs at the cell surface. This article reviews the discovery of CFTR potentiators and correctors, what is known regarding their mechanistic basis, and encouraging results achieved in clinical testing.

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9 The F508del mutation, which is present in at least one allele in 90% ofCFpatients,impairsCFTRfolding,stabilityat the endoplasmic reticulum and plasma membrane, and chloride channel gating (Dalemans etal.1991;Denningetal.1992;Lukacsetal.1993; Duetal.2005a).Othermutationsprimarilyalter channelgating(e.g.,G551D),conductance(e.g., R117H),ortranslation(e.g.,G542X)(Welshand Smith1993).ThefundamentalpremiseofCFTR corrector and potentiator therapy for CF is that Editors: John R. Riordan, Richard C. Boucher, and Paul M. Quinton Additional Perspectives on Cystic Fibrosis available at www.perspectivesinmedicine.org Copyright # 2013 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a009761 Cite this article as Cold Spring Harb Perspect Med 2013;3:a009761 1 correction of the underlying defects in the cellular processing and chloride channel function of CF-causing mutant CFTR alleles will be of clinical benefit. Login to comment

[hide] Genetic testing of sperm donors for cystic fibrosi... Eur J Obstet Gynecol Reprod Biol. 2013 Sep;170(1):183-7. doi: 10.1016/j.ejogrb.2013.06.022. Epub 2013 Jul 15. Landaburu I, Gonzalvo MC, Clavero A, Ramirez JP, Yoldi A, Mozas J, Zamora S, Martinez L, Castilla JA
Genetic testing of sperm donors for cystic fibrosis and spinal muscular atrophy: evaluation of clinical utility.
Eur J Obstet Gynecol Reprod Biol. 2013 Sep;170(1):183-7. doi: 10.1016/j.ejogrb.2013.06.022. Epub 2013 Jul 15., [PMID:23866907]

Abstract [show]
OBJECTIVE: To evaluate the clinical utility of genetic testing for cystic fibrosis (CF) and spinal muscular atrophy (SMA) in sperm donors. STUDY DESIGN: We studied the results of the genetic tests for CF and SMA applied to 372 sperm donor candidates. The CF carrier screening test analysed 32 mutations on the CFTR gene. Regarding SMA, the carrier test studied possible deletions of SMN1/2 by Multiplex Ligation-dependent Probe Amplification (MLPA) methodology. RESULTS: The carrier frequency obtained was greater for SMA than for CF. After adjusting the results obtained for the sensitivity of the tests, and taking into account the prevalence of female carriers in our population, the probability of transmission of the disease to the child from a donor with a negative genetic test was about five times lower in the case of SMA than in CF, although this difference was not statistically significant. The number of donors needed to screen (NNS) to avoid the occurrence of a child being affected by CF and SMA in our population was similar in both cases (1591 vs. 1536). CONCLUSIONS: This study demonstrates the need to include SMA among the diseases for which genetic screening is performed in the process of sperm donor selection. We believe that testing donors for SMA is as important and as useful as doing so for CF.

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51 The panel of mutations studied was: S549N, S549R, R553X, G551D, V520F, I507del, F508del, 3876delA, 1717-1G->A, G542X, R560T, 3120+1G->A, A455E, R117H, 394delTT, 2183AA- >G, 2184delA, 2789+5G->A, 1898+1G->A, 621+1G->T, 711+1G- >T, G85E, R347P, R347H, W1282X, R334W, 1078delT, 3849+10kbC->T, R1162X, N1303K, 3659delC, 3905insT. Login to comment
68 With respect to the relative distribution of the mutations observed, 3 of the 5 cases (60%) were F508del, and the remaining two (40%) were G542X. Login to comment
105 With regard to the relative frequency of mutations detected in our donor candidate population, two different mutations, F508del and G542X, were detected. Login to comment

[hide] Optimizing nasal potential difference analysis for... PLoS One. 2013 Jul 26;8(7):e66955. doi: 10.1371/journal.pone.0066955. Print 2013. Rowe SM, Liu B, Hill A, Hathorne H, Cohen M, Beamer JR, Accurso FJ, Dong Q, Ordonez CL, Stone AJ, Olson ER, Clancy JP
Optimizing nasal potential difference analysis for CFTR modulator development: assessment of ivacaftor in CF subjects with the G551D-CFTR mutation.
PLoS One. 2013 Jul 26;8(7):e66955. doi: 10.1371/journal.pone.0066955. Print 2013., [PMID:23922647]

Abstract [show]
Nasal potential difference (NPD) is used as a biomarker of the cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) activity. We evaluated methods to detect changes in chloride and sodium transport by NPD based on a secondary analysis of a Phase II CFTR-modulator study. Thirty-nine subjects with CF who also had the G551D-CFTR mutation were randomized to receive ivacaftor (Kalydeco; also known as VX-770) in four doses or placebo twice daily for at least 14 days. All data were analyzed by a single investigator who was blinded to treatment assignment. We compared three analysis methods to determine the best approach to quantify changes in chloride and sodium transport: (1) the average of both nostrils; (2) the most-polarized nostril at each visit; and (3) the most-polarized nostril at screening carried forward. Parameters of ion transport included the PD change with zero chloride plus isoproterenol (CFTR activity), the basal PD, Ringer's PD, and change in PD with amiloride (measurements of ENaC activity), and the delta NPD (measuring CFTR and ENaC activity). The average and most-polarized nostril at each visit were most sensitive to changes in chloride and sodium transport, whereas the most-polarized nostril at screening carried forward was less discriminatory. Based on our findings, NPD studies should assess both nostrils rather than a single nostril. We also found that changes in CFTR activity were more readily detected than changes in ENaC activity, and that rigorous standardization was associated with relatively good within-subject reproducibility in placebo-treated subjects (+/- 2.8 mV). Therefore, we have confirmed an assay of reasonable reproducibility for detecting chloride-transport improvements in response to CFTR modulation.

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66 Characteristic Part 1 Part 2 Placebo (n = 4) Ivacaftor TOTAL (n = 20) Placebo (n = 4) Ivacaftor TOTAL (n = 19) 25 mg/75 mg (n = 4) 75 mg/25 mg (n = 4) 75 mg/150 mg (n = 4) 150 mg/75 mg (n = 4) 150 mg (n = 8) 250 mg (n = 7) Sex, n (%) Male 2 (50) 1 (25) 4 (100) 1 (25) 1 (25) 9 (45) 3 (75) 3 (38) 4 (57) 10 (53) Female 2 (50) 3 (75) 0 3 (75) 3 (75) 11 (55) 1 (25) 5 (63) 3 (43) 9 (47) Race, n (%) White 4 (100) 4 (100) 4 (100) 4 (100) 4 (100) 20 (100) 4 (100) 8 (100) 7 (100) 19 (100) Age, yr, median (range) 36 (19-48) 31 (22-51) 41 (22-50) 26 (19-34) 21 (19-33) 30 (19-51) 24 (18-42) 23 (18-40) 21 (20-38) 21 (18-42) BMI, kg/m 2 , median (range) 23 (22-29) 23 (20-24) 24 (19-27) 20 (19-24) 21 (17-26) 23 (17-29) 22 (21-23) 22 (20-23) 23 (20-25) 22 (20-25) CFTR genotype G551D/F508del 3 (75) 4 (100) 4 (100) 2 (50) 3 (75) 16 (80) 4 (100) 7 (88) 5 (71) 16 (84) G551D/1078delT 1 (25) - - - - 1 (5) - - - - G551D/G551D - - - - 1 (25) 1 (5) - - - - G551D/N1303K - - - 1 (25) - 1 (5) - - - - G551D/R553X - - - 1 (25) - 1 (5) - - - - G551D/3849+10 kbC - - - - - - - - 1 (14) 1 (5) G551D/6214R1G+7T - - - - - - - 1 (13) - 1 (5) G551D/G542X - - - - - - - - 1 (14) 1 (5) % Predicted FEV 1 , median (range) 40% to ,70%, n (%) 3 (75) 3 (75) 4 (100) 2 (50) 4 (100) 16 (80) 2 (50) 5 (63) 3 (43) 10 (53) 70% to ,90%, n (%) - - - 1 (25) - 1 (5) .90%, n (%) - 1 (25) - 1 (25) - 3 (15) 2 (50) 1 (13) 1 (14) 4 (21) Sweat chloride, mmol/L, median (range) 105.25 (97.00- 112.00) 107.50 (60.00-117.00) 104.50 (102.00-117.00) 97.50 (92.00-102.50) 99.00 (83.00-104.00) 102.00 (60.00-117.00) 93.75 (88.00- 109.50) 100.13 (86.75- 112.50) 97.25 (84.75- 115.75) 95.50 (84.75- 115.75) BMI, body-mass index; CFTR, cystic fibrosis transmembrane conductance regulator; FEV 1 , forced expiratory volume in one second. Login to comment

[hide] Vasoactive intestinal peptide regulates sinonasal ... FASEB J. 2013 Dec;27(12):5094-103. doi: 10.1096/fj.13-234476. Epub 2013 Aug 9. Lee RJ, Chen B, Doghramji L, Adappa ND, Palmer JN, Kennedy DW, Cohen NA
Vasoactive intestinal peptide regulates sinonasal mucociliary clearance and synergizes with histamine in stimulating sinonasal fluid secretion.
FASEB J. 2013 Dec;27(12):5094-103. doi: 10.1096/fj.13-234476. Epub 2013 Aug 9., [PMID:23934280]

Abstract [show]
Mucociliary clearance (MCC) is the primary physical airway defense against inhaled pathogens and particulates. MCC depends on both proper fluid/mucus homeostasis and epithelial ciliary beating. Vasoactive intestinal peptide (VIP) is a neurotransmitter expressed in the sinonasal epithelium that is up-regulated in allergy. However, the effects of VIP on human sinonasal physiology are unknown, as are VIP's interactions with histamine, a major regulator of allergic disease. We imaged ciliary beat frequency, mucociliary transport, apical Cl(-) permeability, and airway surface liquid (ASL) height in primary human sinonasal air-liquid-interface cultures to investigate the effects of VIP and histamine. VIP stimulated an increase in ciliary beat frequency (EC50 0.5 muM; maximal increase approximately 40% compared with control) and cystic fibrosis transmembrane conductance regulator (CFTR)-dependent and Na(+)K(+)2Cl(-) cotransporter-dependent fluid secretion, all requiring cAMP/PKA signaling. Histamine activated Ca(2+) signaling that increased ASL height but not ciliary beating. Low concentrations of VIP and histamine had synergistic effects on CFTR-dependent fluid secretion, revealed by increased ASL heights. An up-regulation of VIP in histamine-driven allergic rhinitis would likely enhance mucosal fluid secretion and contribute to allergic rhinorrhea. Conversely, a loss of VIP-activated secretion in patients with CF may impair mucociliary transport, contributing to increased incidences of sinonasal infections and rhinosinusitis.

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182 To further test the role of CFTR in this synergistic fluid secretion, we measured basal and stimulated ASL heights in ALI cultures derived from CF patient cells (nafd;3 patients; genotypes were èc;F508/èc;F508, W1282X/N1303K, and èc;F508/G542X). Login to comment

[hide] Cigarette smoke and CFTR: implications in the path... Am J Physiol Lung Cell Mol Physiol. 2013 Oct 15;305(8):L530-41. doi: 10.1152/ajplung.00039.2013. Epub 2013 Aug 9. Rab A, Rowe SM, Raju SV, Bebok Z, Matalon S, Collawn JF
Cigarette smoke and CFTR: implications in the pathogenesis of COPD.
Am J Physiol Lung Cell Mol Physiol. 2013 Oct 15;305(8):L530-41. doi: 10.1152/ajplung.00039.2013. Epub 2013 Aug 9., [PMID:23934925]

Abstract [show]
Chronic obstructive pulmonary disease (COPD) is a progressive respiratory disorder consisting of chronic bronchitis and/or emphysema. COPD patients suffer from chronic infections and display exaggerated inflammatory responses and a progressive decline in respiratory function. The respiratory symptoms of COPD are similar to those seen in cystic fibrosis (CF), although the molecular basis of the two disorders differs. CF is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding a chloride and bicarbonate channel (CFTR), leading to CFTR dysfunction. The majority of COPD cases result from chronic oxidative insults such as cigarette smoke. Interestingly, environmental stresses including cigarette smoke, hypoxia, and chronic inflammation have also been implicated in reduced CFTR function, and this suggests a common mechanism that may contribute to both the CF and COPD. Therefore, improving CFTR function may offer an excellent opportunity for the development of a common treatment for CF and COPD. In this article, we review what is known about the CF respiratory phenotype and discuss how diminished CFTR expression-associated ion transport defects may contribute to some of the pathological changes seen in COPD.

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69 Only six other mutations have a frequency of greater than 1% in the CF population (G542X, W1282X, G551D, 621 af9; G&#a1;T, N1303K, and R553X) (157). Login to comment
123 Alternative approaches have included high-throughput screens to identify small molecules that promote either 1) F508del CFTR rescue and delivery to the cell surface (108), 2) read-through of premature stop mutations to override nonsense mutations and help translation to produce full-length proteins (e.g., G542X, W1282X, etc.) (160), or 3) potentiation of channel-gating mutations such as G551D (151). Login to comment

[hide] Different cystic fibrosis transmembrane conductanc... Urology. 2013 Oct;82(4):824-8. doi: 10.1016/j.urology.2013.06.024. Epub 2013 Aug 14. Lu S, Yang X, Cui Y, Li X, Zhang H, Liu J, Chen ZJ
Different cystic fibrosis transmembrane conductance regulator mutations in Chinese men with congenital bilateral absence of vas deferens and other acquired obstructive azoospermia.
Urology. 2013 Oct;82(4):824-8. doi: 10.1016/j.urology.2013.06.024. Epub 2013 Aug 14., [PMID:23953609]

Abstract [show]
OBJECTIVE: To investigate cystic fibrosis transmembrane conductance regulator (CFTR) gene in Chinese men with congenital bilateral absence of vas deferens (CBAVD) and other obstructive azoospermia. MATERIALS AND METHODS: Four hundred one patients with obstructive azoospermia were included. Patients were grouped as 158 with CBAVD and 243 with other acquired obstructive azoospermia. Another 200 fertile men were used as controls. Genomic deoxyribonucleic acid was isolated from peripheral blood lymphocytes for all men. The exon 10 and 11 CFTR genes were amplified and sequenced. The frequency of CFTR gene mutations was compared among 3 groups. RESULTS: Six heterozygous mutations (+/-), I556V, M469V, E527N, F508del, S485C, and I558S, were found in 30 patients, and 1 homozygous mutation (+/+), I556V, was found in 1 patient. The overall frequency of CFTR mutations was 31 of 401 (7.7%). Of these mutations, I556V was the most common type with 24 of 31 (77.4%). In CBAVD group, 20 of 158 patients were identified with 6 different heterozygous mutations (I556V, M469V, E527N, F508del, S485C, and I558S) and 1 homozygous mutation (I556V). The rate of CFTR mutations was 12.7%. In acquired obstructive group, 11 of 243 patients were identified with 2 different heterozygous mutations, I556V and M469V; the rate of mutations was 4.5%. No CFTR mutations were identified in controls. There was significant difference among 3 groups (P = .000). The frequency of CFTR mutations in CBAVD is 2-fold higher than in other acquired obstructive group. CONCLUSION: Different CFTR mutations are observed in Chinese patients with CBAVD. I556V is the major common type of CFTR mutations in Chinese patients with CBAVD.

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70 In this study, we analyzed exons 10 and 11 CFTR gene in CBAVD and other acquired obstructive azoospermia because common mutations such as F508del, D507, G551D, G542X, R560T, and R553X associated with CF in Caucasians were frequently identified in these exons. Login to comment

[hide] Reactive-oxygen-species-mediated P. aeruginosa kil... PLoS One. 2013 Aug 19;8(8):e71717. doi: 10.1371/journal.pone.0071717. eCollection 2013. Cifani N, Pompili B, Anile M, Patella M, Diso D, Venuta F, Cimino G, Quattrucci S, Di Domenico EG, Ascenzioni F, Del Porto P
Reactive-oxygen-species-mediated P. aeruginosa killing is functional in human cystic fibrosis macrophages.
PLoS One. 2013 Aug 19;8(8):e71717. doi: 10.1371/journal.pone.0071717. eCollection 2013., [PMID:23977124]

Abstract [show]
Pseudomonas aeruginosa is the most common pathogen for chronic lung infection in cystic fibrosis (CF) patients. About 80% of adult CF patients have chronic P. aeruginosa infection, which accounts for much of the morbidity and most of the mortality. Both bacterial genetic adaptations and defective innate immune responses contribute to the bacteria persistence. It is well accepted that CF transmembrane conductance regulator (CFTR) dysfunction impairs the airways-epithelium-mediated lung defence; however, other innate immune cells also appear to be affected, such as neutrophils and macrophages, which thus contribute to this infectious pathology in the CF lung. In macrophages, the absence of CFTR has been linked to defective P. aeruginosa killing, increased pro-inflammatory cytokine secretion, and reduced reactive oxygen species (ROS) production. To learn more about macrophage dysfunction in CF patients, we investigated the generation of the oxidative burst and its impact on bacterial killing in CF macrophages isolated from peripheral blood or lung parenchyma of CF patients, after P. aeruginosa infection. Our data demonstrate that CF macrophages show an oxidative response of similar intensity to that of non-CF macrophages. Intracellular ROS are recognized as one of the earliest microbicidal mechanisms against engulfed pathogens that are activated by macrophages. Accordingly, NADPH inhibition resulted in a significant increase in the intracellular bacteria survival in CF and non-CF macrophages, both as monocyte-derived macrophages and as lung macrophages. These data strongly suggest that the contribution of ROS to P. aeruginosa killing is not affected by CFTR mutations.

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61 Patient Age Gender Genotype CF13 23 F F508del/F508del CF14 30 F F508del/W1282X CF15 16 F F508del/574delA CF16 34 M F508del/unknown CF17 15 M F508del/F508del CF18 24 F F508del/2,3del21Kb CF19 30 M F508del/F508del CF20 35 F N1303K/H119R CF21 52 M F508del/F508del CF22 30 M F508del/F508del CF23 41 M F508del/F508del CF24 33 M F508del/S549R(A_.C) CF25 39 M F508del/G542X CF26 31 F W1282X/W1282X CF27 30 M F508del/N1303K CF28 28 F F508del/F508del CF29 31 F F508del/G542X doi:10.1371/journal.pone.0071717.t001 CFTR expression was analysed on RNA samples isolated from non-CF macrophages using real-time PCR, as previously reported [21]. Login to comment

[hide] Targeting protein-protein interactions to rescue D... EMBO Mol Med. 2013 Oct;5(10):1462-4. doi: 10.1002/emmm.201303301. Epub 2013 Aug 27. Devesa I, Fernandez-Ballester G, Ferrer-Montiel A
Targeting protein-protein interactions to rescue Deltaf508-cftr: a novel corrector approach to treat cystic fibrosis.
EMBO Mol Med. 2013 Oct;5(10):1462-4. doi: 10.1002/emmm.201303301. Epub 2013 Aug 27., [PMID:23983009]

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6 The CF phenotype is caused by more than 1000 mutations of the CFTR gene including missense, such as R117H or G551D that significantly reduce channel activity, and nonsense like G542X, R553X or W1282X, which abrogate protein expression (Kreindler, 2010). Login to comment

[hide] Molecular testing for cystic fibrosis carrier stat... J Genet Couns. 2014 Feb;23(1):5-15. doi: 10.1007/s10897-013-9636-9. Epub 2013 Sep 7. Langfelder-Schwind E, Karczeski B, Strecker MN, Redman J, Sugarman EA, Zaleski C, Brown T, Keiles S, Powers A, Ghate S, Darrah R
Molecular testing for cystic fibrosis carrier status practice guidelines: recommendations of the National Society of Genetic Counselors.
J Genet Couns. 2014 Feb;23(1):5-15. doi: 10.1007/s10897-013-9636-9. Epub 2013 Sep 7., [PMID:24014130]

Abstract [show]
PURPOSE: To provide practice recommendations for genetic counselors whose clients are considering cystic fibrosis (CF) carrier testing or seeking information regarding CF molecular test results. The goals of these recommendations are to: 1) Provide updated information about the natural history, diagnosis, and treatment of CF and related conditions. 2) Supplement genetic counselors' knowledge and understanding of the available carrier screening and diagnostic testing options. 3) Describe the current state of genotype/phenotype correlations for CFTR mutations and an approach to interpreting both novel and previously described variants. 4) Provide a framework for genetic counselors to assist clients' decision-making regarding CF carrier testing, prenatal diagnosis, and pregnancy management. Disclaimer The practice guidelines of the National Society of Genetic Counselors (NSGC) are developed by members of the NSGC to assist genetic counselors and other health care providers in making decisions about appropriate management of genetic concerns; including access to and/or delivery of services. Each practice guideline focuses on a clinical or practice-based issue, and is the result of a review and analysis of current professional literature believed to be reliable. As such, information and recommendations within the NSGC practice guidelines reflect the current scientific and clinical knowledge at the time of publication, are only current as of their publication date, and are subject to change without notice as advances emerge.In addition, variations in practice, which take into account the needs of the individual patient and the resources and limitations unique to the institution or type of practice, may warrant approaches, treatments and/or procedures that differ from the recommendations outlined in this guideline. Therefore, these recommendations should not be construed as dictating an exclusive course of management, nor does the use of such recommendations guarantee a particular outcome. Genetic counseling practice guidelines are never intended to displace a health care provider's best medical judgment based on the clinical circumstances of a particular patient or patient population.Practice guidelines are published by NSGC for educational and informational purposes only, and NSGC does not "approve" or "endorse" any specific methods, practices, or sources of information.

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172 G542X, W1282X II Cause structural alterations to the CFTR protein and prevent it from moving to the cell surface. Login to comment

[hide] Ivacaftor: a review of its use in patients with cy... Drugs. 2013 Sep;73(14):1595-604. doi: 10.1007/s40265-013-0115-2. Deeks ED
Ivacaftor: a review of its use in patients with cystic fibrosis.
Drugs. 2013 Sep;73(14):1595-604. doi: 10.1007/s40265-013-0115-2., [PMID:24030637]

Abstract [show]
Ivacaftor (Kalydeco) is a potentiator of the cystic fibrosis transmembrane conductance regulator (CFTR) and is the first drug that treats an underlying cause of cystic fibrosis to be licensed for use. Ivacaftor increases the open probability (i.e. gating) of CFTR channels with the G551D mutation, thus enhancing chloride transport, and is indicated in a number of countries for the treatment of cystic fibrosis in patients aged >/=6 years who carry this mutation. This review focuses on pharmacological, clinical efficacy and tolerability data relevant to the use of ivacaftor in this indication. In two 48-week, double-blind, phase III trials in patients aged >/=12 (STRIVE) or 6-11 (ENVISION) years with cystic fibrosis and the G551D mutation, oral ivacaftor 150 mg every 12 h significantly improved lung function relative to placebo, when used in combination with standard care. Significant improvements in pulmonary exacerbation risk (in STRIVE) as well as bodyweight and some aspects of health-related quality of life (both studies) were also seen with the drug versus placebo. Moreover, the beneficial effects of ivacaftor on parameters such as lung function and bodyweight were maintained over up to 96 weeks of treatment in an ongoing open-label extension of these studies. Ivacaftor was generally well tolerated, with headache, oropharyngeal pain, upper respiratory tract infection and nasal congestion being among the most common adverse events. Thus, ivacaftor expands the current treatment options for patients with cystic fibrosis who have the G551D mutation. Its potential for use in patients with other CFTR mutations is also of interest.

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37 The in vitro effect of ivacaftor on CFTR mutant proteins with minimal chloride transport, such as those with mutations affecting CFTR synthesis (e.g. G542X) [5] or severely affecting CFTR conductance (e.g. R334W) or processing (e.g. F508del [13]) [5, 16], was minimal [5, 13] or less than its effect on CFTR proteins with mild conductance or processing defects [16]. Login to comment

[hide] Multicenter intestinal current measurements in rec... PLoS One. 2013 Sep 10;8(9):e73905. doi: 10.1371/journal.pone.0073905. eCollection 2013. Clancy JP, Szczesniak RD, Ashlock MA, Ernst SE, Fan L, Hornick DB, Karp PH, Khan U, Lymp J, Ostmann AJ, Rezayat A, Starner TD, Sugandha SP, Sun H, Quinney N, Donaldson SH, Rowe SM, Gabriel SE
Multicenter intestinal current measurements in rectal biopsies from CF and non-CF subjects to monitor CFTR function.
PLoS One. 2013 Sep 10;8(9):e73905. doi: 10.1371/journal.pone.0073905. eCollection 2013., [PMID:24040112]

Abstract [show]
Intestinal current measurements (ICM) from rectal biopsies are a sensitive means to detect cystic fibrosis transmembrane conductance regulator (CFTR) function, but have not been optimized for multicenter use. We piloted multicenter standard operating procedures (SOPs) to detect CFTR activity by ICM and examined key questions for use in clinical trials. SOPs for ICM using human rectal biopsies were developed across three centers and used to characterize ion transport from non-CF and CF subjects (two severe CFTR mutations). All data were centrally evaluated by a blinded interpreter. SOPs were then used across four centers to examine the effect of cold storage on CFTR currents and compare CFTR currents in biopsies from one subject studied simultaneously either at two sites (24 hours post-biopsy) or when biopsies were obtained by either forceps or suction. Rectal biopsies from 44 non-CF and 17 CF subjects were analyzed. Mean differences (microA/cm(2); 95% confidence intervals) between CF and non-CF were forskolin/IBMX=102.6(128.0 to 81.1), carbachol=96.3(118.7 to 73.9), forskolin/IBMX+carbachol=200.9(243.1 to 158.6), and bumetanide=-44.6 (-33.7 to -55.6) (P<0.005, CF vs non-CF for all parameters). Receiver Operating Characteristic curves indicated that each parameter discriminated CF from non-CF subjects (area under the curve of 0.94-0.98). CFTR dependent currents following 18-24 hours of cold storage for forskolin/IBMX, carbachol, and forskolin/IBMX+carbachol stimulation (n=17 non-CF subjects) were 44%, 47.5%, and 47.3%, respectively of those in fresh biopsies. CFTR-dependent currents from biopsies studied after cold storage at two sites simultaneously demonstrated moderate correlation (n=14 non-CF subjects, Pearson correlation coefficients 0.389, 0.484, and 0.533). Similar CFTR dependent currents were detected from fresh biopsies obtained by either forceps or suction (within-subject comparisons, n=22 biopsies from three non-CF subjects). Multicenter ICM is a feasible CFTR outcome measure that discriminates CF from non-CF ion transport, offers unique advantages over other CFTR bioassays, and warrants further development as a potential CFTR biomarker.

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223 In addition, Bedwell and colleagues have shown that transgenic mice carrying the human G542X mutation in CFTR that are treated with suppressors of premature termination codons (aminoglycosides, ataluren) have detectable CFTR function in intestinal tissue studied by ICM [46-49]. Login to comment

[hide] Analysis of CFTR Gene Mutations in Children with C... Iran J Basic Med Sci. 2013 Aug;16(8):917-21. Mehdizadeh Hakkak A, Keramatipour M, Talebi S, Brook A, Tavakol Afshari J, Raazi A, Kianifar HR
Analysis of CFTR Gene Mutations in Children with Cystic Fibrosis, First Report from North-East of Iran.
Iran J Basic Med Sci. 2013 Aug;16(8):917-21., [PMID:24106596]

Abstract [show]
OBJECTIVE(S): More than 1500 registered mutations in cystic fibrosis transmembrane regulator (CFTR) gene are responsible for dysfunction of an ion channel protein and a wide spectrum of clinical manifestations in patients with cystic fibrosis (CF). This study was performed to investigate the frequency of a number of well-known CFTR mutations in North Eastern Iranian CF patients. MATERIAL AND METHODS: A total number of 56 documented CF patients participated in this study. Peripheral blood was obtained and DNA extraction was done by the use of routin methods. Three steps were taken for determining the target mutations: ARMS-PCR was performed for common CFTR mutations based on previous reports in Iran and neighboring countries. PCR-RFLP was done for detection of R344W and R347P, and PCR-Sequencing was performed for exon 11 in patients with unidentified mutation throughout previous steps. Samples which remained still unknown for a CFTR mutation were sequenced for exon 12. RESULTS: Among 112 alleles, 24 mutated alleles (21.42%) were detected: DeltaF508 (10.71%), 1677delTA (3.57%), S466X (3.57%), N1303K (0.89%), G542X (0.89%), R344W (0.89%), L467F (0.89%). Eight out of 56 individuals analyzed, were confirmed as homozygous and eight samples showed heterozygous status. No mutations were detected in exon 12 of sequenced samples. CONCLUSION: Current findings suggest a selected package of CFTR mutations for prenatal, neonatal and carrier screening along with diagnosis and genetic counseling programs in CF patients of Khorasan.

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7 Results: Among 112 alleles, 24 mutated alleles (21.42%) were detected: ƊF508 (10.71%), 1677delTA (3.57%), S466X (3.57%), N1303K (0.89%), G542X (0.89%), R344W (0.89%), L467F (0.89%). Login to comment
20 Although, the prevalence and types of mutations vary in different populations based on their geographic and ethnic origins (9, 10), a few mutations (p.F508del, p.G542X, p.N1303K, p.G551D, p.W1282X) have higher frequencies than others. Login to comment
53 Demographic, clinical, and family characterizations of patients with specific CFTR mutation No of patients Sex Sweat chloride (meq/lit) Pancreatic insufficiency Age of clinical presentation onset (month) First clinical symptom/sign Consanguinity of parents Mutation status 1 M 110 + 6 Steatorrhea/Hepatomegaly First cousin ƊF508/ ƊF508 2 M 115 + 5 Steatorrhea/Cough/ Hepatomegaly First cousin ƊF508/ ƊF508 3 F 130 + 2 Steatorrhea/Cough Wheezing/Skin rash First cousin ƊF508/ ƊF508 4 F 180 + 1 Steatorrhea/Cough/Vomiting/E dema/Hepatomegaly First cousin ƊF508/ ƊF508 5 M 93 + 3.5 FTT/Steatorrhea First cousin once removed ƊF508/ ƊF508 6 M 100 + At birth Wheezing/Meconium ileus - ƊF508/U* 7 M 115 + 2 Steatorrhea/Cough/Fever First cousin once removed ƊF508/U 8 M 90 + 6 Cough/Wheezing - N1303K/U 9 F 70 + At birth Meconium ileus/Crackle First cousin G542X/U 10 F 80 - 5 Cough/Wheezing/Fever - R334W/U 11 M 109 + 1 Fever/Wheezing/Cough Second cousin S466X/ S466X 12 M 120 + 10 Cough/Wheezing/Steatorrhea - S466X/U 13 M 100 + At birth Wheezing/Meconium ileus First cousin S466X/U 14 M 100 + 5.5 Rectal prolapse/Cough/ Wheezing/Steatorrhea First cousin 1677delTA/ 1677delTA 15 M 85 + 3 FTT/Sreatorrhea/Wheezing/ Cough First cousin 1677delTA/ 1677delTA 16 F 93 + 4 Steatorrhea - 1531C/T (L467F)/U * Unknown mutation PCR-RFLP was operated for identification of p.Arg334Trp and p.Arg347Pro mutations and revealed only one heterozygote status for p.Arg334Trp mutation. Login to comment
65 Total chromosomes: 100%, known mutations: 21.42%, unknown mutations: 78.58% cDNA name Protein name Legacy name Number of chromosomes detected Exon/Intron Description Detection method c.1000C>T p.Arg334Trp R334W 1 (0.89* -4.16 &#f0ff; ) Exon 8 C to T at 1132 PCR-RFLP c.1397C>G p.Ser466X S466X 4 (3.57 - 16.66) Exon 11 C to G at 1529 Sequencing c.1399C>T p.Leu467Phe 1531C/T (L467F) 1 (0.89 - 4.16) Exon 11 C or T at 1531 Sequencing c.1521-1523delCTT p.Phe508del ƊF508 12 (10/71 - 50) Exon 11 deletion of 3 bp between 1652 and 1655 ARMS and Sequencing c.1545-1546delTA p.Tyr515X 1677delTA 4 (3.57 - 16.66) Exon 11 deletion of TA from 1677 Sequencing c.1624G>T p.Gly542X G542X 1 (0.89 - 4.16) Exon 12 G to T at 1756 ARMS c.3909C>G p.Asn1303Lys N1303K 1 (0.89 - 4.16) Exon 24 C to G at 4041 ARMS * % of all analyzed chromosomes &#f0ff; % of all mutated chromosomes Alibakhshi et al (2008) (13) explored 69 Iranian CF patients sampled from different geographic areas and ethnic groups around Iran. Login to comment

[hide] Genotype-specific alterations in vascular smooth m... J Cyst Fibros. 2014 May;13(3):251-9. doi: 10.1016/j.jcf.2013.10.009. Epub 2013 Oct 31. Guo JJ, Stoltz DA, Zhu V, Volk KA, Segar JL, McCray PB Jr, Roghair RD
Genotype-specific alterations in vascular smooth muscle cell function in cystic fibrosis piglets.
J Cyst Fibros. 2014 May;13(3):251-9. doi: 10.1016/j.jcf.2013.10.009. Epub 2013 Oct 31., [PMID:24183914]

Abstract [show]
BACKGROUND: The most common CF-causing mutations interfere with CFTR trafficking from the endoplasmic reticulum (CFTR-F508del) or prematurely terminate transcription (CFTR-null). We suspected that genotype-specific patterns of CFTR expression would have differential effects on smooth muscle cell calcium signaling and hence vascular tone. We hypothesized that compared to wild-type or CFTR-null aorta, aorta from CFTR-F508del (dF) piglets will have reduced endoplasmic reticulum calcium mobilization and decreased vasoconstriction. METHODS: Aortic reactivity was assessed by myography, and ratiometric calcium imaging was performed in isolated vascular smooth muscle cells. RESULTS: Aorta from dF piglets had reduced myogenic tone (P<0.001) and decreased constriction to KCl (P<0.05). Combined inhibition of ryanodine and IP3 receptors decreased wild-type and CFTR-null responses to levels seen in dF aorta. Compared to wild-type cells, dF-expressing smooth muscle cells had reduced calcium transients, while CFTR-null cells had decreased baseline intracellular calcium concentrations. CONCLUSIONS: Expression of CFTR-F508del interferes with smooth muscle cell calcium handling and decreases aortic responsiveness.

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12 The second most common CF-causing mutation (G542X) prematurely terminates CFTR transcription [1]. Login to comment

[hide] Sinonasal manifestations of cystic fibrosis: a cor... J Cyst Fibros. 2014 Jul;13(4):442-8. doi: 10.1016/j.jcf.2013.10.011. Epub 2013 Nov 5. Berkhout MC, van Rooden CJ, Rijntjes E, Fokkens WJ, el Bouazzaoui LH, Heijerman HG
Sinonasal manifestations of cystic fibrosis: a correlation between genotype and phenotype?
J Cyst Fibros. 2014 Jul;13(4):442-8. doi: 10.1016/j.jcf.2013.10.011. Epub 2013 Nov 5., [PMID:24210900]

Abstract [show]
BACKGROUND: Patients with Cystic Fibrosis are prone to develop sinonasal disease. Studies in genotype-phenotype correlations for sinonasal disease are scarce and inconclusive. METHODS: In this observational study several aspects of sinonasal disease were investigated in 104 adult patients with CF. In each patient a disease specific quality of life questionnaire (RSOM-31), nasal endoscopy and a CT scan of the paranasal sinuses were performed. Patients were divided into two groups, class I-III mutations and class IV-V mutations, based on their CFTR mutations. RESULTS: The prevalence of rhinosinusitis in adult patients with CF was 63% and the prevalence of nasal polyps 25%. Patients with class I-III mutations had significantly smaller frontal sinuses, sphenoid sinuses, more opacification in the sinonasal area and more often osteitis/neoosteogenesis of the maxillary sinus wall compared to patients with class IV and V mutations. CONCLUSION: These data suggest more severe sinonasal disease in patients with class I-III mutations compared to patients with class IV-V mutations.

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163 Genotype Frequency; N (%) Class of mutation F508del/F508del 61 (58.7) I-III F508del/3849 + 10kbC 2 (1.9) IV-V F508del/N1303K 2 (1.9) I-III F508del/R1162X 2 (1.9) I-III F508del/A455E 12 (11.5) IV-V F508del/3272-26A N G 5 (4.8) IV-V F508del/E528X 1 (1.0) I-III F508del/S1251N 3 (2.9) IV-V F508del/R75Q 1 (1.0) IV-V F508del/G542X 2 (1.9) I-III F508del/1717-1G N A 1 (1.0) I-III F508del/Ser489X 1 (1.0) I-III F508del/4382delA 1 (1.0) -a F508del/L1077 1 (1.0) I-III F508del/1813insC 1 (1.0) -b A455E/S1251N 1 (1.0) IV-V A455E/E60X 1 (1.0) IV-V 3272-26A N G/G970R 1 (1.0) IV-V 3272-26A N G/R1162X 1 (1.0) IV-V F508del/UNK 2 (1.9) -c R117H-7T/UNK 1 (1.0) -d UNK/UNK 1 (1.0) -e Total 104 (100.4) One patient with pancreatic sufficiency and diagnosed at 46 years of age (class IV-V). Login to comment

[hide] Synthetic aminoglycosides efficiently suppress cys... Am J Respir Cell Mol Biol. 2014 Apr;50(4):805-16. doi: 10.1165/rcmb.2013-0282OC. Xue X, Mutyam V, Tang L, Biswas S, Du M, Jackson LA, Dai Y, Belakhov V, Shalev M, Chen F, Schacht J, J Bridges R, Baasov T, Hong J, Bedwell DM, Rowe SM
Synthetic aminoglycosides efficiently suppress cystic fibrosis transmembrane conductance regulator nonsense mutations and are enhanced by ivacaftor.
Am J Respir Cell Mol Biol. 2014 Apr;50(4):805-16. doi: 10.1165/rcmb.2013-0282OC., [PMID:24251786]

Abstract [show]
New drugs are needed to enhance premature termination codon (PTC) suppression to treat the underlying cause of cystic fibrosis (CF) and other diseases caused by nonsense mutations. We tested new synthetic aminoglycoside derivatives expressly developed for PTC suppression in a series of complementary CF models. Using a dual-luciferase reporter system containing the four most prevalent CF transmembrane conductance regulator (CFTR) nonsense mutations (G542X, R553X, R1162X, and W1282X) within their local sequence contexts (the three codons on either side of the PTC), we found that NB124 promoted the most readthrough of G542X, R1162X, and W1282X PTCs. NB124 also restored full-length CFTR expression and chloride transport in Fischer rat thyroid cells stably transduced with a CFTR-G542XcDNA transgene, and was superior to gentamicin and other aminoglycosides tested. NB124 restored CFTR function to roughly 7% of wild-type activity in primary human bronchial epithelial (HBE) CF cells (G542X/delF508), a highly relevant preclinical model with endogenous CFTR expression. Efficacy was further enhanced by addition of the CFTR potentiator, ivacaftor (VX-770), to airway cells expressing CFTR PTCs. NB124 treatment rescued CFTR function in a CF mouse model expressing a human CFTR-G542X transgene; efficacy was superior to gentamicin and exhibited favorable pharmacokinetic properties, suggesting that in vitro results translated to clinical benefit in vivo. NB124 was also less cytotoxic than gentamicin in a tissue-based model for ototoxicity. These results provide evidence that NB124 and other synthetic aminoglycosides provide a 10-fold improvement in therapeutic index over gentamicin and other first-generation aminoglycosides, providing a promising treatment for a wide array of CFTR nonsense mutations.

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1 Using a dual-luciferase reporter system containing the four most prevalent CF transmembrane conductance regulator (CFTR) nonsense mutations (G542X, R553X, R1162X, and W1282X) within their local sequence contexts (the three codons on either side of the PTC), we found that NB124 promoted the most readthrough of G542X, R1162X, and W1282X PTCs. Login to comment
3 NB124 restored CFTR function to roughly 7% of wild-type activity in primary human bronchial epithelial (HBE) CF cells (G542X/ delF508), a highly relevant preclinical model with endogenous CFTR expression. Login to comment
5 NB124 treatment rescued CFTR function in a CF mouse model expressing a human CFTR-G542X transgene; efficacy was superior to gentamicin and exhibited favorable pharmacokinetic properties, suggesting that in vitro results translated to clinical benefit in vivo. Login to comment
28 For example, poly-L-aspartic acid, a compound previously shown to significantly reduce aminoglycoside toxicity, was found to increase both the level and duration of readthrough in a CFTR-G542X transgenic mouse (13). Login to comment
34 One of the early compounds produced, NB54, suppressed PTCs to a level comparable to gentamicin in immortalized and primary human CF cells, as well as in the CFTR-G542X transgenic mouse model, while exhibiting low toxicity, suggesting a potential advantage of this approach (21). Login to comment
43 Dual Luciferase Assay Readthrough cassettes contained the G542X, R553X, R1162X, or W1282X CFTR PTCs (or the corresponding wild-type codon) together with three codons of upstream and downstream human CFTR sequence (Figure 2A; see also Table E1 in the online supplement). Login to comment
62 Cftr knockout mice expressing a human CFTR-G542X transgene under intestine-specific rat fatty acid binding protein were treated by subcutaneous injection once daily for 14 days (6, 13, 15). Login to comment
76 To test suppression of the four most common CFTR PTCs (G542X, R553X, R1162X, and W1282X), we constructed dual-luciferase reporters that each contained a Renilla gene, a firefly gene, and a CFTR readthrough cassette with each PTC, and the context of three additional codons on either side of the PTC (Figure 2A). Login to comment
95 Initially, we treated the CFTR-corrected CFBE41o2 cells expressing the G542X dual-luciferase reporter with NB124. Login to comment
108 To assess the efficacy of synthetic aminoglycosides, FRT-G542X cells were treated for 48 hours, then CFTR activity determined by the change in Gt after forskolin (10 mM) activation. Login to comment
115 When taken together, these results indicated that NB124 suppresses the relatively common CFTR-G542X mutation more efficiently than other tested aminoglycosides. Login to comment
119 We therefore next used CFTR functional assays on primary HBE cells (CFTR genotype G542X/ delF508) to evaluate the PTC suppression efficacy of synthetic aminoglycosides. Login to comment
124 Partial Restoration of CFTR Function in Cftr2/2 Mice Expressing a Human CFTR-G542X Transgene The in vitro results described previously here demonstrate that several synthetic aminoglycosides suppress CFTR PTCs more effectively than gentamicin, with NB124 most effectively restoring CFTR function across several clinically relevant alleles, including CFTR-G542X, the most common disease-causing nonsense allele. Login to comment
125 Based on those results, we next tested the ability of NB124 to suppress CFTR in a Cftr knockout mouse line expressing a human CFTR-G542X transgene under control of the intestine-specific rat fatty acid-binding protein promoter (referred to hereafter as Cftr2/2 hCFTR-G542X). Login to comment
128 Figures 5A and 5B show representative Isc tracings from Cftr1/1 hCFTR-G542X and Cftr2/2 hCFTR-G542X mice (positive and Figure 3. Login to comment
129 Improved CFTR function and expression in CFTR-G542X-expressing Fischer rat thyroid (FRT) monolayers. Login to comment
133 *P , 0.05, ***P , 0.001, ****P , 0.0001 versus vehicle or gentamicin negative controls, respectively), and Figures 5C and 5D show Isc tracings from Cftr2/2 hCFTR-G542X mice treated with 60 mg/kg gentamicin and 30 mg/kg NB124, respectively. Login to comment
134 These data are summarized in Figure 5E, which shows a scatter plot of forskolin-stimulated Isc from ileum samples obtained from untreated and treated Cftr2/2 hCFTR-G542X mice. Login to comment
135 The mean forskolin-stimulated Isc was 5.7 (6 3.7) mA/cm2 in ileum samples from untreated Cftr2/2 hCFTR-G542X mice (negative controls), and 255.7 (6 36.1) mA/cm2 in samples from wild-type mice (positive controls). Login to comment
136 In Cftr2/2 hCFTR-G542X mice treated with 30 mg/kg gentamicin, we did not observe a significant increase in Isc relative to the untreated controls (5.3 6 4.1 mA/cm2 ). Login to comment
138 In contrast, significant increases in forskolin-stimulated Isc were observed in Cftr2/2 hCFTR-G542X mice treated with 30 and 60 mg/kg NB124 (14.7 6 7.8 and 12.4 6 9.2, respectively). Login to comment
141 When taken together, these results indicate that NB124 restores greater CFTR function (and at a 2-fold lower dose) than gentamicin in Cftr2/2 hCFTR-G542X transgenic mice, and has a slightly more favorable dose-response relationship. Login to comment
152 Because reliable readthrough assays in primary HBE cell monolayers and in vivo found that NB124 suppresses the G542X-CFTR nonsense mutation roughly 2.5-fold more effectively than gentamicin in vivo, these results indicate that the efficacy:toxicity ratio of NB124 is approximately 10-fold better than gentamicin. Login to comment
156 Restoration of CFTR function in CF primary human bronchial epithelial (HBE) cells derived from a G542X/F508del donor. Login to comment
170 Representative tracings of (A) untreated Cftr1/1 hCFTR-G542X mice (WT control), (B) untreated Cftr2/2 hCFTR-G542X (negative control), (C) gentamicin-treated Cftr2/2 hCFTR-G542X (60 mg/ kg), and (D) NB124-treated Cftr2/2 hCFTR-G542X (30 mg/kg). Login to comment
171 (E) Scatter plot of all data from Cftr2/2 hCFTR-G542X mice (untreated, gentamicin treated, and NB124 treated). Login to comment
177 Representative images are shown from (F) untreated Cftr2/2 hCFTR-G542X (negative control), (G) gentamicin-treated Cftr2/2 hCFTR-G542X (60 mg/kg), and (H) NB124-treated Cftr2/2 hCFTR-G542X (30 mg/kg). Login to comment
193 We found that three CFTR UGA mutations (G542X, R1162X, and W1282X) exhibited qualitatively similar responses to the compounds and showed maximal suppression with NB124. Login to comment
216 Finally, we used a mouse model that expressed a human CFTR-G542X transgene in a Cftr knockout background to compare the functional results obtained with NB124 and gentamicin (Figure 5). Login to comment

[hide] [Therapeutic update in cystic fibrosis]. Rev Med Interne. 2014 Jun;35(6):388-92. doi: 10.1016/j.revmed.2013.11.003. Epub 2013 Dec 3. Durupt S, Nove Josserand R, Durieu I
[Therapeutic update in cystic fibrosis].
Rev Med Interne. 2014 Jun;35(6):388-92. doi: 10.1016/j.revmed.2013.11.003. Epub 2013 Dec 3., [PMID:24309546]

Abstract [show]
We present the recent therapeutic advances in the cystic fibrosis care. It concerns improvements in symptomatic treatment with the development of dry powder inhaled antibiotics that improved quality of life, and innovative treatments namely the modulators of the cystic fibrosis transmembrane protein conductance regulator (CFTR), molecules which act specifically at the level of the defective mechanisms implied in the disease. The life expectancy of cystic fibrosis patients born after 2000, is estimated now to be about 50 years. This improvement of survival was obtained with the organization of the care within the specialized centers for cystic fibrosis (Centre de ressource et de competences de la mucoviscidose) and remains still based on heavy symptomatic treatments. Dry powder inhaled antibiotics constitute a significant time saving for patients to whom all the care can achieve two hours daily. Since 2012, the modulators of CFTR, molecules allowing a pharmacological approach targeted according to the type of the mutations, allows a more specific approach of the disease. Ivacaftor (Kalydeco((R))) which potentialises the function of the CFTR protein expressed on the cellular surface is now available for patients with the G551D mutation. Lumacaftor is going to be tested in association with ivacaftor in patients with the F508del mutation, that is present in at least 75% of the patients. The ataluren which allows the production of a functional protein CFTR in patients with a no sense mutation is the third representing of this new therapeutic class. We presently have numerous symptomatic treatments for the cystic fibrosis care. The development of CFTR modulators, today available to a restricted number of patients treated with ivacaftor represents a very promising therapeutic avenue. It will represent probably the first step to a personalized treatment according to CFTR genotype.

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110 Classe I II III D&#e9;faut Synth&#e8;se Maturation R&#e9;gulation Exemple G542X F508del G551D Fr&#e9;quence globale 4 % 73 % 3 % Fr&#e9;quence en France 3,5 % 63 % 1 % Correction Lecture forc&#e9;e Prot&#e9;ger la d&#e9;gradation Restaurer la fonction Mol&#e9;cule test&#e9;e PTC 124 Ataluren VX-809 Lumacaftor Lumacaftor + Ivacaftor Ivacaftor r&#e9;alis&#e9;e chez 140 patients &#e2;g&#e9;s de 12 ans et plus, l`ivacaftor n`a pas permis d`am&#e9;lioration significative sur l`ensemble des crit&#e8;res de jugements d&#e9;finis (VEMS, poids, qualit&#e9; de vie) &#e0; l`exception de la concentration en ions chlorures dans la sueur (crit&#e8;re secondaire) [28]. Login to comment
236 [34] Du M, Liu X, Welch EM, Hirawat S, Peltz SW, Bedwell DM. PTC124 is an orally bioavailable compound that promotes suppression of the human CFTR-G542X nonsense allele in a CF mouse model. Login to comment

[hide] Cystic fibrosis carrier screening in a North Ameri... Genet Med. 2014 Jul;16(7):539-46. doi: 10.1038/gim.2013.188. Epub 2013 Dec 19. Zvereff VV, Faruki H, Edwards M, Friedman KJ
Cystic fibrosis carrier screening in a North American population.
Genet Med. 2014 Jul;16(7):539-46. doi: 10.1038/gim.2013.188. Epub 2013 Dec 19., [PMID:24357848]

Abstract [show]
PURPOSE: The aim of this study was to compare the mutation frequency distribution for a 32-mutation panel and a 69-mutation panel used for cystic fibrosis carrier screening. Further aims of the study were to examine the race-specific detection rates provided by both panels and to assess the performance of extended panels in large-scale, population-based cystic fibrosis carrier screening. Although genetic screening for the most common CFTR mutations allows detection of nearly 90% of cystic fibrosis carriers, the large number of other mutations, and their distribution within different ethnic groups, limits the utility of general population screening. METHODS: Patients referred for cystic fibrosis screening from January 2005 through December 2010 were tested using either a 32-mutation panel (n = 1,601,308 individuals) or a 69-mutation panel (n = 109,830). RESULTS: The carrier frequencies observed for the 69-mutation panel study population (1/36) and Caucasian (1/27) and African-American individuals (1/79) agree well with published cystic fibrosis carrier frequencies; however, a higher carrier frequency was observed for Hispanic-American individuals (1/48) using the 69-mutation panel as compared with the 32-mutation panel (1/69). The 69-mutation panel detected ~20% more mutations than the 32-mutation panel for both African-American and Hispanic-American individuals. CONCLUSION: Expanded panels using race-specific variants can improve cystic fibrosis carrier detection rates within specific populations. However, it is important that the pathogenicity and the relative frequency of these variants are confirmed.

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63 This threshold could not be reached Table 1ߒ CFTR allele frequency identified by the CF32 mutation panel Varianta Number of detected alleles Mutation (%) Legacy nomenclature HGVS nomenclature F508delb p.F508del 31,142 68.69 R117Hb p.R117H 5,198 11.46 G542Xb p.G542X 1,162 2.56 G551Db p.G551D 989 2.18 W1282Xb p.W1282X 824 1.82 3120ߙ+ߙ1G>Ab c.2988ߙ+ߙ1G>A 706 1.56 N1303Kb p.N1303K 648 1.43 R553Xb p.R553X 487 1.07 3849ߙ+ߙ10kbC>Tb c.3717ߙ+ߙ12191C>T 436 0.96 621ߙ+ߙ1G>Tb c.489ߙ+ߙ1G>T 410 0.90 1717-1G>Ab c.1585-1G>A 388 0.86 2789ߙ+ߙ5G>Ab c.2657ߙ+ߙ5G>A 382 0.84 I507delb p.I507del 258 0.57 R334Wb p.R334W 257 0.57 R1162Xb p.R1162X 211 0.47 G85Eb p.G85E 199 0.44 1898ߙ+ߙ1G>Ab c.1766ߙ+ߙ1G>A 170 0.37 R347Hc p.R347H 160 0.35 3659delCb c.3528delC 155 0.34 3876delAc c.3744delA 153 0.34 R560Tb p.R560T 132 0.29 S549Nc p.S549N 125 0.28 3905insTc c.3773dupT 121 0.27 R347Pb p.R347P 117 0.26 2184delAb c.2052delA 107 0.24 A455Eb p.A455E 106 0.23 711ߙ+ߙ1G>Tb c.579ߙ+ߙ1G>T 65 0.14 394delTTc c.262_263delTT 56 0.12 V520Fc p.V520F 54 0.12 1078delTc c.948delT 52 0.11 2183AA>Ga,c c.2051_2052delAAinsG 37 0.08 S549Rc p.S549R 31 0.07 Total 45,338 100 a 2183AA>G variant was added to the panel in 2010. b Variants from ACMG/ACOG CF screening panel. c Classified as a CF-causing mutation by the CFTR2 Database. ACMG, American College of Medical Genetics and Genomics; ACOG, American College of Obstetricians and Gynecologists; CF, cystic fibrosis; HGVS, Human Genome Variation Society. Table 2ߒ Continued on next page Table 2ߒ CFTR allele frequency identified by the CF69 mutation panel Varianta Allele frequency Mutation (%) Legacy nomenclature HGVS nomenclature F508delb p.F508del 1,868 60.49 R117Hb p.R117H 274 8.87 D1152Hc p.D1152H 125 4.05 G542Xb p.G542X 98 3.17 L206Wd p.L206W 73 2.36 3120ߙ+ߙ1G>Ab c.2988ߙ+ߙ1G>A 65 2.10 G551Db p.G551D 47 1.52 N1303Kb p.N1303K 42 1.36 W1282Xb p.W1282X 38 1.23 3849ߙ+ߙ10kbC>Tb c.3717ߙ+ߙ12191C>T 28 0.91 3876delAd c.3744delA 28 0.91 F311dele p.F312del 24 0.78 I507delb p.I507del 24 0.78 R553Xb p.R553X 24 0.78 R117Cd p.R117C 22 0.71 621ߙ+ߙ1G>Tb c.489ߙ+ߙ1G>T 21 0.68 1717-1G>Ab c.1585-1G>A 18 0.58 S549Nd p.S549N 18 0.58 R334Wb p.R334W 17 0.55 2789ߙ+ߙ5G>Ab c.2657ߙ+ߙ5G>A 16 0.52 G85Eb p.G85E 14 0.45 3199del6e c.3067_3072delATAGTG 12 0.39 R1066Cd p.R1066C 11 0.36 1898ߙ+ߙ1G>Ab c.1766ߙ+ߙ1G>A 10 0.32 R347Hd p.R347H 10 0.32 R1162 Xb p.R1162X 9 0.29 W1089Xd p.W1089X 9 0.29 2184delAb c.2052delA 8 0.26 2307insAd c.2175dupA 8 0.26 1078delTd c.948delT 7 0.23 R75Xd p.R75X 7 0.23 3120G>Ad c.2988 G>A 6 0.19 3659delCb c.3528delC 6 0.19 Q493Xd p.Q493X 6 0.19 R1158Xd p.R1158X 6 0.19 R560Tb p.R560T 6 0.19 1812-1G>Ad c.1680-1G>A 5 0.16 2055del9>Ad c.1923_1931del9insA 5 0.16 406-1G>Ad c.274-1G>A 5 0.16 A559Td p.A559T 5 0.16 R347Pb p.R347P 5 0.16 S1255Xd p.S1255X 5 0.16 1677delTAd c.1545_1546delTA 4 0.13 711ߙ+ߙ1G>Tb c.579ߙ+ߙ1G>T 4 0.13 E60Xd p.E60X 4 0.13 R352Qd p.R352Q 4 0.13 Y1092Xd p.Y1092X 4 0.13 2183AA>Gd c.2051_2052delAAinsG 3 0.10 3791delCd c.3659delC 3 0.10 3905insTd c.3773dupT 3 0.10 by 10 variants: the 2143delT, A455E, S549R, Y122X, and M1101K mutations, typically observed in Caucasians; 935delA, 2869insG, and Q890X in Hispanics; and 405+3A>C and G480C in the African-American population. Login to comment

[hide] Spectrum and distribution of CFTR gene mutations i... Gene. 2014 Apr 10;539(1):125-31. doi: 10.1016/j.gene.2014.01.022. Epub 2014 Jan 14. Muthuswamy S, Agarwal S, Awasthi S, Singh S, Dixit P, Maurya N, Choudhuri G
Spectrum and distribution of CFTR gene mutations in asthma and chronic pancreatitis cases of North Indian population.
Gene. 2014 Apr 10;539(1):125-31. doi: 10.1016/j.gene.2014.01.022. Epub 2014 Jan 14., [PMID:24440239]

Abstract [show]
BACKGROUND: Cystic fibrosis transmembrane conductance regulator (CFTR) gene accounts for an autosomal recessive condition called cystic fibrosis (CF). In the Indian subcontinent, CF and its related diseases are under-diagnosed by the medical community due to poor knowledge of the disease and its confounding diagnosis, and also due to poor medical facilities available for these patients, thus causing an increased infant mortality rate with a low life expectancy in general. The aim of the study was to document the spectrum and distribution of CFTR mutations in controls, asthma and chronic pancreatitis cases of North India. METHODS: A total of 800 subjects including 400 controls, 250 asthma cases and150 chronic pancreatitis cases were analyzed for 6 mutations (F508del, G542X, G551D, R117H, W1282X, and S549N) and IVS8 Tn polymorphism. RESULTS: Out of 800 subjects, 18% [asthma - 24% (n=250), CP - 29.33% (n=150) cases and controls - 9.3% (n=400)] were positive for heterozygous mutation, 0.8% of the (n=250) asthmatic cases (n=250) were homozygous for IVS8 T5 polymorphism while no subjects were found positive for W1282X mutation. T5 polymorphism was more common in asthmatic cases while F508del mutation in chronic pancreatitis cases. The carrier frequency of F508del, G542X, G551D, R117H, S549N and T5 was 0.015, 0.025, 0.02, 0.005, 0.005, and 0.022 respectively. The cumulative carrier frequency was 0.093. CONCLUSION: CFTR mutations were underestimated in Indian population. The present study will serve in establishment of genetic screening and prenatal setup for Indian population.

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3 Methods: A total of 800 subjects including 400 controls, 250 asthma cases and150 chronic pancreatitis cases were analyzed for 6 mutations (F508del, G542X, G551D, R117H, W1282X, and S549N) and IVS8 Tn polymorphism. Login to comment
6 The carrier frequency of F508del, G542X, G551D, R117H, S549N and T5 was 0.015, 0.025, 0.02, 0.005, 0.005, and 0.022 respectively. Login to comment
44 ARMS PCR Identification of F508del, G551D, G542X, R117H and W1282X mutations were carried out by ARMS PCR (Ferrie et al., 1992). Login to comment
63 Mutation Primer Method Amplicon (size in bp) Reference DF508 C GACTTCACTTCTAATGATGATTATGGGAG ARMS Ferrie et al. (1992) N GTATCTATATTCATCATAGGAAACACCAC 160 M GTATCTATATTCATCATAGGAAACACCAT 157 G551D C TAAAATTTCAGCAATGTTGTTTTTGACC N GCTAAAGAAATTCTTGCTCGTTGCC 285 M AGCTAAAGAAATTCTTGCTCGTTGCT 286 G542X C TAAAATTTCAGCAATGTTGTTTTTGACC N ACTCAGTGTGATTCCACCTTCTAC 256 M CACTCAGTGTGATTCCACCTTCTCA 257 R117H C CACATATGGTATGACCCTCTATATAAACT N CCTATGCCTAGATAAATCGCGATAGAAC 237 M CCTATGCCTAGATAAATCGCGATAGAAT 237 S549N For TTCAGCAATGTTGTTTTGACCAAC RFLP (DdeI) N: 13 + 238 + 174 H: 13 + 238 + 174 + 412 M: 13 + 412 Kerem et al. (1990) Rev CACAGATTCTGAGTAACCATAATC IVS8 Tn For1 TAATGGATCATGGGCCATGT Nested PCR (Xmn1) 5T - 84 7T - 86 9T - 88 Chillon et al. (1995) Rev1 ACAGTGTTGAATGTGGTGCA For2 CCGCCGCTGTGTGTGTGTGTGTGTTTTT Rev2 GGATCCAGCAACCGCCAACA 3. Login to comment
71 Controls (n = 400) Minor allele frequency of F508del, G542X, G551D, R117H and S549N observed to be 0.0075, 0.0125, 0.01, 0.0025 and 0.0025 respectively (Table 2). Login to comment
73 We further stratified data as per their prevalence and found G542X to be the most common mutation with the frequency of 2.49%, followed by G551D, F508del, R117H and S549N with 2.00%, 1.50%, 0.50% and 0.50% respectively and 2.24% for 5T allele (Fig. 1A). Login to comment
76 The frequency of individuals falling in different mutations were 10%, 5.6%, 4.4%, 2.0%, 1.6% and 0.4% with respect to IVS8 T5, G551D, G542X, S549N, F508del and R117H (Fig. 1B). Login to comment
87 G542X mutation The conversion of glycine residue at 542 position to termination codon (G542X) interrupts mRNA processing. Login to comment
89 Among CP patients, G542X mutation prevalence was 6.7% (10/150), followed by asthma patients and controls with 4.4% (11/250) and 2.5% (10/400) respectively. Login to comment
91 However, in asthma patients G542X heterozygosity was slightly higher in relative to controls but statistically insignificant. Login to comment
112 Serial no. Mutation General population (400) Asthma (250) Chronic pancreatitis (150) 1 DF508 0.0075 0.0080 0.0433 2 G542X 0.0125 0.0220 0.0333 3 G551D 0.01 0.0280 0.03 4 R117H 0.0025 0.0020 0.0066 5 S549N 0.0025 0.0100 0.0133 6 IVS8-5T 0.01125 0.0580 0.02 thrive, pancreatic insufficiency, elevated sweat electrolytes, late-stage diabetes and cardiac failure, besides male infertility due to congenital bilateral absence of the vas deferens (CBAVD) (Schrijver et al., 2005). Login to comment
118 A) Among the studied 5 mutation and 1 polymorphism, G542X mutation accounts for 27% (10 out of 37) in controls and the least one is S549N of 5% (2 out of 37). Login to comment
134 Serial No. Mutation General population (n = 400)% Asthma (n = 250)% Chronic pancreatitis (n = 150)% Chi square-testÌe; P value Asth vs ctrl Cp vs ctrl Asth vs CP 1 DF508 1.5 1.6 8.7 NS 0.0002 0.0013 2 G542X 2.5 4.4 6.7 NS 0.03 NS 3 G551D 2.0 5.6 6.0 0.02 0.02 NS 4 R117H 0.5 0.4 1.3 NS NS NS 5 S549N 0.5 2 2.7 NS NS NS 6 IVS8-5T 2.2 10 4.0 b0.0001 NS 0.03 7 Total 9.3 24 29.3 b0.0001 b0.0001 NS Ìe; P b 0.05 is considered significant (Bold values). Login to comment
142 Recently, Shastri and Kabra reported 1161delC, 3849 + 10kbC-T and S549N as other most common mutations in India (Shastri and Kabra 2008), which is in agreement with other reports (Sharma et al., 2009b); however in the control group of the present study 1161delC and 3849+ 10kbC-T mutations were not found (unpublished data) whereas G542X, W1282X, 621 + 1G N T that occur in lesser percentage among Hispanic and non-Hispanic Caucasians (Watson et al., 2004) were not found in earlier studies from India while the present study recorded that G542X mutation is quite common among our controls (2.5%), asthmatic (4.4%) and CP (6.7%) cases in heterozygous nature. Login to comment
145 Based on our data G551D, G542X, F508del, T5, R117H and S549N mutations may be considered for Indian subjects. Login to comment
146 In conclusion, our study indicates that CFTR mutations had been overlooked in our population while the carrier frequency of mutations F508del, G542X, and G551D are 3/200, 1/50 and 1/40, respectively, that is more common with reference to 1/238 of F508del in an earlier report by Kapoor et al. (2006). Login to comment

[hide] Toward a rationale for the PTC124 (Ataluren) promo... Mol Pharm. 2014 Mar 3;11(3):653-64. doi: 10.1021/mp400230s. Epub 2014 Feb 7. Lentini L, Melfi R, Di Leonardo A, Spinello A, Barone G, Pace A, Palumbo Piccionello A, Pibiri I
Toward a rationale for the PTC124 (Ataluren) promoted readthrough of premature stop codons: a computational approach and GFP-reporter cell-based assay.
Mol Pharm. 2014 Mar 3;11(3):653-64. doi: 10.1021/mp400230s. Epub 2014 Feb 7., [PMID:24483936]

Abstract [show]
The presence in the mRNA of premature stop codons (PTCs) results in protein truncation responsible for several inherited (genetic) diseases. A well-known example of these diseases is cystic fibrosis (CF), where approximately 10% (worldwide) of patients have nonsense mutations in the CF transmembrane regulator (CFTR) gene. PTC124 (3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)-benzoic acid), also known as Ataluren, is a small molecule that has been suggested to allow PTC readthrough even though its target has yet to be identified. In the lack of a general consensus about its mechanism of action, we experimentally tested the ability of PTC124 to promote the readthrough of premature termination codons by using a new reporter. The reporter vector was based on a plasmid harboring the H2B histone coding sequence fused in frame with the green fluorescent protein (GFP) cDNA, and a TGA stop codon was introduced in the H2B-GFP gene by site-directed mutagenesis. Additionally, an unprecedented computational study on the putative supramolecular interaction between PTC124 and an 11-codon (33-nucleotides) sequence corresponding to a CFTR mRNA fragment containing a central UGA nonsense mutation showed a specific interaction between PTC124 and the UGA codon. Altogether, the H2B-GFP-opal based assay and the molecular dynamics (MD) simulation support the hypothesis that PTC124 is able to promote the specific readthrough of internal TGA premature stop codons.

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12 The most commonly observed mutations include deletion of a codon at position 508 (delta-F508), a missense mutation at position 551, and a nonsense mutation at position 542 (G542X).34 Cystic fibrosis patients with nonsense-mutation essentially produce no CFTR protein, thus suffering a more severe form of the disease. Login to comment
16 Therefore, we performed the first computational study to investigate putative codon-specific supramolecular interactions between the PTC124 and an 11 codon long sequence corresponding to the CFTR fragment coding mRNA bearing a G542X nonsense mutation (i.e., UGA at position 542). Login to comment
158 Five MD simulations of 100 ns were conducted on the complex between the 1,2,4-oxadiazole ligand (PTC124), and four models of mRNA constituted by a sequence of 33 ribonucleotides of the CFTR gene, that differ in the identity of the central codon normally expressing aminoacid 542: (i) GGA, as found in the wild type mRNA; (ii) UGA, mimicking the G542X nonsense mutation; (iii) UAG, and (iv) UAA, representing the other two types of premature stop codons. Login to comment

[hide] Impact of heterozygote CFTR mutations in COPD pati... Respir Res. 2014 Feb 11;15:18. doi: 10.1186/1465-9921-15-18. Raju SV, Tate JH, Peacock SK, Fang P, Oster RA, Dransfield MT, Rowe SM
Impact of heterozygote CFTR mutations in COPD patients with chronic bronchitis.
Respir Res. 2014 Feb 11;15:18. doi: 10.1186/1465-9921-15-18., [PMID:24517344]

Abstract [show]
BACKGROUND: Cigarette smoking causes Chronic Obstructive Pulmonary Disease (COPD), the 3rd leading cause of death in the U.S. CFTR ion transport dysfunction has been implicated in COPD pathogenesis, and is associated with chronic bronchitis. However, susceptibility to smoke induced lung injury is variable and the underlying genetic contributors remain unclear. We hypothesized that presence of CFTR mutation heterozygosity may alter susceptibility to cigarette smoke induced CFTR dysfunction. Consequently, COPD patients with chronic bronchitis may have a higher rate of CFTR mutations compared to the general population. METHODS: Primary human bronchial epithelial cells derived from F508del CFTR heterozygotes and mice with (CFTR+/-) and without (CFTR+/+) CFTR heterozygosity were exposed to whole cigarette smoke (WCS); CFTR-dependent ion transport was assessed by Ussing chamber electrophysiology and nasal potential difference measurements, respectively. Caucasians with COPD and chronic bronchitis, age 40 to 80 with FEV1/FVC < 0.70 and FEV1 < 60% predicted, were selected for genetic analysis from participants in the NIH COPD Clinical Research Network's Azithromycin for Prevention of Exacerbations of COPD in comparison to 32,900 Caucasian women who underwent prenatal genetic testing. Genetic analysis involved an allele-specific genotyping of 89 CFTR mutations. RESULTS: Exposure to WCS caused a pronounced reduction in CFTR activity in both CFTR (+/+) cells and F508del CFTR (+/-) cells; however, neither the degree of decrement (44.7% wild-type vs. 53.5% F508del heterozygous, P = NS) nor the residual CFTR activity were altered by CFTR heterozygosity. Similarly, WCS caused a marked reduction in CFTR activity measured by NPD in both wild type and CFTR heterozygous mice, but the severity of decrement (91.1% wild type vs. 47.7% CF heterozygous, P = NS) and the residual activity were not significantly affected by CFTR genetic status. Five of 127 (3.9%) COPD patients with chronic bronchitis were heterozygous for CFTR mutations which was not significantly different from controls (4.5%) (P = NS). CONCLUSIONS: The magnitude of WCS induced reductions in CFTR activity was not affected by the presence of CFTR mutation heterozygosity. CFTR mutations do not increase the risk of COPD with chronic bronchitis. CFTR dysfunction due to smoking is primarily an acquired phenomenon and is not affected by the presence of congenital CFTR mutations.

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81 As expected based on genotype-phenotype correlations in the disease [33], HBE cells derived from a F508del CFTR heterozygote had slightly lower CFTR activity at baseline than wild type monolayers as measured by Table 1 List of CFTR mutations analyzed F508del R117H 1717-1G > A R117C G85E R334W 1898 + 1G > A Y122X A455E R347P 2184delA G178R I507del R553X 2789 + 5G > A G314E G542X R560T 3120 + 1G > A G330X G551D W1282X 3659delC R347H N1303K 621 + 1G > T K710X 406-1G > A R1162X 711 + 1G > T E60X G480C R1066C W1089X V520F A559T S1196X Q1238X S1251N S1255X 663delT 935delA 1161delC 1288insTA 2184insA 2307insA 2711delT 2869insG R709X R764X R1158X 574delA Q493X 1898 + 5G > T 3905insT I506T 3849 + 10kbC > T 712-1G > T Q98R Q552X S549N 1078delT H199Y 444delA S549R (T > G) 2143delT P205S 2043delG 1811 + 1.6kbA > G 3272-26A > G L206W 3791delC Y1092X (C > G) 3199del6 F508C 2108delA Y1092X (C > A) D1152H V520I 3667del4 394delTT 3876delA M1101K 1677delTA W1098X (TGA) 1812-1G > A 4016insT 1609delCA 3171delC response to forskolin stimulation (49.3 &#b1; 11.5 bc;A/cm2 in CFTR (+/+) vs. 40.5 &#b1; 5.3 bc;A/cm2 in CFTR (+/-), although this was not statistically significant (Figure 1A,B). Login to comment

[hide] Enhanced F508del-CFTR channel activity ameliorates... Am J Pathol. 2014 Apr;184(4):1132-41. doi: 10.1016/j.ajpath.2013.12.027. Epub 2014 Feb 11. Le Henaff C, Hay E, Velard F, Marty C, Tabary O, Marie PJ, Jacquot JP
Enhanced F508del-CFTR channel activity ameliorates bone pathology in murine cystic fibrosis.
Am J Pathol. 2014 Apr;184(4):1132-41. doi: 10.1016/j.ajpath.2013.12.027. Epub 2014 Feb 11., [PMID:24529904]

Abstract [show]
In patients with cystic fibrosis (CF), rib and thoracic vertebral fractures can have adverse effects on lung health because the resulting pain and debilitation can impair airway clearance. The F508del mutation in the CF transmembrane conductance regulator (Cftr) gene induces an osteopenic phenotype in humans and mice. N-butyldeoxynojyrimicin (miglustat), an approved drug for treating type 1 Gaucher disease, was found to normalize CFTR-dependent chloride transport in human F508del CFTR lung cells and in nasal mucosa of F508del CF mice. Herein, we investigated whether targeting F508del-CFTR may rescue the skeletal osteopenic phenotype in murine CF. We found that oral administration of low-dose miglustat (120 mg/kg once a day for 28 days) improved bone mass and microarchitecture in the lumbar spine and femur in F508del mice. The increased bone density was associated with an increased bone formation rate and reduced bone resorption. This effect was associated with increased 17beta-estradiol but not with insulin-like growth factor 1 serum levels in miglustat-treated F508del mice. Exposure of primary F508del osteoblasts to miglustat partially restored the deficient CFTR-dependent chloride transport in these bone-forming cells. This study provides evidence that reversal of CFTR-dependent chloride transport in osteoblasts normalizes bone mass and microarchitecture in murine CF. These findings may provide a potential therapeutic strategy to prevent or correct the bone disease in patients with CF.

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13 (Am J Pathol 2014, 184: 1132e1141; http://dx.doi.org/10.1016/j.ajpath.2013.12.027) Cystic fibrosis (CF), which is caused by mutations of the CF transmembrane conductance regulator (CFTR), is characterized by multiorgan pathology that begins early in life.1e4 Brittle bones have been reported in children, adolescents, and adults with CF, independently of sex and age; this has been termed CF-related bone disease.5e8 Bone fragility may exclude patients from candidacy for lung transplantation, which imposes limitations on the best therapeutic option for these patients at the advanced stages of the disease.9 Clinical data have suggested a multifactorial etiology of CF-related skeletal deficits, including vitamin K and D insufficiency, calcium malabsorption, malnutrition and pancreatic insufficiency, delayed puberty, sex steroid deficiency, pulmonary infection/systemic inflammation, and frequent glucocorticoid therapy.10e13 Whether Cftr gene dysfunction plays a direct role in bone metabolism remains to be elucidated; however, this has been hypothesized on the basis of animal studies with the Cftr-null14,15 and F508del-Cftr mouse models.16,17 We previously reported that CF mice homozygous for the F508del-CFTR mutation develop a severe osteopenic phenotype in both sexes.16 In humans, the expression of CFTR protein has been identified in bone cells.18 We recently discovered defective CFTR-mediated Cl channel activity and a severe deficit of the release of osteoprotegerin (a key regulator in bone turnover) in primary osteoblasts (cells that form bone) obtained from a 25-year-old man with CF with the F508del/G542X mutation in CFTR.19 These data raise the hypothesis that the mutation in the Cftr gene may play a role in CF-related skeletal disease. Login to comment

[hide] A new era in the treatment of cystic fibrosis. Clin Med (Lond). 2014 Feb;14(1):76-8. doi: 10.7861/clinmedicine.14-1-76. Lane MA, Doe SJ
A new era in the treatment of cystic fibrosis.
Clin Med (Lond). 2014 Feb;14(1):76-8. doi: 10.7861/clinmedicine.14-1-76., [PMID:24532752]

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8 Mutations in the gene encoding the protein CFTR can be divided into five categories on the basis of the effect they have on protein synthesis:6 > Class I defects (eg G542X) disrupt synthesis of CFTR and include nonsense and frameshift mutations that lead to premature termination codons (PTCs) and a lack of protein production. Login to comment

[hide] Cystic fibrosis: toward personalized therapies. Int J Biochem Cell Biol. 2014 Jul;52:192-200. doi: 10.1016/j.biocel.2014.02.008. Epub 2014 Feb 20. Ikpa PT, Bijvelds MJ, de Jonge HR
Cystic fibrosis: toward personalized therapies.
Int J Biochem Cell Biol. 2014 Jul;52:192-200. doi: 10.1016/j.biocel.2014.02.008. Epub 2014 Feb 20., [PMID:24561283]

Abstract [show]
Cystic fibrosis (CF), the most common, life-threatening monogenetic disease in Caucasians, is caused by mutations in the CFTR gene, encoding a cAMP- and cGMP-regulated epithelial chloride channel. Symptomatic therapies treating end-organ manifestations have increased the life expectancy of CF patients toward a mean of 40 years. The recent development of CFTR-targeted drugs that emerged from high-throughput screening and are capable of correcting the basic defect promises to transform the therapeutic landscape from a trial-and-error prescription to personalized medicine. This stratified approach is tailored to a specific functional class of mutations in CFTR, but can be refined further to an individual level by exploiting recent advances in ex vivo drug testing methods. These tests range from CFTR functional measurements in rectal biopsies donated by a CF patient to the use of patient-derived intestinal or pulmonary organoids. Such organoids may serve as an inexhaustible source of epithelial cells that can be stored in biobanks and allow medium- to high-throughput screening of CFTR activators, correctors and potentiators on the basis of a simple microscopic assay monitoring organoid swelling. Thus the recent breakthrough in stem cell biology allowing the culturing of mini-organs from individual patients is not only relevant for future stem cell therapy, but may also allow the preclinical testing of new drugs or combinations that are optimally suited for an individual patient.

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1592 Just four other mutations, notably G551D (class III), W1282X, G542X (class I), and N1303K (class II) have a worldwide prevalence of 1-3% each, whereas only 20 mutations have a frequency above 0.1%. Login to comment

[hide] CFTR genotype and clinical outcomes of adult patie... Gene. 2014 May 1;540(2):183-90. doi: 10.1016/j.gene.2014.02.040. Epub 2014 Feb 26. Bonadia LC, de Lima Marson FA, Ribeiro JD, Paschoal IA, Pereira MC, Ribeiro AF, Bertuzzo CS
CFTR genotype and clinical outcomes of adult patients carried as cystic fibrosis disease.
Gene. 2014 May 1;540(2):183-90. doi: 10.1016/j.gene.2014.02.040. Epub 2014 Feb 26., [PMID:24583165]

Abstract [show]
BACKGROUND: There are nearly 2000 cystic fibrosis transmembrane regulator (CFTR) mutations that cause cystic fibrosis (CF). These mutations are classified into six classes; on the one hand, the first three classes cause severe disease involvement in early childhood, on the other hand, the Class IV, V and VI mutations cause minor severe disease in the same age. Nowadays, with therapeutic advances in CF management and competence of pediatricians, physicians of adults have to deal with two groups of CF patients: (i) adults diagnosed in childhood with severe mutations and (ii) adults who initiated symptoms in adulthood and with Class IV, V and VI mutations. The aim of this study was to analyze adults from a clinical center, treated as CF disease, screening the CFTR genotype and evaluating the clinical characteristics. METHODS: Thirty patients followed as CF disease at the University Hospital were enrolled. After a complete molecular CFTR negative screening and sweat test levels between 40 and 59mEq/L, five patients were characterized as non-CF disease and were excluded. Molecular screening was performed by CFTR gene sequencing/MLPA or by specific mutation screening. Clinical data was obtained from medical records. The patients were divided into three groups: (1) patients with Class I, II and III mutations in two CFTR alleles; (2) genotype with at least one allele of Class IV, V or VI CFTR mutations and, (3) non-identified CFTR mutation+one patient with one allele with CFTR mutation screened (Class I). RESULTS: There was an association of CFTR class mutation and sodium/chloride concentration in the sweat test (sodium: p=0.040; chloride: p=0.016), onset of digestive symptoms (p=0.012), lung function parameter (SpO2 - p=0.016), Bhalla score (p=0.021), age at diagnosis (p=0.008) and CF-related diabetes (p=0.029). There was an association between Pseudomonas aeruginosa chronic colonization (as clinical marker for the lung disease status) and lung impairment (FEV1% - p=0.027; Bhalla score - p=0.021), CF-related diabetes (p=0.040), chloride concentration in the sweat test (p=0.040) and chronic infection by microorganisms (Staphylococcus aureus - p=0.039; mucoid P. aeruginosa - p=0.001). There is no positive association with the status of other clinical markers and the CFTR genotype groups. For clinical association with pancreatic insufficiency (as clinical marker for digestive symptoms), no association was related. CONCLUSION: The adults with CF diagnosed by sweat test have specific clinical and genotypic characteristics, being a population that should be studied to cause better future management. Some patients treated as CF disease by clinical symptoms, showed no disease, taking into account the sweat test and complete exon sequencing/MLPA screening.

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53 2 8 I I s s a l C / I I s s a l C l e d 8 0 5 F / E 5 8 G 1 1 G542X/I618T Class I/Class IV 74.6 72.8 91 97 82.8 84.9 1 G542X/2183AA>G Class I/Class I 111 181 114 105 112.5 143 5 . Login to comment
82 2 7 V I s s a l C / I I s s a l C H 2 5 1 1 D / l e d 8 0 5 F 2 2 F508del/1584-18672pb A>G Class II/Class V 63.7 90.7 61.5 93.1 62.6 91.9 2 P205S/G542X Class IV/Class I 102 136.2 93.1 112.3 97.55 124.25 6 . Login to comment

[hide] CFTR mutations spectrum and the efficiency of mole... PLoS One. 2014 Feb 26;9(2):e89094. doi: 10.1371/journal.pone.0089094. eCollection 2014. Zietkiewicz E, Rutkiewicz E, Pogorzelski A, Klimek B, Voelkel K, Witt M
CFTR mutations spectrum and the efficiency of molecular diagnostics in Polish cystic fibrosis patients.
PLoS One. 2014 Feb 26;9(2):e89094. doi: 10.1371/journal.pone.0089094. eCollection 2014., [PMID:24586523]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane regulator gene (CFTR). In light of the strong allelic heterogeneity and regional specificity of the mutation spectrum, the strategy of molecular diagnostics and counseling in CF requires genetic tests to reflect the frequency profile characteristic for a given population. The goal of the study was to provide an updated comprehensive estimation of the distribution of CFTR mutations in Polish CF patients and to assess the effectiveness of INNOLiPA_CFTR tests in Polish population. The analyzed cohort consisted of 738 patients with the clinically confirmed CF diagnosis, prescreened for molecular defects using INNOLiPA_CFTR panels from Innogenetics. A combined efficiency of INNOLiPA CFTR_19 and CFTR_17_TnUpdate tests was 75.5%; both mutations were detected in 68.2%, and one mutation in 14.8% of the affected individuals. The group composed of all the patients with only one or with no mutation detected (109 and 126 individuals, respectively) was analyzed further using a mutation screening approach, i.e. SSCP/HD (single strand conformational polymorphism/heteroduplex) analysis of PCR products followed by sequencing of the coding sequence. As a result, 53 more mutations were found in 97 patients. The overall efficiency of the CF allele detection was 82.5% (7.0% increase compared to INNOLiPA tests alone). The distribution of the most frequent mutations in Poland was assessed. Most of the mutations repetitively found in Polish patients had been previously described in other European populations. The most frequent mutated allele, F508del, represented 54.5% of Polish CF chromosomes. Another eight mutations had frequencies over 1%, 24 had frequencies between 1 and 0.1%; c.2052-2053insA and c.3468+2_3468+3insT were the most frequent non-INNOLiPA mutations. Mutation distribution described herein is also relevant to the Polish diaspora. Our study also demonstrates that the reported efficiency of mutation detection strongly depends on the diagnostic experience of referring health centers.

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71 Exon / intron (legacy) Exon / intron (Ensembl) Protein change SVM value cDNA (HGVS nomenclature) gDNA (cDNA +132 bp) Number of PL CF chromosomes Reference a Mutations in trans Pathogenic mutations 1 1 L15Ffs10X c.43delC 175delC 1 CFMDB 1717-1G.A 2 2 G27V 21.92 c.80G.T 212G.T 1 Novel F508del 2 2 S18RfsX16 c.54-5940_273 +10250del21kb exon2,3del21kb 66 IL19 various CF mutations i2 i2 IVS2_Donor c.164+1G.A 296+1G.A 3 CFMDB various CF mutations 3 3 G85E 22.61 c.254G.A 386G.A 1 IL17 unknown 3 3 E60X c.178G.T 310G.T 0 IL17 x 3 3 L88IfsX22 c.262_263delTT 394delTT 0 IL17 x 4 4 E92K 21.92 c.274G.A 406G.A 2 CFMDB c.164+1G.A; c.2051- 2AA.G 4 4 L101X c.302T.G 434T.G 1 CFMDB c.3717+12191C.T 4 4 K114IfsX5 c.341_353del13bp 473del13bp 1 Novel F508del 4 4 R117H 20.35 c.350G.A 482G.A 5 IL17 F508del; 2x unknown 4 4 R117C 22.07 c.349C.T 481C.T 2 CFMDB S1206X;1x unknown 4 4 L137_L138insT c.412_413insACT L138ins 1 CFMDB F508del 4 4 R153I 22.61 c.458G.T 590G.T 2 Novel F508del; c.3527delC i4 i4 IVS4_Donor c.489+1G.T 621+1G.T 5 IL17 F508del; c.489+1G.T 5 5 L165X c.494T.A 626T.A 1 Novel F508del i5 i5 IVS5_Donor c.579+1G.T 711+1G.T 0 IL19 x i5 i5 IVS5_Donor c.579+3A.G 711+3A.G 2 CFMDB 2,3del21kb; c.2052-3insA i5 i5 IVS5_Donor c.579+5G.A 711+5G.A 0 IL17 x 7 8 F311L 20.90 c.933C.G 965C.G 2 CFMDB 2x F508 7 8 G314R 20.58 c.940G.A 1072G.A 4 CFMDB various CF mutations 7 8 F316LfsX12 c.948delT 1078delT 1 IL17 unkown 7 8 R334W 22.41 c.1000C.T 1132C.T 6 IL17 various CF mutations 7 8 I336K 22.07 c.1007T.A 1139T.A 2 CFMDB 2,3de21kb; F508del 7 8 R347P 22.27 c.1040G.C 1172G.C 11 IL17 various CF mutations i7 i8 IVS8_Donor c.1116+2T.A 1248+2T.A 1 Novel Q1412X 9 10 A455E 22.61 c.1364C.A 1496C.A 0 IL17 x i9 i10 IVS10_Donor c.1392+1G.A 1524+1G.A 1 CFMDB c.3816-7delGT 10 11 S466X c.1397C.G 1529C.G 1 CFMDB G542X 10 11 I507del c.1519_1521delATC 1651delATC 2 IL19 F508del 10 11 F508del c.1521_1523delCTT 1654delCTT 805 IL19 various CF mutations i10 i11 IVS11_Acceptor c.1585-1G.A 1717-1G.A 27 IL19 various CF mutations 11 12 G542X c.1624G.T 1756G.T 25 IL19 various CF mutations 11 12 G551D 21.24 c.1624G.T 1756G.T 5 IL19 various CF mutations 11 12 Q552X c.1654C.T 1786C.T 0 IL19 x 11 12 R553X c.1657C.T 1789C.T 14 IL19 various CF mutations 11 12 R560T 21.92 c.1679G.C 1811G.C 0 IL19 x i12 i13 IVS13_Donor c.1766+1G.A 1898+1G.A 6 IL19 various CF mutations i12 i13 IVS13_Donor c.1766+1G.C 1898+1G.C 1 CFMDB F508del 13 14 H620P 21.73 c.1859A.C 1991A.C 1 CFMDB F508del 13 14 R668C//G576A 21.61//1.73 c.2002C.T//c.1727G.C 2134C.T// 1859G.C 5 b CFMDB// rs1800098 c.1585-1G.A; 4 unknown 13 14 L671X c.2012delT 2143delT 27 IL17 various CF mutations 13 14 K684SfsX38 c.2051_2052delAAinsG 2183AA.G 10 IL17 various CF mutations 13 14 K684NfsX38 c.2052delA 2184delA 0 IL17 x 13 14 Q685TfsX4 c.2052_2053insA 2184insA 15 CFMDB various CF mutationsc , 1 unknown Table 2. Cont. Exon / intron (legacy) Exon / intron (Ensembl) Protein change SVM value cDNA (HGVS nomenclature) gDNA (cDNA +132 bp) Number of PL CF chromosomes Reference a Mutations in trans 13 14 L732X c.2195T.G 2327T.G 1 CFMDB F508del 14A 15 R851X c.2551C.T 2683C.T 3 CFMDB various CF mutations 14A 15 I864SfsX28 c.2589_2599del11bp 2721del11bp 2 CFMDB F508del; 2,3del21kb i14B i16 IVS16_Donor c.2657+2_2657+3insA 2789+2insA 1 CFMDB F508del i14B i16 IVS16_Donor c.2657+5G.A 2789+5G.A 0 IL17 unkown 15 17 Y919C 21.02 c.2756A.G 2888A.G 1 CFMDB unknown 15 17 H939HfsX27 c.2817_2820delTACTC 2949delTACTC 1 Novel unkown i15 i17 IVS17_Donor c.2908+3A.C 3040+3A.C 1 Novel F508del i16 i18 IVS18_Donor c.2988+1G.A 3120+1G.A 0 IL19 x 17A 19 I1023_V1024del c.3067_3072delATAGTG 3199del6 0 IL19 x i17A i19 IVS19 c.3140-26A.G 3272-26A.G 9 IL19 various CF mutations 17B 20 L1065R 21.90 c.3194T.G 3326T.G 1 CFMDB F508del 17B 20 Y1092X c.3276C.A 3408C.A 1 CFMDB R334W i18 i21 IVS21_Donor c.3468+2_3468+3insT 3600+2insT 11 CFMDB various CF mutationsd , 1 unknown 18 21 E1126EfsX7 c.3376_3379delGAAG 3508delGAAG 1 Novel F508del 19 22 R1158X c.3472C.T 3604C.T 2 CFMDB F508del; R553X 19 22 R1162X c.3484C.T 3616C.T 1 IL17 F508del 19 22 L1177SfsX15 c.3528delC 3659delC 4 IL17 various CF mutations 19 22 S1206X c.3617C.A 3749C.A 1 CFMDB R117C i19 i22 IVS22 c.3717+12191C.T 3849+10kbC.T 58 IL17 various CF mutations 20 23 G1244R 22.62 c.3730G.C 3862G.C 1 CFMDB F508del 20 23 S1251N 22.28 c.3752G.A 3884G.A 0 IL19 x 20 23 L1258FfsX7 c.3773_3774insT 3905insT 0 IL19 x 20 23 V1272VfsX28 c.3816_3817delGT 3944delGT 1 CFMDB c.1392+1G.A 20 23 W1282X c.3846G.A 3978G.A 9 IL19 various CF mutations 21 24 N1303K 22.62 c.3909C.G 4041C.G 18 IL19 various CF mutations 22 25 V1327X c.3979delG 4111delG 1 Novel F508del 22 25 S1347PfsX13 c.4035_4038dupCCTA c.4167dupCCTA 1 CFMDB 2,3del21kb 23 26 Q1382X c.4144C.T 4276C.T 1 CFMDB F508del 23 26 Q1412X c.4234C.T 4366C.T 2 CFMDB F508del; c.1116+2T.A i23 i26 IVS26_Donor c.4242+1G.T 4374+1G.T 1 CFMDB F508del Sequence changes of uncertain pathogenic effect, tentatively counted as mutations 6A 6 E217G 0.30 c.650A.G 782A.G 1 CFMDB; rs1219109046 unknown 7 8 R352Q 20.01 c.1055G.A 1187G.A 1 CFMDB; rs121908753 F508del 7 8 Q359R 0.33 c.1076A.G 1208A.G 1 CFMDB F508del i8 i9 IVS9 c.1210-12T5_1210- 34_35 (TG)12 1332-12Tn_- 34TGm 6 CFMDB F508del; 3x unknown i8 i9 IVS9 c.1210-12T5_1210- 34_35 (TG)13 1332-12Tn_- 34TGm 2 CFMDB 2143delT; 1x unknown i8 i9 IVS9 c.1210-12T8 1332-12Tn 1 Novel unknown 10 11 I506V 20.21 c.1516A.G 1648A.G 1 CFMDB; rs1800091 unknown 12 13 V562L 0.79 c.1684G.C 1816G.C 1 CFMDB; rs1800097 unknown 13 14 G723V 0.44 c.2168G.T 2300G.T 1 CFMDB; rs200531709 unknown 15 17 D924N 0.03 c.2770G.A 2902G.A 1 CFMDB; rs201759207 unknown patient with F508del on another allele) was not supported by the SVM value (+0.35); the patient was PS and had ambiguous chloride values (45, 64 and 83 mmol/L). 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101 The more recent estimates provide much lower values, ranging from 1:5000 [14], 1:6000 cited in WHO 2002 report [15] to 1:7500 for Southeastern Poland estimated for a 1-year period of Table 2. Cont. Exon / intron (legacy) Exon / intron (Ensembl) Protein change SVM value cDNA (HGVS nomenclature) gDNA (cDNA +132 bp) Number of PL CF chromosomes Reference a Mutations in trans 15 17 L967S 0.27 c.2900T.C 3032T.C 1 CFMDB; rs1800110 unknown 18 21 D1152H 0.50 c.3454G.C 3586G.C 1 CFMDB; rs75541969 F508del Sequence changes considered as lacking pathogenic effect 4 4 I148T 2.04 c.443T.U 575T.U 4 IL19e unknown 13 14 I752V 0.35 c.2254A.G 2386A.G 1 Novelf F508 15 17 S912L 2.12 c.2735C.T 2867C.T 1 CFMDBg ; rs121909034 F508 Legend: a IL19 i 17 - mutations included in the INNOLiPA tests (see below); CFMDB - non-INNOLiPA mutations present in the CTFR mutation database; novel - mutations first reported in this study; b in three chromosomes R668C with G576A in trans; c F508del, c.1585-1G.A, G542X, N1303K or c.579+3A.G; d F508del, G542X, R553X or N1303K; e not pathogenic if not in cis with c.3067-72del6 (l.n.3199del6); f not pathogenic - see explanation the text; g not pathogenic if not in cis with G1244V. Login to comment
102 a Mutations detected by two INNOLiPA_CFTR tests (legacy names): IL19 (INNOLiPA_CFTR19): F508del; G542X; N1303K; W1282X; G551D; 1717-1G.A; R553X; CFTRdele2,3(21kb); I507del; 711+1G.T; 3272-26A.G; 3905insT; R560T; 1898+1G.A; S1251N; I148T; 3199del6; 3120+1G.A; Q552X. Login to comment
121 Three mutations in our reduced data set were significantly (p,0.03) less frequent than in Warsaw (F508del: 67.2% vs 71.8%; G542X: 1.98% vs 2.92%; N1303K: 1.47% vs 2.92%), while two mutations were significantly more frequent (c.3468+2_3468+3insT: 0.86% vs 0.10%; c.489+1G.T: 0.43% vs 0%; G314R: 0.34% vs 0%). Login to comment
137 Mutations a Poland Czechs Slovakia c Germany Lithuania W. Ukraine E. Hungary Romania c Bulgaria Serbia Greece Number of chromosomes 1476 1200 856 700 98 264 80 256 208 352 874 F508del 54.54 b 67.42 d 66.80 d 72.00 d 52.0 54.17 70.00 56.3 65.38 d 72.28 d 53.40 exon2,3del21kb (l.n.CFTRdele2,3_21kb) 4.47 5.75 2.26 1.2 f 2.0 4.17 5.00 1.6 NA 0 e 0.34 e c.3717+12191C.T (l.n.3849+10kbC.T) 3.93 1.67 e 4.28 1.00 e NA 0.76 0 0.4 e 1.44 0 e 0.11 e c.2012delT (l.n.2143delT) 1.83 0.92 1.10 0.71 0 1.14 0 0 e 0 0 e 0 e c.1585-1G.A (l.n.1717-1G.A) 1.83 0.33 e NA 0.86 0 0.38 1.25 0.4 0 0 e 0 e G542X 1.69 2.00 4.06 d 1.43 0 2.65 3.75 3.9 3.37 2.57 3.90 d R347P 1.57 0.92 1.10 1.57 0 0 1.25 NA 1.44 0 e 0.11 e N1303K 1.22 2.42 2.03 2.29 2.0 4.92 d 5.00 0.8 6.73 d 0 2.63 c.2052-2053insA (l.n.2184insA) 1.02 0.42 1.58 0.57 0 7.20 d 5.00 d 0 0.48 0.28 0 e R553X 0.95 0.50 0.90 2.29 4.2 d 0.38 0 NA 0 0 0 c.3468+223insT (l.n.3600+2insT) 0.75 0.25 NA 0 e 0 NA 0 NA 0 0 0 e c.2051-2052AA.G (l.n.2183AA.G) 0.68 0.08 NA 0.57 0 0.38 0 0.8 0 0 1.38 W1282X 0.61 0.58 0.50 0.71 1.0 2.27 0 2.3 d 0.96 0 0.67 c.3140-26A.G (l.n.3272-26A.G) 0.61 0.67 0.50 0.86 0 0.76 0 0.4 0 0 0.81 l.n.IVS8 T 5 _TG 12-13 0.54 NA NA NA 0 NA NA NA NA 0 NA R334W 0.41 0.25 NA 0.29 0 0.76 0 0.4 0 0.28 0.81 c.1766+1G.A (l.n.1898+1G.A) 0.41 1.42 d 0.50 0 0 1.14 0 NA 0 0 0.11 c.489+1G.T (l.n.621+1G.T) 0.34 0.42 NA 0.14 0 0.76 0 0.8 0 2.86 d 5.72 d R117H 0.34 NA NA 0.29 0 0 0 0.4 0 0 0.23 G551D 0.34 2.91 d 0.50 1.00 0 0 0 0 0 0 0.34 G314R 0.37 0 NA 0 0 0 0 NA 0 0 0 R668C 0.34 0 NA 0 0 0 0 NA 0 0 0 c.3528delC (l.n.3659delC) 0.27 0.17 NA 0.57 0 0 0 NA 0 0 0 c.164+1G.A (l.n.296+1G.A) 0.20 0.08 NA 0 0 0 0 NA 0 0 0 R851X 0.20 0.08 NA 0 0 0 0 NA 0 0 0 I336K 0.14 0.58 NA 0.45 0 0 0 NA 0 0 0 R1158X 0.14 0.08 NA 0 0 0 0 NA 0 0 1.03 E92K 0.14 0.08 NA 0 0 0.38 0 NA 0 0 0 R153I 0.14 0 NA 0 0 0 0 NA 0 0 0 c.579+3A.G (l.n.711+3A.G) 0.14 0.17 NA 0 0 0 0 NA 0 0 0.69 c.2589-2599del11bp (l.n.2721- 31del11bp) 0.14 0.08 NA 0 0 0.38 0 NA 0 0 0 I507del 0.14 0.08 NA 0.15 0 0 0 0 0 0.28 0.69 R117C 0.14 0.08 NA 0.15 0 0 0 NA 0 0 0.23 of mutation panels [20]), listed in Table 4, were compared to those reported for several Central and Southeastern European countries [21-29]. Login to comment

[hide] Polymorphisms in the glutathione pathway modulate ... BMC Med Genet. 2014 Mar 4;15:27. doi: 10.1186/1471-2350-15-27. Marson FA, Bertuzzo CS, Ribeiro AF, Ribeiro JD
Polymorphisms in the glutathione pathway modulate cystic fibrosis severity: a cross-sectional study.
BMC Med Genet. 2014 Mar 4;15:27. doi: 10.1186/1471-2350-15-27., [PMID:24593045]

Abstract [show]
BACKGROUND: Cystic fibrosis (CF) clinically manifests with various levels of severity, which are thought to be modulated by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR), modifier genes, and the environment. This study verified whether polymorphisms in modifier genes associated with glutathione (GSH) metabolism influence CF severity. METHODS: A cross-sectional study of 180 CF patients was carried out from 2011 to 2012. We analyzed CFTR mutations, polymorphisms (GSTM1 and GSTT1 deletions, GSTP1 + 313A > G, GCLC-129C > T, and GCLC-3506A > G) in modifier genes and CF clinical severity as assessed by 28 clinical and laboratory variables. RESULTS: Significant associations were found between modifier gene polymorphisms and particular phenotypes or genotype changes. These included GCLC-129C > T with a higher frequency of the Pseudomonas aeruginosa mucoid to CC genotype (p = 0.044), and GCLC-3506A > G with a higher frequency of the no-mucoid P. aeruginosa (NMPA) to AA genotype (p = 0.012). The GSTT1 deletion was associated with a higher frequency of the NMPA to homozygous deletion (p = 0.008), GSTP1 + 313A > G with a minor risk of osteoporosis (p = 0.036), and patient age </= 154 months (p = 0.044) with the AA genotype. The Bhalla score was associated with GCLC-3506A > G (p = 0.044) and GSTM1/GSTT1 deletion polymorphisms (p = 0.02), while transcutaneous hemoglobin oxygen saturation levels were associated with GSTT1 deletions (p = 0.048). CONCLUSION: CF severity is associated with polymorphisms in GSH pathways and CFTR mutations.

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47 CFTR mutation identification CFTR mutation identification was performed by polymerase chain reaction (PCR) for F508del and the fragment-length polymorphism method for G542X, R1162X, R553X, G551D, and N1303K mutations. Login to comment
121 An analysis of genotypic combinations for GSTM1 and GSTP1 polymorphic loci showed that changes in GSTP1 activities Table 3 Genotyping of GCLC, GSTM1, GSTT1, and GSTP1 polymorphisms and CFTR mutations Gene Chromosomal position Location Polymorphism MAF HWE p-valuea GCLC, rs17883901 6p12 Promoter region C > T 0.12 9.97 <0.005 GCLC, rs137852340 6p12 Promoter region A > G 0.19 0.04 >0.05 GSTP1, rs1695 11q13 Exon A > G 0.25 1.11 >0.05 GSTM1 1p13.3 Deletion GSTT1 22q11.23 Deletion CFTR mutation N Frequency F508del/F508del 57 31.67% F508del/G542X 12 6.67% F508del/R1162X 5 2.78% F508del/N1303K 4 2.22% F508del/R553X 1 0.56% F508del/S4X 1 0.56% F508del/1717-1G > A 1 0.56% G542X/R1162X 1 0.56% G542X/I618T 1 0.56% G542X/2183A > G 1 0.56% R1162X/R1162X 1 0.56% F508del/- 45 25.00% G542X/- 5 2.78% R1162X/- 1 0.56% -/- 44 24.45% MAF, Minor allele frequency; HWE, Hardy Weinberg Equilibrium; a P-value for Hardy-Weinberg Equilibrium; N, Number of patients; -, No identified CFTR mutation. Login to comment

[hide] Genetics of cystic fibrosis: CFTR mutation classif... Int J Biochem Cell Biol. 2014 Jul;52:94-102. doi: 10.1016/j.biocel.2014.02.023. Epub 2014 Mar 12. Fanen P, Wohlhuter-Haddad A, Hinzpeter A
Genetics of cystic fibrosis: CFTR mutation classifications toward genotype-based CF therapies.
Int J Biochem Cell Biol. 2014 Jul;52:94-102. doi: 10.1016/j.biocel.2014.02.023. Epub 2014 Mar 12., [PMID:24631642]

Abstract [show]
Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an epithelial anion channel. Since the identification of the disease in 1938 and up until 2012, CF patients have been treated exclusively with medications aimed at bettering their respiratory, digestive, inflammatory and infectious symptoms. The identification of the CFTR gene in 1989 gave hopes of rapidly finding a cure for the disease, for which over 1950 mutations have been identified. Since 2012, recent approaches have enabled the identification of small molecules targeting either the CFTR protein directly or its key processing steps, giving rise to novel promising therapeutic tools. This review presents the current CFTR mutation classifications according to their clinical consequences and to their effect on the structure and function of the CFTR channel. How these classifications are essential in the establishment of mutation-targeted therapeutic strategies is then discussed. The future of CFTR-targeted treatment lies in combinatory therapies that will enable CF patients to receive a customized treatment.

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74 23 ACMG recommended panel of classic CF-causing mutations G85E R117H R334W R347P A455E I507del F508del G542X G551D R553X R560T R1162X W1282X N1303K 621 + 1G > T 711 + 1G > T 1717 - 1G > A 1898 + 1G > A 2184delA 2789 + 5G > A 3120 + 1G > A 3659delC 3849 + 10kbC > T Additional or alternative mutations present at significant frequencies in an ethnic population served by a newborn screening program may be assessed. Login to comment

[hide] Biosynthesis of cystic fibrosis transmembrane cond... Int J Biochem Cell Biol. 2014 Jul;52:26-38. doi: 10.1016/j.biocel.2014.03.020. Epub 2014 Mar 28. Pranke IM, Sermet-Gaudelus I
Biosynthesis of cystic fibrosis transmembrane conductance regulator.
Int J Biochem Cell Biol. 2014 Jul;52:26-38. doi: 10.1016/j.biocel.2014.03.020. Epub 2014 Mar 28., [PMID:24685677]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride (Cl(-)) channel. Mutations of its gene lead to the disease of cystis fibrosis (CF) among which the most common is the deletion of phenylalanine at position 508 (Phe508del). CFTR is a multi-domain glycoprotein whose biosynthesis, maturation and functioning as an anion channel involve multi-level post-translational modifications of CFTR molecules and complex folding processes to reach its native, tertiary conformation. Only 20-40% of the nascent chains achieve folded conformation, while the remaining molecules are targeted for degradation by endoplasmic reticulum, lysosomes, or autophagy. A large number of mutations causing CF impair processing of CFTR. Growing knowledge of CFTR biosynthesis has enabled understanding the cellular basis of CF and has brought to light various potential targets for novel, promising therapies.

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1372 The nonsense and frameshift mutations, belonging to the Class I, lead to creation of premature termination codons (PTCs) such as W1282X, G542X, Y122X, and result either in the synthesis of truncated and unstable protein or in the decrease of the half-lives of mutant mRNAs. Login to comment

[hide] Mutation Analysis of Exons 10 and 17a of CFTR Gene... J Reprod Infertil. 2014 Jan;15(1):49-56. Sahami A, Alibakhshi R, Ghadiri K, Sadeghi H
Mutation Analysis of Exons 10 and 17a of CFTR Gene in Patients with Cystic Fibrosis in Kermanshah Province, Western Iran.
J Reprod Infertil. 2014 Jan;15(1):49-56., [PMID:24696795]

Abstract [show]
BACKGROUND: Cystic fibrosis (CF) is the most common genetic disorder with autosomal recessive inheritance among Caucasian populations. So far, more than 1950 different mutations were identified in cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR gene has 27 exons. The type and distribution of mutations vary widely among different countries and/or ethnic groups. Therefore, a comprehensive analysis was performed on exon10 and exon17a of CFTR gene in CF patients in the Kermanshah province, western Iran. METHODS: We tested 27 patients admitted to the medical genetics laboratory of Kermanshah University of Medical Sciences. The patients were from different cities of Kermanshah province. All the patients had the clinical signals and two positive sweat tests. After filling agreement forms and questionnaire, the peripheral blood sampling and DNA extraction were done. DNA samples were extracted. PCR and sequencing special PCR were done. Finally analysis of the results with DNA sequencing analysis version 5.2 software was performed. RESULTS: CFTR mutations analysis identified 4 different mutations in our CF patients. The disease-causing mutations were p.F508del (DeltaF508) (14.81%), p.S466X (1.85%), and p.T1036I (1.85%). M470V polymorphism with frequency of 74.1% was found in 23 patients (17 homozygous and 6 heterozygous). CONCLUSION: Three disease-causing mutations in CF patients in the present study account for approximately 18.51% of mutations. The frequency of p.F508del, the most common mutation was 16-18.1% in Iranian population. The results of the present study can be applied for genetic counseling, population screening and prenatal diagnosis.

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30 In addition to ࢞F508 mutation, these mutations vary greatly in their frequency and distribution, but most of them are very rare. Only four mutations (p.G542X, p.N1303K, p.G551D, and p.W1282X) have overall frequencies greater than 1% (5). Login to comment

[hide] Understanding how cystic fibrosis mutations disrup... Int J Biochem Cell Biol. 2014 Jul;52:47-57. doi: 10.1016/j.biocel.2014.04.001. Epub 2014 Apr 13. Wang Y, Wrennall JA, Cai Z, Li H, Sheppard DN
Understanding how cystic fibrosis mutations disrupt CFTR function: from single molecules to animal models.
Int J Biochem Cell Biol. 2014 Jul;52:47-57. doi: 10.1016/j.biocel.2014.04.001. Epub 2014 Apr 13., [PMID:24727426]

Abstract [show]
Defective epithelial ion transport is the hallmark of the life-limiting genetic disease cystic fibrosis (CF). This abnormality is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), the ATP-binding cassette transporter that functions as a ligand-gated anion channel. Since the identification of the CFTR gene, almost 2000 disease-causing mutations associated with a spectrum of clinical phenotypes have been reported, but the majority remain poorly characterised. Studies of a small number of mutations including the most common, F508del-CFTR, have identified six general mechanisms of CFTR dysfunction. Here, we review selectively progress to understand how CF mutations disrupt CFTR processing, stability and function. We explore CFTR structure and function to explain the molecular mechanisms of CFTR dysfunction and highlight new knowledge of disease pathophysiology emerging from large animal models of CF. Understanding CFTR dysfunction is crucial to the development of transformational therapies for CF patients.

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1947 Common examples include G542X, the second most common CF mutation, prevalent in Mediterranean countries and W1282X, the most common CF mutation in Ashkenazi Jews. Login to comment

[hide] Increasing nontuberculous mycobacteria infection i... J Cyst Fibros. 2015 Jan;14(1):53-62. doi: 10.1016/j.jcf.2014.05.008. Epub 2014 Jun 7. Bar-On O, Mussaffi H, Mei-Zahav M, Prais D, Steuer G, Stafler P, Hananya S, Blau H
Increasing nontuberculous mycobacteria infection in cystic fibrosis.
J Cyst Fibros. 2015 Jan;14(1):53-62. doi: 10.1016/j.jcf.2014.05.008. Epub 2014 Jun 7., [PMID:24917112]

Abstract [show]
BACKGROUND: Nontuberculous mycobacteria (NTM) are emerging infections in the CF population. AIMS: To assess NTM infection prevalence and associated features in our CF clinic population. METHODS: Patient records, 2002-2011, were reviewed for NTM infection. FEV1, pancreatic function, sputum microbiology, and serum cytokines were compared in patients with and without NTM infection. RESULTS: Incidence rate of NTM infection increased from 0 in 2002 to 8.7% in 2011 (p<0.001). NTM infection prevalence increased 3-fold from 5% (4/79) in 2003 to 14.5% (16/110) in 2011 (p=0.05). Prevalence of chronic NTM lung disease has decreased somewhat since a peak in 2009, with institution of aggressive triple therapy. Of NTM-infected compared to uninfected patients, 88.2% vs. 60.3% had a known 'severe' CFTR genotype (p=0.04), 88.2% vs. 58.9% were pancreatic insufficient (p=0.02); 70.6% vs. 43.8% had chronic Pseudomonas aeruginosa (p=0.06); 75% vs. 32% had Aspergillus infection (p=0.007) and 23.5% vs 2.7% had allergic bronchopulmonary aspergillosis (p=0.01). Patients infected with Mycobacterium abscessus had increased TGF-beta, TNF-alpha, IL-1beta, IL-2, IL-4 and IL-5 levels (p<0.05). There was no difference in cytokine levels for all NTM infected compared to uninfected patients. M. abscessus comprised 46% of all NTM infections. Comparing M. abscessus versus other NTM, duration was 10.5 (1-118) months versus 1 (1-70) month, median (range) (p=0.004); lung disease occurred in 69% versus 17% (p=0.0004), with sputum conversion in 4/11 versus 5/6, respectively (NS). CONCLUSIONS: NTM incidence and prevalence have increased dramatically in our CF clinic, associated with a severe CF genotype and phenotype. M. abscessus, the most prevalent NTM, caused prolonged infection despite therapy. There has been some decrease in the prevalence of NTM lung disease since 2009.

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118 The most frequent CFTR mutations in our cohort were W1282X, ƊF508, G542X, D1152H, 3849 + 10kbC࢐T. Login to comment

[hide] New pharmacological approaches for cystic fibrosis... Pharmacol Ther. 2015 Jan;145:19-34. doi: 10.1016/j.pharmthera.2014.06.005. Epub 2014 Jun 14. Bell SC, De Boeck K, Amaral MD
New pharmacological approaches for cystic fibrosis: promises, progress, pitfalls.
Pharmacol Ther. 2015 Jan;145:19-34. doi: 10.1016/j.pharmthera.2014.06.005. Epub 2014 Jun 14., [PMID:24932877]

Abstract [show]
With the discovery of the CFTR gene in 1989, the search for therapies to improve the basic defects of cystic fibrosis (CF) commenced. Pharmacological manipulation provides the opportunity to enhance CF transmembrane conductance regulator (CFTR) protein synthesis and/or function. CFTR modulators include potentiators to improve channel gating (class III mutations), correctors to improve abnormal CFTR protein folding and trafficking (class II mutations) and stop codon mutation read-through drugs relevant for patients with premature stop codons (most class I mutations). After several successful clinical trials the potentiator, ivacaftor, is now licenced for use in adults and children (>six years), with CF bearing the class III G551D mutation and FDA licence was recently expanded to include 8 additional class III mutations. Alternative approaches for class I and class II mutations are currently being studied. Combination drug treatment with correctors and potentiators appears to be required to restore CFTR function of F508del, the most common CFTR mutation. Alternative therapies such as gene therapy and pharmacological modulation of other ion channels may be advantageous because they are mutation-class independent, however progress is less well advanced. Clinical trials for CFTR modulators have been enthusiastically embraced by patients with CF and health care providers. Whilst novel trial end-points are being evaluated allowing CFTR modulators to be efficiently tested, many challenges related to the complexity of CFTR and the biology of the epithelium still need to be overcome.

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492 Common mutations in class I include G542X (common in Brittany and Southern France), R1162X (common in Austria and Northern Italy), or W1282X (reaching 48% amongst Ashkenazi Jews) (Bobadilla et al., 2002). Login to comment
544 Mutation Alternative name Allele frequency (% of total known) in ECFSPR 2010 Allele frequency (% of total known mutations) in 2010 ECFSPR F508del 64.5 Most frequent mutation worldwide Southeast to Northwest increasing prevalence in Europe IL 25.5 to DK 82.6 Mutations with an overall EU prevalence above 1% G542X Mediterranean mutation 2.5 GR 6.7, ES 6.0 N1303K Ancient Phoenician mutation 1.9 IT 4.2 W1282X Jewish Ashkenazi mutation 1.2 IL 22.4 G551D Celtic mutation 1.1 IE 7.3 1717-1GNA Italian mutation 1.0 IT 3.7 Mutations with an overall EU prevalence below 0.5% G85E PT 3.5 A455E Dutch mutation NL 3.5 CFTR dele 2,3 Slavic mutation CZ 5.2, BY 6.7 394delTT Nordic mutation SE 7.9, DK 2.0 3905insT Swiss mutation CH 2.4 R1162X Italian mutation IT 7.8 A561E Portuguese mutation PT 3.2 Abbreviations ECFSPR - European Cystic Fibrosis Society Patient Registry. Login to comment
547 Class Type of defect List of mutations attributed to this class Class I Defective protein production Nonsense mutations: G542X, R1162X, RW1282X Deletions and insertions: CFTRdele2,3; 1078delT; 1717-1G ࢐ A; 3659delC; 621+1G N T Class II Defective protein processing G85E, F508del, I507del, R560T, A561E, R1066C, N1303K Class III Defective protein regulation (gating) G178R, S549N, S549R, G551D, G551S, G970R, G1244E, S1251N, S1255P, G1349D Class IV Defective protein conductance R334W, R347P, R117H Class V Reduced amount of functioning protein 2789+5G ࢐ A, 3272-26ANG, 3849+10KbC ࢐ T, A455E Class VI Reduced cell surface stability Rescued F508del, c.120del23 Unclassified All other mutations, including those unknown a F508del-CFTR pocket (at NBD1:ICL4 interface) (Farinha et al., 2013). Login to comment
577 Class I mutations, which abrogate protein production, often include mutations that generate premature stop codons, class Ia (e.g. G542X) that lead to mRNA degradation by nonsense-mediated decay or those affecting canonical splice sites class Ib (e.g. 1717 - 1GNA). Login to comment

[hide] TNF-alpha polymorphisms as a potential modifier ge... Int J Mol Epidemiol Genet. 2014 May 29;5(2):87-99. eCollection 2014. Coutinho CA, Marson FA, Marcelino AR, Bonadia LC, Carlin MP, Ribeiro AF, Ribeiro JD, Bertuzzo CS
TNF-alpha polymorphisms as a potential modifier gene in the cystic fibrosis.
Int J Mol Epidemiol Genet. 2014 May 29;5(2):87-99. eCollection 2014., [PMID:24959313]

Abstract [show]
Modifier genes, as the TNF-alpha gene, can modulate the cystic fibrosis (CF) severity. Thus, -238G>A and -308G>A polymorphisms of TNF-alpha gene were analyzed as modifiers of CF. In this context, the present study enrolled 49 CF patients (diagnosis performed by sweat test and complete CFTR screening). The -238G>A polymorphism analysis was performed by ARMS-PCR, and -308G>A, by PCR-RFLP. In our data, the -238G>A polymorphism was not associated with clinical variability. The AA genotype for -308G>A polymorphism was a risk factor for early gastrointestinal symptoms (OR=5.98, 95%CI=1.06-49.68) and protection for the first Pseudomonas aeruginosa (OR=0.05, 95%CI=0.0003-0.007). For the first P. aeruginosa, GA genotype was a risk factor (OR=10.2, 95%CI=1.86-84.09); for the same genotype, the diagnosis was made in minor time than the AA genotype (p=0.031). Considering the -308G>A polymorphism alleles, the G allele was a risk factor for early pulmonary symptoms (OR=3.81, 95%CI=1.13-12.97) and P. aeruginosa (OR=66.77, 95%CI=15.18-482.7); however, the same allele showed better transcutaneous oxygen saturation (OR=9.24, 95%CI=1.53-206.1). The A allele was a protective factor for early pulmonary symptoms (OR=12.26, 95%CI=0.08-0.89) and P. aeruginosa (OR=12.15, 95%CI=0002-0007), however, the same allele was a risk factor for worst transcutaneous oxygen saturation (OR=7.01, 95%CI=1.14-157.4). As conclusion, the -308G>A polymorphism of the TNF-alpha gene was associated with the CF severity.

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45 Population characterization for the TNF-b1; and CFTR genotypes, Shwachman-Kulczycki and Kanga scores, transcutaneous oxygen saturation of hemoglobin and forced expiratory volume in the first second (%) (FEV1 ) of forced vital capacity Patient TNF-b1; genotype CFTR genotype Shwachman-Kulczycki Kanga Oxygen saturation FEV1 % -238G>A -308G>A MVG G/G G/A F508del/F508del Moderate Normal Normal Normal JPD G/G G/A F508del/F508del **** Normal Normal Mild RSR G/A A/A F508del/F508del Excellent Normal Mild Normal EVM G/G A/A F508del/R1162X Excellent Normal Mild Severe IFM G/G A/A F508del/F508del **** **** **** **** MLA G/G A/A F508del/F508del **** Exacerbate Normal Moderate VLPC G/G A/A F508del/F508del Moderate Normal Normal Normal MAB G/G A/A F508del/F508del Severe Exacerbate Mild Severe EVSMS G/G A/A F508del/F508del **** Normal Normal Mild YBK G/G A/A F508del/F508del Moderate Normal Normal Normal HB G/A A/A F508del/G542X **** Normal Severe Severe WSP G/G A/A F508del/G542X Moderate Normal Mild Severe DRG G/G A/A F508del/F508del **** **** **** **** BBK G/G G/A F508del/N1303K **** **** **** **** AO G/G A/A F508del/F508del **** Exacerbate Mild Mild LSM G/A A/A F508del/F508del Excellent Normal Normal Moderate VAL G/G A/A F508del/F508del Good Normal Mild Mild LFSA G/G A/A F508del/F508del **** Normal Mild Mild IBN G/G A/A F508del/R553X Excellent Normal Normal Normal CAQ G/G G/G F508del/G542X **** Normal Normal Mild MEMZ G/A A/A F508del/F508del **** Normal Normal Normal TMG G/G G/G F508del/F508del Moderate Normal Normal Normal JMGR G/G G/A F508del/N1303K **** Normal Normal **** MAP G/A G/G F508del/F508del **** **** **** **** AX G/G A/A F508del/F508del Good Normal Normal Normal LPOL G/G G/A F508del/N1303K Good Normal Normal **** EG G/G A/A F508del/R1162X Good Normal Normal Normal NCB G/G G/A F508del/R553X **** Normal Mild Severe FVV G/G A/A F508del/G542X Excellent Normal Normal Normal LSS G/G A/A F508del/F508del **** Exacerbate Moderate **** RNC G/G A/A F508del/F508del **** Normal Normal **** FEL G/G A/A F508del/G542X Moderate Normal Normal Moderate GOV G/G G/A F508del/F508del **** Normal Normal **** CVAR G/G A/A F508del/G542X **** Normal Normal **** EAG G/G A/A F508del/F508del **** Normal Mild **** AVM G/G G/G DF508/G542X Excellent Normal Normal Normal BSG G/G A/A G542X/R1162X Good Exacerbate Mild Mild GPNS G/A A/A F508del/F508del **** Normal Normal **** CAL G/G A/A DF508/G542X Good Normal Normal Normal MOS G/G G/G F508del/F508del Moderate Normal Normal **** FSC G/G A/A F508del/F508del Good Normal Normal Normal ITVS G/G G/A F508del/F508del **** Normal Normal Normal JRL G/G G/G F508del/F508del Mild Normal Normal **** GPT G/A A/A F508del/F508del Excellent Normal Normal Normal some patients were decolonized until the beginning of the study period, being not included in the statistical analysis. Login to comment
69 The F508del allele had the highest prevalence (79.59%), followed by G542X (9.18%), R1162X (6.12%), N1303K (3.06%) and R553X (2.04%). Login to comment

[hide] Increased frequency of CFTR gene mutations identif... Gene. 2014 Sep 10;548(1):43-7. doi: 10.1016/j.gene.2014.07.005. Epub 2014 Jul 7. Sharma H, Mavuduru RS, Singh SK, Prasad R
Increased frequency of CFTR gene mutations identified in Indian infertile men with non-CBAVD obstructive azoospermia and spermatogenic failure.
Gene. 2014 Sep 10;548(1):43-7. doi: 10.1016/j.gene.2014.07.005. Epub 2014 Jul 7., [PMID:25010724]

Abstract [show]
High incidence of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is associated with congenital bilateral absence of the vas deferens (CBAVD) and is considered as the genital form of cystic fibrosis (CF). The CFTR gene may also be involved in the etiology of male infertility in cases other than CBAVD. The present study was conducted to identify the spectrum and frequency of CFTR gene mutations in infertile Indian males with non-CBAVD obstructive azoospermia (n=60) and spermatogenic failure (n=150). Conspicuously higher frequency of heterozygote F508del mutation was detected in infertile males with non-CBAVD obstructive azoospermia (11.6%) and spermatogenic failure (7.3%). Homozygous IVS(8)-5T allele frequency was also significantly higher in both groups in comparison to those in normal healthy individuals. Two mutations in exon 25 viz., R1358I and K1351R were identified as novel mutations in patients with non-CBAVD obstructive azoospermia. Mutation R1358I was predicted as probably damaging CFTR mutation. This is the first report from the Indian population, emphasizing increased frequency of CFTR gene mutations in male infertility other than CBAVD. Thus, it is suggested that screening of CFTR gene mutations may be required in infertile Indian males with other forms of infertility apart from CBAVD and willing for assisted reproduction technology.

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53 Other common mutations viz., 621+1GNT, G542X, G551D and W1282X were screened by multiplex ARMS PCR (Ferrie et al., 1992). Login to comment

[hide] Mechanisms of CFTR functional variants that impair... PLoS Genet. 2014 Jul 17;10(7):e1004376. doi: 10.1371/journal.pgen.1004376. eCollection 2014 Jul. LaRusch J, Jung J, General IJ, Lewis MD, Park HW, Brand RE, Gelrud A, Anderson MA, Banks PA, Conwell D, Lawrence C, Romagnuolo J, Baillie J, Alkaade S, Cote G, Gardner TB, Amann ST, Slivka A, Sandhu B, Aloe A, Kienholz ML, Yadav D, Barmada MM, Bahar I, Lee MG, Whitcomb DC
Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis.
PLoS Genet. 2014 Jul 17;10(7):e1004376. doi: 10.1371/journal.pgen.1004376. eCollection 2014 Jul., [PMID:25033378]

Abstract [show]
CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTRBD) affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens) but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a) screened 984 well-phenotyped pancreatitis cases for candidate CFTRBD mutations from among 81 previously described CFTR variants; (b) conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c) computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d) tested the newly defined CFTRBD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N) not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002). Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTRBD increased risk for rhinosinusitis (OR 2.3, p<0.005) and male infertility (OR 395, p<<0.0001). WNK1-SPAK pathway-activated increases in CFTR bicarbonate permeability are altered by CFTRBD variants through multiple mechanisms. CFTRBD variants are associated with clinically significant disorders of the pancreas, sinuses, and male reproductive system.

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95 CFTR variant %Cases %Uctrls OR p-value %Cases w/N34S OR w/N34S p-value w/N34S CF/BD or BD/BD 2.5 0.1 31.9 ,0.0001 5.5 7.46 0.12 All CF 8.7 3.3 2.76 ,0.0001 16.4 5.65 ,0.0001 F508del CF 6.9 3.1 2.32 ,0.0001 14.5 5.13 ,0.0001 IVS8T5** CF 9.9 8.2 1.24 0.079 10.9 1.37 0.47 2789+5G.A CF 0.3 0.0 0.028 0.0 3849+10kbC.T CF 0.3 0.0 0.028 0.0 N1303K CF 0.3 0.0 0.027 0.0 621+1G.T CF 0.1 0.0 0.13 1.8 ,0.0001 2184delA CF 0.1 0.0 0.13 0.0 3120+1G.A CF 0.1 0.0 0.13 0.0 G551D CF 0.2 0.1 2.50 0.20 0.0 0.00 0.83 W1282X CF 0.2 0.1 2.50 0.20 0.0 0.00 0.83 G542X CF 0.2 0.0 0.059 0.0 R1162X CF 0.1 0.0 0.13 0.0 2183AA.G CF 0.0 0.1 0.17 0.0 0.00 0.83 All BD 14.2 9.8 1.50 0.002 25.5 4.63 ,0.0001 R75Q BD 6.3 6.2 1.02 0.30 16.4 2.97 0.003 S1235R BD 2.4 1.4 1.69 0.052 1.8 1.30 0.80 R117H CF/BD 2.3 0.7 3.49 0.0007 5.5 8.74 0.0002 L967S BD 1.1 0.2 6.87 0.002 1.8 11.17 0.014 L997F BD 0.8 1.0 0.82 0.26 1.8 1.84 0.55 D1152H BD 0.4 0.0 0.014 0.0 D1270N BD 0.3 0.2 1.25 0.29 0.0 0.00 0.71 R170H BD 0.3 0.0 0.028 0.0 R74Q BD 0.3 0.1 3.02 0.17 1.8 21.15 0.002 Other M470V 76.1 74.2 1.11 0.14 70.9 0.85 0.59 T854T 57.3 57.8 0.98 0.29 45.5 0.61 0.071 Q1463Q 39.6 39.5 1.01 0.30 40.0 1.02 0.94 1001+11C.T* 13.4 10.9 1.27 0.016 14.5 1.40 0.42 125G.C 10.3 9.7 1.07 0.26 12.7 1.36 0.45 P1290P 7.6 7.9 0.95 0.28 7.3 0.91 0.86 1716G.A 4.5 4.1 1.10 0.26 1.8 0.43 0.39 R668C 1.0 1.4 0.72 0.19 0.0 0.00 0.38 G576A 0.7 1.2 0.58 0.11 0.0 0.00 0.41 computationally modeled the molecular structure, and studied the dynamics, of wild type (WT) and mutated CFTR channels. Login to comment
124 We identified the R75Q, R117H, L967S, L997F, D1152H, and S1235R CFTRBD variants as well as CFTRCF -associated variants (e.g., F508del, G542X) in cases with rhinosinusitis. Login to comment
129 We identified R75Q, R117H, and S1235R as well as the CFTRCF variants F508del, G542X and 2789+5G,A in male cases with infertility. Login to comment
269 67 SNPs (125GtoC, 1716G.A, 1717-1G.A, 1898+1G.A, 2183AA.G, 2184delA, 2789+5G.A, 3120+1G.A, 3659delC, 3849+10kbC.T, 621+ 1G.T, 711+5G.A, A455E, D110H, D1152H, D1270N, D443Y, D579G, F1052V, F1074L, F508C, F508del, G1069R, G1244E, G1349D, G178R, G542X, G551D, G551S, I1131L/V, I148T, I336K/T, I507del, I807M, IVS8T5, K1180T, L1065P, L967S, L997F, M1V, M470V, M952I, M952T, N1303K, P67L, Q1463Q, R1070Q, R1162X, R117C, R117H, R170H, R258G, R297Q, R31C, R352Q, R553X, R668C, R74W, R75Q, S1235R, S1255P, S485R, S977F, T338I, T854T, V201M, W1282X) were multiplexed into 6 wells; 14 SNPs (S492F, S945L, R74Q, R560T, R1162L, G85E, I1027T, R334W, R347P, G576A, 711+1G.T, 1001+11C.T, P1290P, 3199del6) were ascertained separately via TaqMan Gene Expression Assays, with repeat confirmation of all positive results. Login to comment

[hide] Molecular genetic testing for cystic fibrosis: lab... Genet Med. 2015 Mar;17(3):219-25. doi: 10.1038/gim.2014.93. Epub 2014 Jul 31. Lyon E, Schrijver I, Weck KE, Ferreira-Gonzalez A, Richards CS, Palomaki GE
Molecular genetic testing for cystic fibrosis: laboratory performance on the College of American Pathologists external proficiency surveys.
Genet Med. 2015 Mar;17(3):219-25. doi: 10.1038/gim.2014.93. Epub 2014 Jul 31., [PMID:25077647]

Abstract [show]
BACKGROUND: Molecular testing for cystic fibrosis mutations is widespread and routine in reproductive decision making and diagnosis. Our objective was to assess the level of performance of laboratories for this test. METHODS: The College of American Pathologists administers external proficiency testing with multiple DNA samples distributed biannually. RESULTS are analyzed, reviewed, and graded by the joint College of American Pathologists/American College of Medical Genetics and Genomics Biochemical and Molecular Genetics Committee. Assessment is based on genotype and associated clinical interpretation. RESULTS: Overall, 357 clinical laboratories participated in the proficiency testing survey between 2003 and 2013 (322 in the United States and 35 international). In 2013, US participants reported performing nearly 120,000 tests monthly. Analytical sensitivity and specificity of US laboratories were 98.8% (95% confidence interval: 98.4-99.1%) and 99.6% (95% confidence interval: 99.4-99.7%), respectively. Analytical sensitivity improved between 2003 and 2008 (from 97.9 to 99.3%; P = 0.007) and remained steady thereafter. Clinical interpretation matched the intended response for 98.8, 86.0, and 91.0% of challenges with no, one, or two mutations, respectively. International laboratories performed similarly. DISCUSSION: Laboratory testing for cystic fibrosis in the United States has improved since 2003, and these data demonstrate a high level of quality. Neither the number of samples tested nor test methodology affected performance.

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87 These included 621+1, F508del, A455A, 1717-1, R117H, I507del, 3659delC, G85E, G542X, G551D, R553X, R347P, W1282X, N1303K, and R560T. Login to comment
108 Two other genotypes were also associated with relatively low analytical sensitivities (Figure 2): a heterozygous I507del challenge (2004) and a homozygous G542X sample (2009). Login to comment
110 The most common incorrect responses for the homozygous G542X challenge included reporting heterozygosity for G542X and incomplete entries (not reporting the second allele, rather than entering G542X in both allele fields). Login to comment
118 90 95 A (621 + 1G>T/A455E) (G542X/G542X) (I507 wild type or negative?) Login to comment
139 The homozygous G542X challenge in the 2009 survey was closely examined to determine whether the interpretation matched the given result. Login to comment
140 In all cases for which G542X was not entered in both allele fields, the interpretation was consistent with a result of one mutation or no mutations detected. Login to comment
182 For example, the G542X homozygous genotype and the I507del heterozygous genotype were difficult challenges (Figure 2), but both of these genotypes are rare, especially in the context of population-based screening. Login to comment

[hide] CFTR Modulators for the Treatment of Cystic Fibros... P T. 2014 Jul;39(7):500-11. Pettit RS, Fellner C
CFTR Modulators for the Treatment of Cystic Fibrosis.
P T. 2014 Jul;39(7):500-11., [PMID:25083129]

Abstract [show]
Defects in a single gene lead to the defective proteins that cause cystic fibrosis, making the disease an ideal candidate for mutation-targeted therapy. Although ivacaftor is currently the only FDA-approved CFTR modifier, others are in development.

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11 Table 1 Classification of Gene Mutations That Cause Cystic Fibrosis1,12,14 Class Exemplar Mutation Description I G542X Presence of premature termination codons (PTCs) causes CFTR protein synthesis to be defective or absent. Login to comment

[hide] Defining a mutational panel and predicting the pre... Sultan Qaboos Univ Med J. 2014 Aug;14(3):e323-9. Epub 2014 Jul 24. Fass UW, Al-Salmani M, Bendahhou S, Shivalingam G, Norrish C, Hebal K, Clark F, Heming T, Al-Khusaiby S
Defining a mutational panel and predicting the prevalence of cystic fibrosis in oman.
Sultan Qaboos Univ Med J. 2014 Aug;14(3):e323-9. Epub 2014 Jul 24., [PMID:25097766]

Abstract [show]
OBJECTIVES: Cystic fibrosis transmembrane conductance regulator (CFTR) mutations form distinct mutational panels in different populations and subgroups. The frequency of cystic fibrosis (CF) mutations and prevalence are unknown in Oman. This study aimed to elucidate the mutational panel and prevalence of CF for the North Al Batinah (NAB) region in Oman and to estimate the national prevalence of CF based on the carrier screening of unrelated volunteers. METHODS: The study included retrospective and prospective analyses of CF cases in the NAB region for 1998-2012. Genetic analysis of disease-causing mutations was conducted by screening of the entire coding sequence and exon-intron borders. The obtained mutational panel was used for the carrier screening of 408 alleles of unrelated and unaffected Omani individuals. RESULTS: S549R and F508del were the major mutations, accounting for 89% of mutations in the patient population. Two private mutations, c.1733-1734delTA and c.1175T>G, were identified in the patient cohort. Two carriers, one for F508del and another for S549R, were identified by screening of the volunteer cohort, resulting in a predicted prevalence for Oman of 1 in 8,264. The estimated carrier frequency of CF in Oman was 1 in 94. The carrier frequency in the NAB region was 3.9 times higher. CONCLUSION: The mutational panel for the NAB region and the high proportion of S549R mutations emphasises the need for specific screening for CF in Oman. The different distribution of allele frequencies suggests a spatial clustering of CF in the NAB region.

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23 Other mutations are less frequent and only four, G542X, N1303K, G551D and W1282X, haveallelefrequenciesabove1%inCaucasianpatients.15 Nonetheless, regional and geographical differences of common Caucasian mutations exist in various ethnic subpopulations.15 Similarly comprehensive molecular epidemiological data about CF in Arab populations are missing. Login to comment

[hide] Comprehensive CFTR gene analysis of the French cys... Genet Med. 2015 Feb;17(2):108-16. doi: 10.1038/gim.2014.113. Epub 2014 Aug 14. Audrezet MP, Munck A, Scotet V, Claustres M, Roussey M, Delmas D, Ferec C, Desgeorges M
Comprehensive CFTR gene analysis of the French cystic fibrosis screened newborn cohort: implications for diagnosis, genetic counseling, and mutation-specific therapy.
Genet Med. 2015 Feb;17(2):108-16. doi: 10.1038/gim.2014.113. Epub 2014 Aug 14., [PMID:25122143]

Abstract [show]
PURPOSE: Newborn screening (NBS) for cystic fibrosis (CF) was implemented throughout France in 2002. It involves a four-tiered procedure: immunoreactive trypsin (IRT)/DNA/IRT/sweat test [corrected] was implemented throughout France in 2002. The aim of this study was to assess the performance of molecular CFTR gene analysis from the French NBS cohort, to evaluate CF incidence, mutation detection rate, and allelic heterogeneity. METHODS: During the 8-year period, 5,947,148 newborns were screened for cystic fibrosis. The data were collected by the Association Francaise pour le Depistage et la Prevention des Handicaps de l'Enfant. The mutations identified were classified into four groups based on their potential for causing disease, and a diagnostic algorithm was proposed. RESULTS: Combining the genetic and sweat test results, 1,160 neonates were diagnosed as having cystic fibrosis. The corresponding incidence, including both the meconium ileus (MI) and false-negative cases, was calculated at 1 in 4,726 live births. The CF30 kit, completed with a comprehensive CFTR gene analysis, provides an excellent detection rate of 99.77% for the mutated alleles, enabling the identification of a complete genotype in 99.55% of affected neonates. With more than 200 different mutations characterized, we confirmed the French allelic heterogeneity. CONCLUSION: The very good sensitivity, specificity, and positive predictive value obtained suggest that the four-tiered IRT/DNA/IRT/sweat test procedure may provide an effective strategy for newborn screening for cystic fibrosis.

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53 Because only a limited number of functional studies have assessed the pathogenicity of variants, mutations have been classified in previous studies according to their disease-causing potential.16,22,23 Based on the recommendations and data from these studies (UMD-CFTR-France),24 variants were classified into four groups: A, CF-causing; B, associated with CFTR-RDs; C, no clinical consequences; and D, unknown or Table 1ߒ Allelic frequencies of CF30-kit mutations, identified in neonates with CF, and correspondence between traditional mutation nomenclature and that on the Human Genome Variation Society website Frequency (F) % Mutation Legacy mutation nomenclature Number of alleles/2,320 % of alleles/2,320 Cumulative % ࣙ5 p.Phe508del F508del 1,560 67.24 67.24 p.Gly542* G542X 113 3.19 10.51 p.Asn1303Lys N1303K 81 1.98 c.1585-1G>A 1717-1G>A 48 1.47 1.00ࣙFࣙ4.99 c.2657ߙ+ߙ5G>A 2789ߙ+ߙ5G>A 37 1.42 p.Arg553* R553X 36 1.29 p.Gly551Asp G551D 31 1.16 p.Tyr122* Y122X 26 0.97 6.86 c.2988ߙ+ߙ1G>A 3120ߙ+ߙ1G>A 22 0.82 c.579ߙ+ߙ1G>T 711ߙ+ߙ1G>T 18 0.67 p.Ile507del I507del 17 0.63 c.3140-26A>G 3272-26A>G 16 0.59 0.40ࣙFࣙ0.99 p.Arg347Pro R347P 15 0.56 p.Arg1162* R1162X 15 0.56 p.Trp1282* W1282X 14 0.52 p.Tyr1092* Y1092X 13 0.48 c.2051_2052delinsG 2183AA>G 12 0.45 c.3528delC 3659delC 11 0.41 c.1680-886A>G 1811ߙ+ߙ1.6kbA>G 9 0.39 p.Gly85Glu G85E 8 0.34 3.06 p.Ser1251Asn S1251N 7 0.30 p.Arg334Trp R334W 7 0.30 p.Arg117His R117H 7 0.30 0.1ࣙFࣙ0.39 p.Trp846* W846X 6 0.26 c.489ߙ+ߙ1G>T 621ߙ+ߙ1G>T 6 0.26 c.948delT 1078delT 5 0.22 p.Ala455Glu A455E 5 0.22 p.Glu60* E60X 4 0.17 c.262_263delTT 394delTT 4 0.17 c.3718-2477C>T 3849ߙ+ߙ10kbC>T 3 0.13 Total 2,034 87.67 87.67 Mutations are clustered into four groups of frequency intervals (>5%, 1-4.99%, 0.99-0.4%, and <0.4%). Login to comment

[hide] Mannose-binding lectin gene as a modifier of the c... J Cyst Fibros. 2015 Jan;14(1):78-83. doi: 10.1016/j.jcf.2014.07.012. Epub 2014 Aug 29. Gravina LP, Crespo C, Giugno H, Sen L, Chertkoff L, Mangano A, Castanos C
Mannose-binding lectin gene as a modifier of the cystic fibrosis phenotype in Argentinean pediatric patients.
J Cyst Fibros. 2015 Jan;14(1):78-83. doi: 10.1016/j.jcf.2014.07.012. Epub 2014 Aug 29., [PMID:25178872]

Abstract [show]
BACKGROUND: There is a considerable variation in the phenotype and course of the disease in cystic fibrosis (CF) even in patients with the same CFTR genotype, suggesting that other factors are important for prognosis. Mannose-binding lectin (MBL) has been proposed as one of these factors. We therefore investigated the influence of MBL2 gene variants on disease severity, age at acquisition of Pseudomonas aeruginosa, and survival in CF patients. METHODS: MBL2 variants were studied in 106 Argentinean pediatric CF patients carrying two severe CFTR mutations. Clinical phenotype was defined according to the Shwachman score and lung function tests. Age at infection with P. aeruginosa and age at death were also recorded. RESULTS: MBL insufficiency was associated with a 3.5-fold risk of having a severe phenotype (CI 95%: 1.2-10.3, p=0.03). It was also associated with an earlier onset of infection with P. aeruginosa (p=0.035). No statistically significant differences were found in FEV1 and survival. CONCLUSIONS: MBL insufficiency was associated with detrimental progression of the disease. These results together with previous findings suggest that the effect of MBL2 expression may be a major determinant of the severity of the clinical phenotype in patients with CF.

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116 c Other severe mutations: 1717-1G-NA, G542X, N1303K, W1282X, G551D, DI507, 3659delC. Login to comment

[hide] Intestinal current measurement versus nasal potent... BMC Pulm Med. 2014 Oct 4;14:156. doi: 10.1186/1471-2466-14-156. Bagheri-Hanson A, Nedwed S, Rueckes-Nilges C, Naehrlich L
Intestinal current measurement versus nasal potential difference measurements for diagnosis of cystic fibrosis: a case-control study.
BMC Pulm Med. 2014 Oct 4;14:156. doi: 10.1186/1471-2466-14-156., [PMID:25280757]

Abstract [show]
BACKGROUND: Nasal potential difference (NPD) and intestinal current measurement (ICM) are functional CFTR tests that are used as adjunctive diagnostic tools for cystic fibrosis (CF). Smoking has a systemic negative impact on CFTR function. A diagnostic comparison between NPD and ICM and the impact of smoking on both CFTR tests has not been done. METHODS: The sweat chloride test, NPD, and ICM were performed in 18 patients with CF (sweat chloride >60 mmol/l), including 6 pancreatic sufficient (PS) patients, and 13 healthy controls, including 8 smokers. The NPD CFTR response to Cl-free and isoproterenol perfusion (Delta0Cl- + Iso) was compared to the ICM CFTR response to forskolin/IBMX, carbachol, and histamine (DeltaIsc, forskolin/IBMX+ carbachol+histamine). RESULTS: The mean NPD CFTR response and ICM CFTR response between patients with CF and healthy controls was significantly different (p <0.001), but not between patients with CF who were PS and those who were pancreatic insufficient (PI). Smokers have a decreased CFTR response measured by NPD (p = 0.049). For ICM there is a trend towards decreased CFTR response (NS). Three healthy control smokers had NPD responses within the CF-range. In contrast to NPD, there was no overlap of the ICM response between patients with CF and controls. CONCLUSIONS: ICM is superior to NPD in distinguishing between patients with CF who have a sweat chloride > 60 mmol/l and healthy controls, including smokers. Neither NPD nor ICM differentiated between patients with CF who were PS from those who were PI. Smoking has a negative impact on CFTR function in healthy controls measured by NPD and challenges the diagnostic interpretation of NPD, but not ICM.

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39 For sweat stimulation and collection, the Macroduct&#ae; system (Wescor, Table 1 Characteristics and CFTR response of pancreatic insufficient (CF-PI) and pancreatic sufficient (CF-PS) patients with CF and controls CF-PI (n = 12) CF-PS (n = 6) CF-all (n = 18) Controls (n = 13) Age, years 24.0 &#b1; 6.1 23.3 &#b1; 11.8 22.8 &#b1; 8.0 30.6 &#b1; 10.4 22.0 (19.0 - 26.0) 16.0 (14.5 - 30.5) 20.5 (18.3 - 25.3) 25.0 (23.5 - 35.5) Gender, females:males 3:9 5:1 8:10 7:6 Body mass index Z-score -1.18 &#b1; 0.80 -0.62 &#b1; 1,41 -0.99 &#b1; 1.03* 0.00 &#b1; 0.65* -1.05 (-2.40 - 0.00) 1.41 (-0.20 - 0.70) -0.90 (-2.60 - 0.70) 0.00 (-1.10 - 1.30) Sweat chloride (mmol/l) 110 &#b1; 13** 86 &#b1; 14** 102 &#b1; 17* 19 &#b1; 8* 106 (92 - 140) 90 (70 - 99) 104 (70 - 140) 19 (10 - 36) NPD CFTR response average Ɗ0Cl- + Iso (mV) 4.6 &#b1; 3.9 1.5 &#b1; 4.1 3.6 &#b1; 4.1* -13.6 &#b1; 8.5* 5.1 (-3.0 -11.9) 1.5 (-3.2 - 6.23) 4.5 (-3.2 - 11.9) -12.7 (-26.4 - -1.92) ICM CFTR response average ƊIsc (bc;A/cm2) ) (forskolin/IBMX + carbachol + histamine) -0.3 &#b1; 8.1 5.3 &#b1; 10.9 1.6 &#b1; 9.2* 77.8 &#b1; 34.8* -0.6 (-12.6 - 17.9) 5.0 (-9.7 - 19.0) 0.1 (-12.6 - 19.0) 65.3 (39.6 -140.9) Genotyping F508/F508 (6&#d7;) F508/R347P (2&#d7;) 148 T/R117H-7 T F508/G551D (2&#d7;) F508/3849 + 10 kb C- > T (2&#d7;) F508/- F508/G542X F508/R334W --/-- F508/N1303K F508/? Login to comment

[hide] Analysis of cystic fibrosis gene mutations in chil... J Med Case Rep. 2014 Oct 10;8:339. doi: 10.1186/1752-1947-8-339. Dell'Edera D, Benedetto M, Gadaleta G, Carone D, Salvatore D, Angione A, Gallo M, Milo M, Pisaturo ML, Di Pierro G, Mazzone E, Epifania AA
Analysis of cystic fibrosis gene mutations in children with cystic fibrosis and in 964 infertile couples within the region of Basilicata, Italy: a research study.
J Med Case Rep. 2014 Oct 10;8:339. doi: 10.1186/1752-1947-8-339., [PMID:25304080]

Abstract [show]
INTRODUCTION: Cystic fibrosis is the most common autosomal recessive genetic disease in the Caucasian population. Extending knowledge about the molecular pathology on the one hand allows better delineation of the mutations in the CFTR gene and the other to dramatically increase the predictive power of molecular testing. METHODS: This study reports the results of a molecular screening of cystic fibrosis using DNA samples of patients enrolled from January 2009 to December 2013. Patients were referred to our laboratory for cystic fibrosis screening for infertile couples. In addition, we identified the gene mutations present in 76 patients affected by cystic fibrosis in the pediatric population of Basilicata. RESULTS: In the 964 infertile couples examined, 132 subjects (69 women and 63 men) resulted heterozygous for one of the CFTR mutations, with a recurrence of carriers of 6.85%. The recurrence of carriers in infertile couples is significantly higher from the hypothetical value of the general population (4%). CONCLUSIONS: This study shows that in the Basilicata region of Italy the CFTR phenotype is caused by a small number of mutations. Our aim is to develop a kit able to detect not less than 96% of CTFR gene mutations so that the relative risk for screened couples is superimposable with respect to the general population.

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47 The molecular analysis of the CFTR gene revealed that the two Table 1 Number of subjects tested who were carriers of the cystic fibrosis transmembrane regulator gene Mutation Men Women Total G551D 1 2 3 R553X 0 1 1 F508del 35 32 67 N1303K 7 8 15 I148T 4 9 13 G542X 3 6 9 DI507 2 0 2 L1077P 0 2 2 D1152H 1 6 7 W1282X 2 0 2 2183 AA>G 3 0 3 1259insA 0 1 1 4016insT 1 0 1 I507del 1 0 1 2789+5G>A 1 0 1 4382delA 0 2 2 G1244E 1 0 1 621+3A>G 1 0 1 Total 63 69 132 Figure 1 76 patients with cystic fibrosis and positive sweat test, all have two genes mutated. Login to comment
49 brothers, aged 26 and 29 years old respectively, were both 'compound heterozygotes G542X/D1152H`, while the third one, aged 37 years old, was 'compound heterozygotes G542X/E831X`. Login to comment
59 As mentioned before, molecular screening Table 2 Comparison between the results obtained in this study and those obtained in a previous study Castaldo et al. [14] Mutations observed in the present study F508del 55.8% (29) 48.62% (141) N1303K 3.8% (2) 9.31% (27) G542X 3.8% (2) 8.96% (26) W1282X 3.8% (2) 1.03% (3) 2183AA>G 5.8% (3) 2.76% (8) R1162X 0 0 1717-1G>A 1.9% (1) 0 T338I 0 0 R347P 0 0.69% (2) 711+5G>A 0 0 852del22 5.8% (3) 1.03% (3) 4382delA 0 0.69% (2) 1259insA 0 0.34% (1) 4016insT 0 0.34% (1) R553X 0 0.34% (1) R1158X 0 0 L1077P 0 1.03% (3) I502T 0 0 3849+10kbC>T 1.9% (1) 0.34% (1) D579G 0 0.69% (2) G1244E 3.8% (2) 0 G1349D 0 0.34% (1) 2789+5G>A 0 1.03% (3) 711+1G>T 0 0 L1065P 0 0 2522insC 0 0 E585X 0 0 G85E 0 0 G178R 0 0 D1152H 0 3.10% (9) I148T-3195del6 0 0 I148T (alone) 0 4.48% (13) R334W 0 0 DI507 0 0.69% (2) I1005R 0 0 3272-26A>G 0 0 2711delT 0 0 L558S 1.9% (1) 0.34% (1) W1063X 0 0 D110H 0 0 S549R (A>C) 1.9% (1) 0.69% (2) 2184insA 0 0 3131del22 0 0 Table 2 Comparison between the results obtained in this study and those obtained in a previous study (Continued) R709N 0 0 A349V 0 0 4015insA 0 0 Y849X 1.9% (1) 0.34% (1) G551D 0 1.03% (3) 621+3A>G 0 0.34% (1) E831X 0 0 I507del 0 0.69% (2) IVS8 TG12/t5 0 1.03% (3) H139R (A->G) 0 0.34% (1) 1248+1G>A 0 0.34% (1) R74W;V201M;D1270N 0 0.69% (2) S1455X 0 0.34% (1) dele 2,3 (21kb) 0 0.34% (1) 991del5 0 0.34% (1) UNKNOWN 7 %(4) 4.83% (14) F508C 0 0.69% (2) TOTAL 52 290 of CF is highly recommended in the USA by the National Institutes of Health Consensus Development Conference Statement on genetic testing for cystic fibrosis [17]. Login to comment
79 The test has a sensitivity and a specificity of more than Table 3 List of 60 mutations in the cystic fibrosis transmembrane regulator gene (specificity 100%) F508del I507del F508C 621+1G>T D110H E585X G1349D I502T 1706del17 1677delTA R117H H139R 1898+1G>A 4015delA G542X 1717-1G>A Q552X 852del22 G178R 1898+3A>G G551D S549R(A>C) 2183AA>G T338I 991del5 1898+5G>T N1303K 4016insT 3849+10kb C>T R347P R334W 2184insA G85E 711+5G>A 711+1G>T 1259insA R347H 2522insC 2789+5G>A W1282X G1244E R1066H R352Q 3120+1G>A I148T 3199del6 S912X R1158X 1717-8G>A R1066C R1162X 4382delA D1152H L1077P D579G 3272-26A>G L1065P R553X PoliT: 5T, 7T, 9T 1874insT 3659delC 99%. Login to comment

[hide] Management of endocrine disease: Cystic fibrosis-r... Eur J Endocrinol. 2015 Apr;172(4):R131-41. doi: 10.1530/EJE-14-0644. Epub 2014 Oct 21. Barrio R
Management of endocrine disease: Cystic fibrosis-related diabetes: novel pathogenic insights opening new therapeutic avenues.
Eur J Endocrinol. 2015 Apr;172(4):R131-41. doi: 10.1530/EJE-14-0644. Epub 2014 Oct 21., [PMID:25336504]

Abstract [show]
Cystic fibrosis (CF) is a recessive genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR). CFTR is primarily present in epithelial cells of the airways, intestine and in cells with exocrine and endocrine functions. Mutations in the gene encoding the channel protein complex (CFTR) cause alterations in the ionic composition of secretions from the lung, gastrointestinal tract, liver, and also the pancreas. CF-related diabetes (CFRD), the most common complication of CF, has a major detrimental impact on pulmonary function, nutrition and survival. Glucose derangements in CF seem to start from early infancy and, even when the pathophysiology is multifactorial, insulin insufficiency is clearly a major component. Consistently, recent evidence has confirmed that CFTR is an important regulator of insulin secretion by islet beta-cells. In addition, several other mechanisms were also recognized from cellular and animals models also contributing to either beta-cell mass reduction or beta-cell malfunction. Understanding such mechanisms is crucial for the development of the so-called 'transformational' therapies in CF, including the preservation of insulin secretion. Innovative therapeutic approaches aim to modify specific CFTR mutant proteins or positively modulate their function. CFTR modulators have recently shown in vitro capacity to enhance insulin secretion and thereby potential clinical utility in CFDR, including synergistic effects between corrector and potentiator drugs. The introduction of incretins and the optimization of exocrine pancreatic replacement complete the number of therapeutic options of CFRD besides early diagnosis and implementation of insulin therapy. This review focuses on the recently identified pathogenic mechanisms leading to CFRD relevant for the development of novel pharmacological avenues in CFRD therapy.

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29 The F508del mutation is present in 90% of CF patients worldwide (7) and only four other mutations (G551D, W1282X, G542X, and N1303K) have a minor but substantial prevalence (1-3% each) worldwide (8). Login to comment

[hide] SNaPshot assay for the detection of the most commo... PLoS One. 2014 Nov 11;9(11):e112498. doi: 10.1371/journal.pone.0112498. eCollection 2014. Noveski P, Madjunkova S, Mircevska M, Plaseski T, Filipovski V, Plaseska-Karanfilska D
SNaPshot assay for the detection of the most common CFTR mutations in infertile men.
PLoS One. 2014 Nov 11;9(11):e112498. doi: 10.1371/journal.pone.0112498. eCollection 2014., [PMID:25386751]

Abstract [show]
Congenital bilateral absence of vas deferens (CBAVD) is the most common CFTR-related disorder (CFTR-RD) that explains about 1-2% of the male infertility cases. Controversial data have been published regarding the involvement of CFTR mutations in infertile men with non-obstructive azoospermia and oligozoospermia. Here, we describe single base extension (SNaPshot) assay for detection of 11 common CFTR mutations: F508del, G542X, N1303K, 621+1G->T, G551D, R553X, R1162X, W1282X, R117H, 2184insA and 1717-1G->A and IVS8polyT variants. The assay was validated on 50 previously genotyped samples and was used to screen a total of 369 infertile men with different impairment of spermatogenesis and 136 fertile controls. Our results show that double heterozygosity of cystic fibrosis (CF) and CFTR-related disorder (CFTR-RD) mutations are found in a high percentage (22.7%) of infertile men with obstructive azoospermia, but not in other studied groups of infertile men. The SNaPshot assay described here is an inexpensive, fast and robust method for primary screening of the most common CFTR mutations both in patients with classical CF and CFTR-RD. It can contribute to better understanding of the role of CFTR mutations in impaired spermatogenesis, ultimately leading to improved management of infertile men.

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2 Here, we describe single base extension (SNaPshot) assay for detection of 11 common CFTR mutations: F508del, G542X, N1303K, 621+1G-.T, G551D, R553X, R1162X, W1282X, R117H, 2184insA and 1717-1G-.A and IVS8polyT variants. Login to comment
37 Here, we present a rapid, reliable, cost-effective and simple assay for testing of 11 CFTR (NM_000492.3) mutations: F508del (c.1521_1523delCTT), G542X (c.1624G.T), N1303K (c.3909C.G), 621+1G-.T (c.489+1G.T), G551D (c.1652G. Login to comment
69 Mutation analyzeda Name Sequence 59-.39 Exon/intron amplified (bp)b Length of PCR fragment amplified in bp 621+1G-.T, R117H CFTR ex4/F TCTTGTGTTGAAATTCTCAGGGTA exon4 (216) 374 CFTR ex4/R CCAGCTCACTACCTAATTTATGACA delF508 CFTR ex10/F TGAATCCTGAGCGTGATTTG exon10 (192) 302 CFTR ex10/R TGGGTAGTGTGAAGGGTTCAT G542X, G551D, R553X CFTR ex11/F GCCTTTCAAATTCAGATTGAGC exon11 (95) 288 CFTR ex11/R CTAGCCATAAAACCCCAGGA 2184insA CFTR ex13/F TGCAATAAAACATTAACAAAATGC exon13 (724) 480 CFTR ex13/R GGGAGTCTTTTGCACAATGG R1162X CFTR ex19/F TGTGAAATTGTCTGCCATTCTT exon19 (249) 369 CFTR ex19/R TGCTTCAGGCTACTGGGATT W1282X CFTR ex20/F CTGAATTATGTTTATGGCATGG exon20 (156) 249 CFTR ex20/R TTTTTCTGGCTAAGTCCTTTTG N1303K CFTR ex21/F TGATGGTAAGTACATGGGTGTTTC exon21 (90) 257 CFTR ex21/R CCCCTTTCA AAATCATTTCAG IVS8-5T/7T/9T CFTR intron 8/F GGCCATGTGCTTTTCAAACT intron8 (194) 194 CFTR intron 8/R AAGAAGAGGCTGTCATCACCA a Legacy name. Login to comment
73 CFTR mutation cDNA name according to HGVS (ref. seq. NM_000492.3) Sequence (59-.39) Orientation SNaPshot Result (normal/mutant allele) Size of extended fragment in base pairs (normal allele/mutant allele)a Concentration in mix (mM)b G542X c.1624G.T CAGTGTGATTCCACCTTCTC Reverse C/A (24.9/25.9) 3 N1303K c.3909C.G CCCACTGTTCATAGGGATCCAA Reverse G/C (26.3/26.9) 5 F508del c.1521_1523delCTT CCCCTGGCACCATTAAAG- AAAATATCAT Forward C/T (29.6/31.0) 1 R117H c.350G.A 15(C)GGATAACAAGGAGGAAC Forward G/A (33.6/35.3) 7 IVS8-5T/7T/9T c.1210-12T[5_9] TGTGTGTGTGTGTGTGTGTTTTT Forward A/T 5T - 32.3 7T,9T - 33.4 1 621+1G-.T c.489+1G.T CCCTAGCTATGTTTAGTTTG- ATTTATAAGAAG Forward G/T (37.2/38.2) 5 IVS8-7T/9T c.1210-12T[7_9] 14(C)GTGTGTGTGTGTGT- GTGTTTTTTT Forward A/T 7T - 44.0 9T - 44.9 2 2184insA c.2052_2053insA 13(C)GTCTCCTGGACAGAAAC- AAAAAAA Forward C/A (38.7/39.7) 8 1717-1 G-.A c.1585-1G.A 9(C)GACTCTCTAATTTTC- TATTTTTGGTAATA Forward G/A (41.3/41.7) 2 G551D c.1652G.A 21(C)TGGAATCACACTGAG- TGGAG Forward G/A (43.4/43.9) 4 R553X c.1657C.T 24(C)AATCACACTGAGT- GGAGGTCAA Forward C/T (46.2/47.2) 2 W1282X c.3846G.A 28(C)GGATTCAATA- ACTTTGCAACAGTG Forward G/A (51.6/52.6) 1 R1162X c.3484C.T 29(C)ATTTCAGATG- CGATCTGTGAGC Forward C/T (51.0/52.0) 4 a Data generated on ABI PRISM 3130 Genetic Analyzer with POP-4 polymer, 36-cm capillary array and sized against GeneScan-120 LIZ size standard. Login to comment
98 [1521_1523delCTT];[ = ] 3 100% G542X/[2] c. Login to comment
100 [489+1G.T];[ = ] 2 100% G542X/F508del c. Login to comment
115 Among the oligozoospermic men five patients carried CF mutation (3 with F508del and 2 with G542X) but one of them also carried the CFTR-RD mutation, i.e. 5T allele. Login to comment
120 Compound heterozygosity for CF mutation (F508del or G542X) and CFTR-RD mutation (IVS8-5T) was present in higher frequency among patients with obstructive azoospermia (22.7%), when compared to the fertile controls (0%) p = 3.4261025 and all other groups of infertile men: non-obstructive azoospermia/severe oligozoospermia (1.14%, p = 1.1661023 ), azoospermia (0%, p = 3.3261024 ), oligozoospermia (0.92%, p = 4.8961024 ) and normoasthenoteratozoospermia (0%, p = 4.5461024 ). Login to comment
139 Patients CFTR genotype Intron8 polyT genotype N % Azoospermia [2]/[2] 5T/7T 8 10.1% [2]/[2] 5T/9T 1 1.3% [2]/[2] 7T/7T 56 70.9% [2]/[2] 7T/9T 14 17.7% Total 79 100.0% Oligozoospermia delF508/[2] 7T/9T 2 1.9% delF508/[2] 9T/9T 1 0.9% G542X/[2] 5T/9T 1 0.9% G542X/[2] 7T/9T 1 0.9% [2]/[2] 5T/7T 4 3.7% [2]/[2] 7T/7T 79 73.1% [2]/[2] 7T/9T 20 18.5% Total 108 100.0% Normoasthenoteratozoospermia delF508/[2] 7T/9T 1 1.4% delF508/[2] 9T/9T 2 2.7% [2]/[2] 5T/7T 6 8.2% [2]/[2] 7T/7T 49 67.1% [2]/[2] 7T/9T 14 19.2% [2]/[2] 9T/9T 1 1.4% Total 73 100.0% Fertile Controls delF508/[2] 7T/9T 2 1.5% [2]/[2] 5T/7T 8 5.9% [2]/[2] 5T/9T 1 0.7% [2]/[2] 7T/7T 96 70.6% [2]/[2] 7T/9T 27 19.9% [2]/[2] 9T/9T 2 1.5% Total 136 100.0% Note: [2] means no CF mutation detected on single chromosome. Login to comment
160 We have genotyped one patient to have F508del/ IVS8-5T genotype, with histopathological diagnosis of testicular atrophy and one oligozoospermic patient with G542X/IVS8-5T genotype. Login to comment
162 These results imply that F508del/IVS8-11TG-5T genotype might not be a cause of the infertility in the first patient, while G542X/IVS8-12TG-5T genotype is most probably a cause of infertility in the second patient. Login to comment
163 Similar to F508del, G542X has never been found in linkage disequilibrium with IVS8-5T [39]. Login to comment

[hide] Cystic fibrosis genetics: from molecular understan... Nat Rev Genet. 2015 Jan;16(1):45-56. doi: 10.1038/nrg3849. Epub 2014 Nov 18. Cutting GR
Cystic fibrosis genetics: from molecular understanding to clinical application.
Nat Rev Genet. 2015 Jan;16(1):45-56. doi: 10.1038/nrg3849. Epub 2014 Nov 18., [PMID:25404111]

Abstract [show]
The availability of the human genome sequence and tools for interrogating individual genomes provide an unprecedented opportunity to apply genetics to medicine. Mendelian conditions, which are caused by dysfunction of a single gene, offer powerful examples that illustrate how genetics can provide insights into disease. Cystic fibrosis, one of the more common lethal autosomal recessive Mendelian disorders, is presented here as an example. Recent progress in elucidating disease mechanism and causes of phenotypic variation, as well as in the development of treatments, demonstrates that genetics continues to play an important part in cystic fibrosis research 25 years after the discovery of the disease-causing gene.

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73 The level and/or content of CFTR transcripts can be affected by disease-causing variants in the promoter (for example, c.-234T࢐A (also known as -102T࢐A in legacy nomenclature))140 and splice sites (for example, c.3717ߙ+ߙ12191ߙC࢐T (legacy 3849ߙ+ߙ10ߙkb C࢐T))141 , or by variants that introduce a premature termination codon (PTC) and that lead to RNA decay (for example, p.Gly542X; (legacy G542X)142 . Login to comment
305 Nature Reviews | Genetics * * * G542X G551D 3849+10kbC࢐T Clinical response Clinical response Clinical response Clinical response Function Quantity Function Quantity Function Quantity Function Quantity F508del F508del a b c d Function Quantity Therapy Ivacaftor P5 Potentiators PTC suppressor Splicing corrector Lumacaftor C4 Expression Correctors Figure 3 | Molecular treatments for cystic fibrosis. Login to comment
323 Suppression of the cryptic splice sitecanincreaseCFTRproteinlevelsabove10%ofnormal145 .Thenonsense variant G542X (also known as p.Gly542X) introduces a premature termination codon (PTC) that causes severe reduction in mRNA levels and anabsenceoftheCFTRprotein.UseofPTCsuppressorsincreasestranscript andproteinlevels,leadingtoamodestrecoveryofCFTRfunctionthatfalls short of a clinical response. Login to comment

[hide] Preliminary study of haplotypes linked to the rare... Acta Physiol Hung. 2015 Mar;102(1):86-93. doi: 10.1556/APhysiol.101.2014.013. Oueslati S, Hadj Fredj S, Belhaj R, Siala H, Bibi A, Messaoud T
Preliminary study of haplotypes linked to the rare cystic fibrosis E1104X mutation.
Acta Physiol Hung. 2015 Mar;102(1):86-93. doi: 10.1556/APhysiol.101.2014.013., [PMID:25481366]

Abstract [show]
The analysis of some extra- and intragenic markers within or closely linked to the cystic fibrosis transmembrane regulator (CFTR) gene is useful as a molecular method in clinical linkage analysis. Indeed, knowing that the molecular basis of cystic fibrosis (CF) is highly heterogeneous in our population, the study of haplotype association with normal and CF chromosomes could be very helpful in cases where one or both mutations remain unidentified. In this study, we analysed with PCR-RFLP and capillary electrophoresis some extra (pJ3.11, KM19 and XV2C) and intragenic (IVS8CA, IVS17bTA and IVS17bCA) polymorphic markers in 50 normal and 10 Tunisian patients carrying the rare E1104X mutation in order to determine the haplotype associated with this mutation. For the extragenic markers, 8 haplotypes were identified. The most frequent of them are the 221 and 112 accounting for 80% of total haplotypes. For the intragenic markers, five haplotypes were present on the E1104X chromosomes. One of them 16-31-13 accounted for 50%. To our knowledge, this is the first work to be interested to the haplotypes linked to the E1104X mutation. This preliminary study of haplotypes could be a helpful method to determine the molecular lesions responsible of this pathology.

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62 The most common CF mutations (F508del, G542X and N1303K), provide an exceptional opportunity for the analysis of association between microsatellite allelic systems and SNP`s. Login to comment

[hide] Clinical expression of patients with the D1152H CF... J Cyst Fibros. 2015 Jul;14(4):447-52. doi: 10.1016/j.jcf.2014.12.012. Epub 2015 Jan 10. Terlizzi V, Carnovale V, Castaldo G, Castellani C, Cirilli N, Colombo C, Corti F, Cresta F, D'Adda A, Lucarelli M, Lucidi V, Macchiaroli A, Madarena E, Padoan R, Quattrucci S, Salvatore D, Zarrilli F, Raia V
Clinical expression of patients with the D1152H CFTR mutation.
J Cyst Fibros. 2015 Jul;14(4):447-52. doi: 10.1016/j.jcf.2014.12.012. Epub 2015 Jan 10., [PMID:25583415]

Abstract [show]
BACKGROUND: Discordant results were reported on the clinical expression of subjects bearing the D1152H CFTR mutation, and also for the small number of cases reported so far. METHODS: A retrospective review of clinical, genetic and biochemical data was performed from individuals homozygous or compound heterozygous for the D1152H mutation followed in 12 Italian cystic fibrosis (CF) centers. RESULTS: 89 subjects carrying at least D1152H on one allele were identified. 7 homozygous patients had very mild clinical expression. Over half of the 74 subjects compound heterozygous for D1152H and a I-II-III class mutation had borderline or pathological sweat test and respiratory or gastrointestinal symptoms; one third had pulmonary bacteria colonization and 10/74 cases had complications (i.e. diabetes, allergic bronchopulmonary aspergillosis, and hemoptysis). However, their clinical expression was less severe as compared to a group of CF patients homozygous for the F508del mutation. Finally, 8 subjects compound heterozygous for D1152H and a IV-V class mutation showed very mild disease. CONCLUSIONS: The natural history of subjects bearing the D1152H mutation is widely heterogeneous and is influenced by the mutation in trans.

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85 Legacy name Protein name CDNA name Patients Homozygous for the D1152Ha D1152H p.Asp1152His c.3454GNC 7 Compound heterozygous for class I-II-III mutationsa : 74 F508del p.Phe508del c.1521_1523delCTT 43 G542X p.Gly542X c.1624GNT 7 N1303K p.Asn1303Lys c.3909CNG 4 1717-1GNA No protein name c.1585-1GNA 4 R1158X p.Arg1158X c.3472CNT 4 2183AANG p.Lys684SerfsX38 c.2051_2052delAAinsG 2 W1282X p.Trp1282X c.3846GNA 2 711 + 1GNT No protein name c.579 + 1GNT 1 Y849X p.Tyr849X c.2547CNA 1 L1065P p.Leu1065Pro c.3194 TNC 1 4016insT p.Ser1297PhefsX5 c.3884_3885insT 1 R1066H p.Arg1066His c.3197GNA 2 R1066C p.Arg1066Cys c.3196CNT 1 4382delA p.Glu1418ArgfsX14 c.4251delA 1 Compound heterozygous for class IV-V mutationsa : 8 (TG)12T5 No protein name Not available 2 2789 + 5GNA No protein name c.2657 + 5GNA 1 D579G p.Asp579Gly c.1736ANG 1 [R74W;V201M; D1270N] No protein name Not available 1 3849 + 10KbCNT No protein name c.3717 + 12191CNT 1 R347H p.Arg347His c.1040GNA 1 R347P p.Arg347Pro c.1040GNC 1 a Protein name and cDNA name from the CFTR2 database (http://www.http. com//www.cftr2) and http://www.genet.sickkids.on.ca/Home.html. Login to comment

[hide] Demographic, clinical, and laboratory parameters o... BMC Pulm Med. 2015 Jan 15;15:3. doi: 10.1186/1471-2466-15-3. Marson FA, Hortencio TD, Aguiar KC, Ribeiro JD
Demographic, clinical, and laboratory parameters of cystic fibrosis during the last two decades: a comparative analysis.
BMC Pulm Med. 2015 Jan 15;15:3. doi: 10.1186/1471-2466-15-3., [PMID:25592785]

Abstract [show]
BACKGROUND: In recent years, patients with cystic fibrosis (CF) have tended to experience a longer life expectancy and higher quality of life. In this context, the aim of the present study was to evaluate and compare the demographic, clinical, and laboratory markers of patients with CF during the last two decades at a CF referral center. METHODS: A retrospective study of the demographic, clinical, and laboratory markers for CF treatment at a CF referral center was performed during two decades: 2000 (DI, 1990-2000, n = 104 patients) and 2010 (DII, 2000-2010, n = 181 patients). RESULTS: The following variables were less common in DI than in DII: (i) pancreatic insufficiency, (ii) meconium ileus, (iii) diabetes mellitus, (iv) Burkholderia cepacia colonization, (v) moderate and severe Shwachman-Kulczycki score (SKS), (vi) F508del mutation screening, (vii) patients without an identified CFTR mutation (class IV, V, or VI mutation), (viii) patients above the 10th percentile for weight and height, (ix) restrictive lung disease, and (x) older patients (p < 0.01). The following variables were more common in DI than in DII: (i) excellent and good SKS, (ii) F508del heterozygous status, (iii) colonization by mucoid and nonmucoid Pseudomonas aeruginosa, (iv) obstructive lung disease, and (v) minimal time for CF diagnosis (p < 0.01). CONCLUSION: Clinical outcomes differed between the two decades. Demographic, clinical, and laboratory markers in patients with CF are useful tools and should be encouraged in CF referral centers to determine the results of CF management and treatment, enabling a better understanding of this disease and its clinical evolution. Early diagnosis and management of CF will improve patients' quality of life and life expectancy until personalized drug therapy is possible for all patients with CF.

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27 Demographic, clinical, and laboratory markers The demographic, clinical, and laboratory variables analyzed in this study were sex (male/female), ethnicity (Caucasian or non-Caucasian), age, age range, number of deaths, clinical manifestations (respiratory and digestive), age at diagnosis, comorbidities [pancreatic insufficiency (PI), meconium ileus (MI), and diabetes mellitus (DM)], nutritional status as determined by weight and height on a growth curve (weight and height below the 10th percentile), oxygen saturation (SpO2) (>95%, 91%-95%, or <91%), sweat chloride level, microorganisms in the sputum (Staphylococcus aureus, mucoid and nonmucoid Pseudomonas aeruginosa, and Burkholderia cepacia), spirometry findings (normal, restrictive lung disease, obstructive lung disease, or mixed respiratory disorder) [14], genetic screening for the CFTR mutations [F508del (rs113993960, c.1521_ 1523delCTT), G542X (rs113993959, c.1624G > T), N1303K (rs80034486, c.3909C > G), G551D, R553X (rs74597325, c.1657C > T), and W1282X (rs77010898, c.3846G > A)], Shwachman-Kulczycki score (SKS) (excellent or good, mild, or moderate or severe) [15], and fecal fat. Login to comment
82 Older patients constitute a major proportion of the Table 1 Comparison of data (demographic, clinical, and laboratory markers) of patients with cystic fibrosis from a Brazilian referral center during the decades of 1990 to 2000 and 2000 to 2010 Demographic and clinical markers Category DI (1990-2000) DII (2000-2010) p Number of patients 104 181 Age Mean: 10 years 9 months &#b1; 6.33 months Mean :16 years 7 months &#b1; 13.16 months <0.001 Median: 9 years Median: 12 years 10 months Range: 11 months to 31 years 2 months Range: 6 months to 73 years 10 months Sex Male 53.8% 49.7% 0.539 Female 46.2% 50.3% Ethnicity Caucasian 93.3% 92.0% 0.818 NonCaucasian 6.7% 8.0% Consanguineous parents 6.2% 1.1% 0.054 Manifestation Respiratory 89.4% 91.7% 0.528 Digestive 59.6% 83.3% <0.001 Onset of symptoms Mean: 16 months Mean: 91.75 months <0.001 Median: 3 months Median: 3 months Range: 0-20 years Range: 0-60 years Age at diagnosis Mean: 4 years 2 months Mean: 2 years 10 months <0.001 Median: 2 years 4 months Median: 2 years Range: 0 to 29 years 11 months Range: 0-60 years Meconium ileus 5.8% 15.0% 0.021 Diabetes mellitus 4.8% 18.5% 0.001 Nutritional status Weight below 10th percentile 69.9% 35.71% <0.001 Height below 10th percentile 56.6% 40.82% 0.025 SpO2 >95% 59.5% 55.5% 0.713 91%-95% 32.9% 34.7% <91% 7.6% 9.8% Sweat test <60 mEq/L* 10.6% - 60-100 mEq/L 28.8% 40.51% >100 mEq/L 60.6% 59.49% Bacteria Staphylococcus aureus 80.2% 78.5% 0.880 Pseudomonas aeruginosa 76.0% 55.8% 0.001 Mucoid P. aeruginosa 53.1% 42.0% 0.085 Burkholderia cepacia 5.2% 14.4% 0.016 Mucoid and nonmucoid P. aeruginosa 51.0% 21.85% <0.001 Spirometry Normal 27.3% 34.4% <0.001 Restrictive ventilatory disorder 18.2% 48.9% Obstructive lung disorder 25.4% 14.5% Mixed respiratory disorder 29.1% 2.3% CFTR mutation F508del homozygotes 18.75% 26.5% <0.001 F508del heterozygotes 62.5% 22.7% G542X 4.17% 6.45% N1303K 2.08% 1.1% G551D 1.04% - patients seen at CF centers, and older age can be associated with variations in CF diagnosis and treatment [22]. Login to comment

[hide] Improving newborn screening for cystic fibrosis us... Genet Med. 2015 Feb 12. doi: 10.1038/gim.2014.209. Baker MW, Atkins AE, Cordovado SK, Hendrix M, Earley MC, Farrell PM
Improving newborn screening for cystic fibrosis using next-generation sequencing technology: a technical feasibility study.
Genet Med. 2015 Feb 12. doi: 10.1038/gim.2014.209., [PMID:25674778]

Abstract [show]
Purpose:Many regions have implemented newborn screening (NBS) for cystic fibrosis (CF) using a limited panel of cystic fibrosis transmembrane regulator (CFTR) mutations after immunoreactive trypsinogen (IRT) analysis. We sought to assess the feasibility of further improving the screening using next-generation sequencing (NGS) technology.Methods:An NGS assay was used to detect 162 CFTR mutations/variants characterized by the CFTR2 project. We used 67 dried blood spots (DBSs) containing 48 distinct CFTR mutations to validate the assay. NGS assay was retrospectively performed on 165 CF screen-positive samples with one CFTR mutation.Results:The NGS assay was successfully performed using DNA isolated from DBSs, and it correctly detected all CFTR mutations in the validation. Among 165 screen-positive infants with one CFTR mutation, no additional disease-causing mutation was identified in 151 samples consistent with normal sweat tests. Five infants had a CF-causing mutation that was not included in this panel, and nine with two CF-causing mutations were identified.Conclusion:The NGS assay was 100% concordant with traditional methods. Retrospective analysis results indicate an IRT/NGS screening algorithm would enable high sensitivity, better specificity and positive predictive value (PPV). This study lays the foundation for prospective studies and for introducing NGS in NBS laboratories.Genet Med advance online publication 12 February 2015Genetics in Medicine (2015); doi:10.1038/gim.2014.209.

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15 Correspondence: Mei W. Baker (mwbaker@wisc.edu) Improving newborn screening for cystic fibrosis using next-generation sequencing technology: a technical feasibility study Mei W. Baker, MD1,2 , Anne E. Atkins, MPH2 , Suzanne K. Cordovado, PhD3 , Miyono Hendrix, MS3 , Marie C. Earley, PhD3 and Philip M. Farrell, MD, PhD1,4 Table 1ߒ CF-causing or varying consequences mutations in the MiSeqDx IUO Cystic Fibrosis System c.1521_1523delCTT (F508del) c.2875delG (3007delG) c.54-5940_273ߙ+ߙ10250del21kb (CFTRdele2,3) c.3909C>G (N1303K) c.3752G>A (S1251N) Mutations that cause CF when combined with another CF-causing mutation c.1624G>T (G542X) c.2988ߙ+ߙ1G>A (3120ߙ+ߙ1G->A) c.3964-78_4242ߙ+ߙ577del (CFTRdele22,23) c.613C>T (P205S) c.1021T>C (S341P) c.948delT (1078delT) c.2988G>A (3120G->A) c.328G>C (D110H) c.200C>T (P67L) c.1397C>A (S466X(C>A)) c.1022_1023insTC (1154insTC) c.2989-1G>A (3121-1G->A) c.3310G>T (E1104X) c.3937C>T (Q1313X) c.1397C>G (S466X(C>G)) c.1081delT (1213delT) c.3140-26A>G (3272-26A->G) c.1753G>T (E585X) c.658C>T (Q220X) c.1466C>A (S489X) c.1116ߙ+ߙ1G>A (1248ߙ+ߙ1G->A) c.3528delC (3659delC) c.178G>T (E60X) c.115C>T (Q39X) c.1475C>T (S492F) c.1127_1128insA (1259insA) c.3659delC (3791delC) c.2464G>T (E822X) c.1477C>T (Q493X) c.1646G>A (S549N) c.1209ߙ+ߙ1G>A (1341ߙ+ߙ1G->A) c.3717ߙ+ߙ12191C>T (3849ߙ+ߙ10kbC->T) c.2491G>T (E831X) c.1573C>T (Q525X) c.1645A>C (S549R) c.1329_1330insAGAT (1461ins4) c.3744delA (3876delA) c.274G>A (E92K) c.1654C>T (Q552X) c.1647T>G (S549R) c.1393-1G>A (1525-1G->A) c.3773_3774insT (3905insT) c.274G>T (E92X) c.2668C>T (Q890X) c.2834C>T (S945L) c.1418delG (1548delG) c.262_263delTT (394delTT) c.3731G>A (G1244E) c.292C>T (Q98X) c.1013C>T (T338I) c.1545_1546delTA (1677delTA) c.3873ߙ+ߙ1G>A (4005ߙ+ߙ1G->A) c.532G>A (G178R) c.3196C>T (R1066C) c.1558G>T (V520F) c.1585-1G>A (1717-1G->A) c.3884_3885insT (4016insT) c.988G>T (G330X) c.3197G>A (R1066H) c.3266G>A (W1089X) c.1585-8G>A (1717-8G->A) c.273ߙ+ߙ1G>A (405ߙ+ߙ1G->A) c.1652G>A (G551D) c.3472C>T (R1158X) c.3611G>A (W1204X) c.1679ߙ+ߙ1.6kbA>G (1811ߙ+ߙ1.6kbA->G) c.274-1G>A (406-1G->A) c.254G>A (G85E) c.3484C>T (R1162X) c.3612G>A (W1204X) c.1680-1G>A (1812-1G->A) c.4077_4080delTGTTinsAA (4209TGTT->AA) c.2908G>C (G970R) c.349C>T (R117C) c.3846G>A (W1282X) c.1766ߙ+ߙ1G>A (1898ߙ+ߙ1G->A) c.4251delA (4382delA) c.595C>T (H199Y) c.1000C>T (R334W) c.1202G>A (W401X) c.1766ߙ+ߙ3A>G (1898ߙ+ߙ 3A->G) c.325_327delTATinsG (457TAT->G) c.1007T>A (I336K) c.1040G>A (R347H) c.1203G>A (W401X) c.2012delT (2143delT) c.442delA (574delA) c.1519_1521delATC (I507del) c.1040G>C (R347P) c.2537G>A (W846X) c.2051_2052delAAinsG (2183AA->G) c.489ߙ+ߙ1G>T (621ߙ+ߙ 1G->T) c.2128A>T (K710X) c.1055G>A (R352Q) c.3276C>A (Y1092X (C>A)) c.2052delA (2184delA) c.531delT (663delT) c.3194T>C (L1065P) c.1657C>T (R553X) c.3276C>G (Y1092X (C>G)) c.2052_2053insA (2184insA) c.579ߙ+ߙ1G>T (711ߙ+ߙ 1G->T) c.3230T>C (L1077P) c.1679G>A (R560K) c.366T>A (Y122X) c.2175_2176insA (2307insA) c.579ߙ+ߙ3A>G (711ߙ+ߙ 3A->G) c.617T>G (L206W) c.1679G>C (R560T) - c.2215delG (2347delG) c.579ߙ+ߙ5G>A (711ߙ+ߙ 5G->A) c.1400T>C (L467P) c.2125C>T (R709X) - c.2453delT (2585delT) c.580-1G>T (712-1G->T) c.2195T>G (L732X) c.223C>T (R75X) - c.2490ߙ+ߙ1G>A (2622ߙ+ߙ1G->A) c.720_741delAGGGAG AATGATGATGAAGTAC (852del22) c.2780T>C (L927P) c.2290C>T (R764X) - c.2583delT (2711delT) c.1364C>A (A455E) c.3302T>A (M1101K) c.2551C>T (R851X) - c.2657ߙ+ߙ5G>A (2789ߙ+ߙ5G->A) c.1675G>A (A559T) c.1A>G (M1V) c.3587C>G (S1196X) - Mutations/variants that were validated in this study are in bold. CF, cystic fibrosis. Table 1ߒ Continued on next page reduce carrier detection and potentially improve the positive predictive value (PPV), the NBS goals of equity and the highest possible sensitivity become more difficult to achieve. Login to comment

[hide] [CFTR gene sequencing in a group of Chilean patien... Rev Chil Pediatr. 2014 Jul;85(4):448-54. doi: 10.4067/S0370-41062014000400007. Lay-Son R G, Vasquez D M, Puga Y A, Manque M P, Repetto L G
[CFTR gene sequencing in a group of Chilean patients with cystic fibrosis].
Rev Chil Pediatr. 2014 Jul;85(4):448-54. doi: 10.4067/S0370-41062014000400007., [PMID:25697318]

Abstract [show]
INTRODUCTION: Cystic fibrosis (CF) is an autosomal recessive genetic disorder caused by mutations of the CFTR gene, in which over 1,900 different mutations have been identified. In Chile, the diagnosis panel with the 36 most common mutations detects approximately 50% of all alleles, while for Caucasians, it is nearly 90%. The objective of this study is to expand the capacity of mutational screening in Chilean patients and look for recurrent mutations at the national level. METHOD: The detection of unknown pathogenic alleles was assessed by CFTR gene sequencing in a selected group of patients from the National Cystic Fibrosis Foundation (NCFF). 39 patients, who met the CF diagnostic criteria and had only one allele identified according to the mutational panel, were studied. Massive sequencing was performed throughout the investigation and the main CFTR databases were used for analysis. RESULTS: The second pathogenic allele was identified in 16 of 39 patients of this study (41%), finding eleven different mutations that had not been reported in our population. We believe that the reason is that one of the variants had not been previously described. CONCLUSIONS: Mutations that had been described mainly in Hispanic and/or Mediterranean populations were identified. We found a variation that had not been previously reported, but not enough recurrent mutations that could explain the low rate of detection were found. Knowledge about mutations can provide appropriate genetic counseling and will be critical to evaluate the potential use of new targeted therapies for treating them.

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24 Otras mutaciones llamadas "comunes" a nivel mundial, es decir con frecuencias > 1%, incluyen p.G542X, p.G551D, p.N1303K y p.W1282X. Login to comment

[hide] Benign outcome among positive cystic fibrosis newb... J Cyst Fibros. 2015 Nov;14(6):714-9. doi: 10.1016/j.jcf.2015.03.006. Epub 2015 Mar 29. Salinas DB, Sosnay PR, Azen C, Young S, Raraigh KS, Keens TG, Kharrazi M
Benign outcome among positive cystic fibrosis newborn screen children with non-CF-causing variants.
J Cyst Fibros. 2015 Nov;14(6):714-9. doi: 10.1016/j.jcf.2015.03.006. Epub 2015 Mar 29., [PMID:25824995]

Abstract [show]
BACKGROUND: The Clinical and Functional Translation of CFTR project (CFTR2) classified some cystic fibrosis transmembrane conductance regulator (CFTR) gene variants as non-cystic fibrosis (CF)-causing. To evaluate this, the clinical status of children carrying these mutations was examined. METHODS: We analyzed CF disease-defining variables over 2-6 years in two groups of California CF screen- positive neonates born from 2007 to 2011: (1) children with two CF-causing variants and (2) children with one CF-causing and one non-CF-causing variant, as defined by CFTR2. RESULTS: Children carrying non-CF-causing variants had significantly higher birth weight, lower immunoreactive trypsinogen and sweat chloride values, higher first year growth curves, and a lower rate of persistent Pseudomonas aeruginosa colonization compared to children with two CF-causing variants. CONCLUSIONS: The outcomes in children 2-6 years of age with the L997F, G576A, R1162L, V754M, R668C, R31C, and S1235R variants are consistent with the CFTR2 non-CF-causing classification.

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55 There were no sweat chloride results meeting CF diagnostic criteria of ࣙ60 mmol/L in the N-CF group, and only one subject with three variants (G542X, G576A, R668C) had values in the "possible CF" category (ࣙ40 mmol/L) beyond 6 months. Login to comment

[hide] Mutation analysis of PRSS1, SPINK1 and CFTR gene i... Turk J Gastroenterol. 2015 Mar;26(2):176-80. doi: 10.5152/tjg.2015.4287. Sisman G, Tugcu M, Ayla K, Sebati O, Senturk H
Mutation analysis of PRSS1, SPINK1 and CFTR gene in patients with alcoholic and idiopathic chronic pancreatitis: A single center study.
Turk J Gastroenterol. 2015 Mar;26(2):176-80. doi: 10.5152/tjg.2015.4287., [PMID:25835118]

Abstract [show]
BACKGROUND/AIMS: A relation between some genetic mutations and chronic pancreatitis (CP) has been reported. However, the relation of genetic mutation to alcoholic CP (ACP) and idiopathic CP (ICP) still remains controversial. In this study, we investigated the prevalence of protease serine 1 (PRSS1), serine protease inhibitor, Kazal type 1 (SPINK1) SPINK1 and cystic fibrosis transmembrane conductance regulator (CFTR) mutations in ACP and ICP patients in Turkey. MATERIALS AND METHODS: Forty-one patients with ACP and 38 patients with ICP were enrolled, and 35 healthy individuals served as controls. The PRSS1 and SPINK1 mutations were investigated by the polymerase chain reaction (PCR)-restriction fragment-length polymorphism (RFLP) technique. The CFTR mutation was examined with PCR direct sequencing. RESULTS: The mean ages of the ACP, ICP and healthy control groups were 53.2, 40.4 and 46.3 years, respectively. A CFTR F508 mutation was detected as a heterozygote in one (2.4%) patient with ACP. In the ICP and control populations, PRSS1, SPINK1 and CFTR mutations were not detected. CONCLUSION: This study shows that PRSS1, SPINK1 and CFTR mutations do not play a role in ACP and ICP patients.

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45 DNA samples were multiplied by multiplex PCR with a CF 22Mut and CF 14Mut+Tn strip assay kit which has 36 common mutations of the CFTR gene (DF508, DI507, F508C, I502T, 1706del17, 1677del TA, G542X, 1717-1G>A, R553X, Q552X, G551D, S549R(A>C), N1303K, 4016insT, R1162X, R1158X, W1282X, G1244E, 2789+5G>A, 2183AA>G, 711+5G>A, 711+1G>T, G85E, 3849+10kbC>T, 621+1G>T, R117H, D1152H, L1065P, R1066H, L1077P, 4382delA, 1259insA, 852del22, R347P, T338I, S912X and Allele5T-7T-9T). Login to comment

[hide] Clinical diagnostic Next-Generation sequencing: th... Scand J Clin Lab Invest. 2015 Sep;75(5):374-81. doi: 10.3109/00365513.2015.1031689. Epub 2015 Apr 15. Loukas YL, Thodi G, Molou E, Georgiou V, Dotsikas Y, Schulpis KH
Clinical diagnostic Next-Generation sequencing: the case of CFTR carrier screening.
Scand J Clin Lab Invest. 2015 Sep;75(5):374-81. doi: 10.3109/00365513.2015.1031689. Epub 2015 Apr 15., [PMID:25874479]

Abstract [show]
A 23-mutation panel for CFTR carrier screening is recommended to women of reproductive age by the American College of Obstetricians and Gynecologists. In the present study the optimized efficiency regarding the carrier rate of Next-Generation sequencing (NGS) technology is compared to the one of limited mutation detection panels. A total of 824 consequent cases were subjected to the commercial Cystic Fibrosis Genotyping Assay. Some 188 negative samples randomly selected from the initial group of probands were further subjected to an extended mutation panel characterized by 92% detection rate, as well as to massive parallel sequencing. Twenty-two probands subjected to the commercial assay proved to carry one mutation included in the ACOG panel (carrier rate 0.0267). The latter panels revealed the presence of mutations not included in the ACOG panel in four probands, resulting to an increase of carrier rate of 0.0106 in the case of in-house panel and an increase of rate of 0.0213 if NGS was used. The above data seem to support the implementation of NGS in the routine CFTR carrier screening.

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36 Sample code Source Genotype NA18668*2 Coriell Cell Repositories* CFTR, CFdelex2,3/p.F508del NA07830 Coriell Cell Repositories* CFTR, F508del/556delA NA11275 Coriell Cell Repositories* CFTR, 3659delC/F508del NA11277 Coriell Cell Repositories* CFTR, I507del/wt NA11860 Coriell Cell Repositories* CFTR, 3849af9;10kb,Cb0e;T/3849af9;10kb,Cb0e;T 40C2 CDC** CFTR, F508del/R334W 10C4 CDC** CFTR, 2184delA/394delTT CDC2 CDC** CFTR, F508del/Exon 17&#aa;-17b-18del 212C4 CDC** CFTR, F508del/3659delC 412C2 CDC** CFTR, F508del/R334W 213C4 CDC** CFTR, W1282X/W1282X 21C2 CDC** CFTR, 1717-1Gb0e;A/1154insTC 412C5 CDC** CFTR, F508del/2183AAb0e;G 412C1 CDC** CFTR, 2184delA/394delTT 212C5 CDC** CFTR, F508del/3849af9;10KbCb0e;T 38C4 CDC** CFTR, R553X/wt 48C1 CDC** CFTR, F508del/G542X 48C3 CDC** CFTR, F508del/G551D 19C4 CDC** CFTR, F508del/R560T 19C5 CDC** CFTR, G551D/G551D 29C3 CDC** CFTR, 621af9;1Gb0e;T/N1303K 29C5 CDC** CFTR, F508del/2789af9;5Gb0e;A 49C1 CDC** CFTR, 3120af9;1Gb0e;A/L467P# 49C3 CDC** CFTR, 621af9;1Gb0e;T/R1162X 40C5 CDC** CFTR, 711af9;1Gb0e;T/wt 21C1 CDC** CFTR, A455E/F508del 112C2 CDC** CFTR, 1898af9;1Gb0e;A/F508del 214C5 CDC** CFTR, F508del/3140-26Ab0e;G *http://ccr.coriell.org/; **CDC, Center for Disease Control & Prevention, http://www.cdc.gov/; #According to CDC report, its clinical significance is unknown. Login to comment
94 Mutation cDNA Coverage Score Reference allele (F/R strand) Mutant allele (F/R strand) Genotype F508del* c.1521_1523delCTT 2080 26.5 491/557 523/504 HET 556delA c.424delA 2168 26.7 524/557 547/536 HET 3659delC* c.3528delC 2359 27.0 573/605 566/609 HET I507del c.1519_1521delATC 2246 26.8 508/612 619/501 HET 3849af9;10kb,Cb0e;T c.3717af9;12191Cb0e;T 3596 28.4 - 1834/1756 HOM 3849af9;10kb,Cb0e;T c.3717af9;12191Cb0e;T 4169 29.0 1023/1059 1116/967 HET R334W* c.1000Cb0e;T 2473 27.1 636/599 626/609 HET 2184delA* c.2052delA 3069 27.9 734/801 792/738 HET 394delTT* c.262_263delTT 3176 28.0 775/811 819/766 HET W1282X c.3846Gb0e;A 4268 29.0 - 2168/2096 HOM 1717-1Gb0e;A c.1585-1Gb0e;A 3863 28.7 922/1007 985/944 HET 1154insTC c.1022_1023insTC 4021 28.8 1058/1039 979/941 HET 2183AAb0e;G c.2051_2052delAAinsG 3927 28.8 1023/996 974/926 HET R553X c.1657Cb0e;T 6027 30.2 1532/1480 1476/1534 HET G542X c.1624Gb0e;T 3862 28.7 933/996 925/1002 HET G551D c.1652Gb0e;A 5225 29.7 1257/1351 1341/1268 HET G551D c.1652Gb0e;A 4862 29.5 - 2487/2369 HOM R560T c.1679Gb0e;C 3542 28.4 861/908 915/853 HET 621af9;1Gb0e;T* c.489af9;1Gb0e;T 2256 26.8 534/592 606/519 HET N1303K c.3909Cb0e;G 2126 26.6 534/528 492/568 HET 2789af9;5Gb0e;A c.2657af9;5Gb0e;A 3453 28.3 824/901 895/828 HET 3120af9;1Gb0e;A c.2988af9;1Gb0e;A 3021 27.8 721/787 802/707 HET L467P c.1400Cb0e;T 3848 28.7 928/993 1003/920 HET R1162X c.3484Cb0e;T 4180 29.0 1021/1065 1112/976 HET 711af9;1Gb0e;T c.579af9;1Gb0e;T 4222 29.0 1036/1072 1001/1108 HET A455E c.1364Cb0e;A 5621 30.0 1365/1443 1438/1370 HET 1898af9;1Gb0e;A c.1766af9;1Gb0e;A 2934 27.7 683/782 702/762 HET 3272-26Ab0e;G 3140-26Ab0e;G 3755 28.6 902/973 1008/867 HET of the majority of CFTR mutations carriers in our region. Login to comment

[hide] A Genotypic-Oriented View of CFTR Genetics Highlig... Mol Med. 2015 Apr 21;21:257-75. doi: 10.2119/molmed.2014.00229. Lucarelli M, Bruno SM, Pierandrei S, Ferraguti G, Stamato A, Narzi F, Amato A, Cimino G, Bertasi S, Quattrucci S, Strom R
A Genotypic-Oriented View of CFTR Genetics Highlights Specific Mutational Patterns Underlying Clinical Macrocategories of Cystic Fibrosis.
Mol Med. 2015 Apr 21;21:257-75. doi: 10.2119/molmed.2014.00229., [PMID:25910067]

Abstract [show]
Cystic fibrosis (CF) is a monogenic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The genotype-phenotype relationship in this disease is still unclear, and diagnostic, prognostic and therapeutic challenges persist. We enrolled 610 patients with different forms of CF and studied them from a clinical, biochemical, microbiological and genetic point of view. Overall, there were 125 different mutated alleles (11 with novel mutations and 10 with complex mutations) and 225 genotypes. A strong correlation between mutational patterns at the genotypic level and phenotypic macrocategories emerged. This specificity appears to largely depend on rare and individual mutations, as well as on the varying prevalence of common alleles in different clinical macrocategories. However, 19 genotypes appeared to underlie different clinical forms of the disease. The dissection of the pathway from the CFTR mutated genotype to the clinical phenotype allowed to identify at least two components of the variability usually found in the genotype-phenotype relationship. One component seems to depend on the genetic variation of CFTR, the other component on the cumulative effect of variations in other genes and cellular pathways independent from CFTR. The experimental dissection of the overall biological CFTR pathway appears to be a powerful approach for a better comprehension of the genotype-phenotype relationship. However, a change from an allele-oriented to a genotypic-oriented view of CFTR genetics is mandatory, as well as a better assessment of sources of variability within the CFTR pathway.

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167 The W19X(TAG) (p.Trp19*) mutation was found in a CF-PI male patient with a G542X/W19X(TAG) (p. Login to comment
191 [Trp1282*];[Thr465Asn] 5 G542X/W19X(TAG) c. Login to comment
276 These patients had the following mutations on the other allele: F508del (p.Phe508del) (1 CF-PI), G542X (p.Gly542*) (1 CF-PI), 2789+5G>A (c.2657+5G>A) (1 CF-PS). Login to comment
318 These patients had the following mutations on the other allele: F508del (p.Phe508del) (3 CF-PS, 5 CFTR-RD and 10 CBAVD), N1303K (p.Asn1303Lys) (1 CF-PS, 3 CFTR-RD and 1 CBAVD), 1717-1G>A (c.1585-1G>A) (3 CF-PS and 1 CFTR-RD), W1282X (p.Trp1282*) (3 CFTR-RD), G542X (p.Gly542*) (1 CF-PS, 1 CFTR-RD and 1 CBAVD), Y849X (p.Tyr849*) (1 CFTR-RD), 3849+10kbC>T (c.3717+12191C>T) (1 CFTR-RD), R1162X (p.Arg1162*) (1 CBAVD), S549R(A>C) (p.Ser549Arg) (1 CFTR-RD) and unknown (1 CF-PS and 3 CBAVD). Login to comment
381 [Glu479*;Val754Met] F508del c.1521_1523delCTT CF-PI CF-causing p.Phe508del 1717-8G>A c.1585-8G>A CF-PI CF-causing 1717-1G>A c.1585-1G>A CF-PI CF-causing D529N c.1585G>A CF-PI nd p.Asp529Asn G542X c.1624G>T CF-PI CF-causing p.Gly542* S549R(A>C) c.1645A>C CF-PI CF-causing p.Ser549Arg S549N c.1646G>A CF-PI CF-causing p.Ser549Asn S549R(T>G) c.1647T>G CF-PI CF-causing p.Ser549Arg G551D c.1652G>A CF-PI CF-causing p.Gly551Asp Q552X c.1654C>T CF-PI CF-causing p.Gln552* R553X c.1657C>T CF-PI CF-causing p.Arg553* L558S c.1673T>C CF-PI unknown significance p.Leu558Ser Y569D c.1705T>G CFTR-RD,CBAVD unknown significance p.Tyr569Asp Continued on next page 2 0 | L U C A R E L L I E T A L . Login to comment

[hide] Translating the genetics of cystic fibrosis to per... Transl Res. 2015 Apr 15. pii: S1931-5244(15)00131-0. doi: 10.1016/j.trsl.2015.04.008. Corvol H, Thompson KE, Tabary O, le Rouzic P, Guillot L
Translating the genetics of cystic fibrosis to personalized medicine.
Transl Res. 2015 Apr 15. pii: S1931-5244(15)00131-0. doi: 10.1016/j.trsl.2015.04.008., [PMID:25940043]

Abstract [show]
Cystic fibrosis (CF) is the most common life-threatening recessive genetic disease in the Caucasian population. This multiorgan disease is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, a chloride channel recognized as regulating several apical ion channels. The gene mutations result either in the lack of the protein at the apical surface or in an improperly functioning protein. Morbidity and mortality because of the mutation of CFTR are mainly attributable to lung disease resulting from chronic infection and inflammation. Since its discovery as the causative gene in 1989, much progress has been achieved not only in clinical genetics but also in basic science studies. Recently, combinations of these efforts have been successfully translated into development and availability for patients of new therapies targeting specific CFTR mutations to correct the CFTR at the protein level. Current technologies such as next gene sequencing and novel genomic editing tools may offer new strategies to identify new CFTR variants and modifier genes, and to correct CFTR to pursue personalized medicine, which is already developed in some patient subsets. Personalized medicine or P4 medicine ("personalized," "predictive," "preventive," and "participatory") is currently booming for CF. The various current and future challenges of personalized medicine as they apply to the issues faced in CF are discussed in this review.

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48 T p.Gly542X (G542X) 2.6% c.1521_1523del p.Phe508del (F508del) 70% c.1652G . Login to comment

[hide] Inconclusive diagnosis of cystic fibrosis after ne... Pediatrics. 2015 Jun;135(6):e1377-85. doi: 10.1542/peds.2014-2081. Epub 2015 May 11. Ooi CY, Castellani C, Keenan K, Avolio J, Volpi S, Boland M, Kovesi T, Bjornson C, Chilvers MA, Morgan L, van Wylick R, Kent S, Price A, Solomon M, Tam K, Taylor L, Malitt KA, Ratjen F, Durie PR, Gonska T
Inconclusive diagnosis of cystic fibrosis after newborn screening.
Pediatrics. 2015 Jun;135(6):e1377-85. doi: 10.1542/peds.2014-2081. Epub 2015 May 11., [PMID:25963003]

Abstract [show]
OBJECTIVES: To prospectively study infants with an inconclusive diagnosis of cystic fibrosis (CF) identified by newborn screening (NBS; "CF screen positive, inconclusive diagnosis" [CFSPID]) for disease manifestations. METHODS: Infants with CFSPID and CF based on NBS from 8 CF centers were prospectively evaluated and monitored. Genotype, phenotype, repeat sweat test, serum trypsinogen, and microbiology data were compared between subjects with CF and CFSPID and between subjects with CFSPID who did (CFSPID-->CF) and did not (CFSPID-->CFSPID) fulfill the criteria for CF during the first 3 years of life. RESULTS: Eighty-two subjects with CFSPID and 80 subjects with CF were enrolled. The ratio of CFSPID to CF ranged from 1:1.4 to 1:2.9 in different centers. CFTR mutation rates did not differ between groups; 96% of subjects with CFSPID and 93% of subjects with CF had 2 mutations. Subjects with CFSPID had significantly lower NBS immunoreactive trypsinogen (median [interquartile range]:77 [61-106] vs 144 [105-199] mug/L; P < .0001) than did subjects with CF. Pseudomonas aeruginosa and Stenotrophomonas maltophilia were isolated in 12% and 5%, respectively, of subjects with CFSPID. CF was diagnosed in 9 of 82 (11%) subjects with CFSPID (genotype and abnormal sweat chloride = 3; genotype alone = 4; abnormal sweat chloride only = 2). Sweat chloride was abnormal in CFSPID-->CF patients at a mean (SD) age of 21.3 (13.8) months. CFSPID-->CF patients had significantly higher serial sweat chloride (P < .0001) and serum trypsinogen (P = .009) levels than did CFSPID-->CFSPID patients. CONCLUSIONS: A proportion of infants with CFSPID will be diagnosed with CF within the first 3 years. These findings underscore the need for clinical monitoring, repeat sweat testing at age 2 to 3 years, and extensive genotyping.

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108 TABLE 3 Characteristics of Subjects With CFSPID Who Later Met Diagnostic Criteria of CF Subject Number Allele 1 Allele 2 Ethnicity NBS IRT, mg/L Initial Sweat Chloride, mmol/L Highest Sweat Chloride, mmol/L Country 1 F508del R117C White 105.8 36 61 Canada 2 F508del S1455X White 66.6 46 74 Canada 3 F508del P67L White 151.2 38 38 Canada 4 F508del L206W White 83.8 58 64 Canada 5 G542X L206W White 67 49 66 Canada 6 F508del L206W White 59.9 45 45 Canada 7 R1162X R117H-7T White 126 36 70 Italy 8 2183AA.G R117C White 129 32 32 Italy 9 F508del R117C White 80.4 48 56 Canada e OOI et al including in newborn-screened infants with equivocal CF diagnosis and in older individuals with single-organ manifestations of CF.17,18,20-22 As in the case of the 7 subjects who were initially classified as CFSPID but who were subsequently recognized to carry 2 disease-causing mutations on the basis of the CFTR2 project, the diagnostic consequences (benign versus disease-causing) of the CFTR mutations identified in all of the other subjects with CFSPID may not be apparent until later on, when new genetic information becomes available and classification of CFTR mutations currently considered to be of "unknown" consequences is updated. Login to comment

[hide] Prenatal diagnosis of cystic fibrosis: 10-years ex... Pathol Biol (Paris). 2015 Jun;63(3):126-9. doi: 10.1016/j.patbio.2015.04.002. Epub 2015 May 20. Hadj Fredj S, Ouali F, Siala H, Bibi A, Othmani R, Dakhlaoui B, Zouari F, Messaoud T
Prenatal diagnosis of cystic fibrosis: 10-years experience.
Pathol Biol (Paris). 2015 Jun;63(3):126-9. doi: 10.1016/j.patbio.2015.04.002. Epub 2015 May 20., [PMID:26002249]

Abstract [show]
PURPOSE: We present in this study our 10years experience in prenatal diagnosis of cystic fibrosis performed in the Tunisian population. PATIENTS AND METHODS: Based on family history, 40 Tunisian couples were selected for prenatal diagnosis. Fetal DNA was isolated from amniotic fluid collected by transabdominal amniocentesis or from chronic villi by transcervical chorionic villus sampling. The genetic analysis for cystic fibrosis mutations was performed by denaturant gradient gel electrophoresis and denaturing high-pressure liquid phase chromatography. We performed microsatellites analysis by capillary electrophoresis in order to verify the absence of maternal cell contamination. RESULTS: Thirteen fetuses were affected, 21 were heterozygous carriers and 15 were healthy with two normal alleles of CFTR gene. Ten couples opted for therapeutic abortion. The microsatellites genotyping showed the absence of contamination of the fetal DNA by maternal DNA in 93.75%. CONCLUSION: Our diagnostic strategy provides rapid and reliable prenatal diagnosis at risk families of cystic fibrosis.

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77 Ten different CFTR mutations were identified, including F508del (51.28%), E1104X (12.82%), N1303K (8.97%), G542X (8.97%), 711 + 1 G!T (6.41%), W1282X (5.12 %), R785X (1.28 %) and V754M (1.28%). Login to comment
91 Fetus genotype Number Percentage (%) F508del/- 14 28.57 F508del/F508del 6 12.24 E1104X/- 3 6.12 N1303K/- 3 6.12 E1104X/N1303K 2 4.08 F508del/711 + 1 G!T 1 2.04 E1104X/E1104X 1 2.04 W1282X/W1282X 1 2.04 711 + 1 G!T/711 + 1 G!T 1 2.04 4268 + 2T!G/4268 + 2T!G 1 2.04 G542X/- 1 2.04 -/- 15 30.61 ''-``: absence of mutation. Login to comment

[hide] The improvement of the best practice guidelines fo... Eur J Hum Genet. 2015 May 27. doi: 10.1038/ejhg.2015.99. Girardet A, Viart V, Plaza S, Daina G, De Rycke M, Des Georges M, Fiorentino F, Harton G, Ishmukhametova A, Navarro J, Raynal C, Renwick P, Saguet F, Schwarz M, SenGupta S, Tzetis M, Roux AF, Claustres M
The improvement of the best practice guidelines for preimplantation genetic diagnosis of cystic fibrosis: toward an international consensus.
Eur J Hum Genet. 2015 May 27. doi: 10.1038/ejhg.2015.99., [PMID:26014425]

Abstract [show]
Cystic fibrosis (CF) is one of the most common indications for preimplantation genetic diagnosis (PGD) for single gene disorders, giving couples the opportunity to conceive unaffected children without having to consider termination of pregnancy. However, there are no available standardized protocols, so that each center has to develop its own diagnostic strategies and procedures. Furthermore, reproductive decisions are complicated by the diversity of disease-causing variants in the CFTR (cystic fibrosis transmembrane conductance regulator) gene and the complexity of correlations between genotypes and associated phenotypes, so that attitudes and practices toward the risks for future offspring can vary greatly between countries. On behalf of the EuroGentest Network, eighteen experts in PGD and/or molecular diagnosis of CF from seven countries attended a workshop held in Montpellier, France, on 14 December 2011. Building on the best practice guidelines for amplification-based PGD established by ESHRE (European Society of Human Reproduction and Embryology), the goal of this meeting was to formulate specific guidelines for CF-PGD in order to contribute to a better harmonization of practices across Europe. Different topics were covered including variant nomenclature, inclusion criteria, genetic counseling, PGD strategy and reporting of results. The recommendations are summarized here, and updated information on the clinical significance of CFTR variants and associated phenotypes is presented.European Journal of Human Genetics advance online publication, 27 May 2015; doi:10.1038/ejhg.2015.99.

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78 Table 1 Examples of common CF-causing, indetermined, and non CF-causing variants (modified from5,8,17) HGVS nomenclature Legacy name cDNA nucleotide name Protein name CF-causing variantsa F508del c.1521_1523delCTT p.Phe508del G542X c.1624G4T p.Gly542* G551D c.1652G4A p.Gly551Asp N1303K c.3909C4G p.Asn1303Lys W1282X c.3846G4A p.Trp1282* 621+1G4T c.489+1G4T CFTRdele2,3 c.54-5940_273 +10250del21080 p.Ser18Argfs*16 E60X c.178G4T p.Glu60* G85E c.254G4A p.Gly85Glu 394delTT c.262_263delTT p.Leu88Ilefs*22 711+1G4T c.579+1G4T R347P c.1040G4C p.Arg347Pro A455E c.1364C4A p.Ala455Glu Q493X c.1477C4T p.Gln493* I507del c.1519_1521delATC p.Ile507del R553X c.1657C4T p.Arg553* R560T c.1679G4C p.Arg560Thr 1898+1G4A c.1766+1G4A 2183AA4G c.2051_2052delAAinsG p.Lys684Serfs*38 2789+5G4A c.2657+5G4A 3120+1G4A c.2988+1G4A M1101K c.3302 T4A p.Met1101Lys R1162X c.3484C4T p.Arg1162* 3659delC c.3528delC p.Lys1177Serfs*15 M1V c.1 A4G p.? Login to comment

[hide] [Challenges of personalized medicine for cystic fi... Arch Pediatr. 2015 Jul;22(7):778-86. doi: 10.1016/j.arcped.2015.04.015. Epub 2015 May 26. Corvol H, Taytard J, Tabary O, Le Rouzic P, Guillot L, Clement A
[Challenges of personalized medicine for cystic fibrosis].
Arch Pediatr. 2015 Jul;22(7):778-86. doi: 10.1016/j.arcped.2015.04.015. Epub 2015 May 26., [PMID:26021452]

Abstract [show]
Personalized medicine, or P4 medicine for "Personalized", "Predictive", "Preventive" and "Participatory", is currently booming for cystic fibrosis, with the development of therapies targeting specific CFTR mutations. The various challenges of personalized medicine applied to cystic fibrosis issues are discussed in this paper.

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36 Cette classe inclut les mutations non-sens et celles qui produisent un codon stop pre &#b4;mature &#b4;, comme la mutation G542X ; classe II : mutations perturbant le processus de &#ab; maturation &#bb; cellulaire de la prote &#b4;ine et son ciblage vers la membrane plasmique. La prote &#b4;ine mutante n`est pas adresse &#b4;e au bon endroit et reste en ge &#b4;ne &#b4;ral localise &#b4;e dans le cytoplasme ou ` elle est de &#b4;grade &#b4;e. Login to comment
273 Aminoglycoside suppression of a premature stop mutation in a Cftr/ mouse carrying a human CFTR-G542X transgene. Login to comment
283 [28] Du M, Liu X, Welch EM, et al. PTC124 is an orally bioavailable compound that promotes suppression of the human CFTR-G542X nonsense allele in a CF mouse model. Login to comment

[hide] Prevalence of meconium ileus marks the severity of... Genet Med. 2015 Jun 18. doi: 10.1038/gim.2015.79. Dupuis A, Keenan K, Ooi CY, Dorfman R, Sontag MK, Naehrlich L, Castellani C, Strug LJ, Rommens JM, Gonska T
Prevalence of meconium ileus marks the severity of mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene.
Genet Med. 2015 Jun 18. doi: 10.1038/gim.2015.79., [PMID:26087176]

Abstract [show]
RATIONALE: Meconium ileus (MI) is a perinatal complication in cystic fibrosis (CF), which is only minimally influenced by environmental factors. We derived and examined MI prevalence (MIP) scores to assess CFTR phenotype-phenotype correlation for severe mutations. METHOD: MIP scores were established using a Canadian CF population (n = 2,492) as estimates of the proportion of patients with MI among all patients carrying the same CFTR mutation, focusing on patients with p.F508del as the second allele. Comparisons were made to the registries from the US CF Foundation (n = 43,432), Italy (Veneto/Trentino/Alto Adige regions) (n = 1,788), and Germany (n = 3,596). RESULTS: The prevalence of MI varied among the different registries (13-21%). MI was predominantly prevalent in patients with pancreatic insufficiency carrying "severe" CFTR mutations. In this severe spectrum MIP scores further distinguished between mutation types, for example, G542X (0.31) with a high, F508del (0.22) with a moderate, and G551D (0.08) with a low MIP score. Higher MIP scores were associated with more severe clinical phenotypes, such as a lower forced expiratory volume in 1 second (P = 0.01) and body mass index z score (P = 0.04). CONCLUSIONS: MIP scores can be used to rank CFTR mutations according to their clinical severity and provide a means to expand delineation of CF phenotypes.Genet Med advance online publication 18 June 2015Genetics in Medicine (2015); doi:10.1038/gim.2015.79.

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11 In this severe spectrum MIP scores further distinguished between mutation types, for example, G542X (0.31) with a high, F508del (0.22) with a moderate, and G551D (0.08) with a low MIP score. Login to comment
63 Canadian studies for CF modfier genes 2,492 3,153 43,432 3,596 1,788 2,230 23,397 16,023 3 716 3,438 860 15% (19%) 1,902 2,576 PIP and MIP derivation FEV1 and zBMI modeling MIP calculation following correction of MI variable 23,301 2,413 510 21% (25%) 20% (23%) 13% (15%) Total F508del/others MI prevalence uncorrected (estimated) Missing or incomplete genotype Available for analysis Canadian CF patient registry, born after 1980 US CF patient registry German CF patient registry CF patient registry, North Italy Table 1ߒ Meconium ileus prevalence scores for the most common cystic fibrosis-causing variants p. F508del/other variants Class PIP Canada, (n) MIP, (n) Canada United States Germany Italy HGVS Legacy name c.262_263delTT 394delTT I 0.38 (50) c.3472C>T R1158X I 0.37 (35) c.1558G>T V520F 0.35 (43) c.3484C>T R1162X I 0.34 (135) 0.17 (14) 0.22 (45) c.2012delT 2143delT I 0.33 (13) c.3276C>A or G Y1092X I 0.92 (13) 0.09 (12) 0.33 (55) c.3846G>A W1282X I 1.00 (13) 0.29 (13) 0.32 (442) 0.17 (20) c.1477C>T Q493X I 1.00 (11) 0.19 (11) 0.32 (102) c.3528delC 3659delC I 0.31 (139) c.579ߙ+ߙ1G>T 711ߙ+ߙ1G>T 0.97 (39) 0.30 (38) 0.31 (54) c.178G>T E60X I 0.30 (66) c.1657C>T R553X I 1.00 (16) 0.28 (16) 0.30 (415) 0.24 (107) c.1585-1G>A 1717-1G>A I 1.00 (12) 0.23 (12) 0.29 (367) 0.22 (38) 0.16 (22) c.1766ߙ+ߙ1G>A 1898ߙ+ߙ1G>A 0.29 (139) c.1624G>T G542X I 0.99 (73) 0.31 (72) 0.29 (976) 0.21 (79) 0.22 (33) c.1521_1523delCTT F508del II 0.99 (1292) 0.22 (1260) 0.27 (15391) 0.21 (1910) 0.20 (230) c.1679G>C R560T II 0.27 (123) c.3744delA 3876delA 0.27 (22) c.2128A>T K710X I 0.26 (12) c.1519_1521delATC I507del II 1.00 (20) 0.21 (19) 0.25 (162) c.3909C>G N1303K II 0.98 (40) 0.13 (39) 0.25 (534) 0.23 (80) 0.14 (62) c.489ߙ+ߙ1G>T 621ߙ+ߙ1G>T I 1.00 (90) 0.24 (88) 0.25 (369) 0.21 (11) c.3266G>A W1089X I 0.25 (17) c.1675G>A A559T 0.24 (21) c.988G>T G330X 0.24 (10) c.3773_3774insT 3905insT 0.23 (78) c.2988ߙ+ߙ1G>A 3120ߙ+ߙ1G>A 0.22 (121) c.443T>C I148T;3199del6 1.00 (15) 0.22 (15) c.2052delA 2184delA I 0.21 (89) 0.22 (10) c.2051_2052delAAinsG 2183AA>G 0.20 (73) 0.20 (42) c.948delT 1078delT 0.19 (20) c.1652G>A G551D III 0.96 (54) 0.08 (53) 0.15 (979) 0.09 (84) c.254G>A G85E 0.50 (24) 0.06 (24) 0.14 (137) 0.00 (10) c.3196C>T R1066C 0.14 (42) c.1466C>A S489X 1.00 (14) 0.14 (14) c.3808G>A D1270N 0.13 (19) c.1055G>A R352Q 0.12 (18) c.579ߙ+ߙ5G>A 711ߙ+ߙ5G>A 0.12 (30) c.2175_2176insA 2307insA 0.11 (24) c.349C>T R117C 0.10 (37) c.1040G>C R347P IV 0.18 (11) 0.19 (11) 0.10 (130) 0.02 (56) c.350G>A R117H IV 0.05 (21) 0.00 (21) 0.07 (666) 0.02 (19) c.2657ߙ+ߙ5G>A 2789ߙ+ߙ5G>A V 0.25 (20) 0.00 (20) 0.06 (271) 0.01 (21) c.1040G>A R347H 0.06 (55) c.2988G>A 3120G->A 0.06 (36) c.328G>C D1152H IV 0.06 (124) c.3717ߙ+ߙ12191C>T 3849ߙ+ߙ10kbC>T V 0.07 (14) 0.00 (14) 0.05 (299) 0.01 (42) 0.00 (15) c.1364C>A A455E V 0.16 (45) 0.01 (41) 0.05 (109) c.1000C>T R334W IV 0.18 (11) 0.00 (10) 0.05 (92) c.617T>G L206W 0.06 (18) 0.05 (17) 0.04 (52) c.3302T>A M1101K 0.04 (17) c.200C>T P67L V 0.07 (14) 0.00 (14) Meconium ileus prevalence (MIP) and pancreas insufficiency prevalence (PIP) scores are presented. Login to comment
74 The highest MIP scores were calculated for mutations with premature termination, such as G542X (class I mutation, MIP 0.31), followed by F508del (class II mutation, MIP 0.22); the lowest MIP score was for G551D (class III mutation, MIP 0.08) (P = 0.0009). Login to comment

[hide] Targeting ion channels in cystic fibrosis. J Cyst Fibros. 2015 Sep;14(5):561-70. doi: 10.1016/j.jcf.2015.06.002. Epub 2015 Jun 23. Mall MA, Galietta LJ
Targeting ion channels in cystic fibrosis.
J Cyst Fibros. 2015 Sep;14(5):561-70. doi: 10.1016/j.jcf.2015.06.002. Epub 2015 Jun 23., [PMID:26115565]

Abstract [show]
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause a characteristic defect in epithelial ion transport that plays a central role in the pathogenesis of cystic fibrosis (CF). Hence, pharmacological correction of this ion transport defect by targeting of mutant CFTR, or alternative ion channels that may compensate for CFTR dysfunction, has long been considered as an attractive approach to a causal therapy of this life-limiting disease. The recent introduction of the CFTR potentiator ivacaftor into the therapy of a subgroup of patients with specific CFTR mutations was a major milestone and enormous stimulus for seeking effective ion transport modulators for all patients with CF. In this review, we discuss recent breakthroughs and setbacks with CFTR modulators designed to rescue mutant CFTR including the common mutation F508del. Further, we examine the alternative chloride channels TMEM16A and SLC26A9, as well as the epithelial sodium channel ENaC as alternative targets in CF lung disease, which remains the major cause of morbidity and mortality in patients with CF. Finally, we will focus on the hurdles that still need to be overcome to make effective ion transport modulation therapies available for all patients with CF irrespective of their CFTR genotype.

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607 Class I mutations such as G542X can be targeted with so called read-through agents. Login to comment

[hide] Measurements of Functional Responses in Human Prim... EBioMedicine. 2014 Dec 17;2(2):147-53. doi: 10.1016/j.ebiom.2014.12.005. eCollection 2015 Feb. Awatade NT, Uliyakina I, Farinha CM, Clarke LA, Mendes K, Sole A, Pastor J, Ramos MM, Amaral MD
Measurements of Functional Responses in Human Primary Lung Cells as a Basis for Personalized Therapy for Cystic Fibrosis.
EBioMedicine. 2014 Dec 17;2(2):147-53. doi: 10.1016/j.ebiom.2014.12.005. eCollection 2015 Feb., [PMID:26137539]

Abstract [show]
BACKGROUND: The best investigational drug to treat cystic fibrosis (CF) patients with the most common CF-causing mutation (F508del) is VX-809 (lumacaftor) which recently succeeded in Phase III clinical trial in combination with ivacaftor. This corrector rescues F508del-CFTR from its abnormal intracellular localization to the cell surface, a traffic defect shared by all Class II CFTR mutants. Our goal here is to test the efficacy of lumacaftor in other Class II mutants in primary human bronchial epithelial (HBE) cells derived from CF patients. METHODS: The effect of lumacaftor was investigated in primary HBE cells from non-CF and CF patients with F508del/F508del, A561E/A561E, N1303K/G542X, F508del/G542X and F508del/Y1092X genotypes by measurements of Forskolin plus Genistein-inducible equivalent short-circuit current (Ieq-SC-Fsk + Gen) in perfused open-circuit Ussing chambers. Efficacy of corrector C18 was also assessed on A561E/A561E and F508del/F508del cells. RESULTS: Our data indicate that A561E (when present in both alleles) responds positively to lumacaftor treatment at equivalent efficacy of F508del in primary HBE cells. Similarly, lumacaftor has a positive impact on Y1092X, but not on N1303K. Our data also show that cells with only one copy of F508del-CFTR respond less to VX-809. Moreover, there is great variability in lumacaftor responses among F508del-homozygous cells from different donors. Compound C18 failed to rescue A561E-CFTR but not in F508del-CFTR, thus plausibly it has a different mechanism of action distinct from lumacaftor. CONCLUSIONS: CF patients with A561E (and likely also those with Y1029X) can potentially benefit from lumacaftor. Moreover, the methodology used here exemplifies how ex vivo approaches may apply personalized therapies to CF and possibly other respiratory diseases.

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3 Methods: The effect of lumacaftor was investigated in primary HBE cells from non-CF and CF patients with F508del/F508del, A561E/A561E, N1303K/G542X, F508del/G542X and F508del/Y1092X genotypes by measurements of Forskolin plus Genistein-inducible equivalent short-circuit current (Ieq-SC-Fsk + Gen) in perfused open-circuit Ussing chambers. Login to comment
35 In addition we tested VX-809 in HBE cells bearing 2 nonsense mutations: G542X and Y1092X, both in heterozygosity with F508del. Login to comment
42 Culture Conditions of Primary Human Bronchial Epithelial Cells Human lung tissues from CF donors with the F508del/F508del (2 donors), A561E/A561E, N1303K/G542X, F508del/G542X and F508del/ Y1092X genotypes, were obtained from the Cardio-Thoracic Surgery Department (University Hospital la Fe, Valencia, Spain) after receiving patient's written consent and approval by the hospital Ethics Committee. Login to comment
58 Response to lumacaftor for Class II Mutants Assessed by CFTR-Mediated Chloride Secretion The effects of 24 h-treatment with lumacaftor were assessed here by determining CFTR-mediated Cl-secretion in HBE cells from CF donors with the following genotypes (Fig. 1): wt/wt control (a, b); F508del/F508del-Donor 1 (c, d); F508del/F508del-Donor 2 (e, f); A561E/A561E (Fig. 1g, h) and also on the additional genotypes (Fig. 2): N1303K/G542X (a, b), F508del/G542X (c, d); F508del/ Y1092X (e, f). Login to comment
59 Since G542X is a "null" variant (i.e., generating no protein) results on the latter are representative of the N1303K variant, albeit in a single dose. Login to comment
61 These results show that Fsk + Gen responses of F508del/F508del (2 donors), A561E/A561E F508del/G542X and F508del/Y1092X cells after VX-809/lumacaftor treatment were significantly different from those under DMSO, while that of N1303K/G542X cells was not significantly different (Fig. 3b). Login to comment
69 These data again clearly show a positive effect of VX-809 on HBE cells with genotypes F508del/ F508del (both donors), A561E/A561E, F508del/G542X and F508del/ Y1092X but not on N1303K/G542X cells. Login to comment
71 It is also interesting to note the difference in responses by the F508del/G542X and F508del/Y1092X cells. Login to comment
72 Since F508del/ Y1092X cells (but not F508del/G542X cells) already exhibit levels of Ieq-sc-Fsk or Ieq-sc-Fsk + Gen before VX-809, we assessed Fig. 2. Login to comment
73 Effect of lumacaftor (VX-809) on cAMP-induced Isc-eq in primary cultures of HBE cells from CF patients with different CFTR mutations. Original Ussing chamber (open-circuit) recordings showing transepithelial voltage measurements (Vte) obtained for CF primary airway HBE monolayers with different genotypes: N1303K/G542X (a, b); F508del/G542X (c, d); and F508del/Y1092X (e, f). Login to comment
77 Data, show that the levels of Y1092X-transcripts are higher than those from G542X (Table S3), indicating that Y1092X transcripts are less prone to degradation through nonsense-mediated decay. Login to comment
110 Data presented here also show a positive effect of VX-809 on HBE cells with genotypes F508del/G542X (~4% vs non-CF) and F508del/Y1092X (~7% vs non-CF) but not on N1303K/G542X cells. Login to comment
117 In contrast to the effect on A561E/A561E HBE cells, the magnitude of the response of lumacaftor-treated N1303K/G542X cells was just slightly higher by ~2-fold (both under Fsk and Gen) and not statistically different from that in DMSO-treated cells. Login to comment
123 Secondly, it is possible that the response of a single copy of N1303K (the other CFTR allele is G542X, a "null" variant) may be insufficient to observe an effect similar in magnitude to that of A561E/A561E or F508del/F508del cells. Login to comment
124 Contradicting the latter hypothesis are the positive responses of the F508del/G542X and F508del/Y1092X cells, showing that VX-809 can elicit a detectable effect on a single dose of F508del, in contrast to N1303K. Login to comment
125 Interestingly, the response of F508del/Y1092X cells is almost double to that of F508del/G542X. Login to comment
126 While difference could be due to intrinsic responses of the F508del alleles from each of these donors, it is also plausible that the Y1092X mutation, given its localization towards the C-terminus of the protein, does not totally abolish the production of functional CFTR protein, in contrast to G542X. Login to comment
128 Moreover, the levels of Y1092X-transcripts (Table S3) are higher than those from G542X, again indicating that Y1092X transcripts are less prone to degradation through nonsense-mediated decay. Login to comment

[hide] Predicting CFTR activity with front-runner cystic ... EBioMedicine. 2015 Jan 27;2(2):100-1. doi: 10.1016/j.ebiom.2015.01.013. eCollection 2015 Feb. Becq F
Predicting CFTR activity with front-runner cystic fibrosis drugs.
EBioMedicine. 2015 Jan 27;2(2):100-1. doi: 10.1016/j.ebiom.2015.01.013. eCollection 2015 Feb., [PMID:26137547]

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20 The authors evaluated and compared the efficacy of correctors lumacaftor (VX-809) and its analogue C18 on epithelial cell preparations obtained from donors with different CF genotypes - homozygous for F508del, A561E or heterozygous N1303K/ G542X, F508del/G542X, F508del/Y1092X. Login to comment

[hide] Enhancement of premature stop codon readthrough in... Eur J Med Chem. 2015 Aug 28;101:236-44. doi: 10.1016/j.ejmech.2015.06.038. Epub 2015 Jun 21. Pibiri I, Lentini L, Melfi R, Gallucci G, Pace A, Spinello A, Barone G, Di Leonardo A
Enhancement of premature stop codon readthrough in the CFTR gene by Ataluren (PTC124) derivatives.
Eur J Med Chem. 2015 Aug 28;101:236-44. doi: 10.1016/j.ejmech.2015.06.038. Epub 2015 Jun 21., [PMID:26142488]

Abstract [show]
Premature stop codons are the result of nonsense mutations occurring within the coding sequence of a gene. These mutations lead to the synthesis of a truncated protein and are responsible for several genetic diseases. A potential pharmacological approach to treat these diseases is to promote the translational readthrough of premature stop codons by small molecules aiming to restore the full-length protein. The compound PTC124 (Ataluren) was reported to promote the readthrough of the premature UGA stop codon, although its activity was questioned. The potential interaction of PTC124 with mutated mRNA was recently suggested by molecular dynamics (MD) studies highlighting the importance of H-bonding and stacking pi-pi interactions. To improve the readthrough activity we changed the fluorine number and position in the PTC124 fluoroaryl moiety. The readthrough ability of these PTC124 derivatives was tested in human cells harboring reporter plasmids with premature stop codons in H2BGFP and FLuc genes as well as in cystic fibrosis (CF) IB3.1 cells with a nonsense mutation. Maintaining low toxicity, three of these molecules showed higher efficacy than PTC124 in the readthrough of the UGA premature stop codon and in recovering the expression of the CFTR protein in IB3.1 cells from cystic fibrosis patient. Molecular dynamics simulations performed with mutated CFTR mRNA fragments and active or inactive derivatives are in agreement with the suggested interaction of PTC124 with mRNA.

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17 The most commonly observed mutations include three base pair deletion causing the loss of phenylalanine at position 508 (delta-F508), a missense mutation at position 551 and a nonsense mutation at position 542 (G542X) known as a class I mutation type. Login to comment

[hide] Exogenous and endogenous determinants of vitamin K... Sci Rep. 2015 Jul 10;5:12000. doi: 10.1038/srep12000. Krzyzanowska P, Pogorzelski A, Skorupa W, Moczko J, Grebowiec P, Walkowiak J
Exogenous and endogenous determinants of vitamin K status in cystic fibrosis.
Sci Rep. 2015 Jul 10;5:12000. doi: 10.1038/srep12000., [PMID:26160248]

Abstract [show]
Cystic fibrosis (CF) patients are at high risk for vitamin K deficiency. The effects of vitamin K supplementation are very ambiguous. Therefore, we aimed to define the determinants of vitamin K deficiency in a large cohort of supplemented - 146 (86.9%) and non-supplemented - 22 (13.1%) CF patients. Vitamin K status was assessed using prothrombin inducted by vitamin K absence (PIVKA-II) and undercarboxylated osteocalcin (u-OC). The pathological PIVKA-II concentration (>/= 2 ng/ml) and abnormal percentage of osteocalcin (>/= 20%) were found in 72 (42.8%) and 60 (35.7%) subjects, respectively. We found that liver involvement, diabetes, and glucocorticoid therapy were potential risk factors for vitamin K deficiency. Pathological concentrations of PIVKA-II occurred more frequently in patients with pancreatic insufficiency and those who have two severe mutations in both alleles of the CFTR gene. Pathological percentage of u-OC was found more frequently in adult CF patients and those not receiving vitamin K. However, it seems that there are no good predictive factors of vitamin K deficiency in CF patients in everyday clinical care. Early vitamin K supplementation in CF patients seems to be warranted. It is impossible to clearly determine the supplementation dose. Therefore, constant monitoring of vitamin K status seems to be justified.

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122 The genotypes of the studied patients were as follows: F508del/F508del (nߙ=Èa;ߙ74); F508del/- (nߙ=Èa;ߙ23); F508del/3849ߙ+Èa;ߙ10ߙkbCߙ>Èa;ߙT (nߙ=Èa;ߙ6); F508del/2143delT (nߙ =Èa;ߙ 6); F508del/R553X (nߙ =Èa;ߙ4); F508del/2183AAߙ>Èa;ߙG (nߙ=Èa;ߙ3); F508del/1717-1G>Èa;A (nߙ=Èa;ߙ3); F508del/CFTRdele2,3(21ߙkb) (nߙ=Èa;ߙ3); F508del/3272-26Aߙ>Èa;ߙG (nߙ=Èa;ߙ 2); F508del/N1303K (nߙ =Èa;ߙ2); F508del/4374ߙ+Èa;ߙ1Gߙ>Èa;ߙT (nߙ=Èa;ߙ1); F508del/621ߙ+Èa;ߙ1Gߙ>Èa;ߙT (nߙ=Èa;ߙ 1); F508del/3659delC (nߙ =Èa;ߙ1); F508del/ G1244R (nߙ =Èa;ߙ 1); F508del/G542X (nߙ =Èa;ߙ 1); F508del/R117H (nߙ =Èa;ߙ 1); F508del/R334W (nߙ =Èa;ߙ1); G542X/- (nߙ=Èa;ߙ2); CFTRdele2,3(21ߙkb)/- (nߙ=Èa;ߙ2); CFTRdele2,3(21ߙkb)/CFTRdele2,3(21ߙkb) (nߙ=Èa;ߙ1); 1717-1-Gߙ>Èa;ߙA/ CFTRdele2,3(21ߙkb) (nߙ=Èa;ߙ1); 3849ߙ+Èa;ߙ10ߙkbCߙ>Èa;ߙT/- (nߙ=Èa;ߙ1); 3849ߙ+Èa;ߙ10ߙkbCߙ>Èa;ߙT/1717ߙ-Èa;ߙ1Aߙ>Èa;ߙG (nߙ=Èa;ߙ1); N1303K/- (nߙ=Èa;ߙ1); N1303K/3272-26Aߙ>Èa;ߙG (nߙ=Èa;ߙ1); G542X/R553X (nߙ=Èa;ߙ1); 1524ߙ+Èa;ߙ1Gߙ>Èa;ߙA/E585X (nߙ=Èa;ߙ1); 2183AAߙ>Èa;ߙG/- (nߙ=Èa;ߙ1); 2184insA/622-1Gߙ>Èa;ߙA (nߙ=Èa;ߙ1); 2143delT/R1102X (nߙ=Èa;ߙ1); 3272-26Aߙ>Èa;ߙG/- (nߙ=Èa;ߙ1); 3659delC/- (nߙ=Èa;ߙ1); R347P/R347P (nߙ=Èa;ߙ1); S1196X/Q1382X (nߙ=Èa;ߙ1). Login to comment

[hide] Limited premature termination codon suppression by... J Cyst Fibros. 2015 Aug 5. pii: S1569-1993(15)00169-1. doi: 10.1016/j.jcf.2015.07.007. Zomer-van Ommen DD, Vijftigschild LA, Kruisselbrink E, Vonk AM, Dekkers JF, Janssens HM, de Winter-de Groot KM, van der Ent CK, Beekman JM
Limited premature termination codon suppression by read-through agents in cystic fibrosis intestinal organoids.
J Cyst Fibros. 2015 Aug 5. pii: S1569-1993(15)00169-1. doi: 10.1016/j.jcf.2015.07.007., [PMID:26255232]

Abstract [show]
Premature termination codon read-through drugs offer opportunities for treatment of multiple rare genetic diseases including cystic fibrosis. We here analyzed the read-through efficacy of PTC124 and G418 using human cystic fibrosis intestinal organoids (E60X/4015delATTT, E60X/F508del, G542X/F508del, R1162X/F508del, W1282X/F508del and F508del/F508del). G418-mediated read-through induced only limited CFTR function, but functional restoration of CFTR by PTC124 could not be confirmed. These studies suggest that better read-through agents are needed for robust treatment of nonsense mutations in cystic fibrosis.

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31 Human organoid culture and functional CFTR measurements Crypts were isolated from rectal biopsies of six subjects with cystic fibrosis (E60X/4015delATTT, E60X/F508del, G542X/ F508del, R1162X/F508del, W1282X/F508del and F508del/ F508del) as previously described (20). Login to comment
46 We next assessed read-through by G418 and PTC124 in organoids compound heterozygous for F508del and a nonsense mutation (E60X, G542X, R1162X and W1282X) (Fig. 2A). Login to comment
70 3 of PTC124, and included CFTR mutations that were previously associated with PTC124 read-through, such as G542X (c.1624G- N T) and W1282X (c.3846G- N A) (cftr2.org). Login to comment

[hide] Contribution of M470V variant to cystic fibrosis: ... Pathol Biol (Paris). 2015 Sep;63(4-5):169-74. doi: 10.1016/j.patbio.2015.07.004. Epub 2015 Sep 8. Nefzi M, Hadj Fredj S, Tebib N, Barsaoui S, Boussetta K, Siala H, Messaoud T
Contribution of M470V variant to cystic fibrosis: First study in CF and normal Tunisian population.
Pathol Biol (Paris). 2015 Sep;63(4-5):169-74. doi: 10.1016/j.patbio.2015.07.004. Epub 2015 Sep 8., [PMID:26358851]

Abstract [show]
PURPOSE: Determining the frequency of M470V polymorphism in cystic fibrosis and healthy cohort in Tunisia to establish the contribution of M470V polymorphism in cystic fibrosis variable presentation and course. Additionally, studying the origin of cystic fibrosis transmembrane conductance regulator gene in Tunisian population and its evolution among populations worldwide. PATIENTS AND METHODS: The genotyping of M470V marker was realized by PCR-RFLP technique in 34 unrelated patients and 50 healthy subjects. RESULTS: Statistical difference was found in the genotype and allelic distribution between CF and control groups. Exclusive association between F508del allele and M470 allele was noted. CONCLUSION: This study has contributed to better understanding involvement of the M470V polymorphism in the CF clinical expression in the Tunisian population and has confirmed the utility of this marker in the study of the origin and evolution of the CFTR locus in the human history.

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106 Genotype analysis We identified five CF mutations in our cohort: F508del mutation (63.20%), which is the most frequent mutation N1303K (4.4%), G542X (2.94%), E1104X (2.94%) and 1524 + 5insC (1.47%) utilizing the obtained data from previously carried out molecular study using different techniques (DGGE, DHPLC and sequencing) [7]. Login to comment

[hide] The impact of a national population carrier screen... J Cyst Fibros. 2015 Sep 16. pii: S1569-1993(15)00203-9. doi: 10.1016/j.jcf.2015.08.007. Stafler P, Mei-Zahav M, Wilschanski M, Mussaffi H, Efrati O, Lavie M, Shoseyov D, Cohen-Cymberknoh M, Gur M, Bentur L, Livnat G, Aviram M, Alkrinawi S, Picard E, Prais D, Steuer G, Inbar O, Kerem E, Blau H
The impact of a national population carrier screening program on cystic fibrosis birth rate and age at diagnosis: Implications for newborn screening.
J Cyst Fibros. 2015 Sep 16. pii: S1569-1993(15)00203-9. doi: 10.1016/j.jcf.2015.08.007., [PMID:26386752]

Abstract [show]
BACKGROUND: Population carrier screening (PCS) has been available in Israel since 1999 and universally subsidized since 2008. We sought to evaluate its impact. METHODS: A retrospective review of governmental databanks, the national CF registry and CF centers. RESULTS: CF rate per 100,000 live births has decreased from 14.5 in 1990 to 6 in 2011. From 2004-2011 there were 95 CF births: 22 utilized PCS; 68 (72%) had 2 known CFTR mutations; 37% were pancreatic sufficient. At diagnosis, age was 6 (0-98) months; 53/95 had respiratory symptoms, 41/95 failure to thrive and 19/95 pseudomonas. Thirty-four (36%) were Arabs and 19 (20%) orthodox Jews, compared to 20% and 8% respectively, in the general population. CONCLUSIONS: PCS markedly reduced CF birth rates with a shift towards milder mutations, but was often avoided for cultural reasons. As children regularly have significant disease at diagnosis, we suggest a balanced approach, utilizing both PCS and newborn screening.

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55 Mutation DF508 G542X W1282X N1303K 3849 + 10kbC- N T D1152H 405 + 1G࢐A G85E S549R W1089X 1717 + 1G࢐A I1234Va Y1092Xb 3121-1G N Ab 3120 + 1kbdel8.6 kbc 2183AA N Gc 4010delTATTc The first 14 mutations served as the panel used for Jewish population carrier screening program during the study period. Login to comment

[hide] Bone disease in cystic fibrosis: new pathogenic in... Osteoporos Int. 2015 Oct 2. Jacquot J, Delion M, Gangloff S, Braux J, Velard F
Bone disease in cystic fibrosis: new pathogenic insights opening novel therapies.
Osteoporos Int. 2015 Oct 2., [PMID:26431978]

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Mutations within the gene encoding for the chloride ion channel cystic fibrosis transmembrane conductance regulator (CFTR) results in cystic fibrosis (CF), the most common lethal autosomal recessive genetic disease that causes a number of long-term health problems, as the bone disease. Osteoporosis and increased vertebral fracture risk associated with CF disease are becoming more important as the life expectancy of patients continues to improve. The etiology of low bone density is multifactorial, most probably a combination of inadequate peak bone mass during puberty and increased bone losses in adults. Body mass index, male sex, advanced pulmonary disease, malnutrition and chronic therapies are established additional risk factors for CF-related bone disease (CFBD). Consistently, recent evidence has confirmed that CFTR plays a major role in the osteoprotegerin (OPG) and COX-2 metabolite prostaglandin E2 (PGE2) production, two key regulators in the bone formation and regeneration. Several others mechanisms were also recognized from animal and cell models contributing to malfunctions of osteoblast (cell that form bone) and indirectly of bone-resorpting osteoclasts. Understanding such mechanisms is crucial for the development of therapies in CFBD. Innovative therapeutic approaches using CFTR modulators such as C18 have recently shown in vitro capacity to enhance PGE2 production and normalized the RANKL-to-OPG ratio in human osteoblasts bearing the mutation F508del-CFTR and therefore potential clinical utility in CFBD. This review focuses on the recently identified pathogenic mechanisms leading to CFBD and potential future therapies for treating CFBD.

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79 In human osteoblasts obtained from a 25-year-old CF male with the F508del/ G542X mutation in CFTR, we discovered a defective CFTR-mediated Cl-channel activity and a severe deficit of the production of OPG [59] which is a key regulator in bone density [60]. Login to comment

[hide] Identification and frequencies of cystic fibrosis ... Clin Biochem. 2015 Oct 21. pii: S0009-9120(15)00473-7. doi: 10.1016/j.clinbiochem.2015.10.007. Pepermans X, Mellado S, Chialina S, Wagener M, Gallardo L, Lande H, Bordino W, Baran D, Bours V, Leal T
Identification and frequencies of cystic fibrosis mutations in central Argentina.
Clin Biochem. 2015 Oct 21. pii: S0009-9120(15)00473-7. doi: 10.1016/j.clinbiochem.2015.10.007., [PMID:26500004]

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99 rs name HGVS p. name HGVS c. name Legacy name n (%) Screening panel CFTR1 database CFTR2 database rs199826652 p.Phe508del c.1521_1523delCTT F508del 94 (56.6) Yes Yes CF-causing rs113993959 p.Gly542* c.1624G N T G542X 7 (4.2) Yes Yes CF-causing No p.Asn1303Lys c.3909C N G N1303K 5 (3) Yes Yes CF-causing rs74767530 p.Arg1162* c.3484C N T R1162X 4 (2.4) Yes Yes CF-causing rs75961395 p.Gly85Glu c.254G N A G85E 3 (1.8) Yes Yes CF-causing rs78756941 NA c.489 + 1G N T 621 + 1G N T 3 (1.8) Yes Yes CF-causing rs76713772 NA c.1585-1G N A 1717-1G N A 3 (1.8) Yes Yes CF-causing No p.Lys684Serfs*38 c.2051_2052delAAinsG 2183AA N G 3 (1.8) Yes Yes CF-causing rs397508173 p.Ser4* c.11C N A S4X 2 (1.2) No Yes No rs121909011 p.Arg334Trp c.1000C N T R334W 2 (1.2) Yes Yes CF-causing rs77010898 p.Trp1282* c.3846G N A W1282X 2 (1.2) Yes Yes CF-causing rs397508141 p.Leu34_Gln39del c.100_117delTTGTCAGACATATACCAA 232del18 1 (0.6) No Yes No No p.Leu49Pro c.146 T N C L49P &#a7; 1 (0.6) No No No rs77834169 p.Arg117Cys c.349C N T R117C 1 (0.6) Yes Yes CF-causing No p.Arg117Pro c.350G N C R117P 1 (0.6) No Yes No rs80282562 p.Gly178Arg c.532G N A G178R 1 (0.6) Yes Yes CF-causing rs121908803 p.Pro205Ser c.613C N T P205S 1 (0.6) No Yes CF-causing rs121908752 p.Leu206Trp c.617 T N G L206W 1 (0.6) Yes Yes CF-causing No p.Arg347Pro c.1040G N C R347P 1 (0.6) Yes Yes CF-causing rs397508155 p.Tyr362* c.1086 T N A Y362X 1 (0.6) No Yes No rs74597325 p.Arg553* c.1657C N T R553X 1 (0.6) Yes Yes CF-causing rs1800098 + rs1800100 p.[Gly576Ala(;)Arg668Cys] c. Login to comment

[hide] Delta F508 genotype does not predict disease sever... Pediatrics. 1994 Jan;93(1):114-8. Lester LA, Kraut J, Lloyd-Still J, Karrison T, Mott C, Billstrand C, Lemke A, Ober C
Delta F508 genotype does not predict disease severity in an ethnically diverse cystic fibrosis population.
Pediatrics. 1994 Jan;93(1):114-8., [PMID:7505422]

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OBJECTIVE: As part of a study to determine population-based frequencies of CFTR mutations in an ethnically diverse, midwestern cystic fibrosis (CF) population, clinical histories were studied in 119 CF patients. METHODOLOGY: We sought to examine the association between genotype as characterized by the delta F508 and 11 other commonly occurring mutations and clinical parameters including age at diagnosis, clinical presentation, sweat chloride level, chest roentgenogram score, clinical scores, pulmonary function test results, percent weight for height, and presence of associated CF complications. RESULTS: Age at diagnosis of CF was significantly associated with homozygosity for delta F508 (mean age at diagnosis +/- SE: 1.7 +/- 0.3 years for delta F508/delta F508 vs 3.9 +/- 0.9 years for delta F508/other and other/other; P = .03). No other age-adjusted clinical parameter was significantly associated with delta F508 or any other genotype. CONCLUSION: These data suggest that in this sample of CF patients, delta F508 genotype is not predictive of disease severity. The lack of association between disease severity and genotype in this ethnically diverse sample may reflect the presence of more severe undetected mutations in our sample, or the effects of modifying genes at other, non-CF loci.

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41 CFTR Mutation Analysis All persons were tested for the following mutations: SF508, G542X, G551D, G553X, W1282X, N1303K, 621 +IG-*T, R117H, S549N, 3849+lOkbC-T, l6O9delCA, and R1162X. Login to comment
59 Frequencies of 12 CF With Cystic Fibrosis TR Mutations in 119 Patients Mutation Frequency SF508 0.559 G542X 0.092 G5SID 0.029 R553X 0.004 W1282X 0.012 N1303K 0.021 621 + IG -p 0.012 RII7H 0.004 R1162.X 0.008 3849 + lOkbC T 0.008 S549N 0 l6O9delCA 0 Unidentified 0.248 groups were not significantly different with respect to current age, presence of pancreatic insufficiency, clinical presentation, or associated complications common in CF patients. Login to comment

[hide] Gene therapy for the respiratory manifestations of... Am J Respir Crit Care Med. 1995 Mar;151(3 Pt 2):S75-87. Korst RJ, McElvaney NG, Chu CS, Rosenfeld MA, Mastrangeli A, Hay J, Brody SL, Eissa NT, Danel C, Jaffe HA, et al.
Gene therapy for the respiratory manifestations of cystic fibrosis.
Am J Respir Crit Care Med. 1995 Mar;151(3 Pt 2):S75-87., [PMID:7533609]

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Cystic fibrosis (CF) is caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The major manifestations are on the airway epithelial surface, with purulent mucus, recurrent infections, chronic inflammation, and loss of lung function. Consequent to mutations in both parental genes, airway epithelial cells have insufficient CFTR function. Because this can be corrected in vitro by transfer of the normal CFTR gene into airway epithelial cells, it is reasonable to hypothesize that the respiratory manifestations of CF could be prevented by transfer of the normal human CFTR cDNA to the airway epithelium in vivo. Over the past 6 years, our laboratory has developed a strategy to accomplish this goal using a replication deficient E1-E3- recombinant adenovirus (Ad) serotype 5 vector containing the normal human CFTR cDNA (AdCFTR). Studies with experimental animals demonstrate that with administration of such a vector to the airways, the human CFTR cDNA could be transferred to the airway epithelium, with expression of the human CFTR cDNA for at least 6 weeks. Extensive preclinical studies in vitro and in vivo demonstrated that the risks to humans were sufficiently low to initiate a Phase I trial using the AdCFTR vector to treat the respiratory manifestations of CF in humans. Following approval by the National Heart, Lung, and Blood Institute Institutional Review Board, the National Institutes of Health Biosafety Committee, the National Institutes of Health Recombinant DNA Advisory Committee, and the Food and Drug Administration, we initiated the first human trial of gene therapy for CF on April 17, 1993. The clinical study is still ongoing, with safety and efficacy data being evaluated, but there is clear evidence that it is feasible to transfer and express the normal CFTR cDNA to the airway epithelium in vivo in individuals with CF.

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99 Alleles G551D, S5491, A455E, and G542X account for 10-20% of the non-L\F508 mutations. Login to comment

[hide] Pulmonary function and clinical observations in me... Chest. 1996 Aug;110(2):440-5. Colin AA, Sawyer SM, Mickle JE, Oates RD, Milunsky A, Amos JA
Pulmonary function and clinical observations in men with congenital bilateral absence of the vas deferens.
Chest. 1996 Aug;110(2):440-5., [PMID:8697849]

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Congenital bilateral absence of the vas deferens (CBAVD) was once thought to be a distinct clinical entity, but genetic similarities in men with cystic fibrosis (CF) and CBAVD are described increasingly. We evaluated the clinical status, growth and nutritional state, and respiratory function of 18 men with CBAVD to determine whether these men with different CF transmembrane regulator (CFTR) genotypes may have clinical evidence of mild CF. Following a thorough history and examination, pulmonary function tests, sweat test, and renal ultrasound were performed. Genetic evaluation for 50 known CF mutations, screening for private mutations (single-strand conformational polymorphism and direct sequencing), and assay of the length of the polypyrimidine tract in the splice site acceptor of intron 8 was performed. A history of pulmonary disease was present in three, and an additional man had some features suggestive of malabsorption. Results of general physical examination and anthropomorphic measurements were unremarkable in all patients, with a mean (SD) body mass index of 26 (3). Pulmonary function tests of large and small airway function as well as lung volumes were normal in all except one whose results were consistent with moderate asthma. Five men were compound heterozygotes for CFTR mutations, four of whom had positive sweat tests (sweat chloride > 60 mEq/L). Twelve men were heterozygotes for CFTR mutations while no mutations were identified in one man. Although putative etiologic factors may suggest that men with CBAVD and CFTR mutations could be considered within the spectrum of clinical CF, the authors suggest that in men with CBAVD without any other clinical features of CF, the diagnosis of CF may not be made.

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38 His stool description Table 1.Age, Sweat Sodium and Chloride Concentration and Ratio, and Genotype Subject/Age, yr Symptoms Sweat Na+: mEq/L Sweat Cl mEq/L Na:Cl Ratio CFTR Genotype Polythymidine Splice Variant l*/32 2*/27 3f/30 4/23 5*/22 6*/31 7*/37 8/30 9/41 10/31 11/31 12/38 13/40 14/32 15/31 16/43 17/40 18/27 Asthma Malabsorption Bronchitis Asthma 100 39 53 37 36 36 26 46 42 35 83 74 44 44 44 28 21 106 80 37 51 35 34 34 24 45 43 32 79 75 46 44 40 32 22 0.94 1.05 1.03 1.06 1.05 1.05 1.08 1.00 0.98 1.09 1.05 0.99 0.96 1.00 1.10 0.90 0.95 AF508/R117H AF508/R117H AF508/- AF508/- AF508/- AF508/- AF508/- R117H/- Q493X/- AF508/- AF508/- AF508/R117H G551D/R117H G551D/D1152H AF508/- G542X/- R117H/- -/- 7/9 7/9 5/9 5/9 7/7 7/9 7/9 NT$ 7/7 NT NT 7/9 7/7 7/7 5/9 5/9 7/7 5/9 * Subject 1 and 2 are brothers. Login to comment

[hide] Recent advances in capillary electrophoresis of DN... Biochem Soc Trans. 1997 Feb;25(1):267-73. Righetti PG, Gelfi C
Recent advances in capillary electrophoresis of DNA fragments and PCR products.
Biochem Soc Trans. 1997 Feb;25(1):267-73., [PMID:9056882]

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58 Thus the experimental parameters Biochemical Society Transactions M/M I 270 ' G542X Figure 2 CZE analysis of three mutants [I7 17- IG+A (a), G542X (c) and I784delG (d)] in exon I I of the CFTR gene All conditions were as in Figure I , except that the buffer was 8.9 mM Trisborate. Login to comment
68 Figure 2 shows the analysis of a set of intermediate-low-melting fragments, amplified from patients with cystic fibrosis heterozygous for different mutations in exon 11 of the CFTR gene [1717-1G+A (Figure 2a), G542X (G-T at 1756; Figure 2c) and 1784delG (Figure 2 4 1 with their respective normal control (Figure 2e). Login to comment

[hide] [Male infertility caused by bilateral agenesis of ... Rev Med Interne. 1997;18(2):114-8. Durieu I, Bey-Omar F, Rollet J, Boggio D, Bellon G, Morel Y, Vital Durand D
[Male infertility caused by bilateral agenesis of the vas deferens: a new clinical form of cystic fibrosis?].
Rev Med Interne. 1997;18(2):114-8., [PMID:9092029]

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Congenital bilateral absence of vas deferens causes male excretory infertility and represents 1 to 2% of male infertility. Because of a genotypic similarity with cystic fibrosis, the possible in vitro fertilization with epididymal sperm requires careful genetic counselling. We studied genotype, sweat chloride concentration, respiratory function tests, sinus abnormalities, pancreatic and hepatic functions in 22 subjects with congenital bilateral absence of vas deferens. Among them, four were compound heterozygotus, all of them with the R117H mutation. Ten had a positive sweat test, one of them also being compound heterozygotus. Congenital bilateral absence of vas deferens and double mutation or positive sweat test led to high probable cystic fibrosis diagnosis in 13 subjects. Six subjects were heterozygotus for one cystic fibrosis mutation, criterium which is not sufficient for cystic fibrosis diagnosis; five of them had sinus abnormalities, present in 11 of the 22 subjects. Only three patients had no mutation nor sweat chloride abnormalities. This work confirms the high frequency of cystic fibrosis mutations in males with congenital bilateral absence of vas deferens, with a higher frequency of positive sweat test than in other publications, and a high frequency of sinus abnormalities. This monosymptomatic phenotype of cystic fibrosis suggests new hypotheses for a relationship between genotype and phenotype.

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46 Vingt-deux mutations du gene CFTR ont Cte recher- chtes : les cinq plus frequentes (AF508, G542X, N1303K, 1717-G--A, G85E) et les 17 suivantes : R117H, 556delA, R334W, R347H, R347P, S549N, S5491, S549R, G551D, R553X,R560T,G1244E3,S1255X,W1282X,R1283K,3898 ins C, D1270N. Login to comment
55 Quatorze sujetsCtaienth&&o- zygotespourlesmutationsAF508 (12), G542X (un) et D1270N (un). Login to comment
76 Le testdela sueurest positif chez dix sujets.Six sujetssont heterozygotes pour la mutationAF508 ou G542X et ont un testdela sueurntgatif. Login to comment
120 DF508, G542X and Nl303K are the most common of the 36 mutations screened in the CFTR gene of 427 french CF chromosomes. Login to comment