ABCMdb

ABCC7 p.Gly551Asp

Admin's notes:	Class III (gating defect) Veit et al.
ClinVar:	c.1651G>A , p.Gly551Ser D , Pathogenic c.1652G>A , p.Gly551Asp D , Pathogenic
CF databases:	c.1652G>A , p.Gly551Asp D , CF-causing ; CFTR1: This mutation has been found in six Caucasian CF chromosomes out of 155 eamined for a frequency of 4 %. It has not been found on any Black CF chromosomes. This mutation appears to be associated with a particular ten site haplotype shown on the following pages. We have not detected this mutation on any normal Caucasian chromosomes with similar haplotypes or other haplotypes. c.1651G>A , p.Gly551Ser D , CF-causing ; CFTR1: This mutation can be detected using ASOs: normal 5' GAGTGGAGGTCAACG 3', mutant 5' GAGTGGAAGTCAACG 3' with a final wash at 42 degrees celsius in 40 mM NaHPO4, 1 mM EDTA, 0.5 % SDS for 15 minutes. Two patients were found to be homozygous for this mutation. Their parents are second cousins and each carries the G551S mutation. These patients are remarkable in that they have a mild disease without elevated Na+ levels. One patient had decreased lung function, Pseudomonas infections, chronic pancreatitis, clubbing, and is currently 49 years old. This mutation was not found in 363 non-[delta]F508 CF chromosomes, nor in over 700[delta]F508 chromosomes, nor in a small number of normal chromosomes.
Predicted by SNAP2:	A: D (95%), C: D (95%), D: D (71%), E: D (95%), F: D (95%), H: D (95%), I: D (95%), K: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: N (61%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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III (gating defect) <a href="https://www.ncbi.nlm.nih.gov/pubmed/26823392">Veit et al.</a>
admin on 2016-08-19 15:16:22

Publications
[hide] Function of the ABC signature sequences in the hum... Mol Pharmacol. 2004 Jun;65(6):1536-42. Ren XQ, Furukawa T, Haraguchi M, Sumizawa T, Aoki S, Kobayashi M, Akiyama S
Function of the ABC signature sequences in the human multidrug resistance protein 1.
Mol Pharmacol. 2004 Jun;65(6):1536-42., [PMID:15155846]

Abstract [show]
Human multidrug resistance protein 1 (MRP1) is a membrane ATP-binding cassette transporter that confers multidrug resistance to tumor cells by effluxing intracellular drugs in an ATP-dependent manner. The mechanisms by which transport occurs and by which ATP hydrolysis is coupled to drug transport are not fully elucidated. In particular, the function of the signature sequences in the nucleotide binding domains (NBDs) of MRP1 is unknown. We therefore investigated the effect of mutation of the signature sequences (G771D and G1433D) and of the Walker A motifs (K684M and K1333M) in the NBDs on the 8-azido-[alpha-32P]ATP photolabeling and 8-azido-[alpha-32P]ADP vanadate trapping of MRP1. Both mutations in the Walker A motif almost completely inhibited the labeling of the mutated NBD with 8-azido-[alpha-32P]ATP but not the labeling of the other intact NBD. In contrast, the G771D mutation in the signature sequence of NBD1 enhanced the labeling of NBD1 but slightly decreased the labeling of NBD2. The G1433D mutation in the signature motif of NBD2 enhanced the labeling of NBD2 but did not affect the labeling of NBD1. These effects were all substrate-independent. Photolabeling of NBD2 and a very slight photolableing of NBD1 were detectable under vanadate trapping conditions with 8-azido-[alpha-32P]ATP. Trapping at both NBD1 and NBD2 was almost completely inhibited by K684M and K1333M mutations and by the K684M/K1333M double mutation. The G771D mutation completely inhibited trapping at NBD2 and considerably inhibited trapping at NBD1. However, whereas the G1433D mutation also considerably inhibited trapping at NBD1, it only partially inhibited trapping of NBD2, and the trapping could still be enhanced by leukotriene C4. Our findings suggest that both signature sequences of MRP1 are involved in ATP hydrolysis and must be intact for the ATP hydrolysis and the transport by MRP1.

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31 Likewise, mutation of the CFTR signature sequence at G551D did not change the ATP-binding of purified NBD1 but reduced the ATPase activity (Li et al., 1996; Qu et al., 1997). Login to comment

[hide] Mutation of the aromatic amino acid interacting wi... J Biol Chem. 2004 Nov 19;279(47):48505-12. Epub 2004 Sep 7. Zhao Q, Chang XB
Mutation of the aromatic amino acid interacting with adenine moiety of ATP to a polar residue alters the properties of multidrug resistance protein 1.
J Biol Chem. 2004 Nov 19;279(47):48505-12. Epub 2004 Sep 7., 2004-11-19 [PMID:15355964]

Abstract [show]
Structural analyses of several bacterial ATP-binding cassette (ABC) transporters indicate that an aromatic amino acid residue in a nucleotide-binding domain (NBD) interacts with the adenine ring of the bound ATP and contributes to the ATP binding. Substitution of this aromatic residue with a polar serine residue in bacterial histidine transporter completely abolished both ATP binding and ATP-dependent histidine transport. However, substitution of the aromatic amino acid residue in the human cystic fibrosis transmembrane conductance regulator with a polar cysteine residue did not have any effect on the ATP-dependent chloride channel function of the protein. To determine whether the other eucaryotic ABC transporters use the strategy analogous to that in some bacterial ABC transporters, the aromatic Trp653 residue in NBD1 and the Tyr1302 residue in NBD2 of human multidrug resistance-associated protein 1 (MRP1) was mutated to either a different aromatic residue or a polar cysteine residue. Substitution of the aromatic residue with a different aromatic amino acid, such as W653Y or Y1302W, did not affect ATP-dependent leukotriene C4 (LTC4) transport. In contrast, substitution of the aromatic residue with a polar cysteine residue, such as W653C or Y1302C, decreased the affinity for ATP, resulting in greatly increased Kd values for ATP binding or Km values for ATP in ATP-dependent LTC4 transport. Interestingly, although substitution of the aromatic Trp653 in NBD1 of MRP1 with a polar cysteine residue greatly decreases the affinity for ATP, the ATP-dependent LTC4 transport activities are much higher than that of wild-type MRP1, supporting our hypothesis that the increased release rate of the bound ATP from the mutated NBD1 facilitates the protein to start a new cycle of ATP-dependent solute transport.

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13 Additional evidence comes from the naturally occurring CFTR mutant G551D, a mutation of the Gly551 residue in the ABC signature motif of NBD1 causing cystic fibrosis (21-23). Login to comment

[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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203 G551D is known to alter channel activity without affecting its trafficking, to reduce nucleotide binding and ATPase activity of NBD1 [60,110,111]. Login to comment
207 Genistein chemically complemented the defective function of G551D [113]. Login to comment

[hide] Novel pharmacologic therapies for cystic fibrosis. J Clin Invest. 1999 Feb;103(4):447-52. Zeitlin PL
Novel pharmacologic therapies for cystic fibrosis.
J Clin Invest. 1999 Feb;103(4):447-52., [PMID:10021451]

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30 Mutations, such as G551D, tend to be associated with a severe phenotype. Class IV mutations affect chloride conductance or channel gating and thus result in reduced chloride current. Login to comment
116 On the other hand, genistein alone induced a small chloride response in CF patients carrying one copy of the G551D mutation. Login to comment
117 G551D is a Class III mutation that is synthesized, resides in the plasma membrane but is not activated with isoproterenol. Login to comment
118 The ability of genistein to activate G551D in the intestine of the G551D CF mouse has also been observed (47). Login to comment
121 G551D is a common mutation associated with pancreatic insufficiency and the severe phenotype. Login to comment
122 G551D CFTR does not conduct chloride in response to elevated cAMP but is resident in the plasma membrane. Login to comment
123 Genistein and related compounds are the first chemicals ever shown to stimulate G551D chloride conductance in vitro. Login to comment
339 Genistein stimulates chloride secretion in normal volunteers and CF patients with a G551D mutation. Login to comment
345 Effect of IBMX and alkaline phosphatase inhibitors on Cl-secretion in G551D cystic fibrosis mutant mice. 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
53 In this patient, normal bands were visible at the sites of the other mutations localized in exon 11 (1717-1G→A, G551D, R553X) thereby excluding a deletion of exon 11. 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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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 cystic fibrosis transmembrane conductance regu... J Biol Chem. 1999 Apr 23;274(17):11811-6. Schreiber R, Nitschke R, Greger R, Kunzelmann K
The cystic fibrosis transmembrane conductance regulator activates aquaporin 3 in airway epithelial cells.
J Biol Chem. 1999 Apr 23;274(17):11811-6., 1999-04-23 [PMID:10206998]

Abstract [show]
Enhanced osmotic water permeability has been observed in Xenopus oocytes expressing cystic fibrosis transmembrane conductance regulator (CFTR) protein. Subsequent studies have shown that CFTR activates an endogenous water permeability in oocytes, but that CFTR itself is not the water channel. Here, we show CFTR-dependent activation of endogenous water permeability in normal but not in cystic fibrosis human airway epithelial cells. Cell volume was measured by novel confocal x-z laser scanning microscopy. Glycerol uptake and antisense studies suggest CFTR-dependent regulation of aquaporin 3 (AQP3) water channels in airway epithelial cells. Regulatory interaction was confirmed by coexpression of CFTR and AQP3 cloned from human airways in Xenopus oocytes and of CFTR and rat AQP3 in Chinese hamster ovary cells. These findings indicate that CFTR is a regulator of AQP3 in airway epithelial cells.

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60 Oocytes of identical batches were injected with 10-50 ng of cRNA (wild-type CFTR, G551D-CFTR, and hAQP3 cloned from human airway cells). Login to comment
130 A mutation in the first nucleotide-binding fold of CFTR (G551D-CFTR) abolished the effects of IBMX on Pf (Fig. 6B), confirming impaired regulation of hAQP3 by mutant forms of CFTR. Login to comment
175 Pf was increased in AQP3/CFTR-coexpressing oocytes and was further enhanced by IBMX, whereas oocytes coexpressing AQP3 and G551D-CFTR did not show an increase in Pf upon stimulation by IBMX. 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

[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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59 In 5 compound heterozygotes, G551D/DF508 (3 cases), G551D/1078delT and G551D/574delA, the neonatal IRT concentrations were highly increased, to 1490-2000 mg LÀ1 (mean Æ SD: 1653 Æ 208 mg LÀ1 ). Login to comment
60 Two CF newborns carrying mutation R117H and G551D or DF508 had lower IRT concentrations (1010 and 1070 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] Murine submucosal glands are clonally derived and ... Am J Respir Cell Mol Biol. 1999 Jun;20(6):1181-9. Borthwick DW, West JD, Keighren MA, Flockhart JH, Innes BA, Dorin JR
Murine submucosal glands are clonally derived and show a cystic fibrosis gene-dependent distribution pattern.
Am J Respir Cell Mol Biol. 1999 Jun;20(6):1181-9., [PMID:10340937]

Abstract [show]
Submucosal glands (SMGs) are the major site of expression of the cystic fibrosis (CF) transmembrane conductance regulator gene (CFTR) in the human lung. As such, SMGs may be a critical component of CF lung disease pathogenesis and an important target for gene therapy. Gene-targeted mouse models exist for CF and these are used to validate gene therapy or other interventions and to dissect CF phenotypes. It is important, therefore, to compare human and mouse SMGs. We show that SMGs in the mouse are similar in structure, cell types, and Cftr expression to those in the human. Murine SMGs were found to be present in the proximal regions of the trachea at the same density as in humans but, unlike in humans, did not extend below the trachea. Upon investigation of homozygous Cftr tm1HGU and Cftr tm1G551D mutant mice, SMGs were found to extend more distally than those in wild-type control mice (P < 0.05). To investigate the development of SMGs we generated aggregation chimeric mice. Chimeric offspring contained a contribution of transgenic cells that were detectable either by DNA in situ hybridization (reiterated beta-globin transgene TgN[Hbb-bl]83Clo) or beta-galactosidase histochemistry (Lac Z reporter gene TgR[ROSA26]- 26Sor). Analysis of the distribution of transgenic cells in chimeric SMGs suggests that SMGs are clonally derived.

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166 SMG Distribution in cftr-Deficient Mice Is Abnormal In an experiment blinded to genotype, the distribution of PAS-stained SMGs was studied by using whole-mount preparation of tracheas measured against trachea ring number in wild-type littermates and CF mice homozygous for the G551D mutation or the HGU insertional gene disruption. 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
60 Restriction digestion and electrophoresis (Figures 2 and 3) Detection of 621ϩ1G→T, R117H, G551D, R553X, W1282X and N1303K were performed using appropriate restriction enzymes (New England Biolabs, Ozyme, Saint Quentin Yvelines, France) as described in Table IV. 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] Downregulation of epithelial sodium channel (ENaC)... J Membr Biol. 1999 Jun 1;169(3):175-88. Chabot H, Vives MF, Dagenais A, Grygorczyk C, Berthiaume Y, Grygorczyk R
Downregulation of epithelial sodium channel (ENaC) by CFTR co-expressed in Xenopus oocytes is independent of Cl- conductance.
J Membr Biol. 1999 Jun 1;169(3):175-88., 1999-06-01 [PMID:10354464]

Abstract [show]
Defective regulatory interactions between the cystic fibrosis conductance regulator (CFTR) and the epithelial sodium channel (ENaC) have been implicated in the elevated Na+ transport rates across cystic fibrosis airway epithelium. It has recently been proposed that ENaC downregulation by CFTR depends on the ability of CFTR to conduct Cl- into the cell and is negligible when Cl- flows out of the cell. To study the mechanisms of this downregulation we have measured amiloride-inhibitable Na+ current (Iamil) in oocytes co-expressing rat ENaC and human wild-type CFTR. In oocytes voltage-clamped to -60 mV, stimulating CFTR with 1 mm IBMX reduced Iamil by up to 80%, demonstrating that ENaC is inhibited when Cl- is conducted out of the cell. Decreasing the level of CFTR stimulation in a single oocyte, decreased both the degree of Iamil downregulation and the CFTR-mediated plasma membrane Cl- conductance, suggesting a direct correlation. However, Iamil downregulation was not affected when Cl- flux across oocyte membrane was minimized by holding the oocyte membrane potential near the Cl- reversal potential (67% +/- 10% inhibition at -20 mV compared to 79% +/- 4% at -60 mV) demonstrating that Iamil downregulation was independent of the amount of current flow through CFTR. Studies with the Ca2+-sensitive photoprotein aequorin showed that Ca2+ is not involved in Iamil downregulation by CFTR, although Ca2+ injection into the cytoplasm did inhibit Iamil. These results demonstrate that downregulation of ENaC by CFTR depends on the degree of CFTR stimulation, but does not involve Ca2+ and is independent of the direction and magnitude of Cl- transport across the plasma membrane.

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31 This hypothesis was based on the observation that the magnitude of Iamil downregulation was correlated with the ability of CFTR to conduct Cl- , was reduced for CFTR mutants that had diminished membrane Cl- permeability (G551D, ⌬F508, R117H) and was abolished by inhibition of CFTR Cl-conductance using diphenylamine-carboxylate (DPC; Mall et al., 1996; Briel et al., 1998). Login to comment
255 The interaction was not observed if the CFTR peptide fragment contained the CF mutation G551D, and coexpression of ENaC with each CFTR peptide fragment in Xenopus oocytes suggested that ENaC was downregulated by wild type but not G551D peptide. 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

[hide] The cystic fibrosis conductance regulator gene exo... Am J Rhinol. 1999 May-Jun;13(3):221-3. Bucholtz GA, Ejercito VS, Burmester JK
The cystic fibrosis conductance regulator gene exon sequence is normal in a patient with edematous eosinophilic nasal polyps.
Am J Rhinol. 1999 May-Jun;13(3):221-3., [PMID:10392242]

Abstract [show]
Nasal polyps are the most common mass lesions found in the nose and their etiology is unknown. Nasal polyps from cystic fibrosis (CF) patients are histologically distinct from nasal polyps from patients without CF. It has been suggested that a mutation (G551D) of the cystic fibrosis transmembrane conductance regulator (CFTR) gene may play a role in nasal polyp formation in patients without CF. To investigate the possibility that this or other CFTR gene exon mutations are required for nasal polyp formation, the CFTR gene exons were sequenced from peripheral blood DNA derived from an adult patient with edematous eosinophilic nasal polyps and no personal or family history of CF. No mutations or deletions were identified in any of the CFTR exons. A single polymorphism (A or G) was found in exon 10, base pair 1540, amino acid 470. This polymorphism was detected in 11 of 16 subjects (69%) with edematous eosinophilic nasal polyps and 10 of 21 normal subjects (48%) without nasal polyps and was not statistically significant (p = 0.316). These results demonstrate that mutations of the CFTR coding region are not a prerequisite for the formation of edematous eosinophilic nasal polyps.

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11 12,13 Burger et al. analyzed 112 nasal polyp specimens from non-CF patients for 8 cystic fibrosis conductance regulator (CFTR) gene mutations (Ll508, Ll] 507, D 11OH, TI17H, 621+ IG---7T, N1303K, G551D, R553X).14 Ho- Delivered by Publishing Technology to: University of North Carolina IP: 152.19.83.63 On: Mon, 08 Aug 2011 14:07:01 Copyright (c) Oceanside Publications, Inc. All rights reserved. For permission to copy go to www.copyright.com mozygous or compound heterozygous individuals were not found. 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

[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

[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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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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20 While ⌬F508 and other CFTR gene alleles (e.g., W1282X, and G551D) can be categorized as severe with respect to the level of exocrine pancreatic dysfunction (20), correlations of genotype with the severity of pulmonary disease have been less clear. Login to comment
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
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] Defective function of the cystic fibrosis-causing ... Am J Physiol. 1999 Oct;277(4 Pt 1):C833-9. Illek B, Zhang L, Lewis NC, Moss RB, Dong JY, Fischer H
Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein.
Am J Physiol. 1999 Oct;277(4 Pt 1):C833-9., [PMID:10516113]

Abstract [show]
The patch-clamp technique was used to investigate the effects of the isoflavone genistein on disease-causing mutations (G551D and DeltaF508) of the cystic fibrosis transmembrane conductance regulator (CFTR). In HeLa cells recombinantly expressing the trafficking-competent G551D-CFTR, the forskolin-stimulated Cl currents were small, and average open probability of G551D-CFTR was P(o) = 0.047 +/- 0.019. Addition of genistein activated Cl currents approximately 10-fold, and the P(o) of G551D-CFTR increased to 0.49 +/- 0.12, which is a P(o) similar to wild-type CFTR. In cystic fibrosis (CF) epithelial cells homozygous for the trafficking-impaired DeltaF508 mutation, forskolin and genistein activated Cl currents only after 4-phenylbutyrate treatment. These data suggested that genistein activated CFTR mutants that were present in the cell membrane. Therefore, we tested the effects of genistein in CF patients with the G551D mutation in nasal potential difference (PD) measurements in vivo. The perfusion of the nasal mucosa of G551D CF patients with isoproterenol had no effect; however, genistein stimulated Cl-dependent nasal PD by, on average, -2.4 +/- 0.6 mV, which corresponds to 16.9% of the responses (to beta-adrenergic stimulation) found in healthy subjects.

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6 It is published 12 times aAJP - Cell Physiology rapid communication Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein BEATE ILLEK,1 LEI ZHANG,2 NANCY C. LEWIS,1 RICHARD B. MOSS,3 JIAN-YUN DONG,2 AND HORST FISCHER1 1Research Institute and Pulmonary Center, Children`s Hospital Oakland, Oakland 94609; 2Laboratory Medicine, University of California at San Francisco, San Francisco 94143; and 3Pediatric Pulmonary Medicine, Stanford University Medical Center, Stanford, California 94305 Illek, Beate, Lei Zhang, Nancy C. Lewis, Richard B. Moss, Jian-Yun Dong, and Horst Fischer. Login to comment
7 Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein. Login to comment
8 Am. J. Physiol. 277 (Cell Physiol. 46): C833-C839, 1999.-The patch-clamp technique was used to investigate the effects of the isoflavone genistein on disease-causing mutations (G551D and ⌬F508) of the cystic fibrosis transmembrane conductance regulator (CFTR). Login to comment
9 In HeLa cells recombinantly expressing the trafficking-competent G551D-CFTR, the forskolin-stimulated Cl currents were small, and average open probability of G551D-CFTR was Po ϭ 0.047 Ϯ 0.019. Login to comment
10 Addition of genistein activated Cl currents ϳ10-fold, and the Po of G551D-CFTR increased to 0.49 Ϯ 0.12, which is a Po similar to wild-type CFTR. Login to comment
13 Therefore, we tested the effects of genistein in CF patients with the G551D mutation in nasal potential difference (PD) measurements in vivo. Login to comment
14 The perfusion of the nasal mucosa of G551D CF patients with isoproterenol had no effect; however, genistein stimulated Cl-dependent nasal PD by, on average, -2.4 Ϯ 0.6 mV, which corresponds to 16.9% of the responses (to beta-adrenergic stimulation) found in healthy subjects. Login to comment
18 The glycine-to- aspartic acid missense mutation at codon 551 (G551D) is the third commonest CF mutation, with a worldwide frequency of ϳ2% on CF chromosomes (25, 27). Login to comment
19 Higher frequencies of G551D are found in populations of Celtic descent, with incidences of 6.9% and 5.5% in the Irish and Australian CF populations, respectively (3, 12, 25). Login to comment
22 The G551D-CFTR protein traffics to the apical membrane, but Cl channel function is markedly reduced (class III mutation) (27). Login to comment
23 Compared with CF patients homozygous for the ⌬F508 mutation, patients carrying the G551D mutation show a threefold reduction in the incidence of meconium ileus (neonatal intestinal blockage), a trend toward later age at diagnosis of pancreatic insufficiency (12), and milder disease in homozygotes (22). Login to comment
24 Interestingly, both the ⌬F508 mutation and the G551D mutation are in NBD1 of CFTR; however, ATP binding of NBD1 is not affected by the ⌬F508 mutation but is significantly impaired in G551D-CFTR (21). Login to comment
28 We therefore tested its effects on G551D-CFTR, which is present in the cell membrane, and on ⌬F508-CFTR, once it was translocated in sufficient quantity to the membrane after 4-PB treatment. Login to comment
29 MATERIALS AND METHODS Generation of recombinant adenovirus carrying the G551D gene. Login to comment
30 Recombinant adenovirus carrying the CFTR G551D mutation was generated by a modified ligation procedure. Login to comment
31 With the use of PCR-mediated point mutation, G551D was generated by converting the codon for glycine (GGT) at position 551 to an aspartic acid codon (GAT). Login to comment
35 The G551D cDNA was inserted into a shuttle vector, pLadC, which contains the left-hand terminal of adenovirus sequence and a cytomegalovirus promoter. Login to comment
36 The recombinant adenovirus, AdcG551D, was generated by ligation of the linearized pLadC-G551D to the right-hand portion of the adenovirus sub360, which contains the deletion in the E1a and E3 regions. Login to comment
40 The inserted G551D cDNA was confirmed using PCR protocols. Login to comment
45 Twenty-four hours before use, cells were infected with viral vectors containing G551D-CFTR, wtCFTR, or LacZ as control at a multiplicity of infection of ϳ100. Login to comment
63 Patients with at least one G551D-CFTR allele were recruited from Children`s Hospital Oakland and from the Stanford CF Center at Lucile Salter Packard Children`s Hospital. Login to comment
70 G551D-CFTR-mediated current is stimulated by genistein. Login to comment
71 A: whole cell recording from a HeLa cell expressing G551D-CFTR. Login to comment
89 F: average calculated Po values during forskolin treatment (fsk, open bars) and forskolin ϩ genistein treatment (geni, solid bars) in HeLa cells expressing G551D or wild-type (wt) CFTR. Login to comment
90 Genistein significantly increased Po of G551D-CFTR (P ϭ 0.01, n ϭ 4) and wtCFTR (P ϭ 0.02, n ϭ 4, paired t-tests). Login to comment
94 RESULTS Effects of genistein on G551D-CFTR in patch-clamp recordings. Login to comment
95 HeLa cells infected with the G551D-CFTR-containing adenovirus were patch clamped in the whole cell and the cell-attached mode. Login to comment
96 Figure 1 shows a whole cell recording from a G551D-CFTR-expressing cell. Login to comment
101 Current variance to mean current plots was used to calculate the average Po of G551D-CFTR-mediated whole cell current. Login to comment
106 Figure 1F shows average Po values of G551D-CFTR compared with wtCFTR. Login to comment
107 Note the low Po of G551D-CFTR during forskolin stimulation (0.047 Ϯ 0.19, n ϭ 4), which was increased by genistein to 0.49 Ϯ 0.12, a value not different from wtCFTR. Login to comment
109 Figure 2 shows the effects of genistein on the G551D-CFTR channel in a cell-attached patch-clamp recording. Login to comment
114 Figure 2B shows the average single channel I-V relation for G551D-CFTR recorded in the cell-attached mode. Single channel I-V relations were typically outwardly rectifying in the cell-attached mode (7), with a conductance in the positive voltage range of g ϭ 8.7 Ϯ 1.0 pS (n ϭ 4, forskolin) and 9.6 Ϯ 0.8 pS (forskolin ϩ genistein), which were similar to wtCFTR (g ϭ 8.6 Ϯ 0.91 pS, n ϭ 3) recorded under the same conditions. Login to comment
116 Genistein showed no effects on Cl current, indicating that 1) HeLa cells had no endogenous genistein-activated Cl current, and 2) unstimulated G551D-CFTR could not be activated by genistein alone. Login to comment
118 This indicated that genistein was not a direct channel opener of G551D-CFTR but relied on regulation by the cAMP/protein kinase A (PKA) pathway and that G551D-CFTR was only active when both genistein was present and PKA was active. Login to comment
119 In summary, these patch-clamp data show that G551D-CFTR is a channel with markedly reduced open probability. Login to comment
120 Treatment of G551D-CFTR with genistein effectively restored regulation of Po by the cAMP pathway similar to wtCFTR. Login to comment
127 Cell-attached recording of G551D-CFTR. Login to comment
138 We performed nasal potential difference (PD) measurements in CF patients bearing the G551D mutation on at least one allele and in healthy human subjects. Login to comment
141 Figure 5 shows measurements of nasal PD from a healthy volunteer (Fig. 5A) and a G551D CF patient (Fig. 5B). Login to comment
143 Perfusion with Cl-free solution and subsequent addition of isoproterenol hyperpolarized nasal PD in healthy subjects, whereas nasal PD was depolarized in G551D CF patients, indicating a lack of a beta-adrenergic- stimulated Cl conductance in CF patients. Login to comment
144 However, perfusion of the nasal mucosa with genistein significantly hyperpolarized nasal PD both in G551D CF patients and in healthy subjects, indicative of a genistein-induced Cl conductance in both groups. Login to comment
146 In G551D CF patients, genistein caused a hyperpolarization of, on average, -2.4 Ϯ 0.6 mV (n ϭ 5), which corresponds to 16.9% of the average response of normal subjects to treatment with Cl-free solution and isoproterenol (-14.0 Ϯ 2.1 mV, n ϭ 8). Login to comment
147 Another tested flavonoid, quercetin, was also active but less potent than genistein in stimulating nasal PD in G551D CF patients (by -0.9 Ϯ 0.3 mV, n ϭ 4, significantly different from 0, one sample t-test). Login to comment
148 DISCUSSION Genistein restores cAMP-dependent G551D-CFTR activity. Login to comment
149 For this study we selected the G551D mutation because the G551D-CFTR protein was described as a trafficking-competent mutant (23). Login to comment
150 We found that acute treatment of cells with genistein in the presence of forskolin resulted in G551D-CFTR activity that was functionally similar to wtCFTR during stimulation with forskolin. Login to comment
151 The genistein-stimulated G551D-CFTR had a single channel conductance of 9.6 Ϯ 0.8 pS, showed time-and voltage-independent currents, and Po was 0.49 Ϯ 0.12, which are typical biophysical characteristics of wtCFTR. Login to comment
152 In addition, stimulation of G551D-CFTR by genistein required intrinsic regulation of the channel by the cAMP/PKA system. Login to comment
155 Genistein has no effect on G551D-CFTR-mediated Cl currents in unstimulated cells. Login to comment
156 Whole cell recording of a HeLa cell expressing G551D-CFTR. Login to comment
171 Effects of genistein in G551D CF patients. Login to comment
172 Our nasal PD measurements in G551D CF patients showed the typical abnormalities present in other CF patients, which are: a hyperpolarized basal nasal PD, an increased amiloride-sensitive PD, a depolarization of nasal PD with Cl-free solutions, and a lack of response to the beta-adrenergic agonist isoproterenol (20). Login to comment
173 In contrast, perfusion of the nasal epithelium of G551D CF patients with genistein resulted in a hyperpolarization of nasal PD in all patients, which under the experimental conditions corresponds to an activation of a Cl conductance. Login to comment
183 Thus the responses to genistein of nasal PD in G551D CF patients correspond to 13.6% of normal in terms of Cl current, which may have therapeutic significance. Login to comment
189 Genistein activates Cl-selective potentials in CF patients with G551D mutation. Login to comment
194 B: measurement of nasal PD in a G551D CF patient. Login to comment
195 Patient was a 14-year-old Caucasian female with genotype G551D/⌬F508. Login to comment
200 C: average responses of G551D CF patients (n ϭ 5) to treatment with amiloride (amil, 50 µM), Cl-free solution (exchanged for gluconate) and 10 µM isoproterenol (Cl free/iso), and 30 µM genistein (geni), in comparison to measurements in healthy subjects (n ϭ 8). Login to comment
202 Genotypes (and responses of PD to genistein) of patients were: three G551D/⌬F508 (-0.8, -2.4, and -2.3 mV), one G551D/G551D (-2.0 mV), and one G551D/unknown (-4.3 mV). Login to comment
205 In G551D CF patients average nasal PD values were: NaCl, -33.8 Ϯ 3.2 mV; amiloride, -11.7 Ϯ 0.9 mV; isoproterenol/Cl free, -6.4 Ϯ 1.7 mV; and genistein, -8.7 Ϯ 1.6 mV. Login to comment

[hide] Interaction between calcium-activated chloride cha... Pflugers Arch. 1999 Oct;438(5):635-41. Wei L, Vankeerberghen A, Cuppens H, Eggermont J, Cassiman JJ, Droogmans G, Nilius B
Interaction between calcium-activated chloride channels and the cystic fibrosis transmembrane conductance regulator.
Pflugers Arch. 1999 Oct;438(5):635-41., [PMID:10555560]

Abstract [show]
We investigated interactions between cystic fibrosis conductance regulator (CFTR) and endogenous Ca2+-activated Cl- channels (CaCC) in bovine pulmonary artery endothelium (CPAE). CPAE cells, which do not express CFTR, were transiently transfected with wild-type (WT) CFTR and the deletion mutant deltaF508 CFTR. Currents through CaCC were significantly reduced after expression of WT CFTR. This inhibition was increased by stimulation (isobutylmethylxanthine, forskolin) of CFTR in cells expressing WT CFTR. There were no such effects when deltaF508 mutant CFTR, which is retained in the endoplasmic reticulum, was expressed. It is concluded that CFTR and CaCC are functionally coupled probably through a direct channel-channel interaction.

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164 Bilayer experiments showed diminished activation of ORCC in the absence of a functional CFTR protein [18] or when a mutant CFTR channel (G551D) was inserted. Login to comment

[hide] Activation of cystic fibrosis transmembrane conduc... Biol Reprod. 2000 Jan;62(1):143-9. Leung GP, Wong PY
Activation of cystic fibrosis transmembrane conductance regulator in rat epididymal epithelium by genistein.
Biol Reprod. 2000 Jan;62(1):143-9., [PMID:10611078]

Abstract [show]
The effect of genistein on anion secretion via cystic fibrosis transmembrane conductance regulator (CFTR) in cultured rat cauda epididymal epithelia was studied by short-circuit current (Isc) technique. Genistein added apically stimulated a concentration-dependent rise in Isc due to Cl(-) and HCO(3)(-) secretion. The genistein-induced Isc was observed in basolaterally permeabilized monolayers, suggesting that the Isc response was mediated by the apical anion channel. The response could be blocked by the nonspecific Cl(-) channel blocker, diphenylamine-2-carboxylate (DPC), but not by the Ca(2+)-activated Cl(-) channel blocker, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Genistein did not increase intracellular cAMP, but H-89, a protein kinase A inhibitor, completely abolished the Isc response to genistein. Moreover, pretreatment of the tissues with MDL-12330A, an adenylate cyclase inhibitor, markedly attenuated the Isc response to genistein, but the response was restored upon the addition of exogenous cAMP. Ca(2+), protein kinase C, tyrosine kinase, and protein phosphatase signalling pathways were not involved in the action of genistein. It is speculated that genistein stimulates anion secretion by direct interaction with CFTR. This requires a low level of phosphorylation of CFTR by basal protein kinase A activity. It is suggested that genistein may provide therapeutic benefit to male infertility associated with cystic fibrosis.

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180 In heterologous cell expression systems, genistein has been shown to potentiate the cAMP-dependent activation of G551D-CFTR [35] and ⌬F508-CFTR [19,36], the most common mutations in CF. 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] Stimulation of Cl(-) secretion by chlorzoxazone. J Pharmacol Exp Ther. 2000 Feb;292(2):778-87. Singh AK, Devor DC, Gerlach AC, Gondor M, Pilewski JM, Bridges RJ
Stimulation of Cl(-) secretion by chlorzoxazone.
J Pharmacol Exp Ther. 2000 Feb;292(2):778-87., [PMID:10640318]

Abstract [show]
We previously demonstrated that 1-ethyl-2-benzimidazolone (1-EBIO) directly activates basolateral membrane calcium-activated K(+) channels (K(Ca)), thereby stimulating Cl(-) secretion across several epithelia. In our pursuit to identify potent modulators of Cl(-) secretion that may be useful to overcome the Cl(-) secretory defect in cystic fibrosis (CF), we have identified chlorzoxazone [5-chloro-2(3H)-benzoxazolone], a clinically used centrally acting muscle relaxant, as a stimulator of Cl(-) secretion in several epithelial cell types, including T84, Calu-3, and human bronchial epithelium. The Cl(-) secretory response induced by chlorzoxazone was blocked by charybdotoxin (CTX), a known blocker of K(Ca). In nystatin-permeabilized monolayers, chlorzoxazone stimulated a basolateral membrane I(K), which was inhibited by CTX and also stimulated an apical I(Cl) that was inhibited by glibenclamide, indicating that the G(Cl) responsible for this I(Cl) may be cystic fibrosis transmembrane conductance regulator (CFTR). In membrane vesicles prepared from T84 cells, chlorzoxazone stimulated (86)Rb(+) uptake in a CTX-sensitive manner. In excised, inside-out patches, chlorzoxazone activated an inwardly-rectifying K(+) channel, which was inhibited by CTX. 6-Hydroxychlorzoxazone, the major metabolite of chlorzoxazone, did not activate K(Ca), whereas zoxazolamine (2-amino-5-chlorzoxazole) showed a similar response profile as chlorzoxazone. In normal human nasal epithelium, chlorzoxazone elicited hyperpolarization of the potential difference that was similar in magnitude to isoproterenol. However, in the nasal epithelium of CF patients with the DeltaF508 mutation of CFTR, there was no detectable Cl(-) secretory response to chlorzoxazone. These studies demonstrate that chlorzoxazone stimulates transepithelial Cl(-) secretion in normal airway epithelium in vitro and in vivo, and suggest that stimulation requires functional CFTR in the epithelia.

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95 Eight nonsmoking healthy subjects (six males, two females) with no history of respiratory disease, and five CF patients (four males, one female), four homozygous with ⌬F508 and one with G551D/ ⌬F508 mutations were studied. Login to comment
285 Although the G551D mutation does reach the plasma membrane, its channel function is significantly impaired and this may explain why we did not observe a Cl-secretory response in the G551D/ ⌬F508-CFTR CF patient. Login to comment

[hide] Congenital bilateral absence of the vas deferens, ... Hum Reprod. 2000 Feb;15(2):431-5. Phillipson GT, Petrucco OM, Matthews CD
Congenital bilateral absence of the vas deferens, cystic fibrosis mutation analysis and intracytoplasmic sperm injection.
Hum Reprod. 2000 Feb;15(2):431-5., [PMID:10655317]

Abstract [show]
The aim of this study was to assess the outcome of intracytoplasmic sperm injection (ICSI) with fresh and frozen-thawed surgically retrieved spermatozoa from men diagnosed with congenital bilateral absence of the vas deferens (CBAVD). Twenty-seven azoospermic men with their partners were treated [25 with CBAVD and two with clinical cystic fibrosis (CF)]. CF gene mutation analysis and genetic counselling was provided. Spermatozoa were aspirated by microsurgical epididymal sperm aspiration (MESA), percutaneous epididymal sperm aspiration (PESA) or open testis biopsy. Of the men with CBAVD, 60% carried a single mutation, 20% were compound heterozygotes, and 20% had no CF mutation identified. Of the 28 sperm aspiration procedures, 86% had supplementary spermatozoa for cryopreservation with 83% of those samples assessed as satisfactory when thawed. Of 29 cycles with fresh spermatozoa a fertilization rate of 76% of oocytes injected and 17% embryo implantation rate occurred. Twenty-four cycles in which cryopreserved spermatozoa were used resulted in an oocyte fertilization rate of 69% and embryo implantation rate of 20%. Eighteen clinical pregnancies occurred with 14 live births without congenital anomaly. Two pregnancies were achieved following pre-implantation genetic diagnosis. It is concluded that the presence of CF mutations in the male partner does not compromise in-vitro fertilization treatment outcomes or the opportunity for healthy live births.

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109 Her genotype (G551D/R117H) carrier of a severe CF gene mutation and the female tests comprised a severe CF mutation (G551D) combined with negative for the group of mutations screened. Login to comment
117 Sperm recovery, sperm cryopreservation, and the ICSI G551D and likely to result in a child with CF. Login to comment
121 without the ∆F508 mutation would confer a G551D or R117H Historically a few reports of fertile CF males have been heterozygote genotype. Login to comment

[hide] Pharmacologic restoration of delta F508 CFTR-media... Kidney Int. 2000 Mar;57(3):832-7. Zeitlin PL
Pharmacologic restoration of delta F508 CFTR-mediated chloride current.
Kidney Int. 2000 Mar;57(3):832-7., [PMID:10720936]

Abstract [show]
Cystic fibrosis (CF) is an autosomal inherited disorder caused by over 800 different mutations in the CFTR gene. The most common mutation, delta F508, causes a trafficking arrest in the endoplasmic reticulum and the CFTR protein is degraded. Restoration of CFTR trafficking in vitro restores cAMP-mediated chloride transport at the cell surface. The hypothesis of this discussion is that the short chain fatty acids, butyrate and 4-phenylbutyrate, up-regulate mature CFTR at the plasma membrane. Evidence that these compounds regulate CFTR production and maturation in part through effects on molecular chaperones in CF cells in culture is discussed. The oral drug, 4-phenylbutyrate, was tested in a Phase I clinical trial in CF subjects and further trials are underway. Other new therapeutic approaches directed at different classes of mutations in CFTR are also discussed. Chemical and pharmacologic agents that regulate endogenous gene expression at different steps in the biosynthetic processing pathway of a membrane glycoprotein will be needed to comprehensively treat a complex inherited disorder like cystic fibrosis.

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70 G551D is a Class III mutation that is synthe- and Hsp90. Login to comment
72 The ability of gen- CFTR chloride channel activity at the cell surface in istein to activate G551D in the intestine of the G551D both immortalized human CF airway and biliary epithe- CF mouse has also been observed [40]. Login to comment
94 G551D is a common ence response to isoproterenol [38]. Login to comment
104 Defects instead lie in chlorideother hand, genistein alone induced a small chloride response in CF patients carrying one copy of the G551D conductance or channel gating (Fig. 1E). 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
78 No correlation of myocardial complications with CFTR mutations has been performed so far and, in the literature, only one case was recognised as a DF508/M compound heterozygote, negative for R347P, G551D, R553X and N1303K as a second mutation (9). 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
122 In this subgroup, eight out of nine patients In summary, CFTR mutations are the molecu- had one of the following mutations detected on lar cause for a variety of different forms of male one of their two CFTR alleles: DF508 (n=4), infertility due to obstructive azoospermia, ranging R117H (n=2), G551D (n=1) and N1303K (n=1) from CBAVD and CUAVD with contralateral (Mickle et al., 1995). Login to comment
134 Five of the patients with obstructive reduced sperm quality and two of 21 men (9.5%) with azoospermia carried one CFTR mutation.azoospermia were heterozygous for IVS8-5T, two were carriers of R117H, and one was heterozygous One azoospermic male was compound heterozygous for G551D and R117H. 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] Two mechanisms of genistein inhibition of cystic f... J Physiol. 2000 Apr 15;524 Pt 2:317-30. Lansdell KA, Cai Z, Kidd JF, Sheppard DN
Two mechanisms of genistein inhibition of cystic fibrosis transmembrane conductance regulator Cl- channels expressed in murine cell line.
J Physiol. 2000 Apr 15;524 Pt 2:317-30., 2000-04-15 [PMID:10766914]

Abstract [show]
1. The isoflavone genistein may either stimulate or inhibit cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. To investigate how genistein inhibits CFTR, we studied CFTR Cl- channels in excised inside-out membrane patches from cells expressing wild-type human CFTR. 2. Addition of genistein (100 microM) to the intracellular solution caused a small decrease in single-channel current amplitude (i), but a large reduction in open probability (Po). 3. Single-channel analysis of channel block suggested that genistein (100 microM) may inhibit CFTR by two mechanisms: first, it may slow the rate of channel opening and second, it may block open channels. 4. Acidification of the intracellular solution relieved channel block, suggesting that the anionic form of genistein may inhibit CFTR. 5. Genistein inhibition of CFTR Cl- currents was weakly voltage dependent and unaffected by changes in the extracellular Cl- concentration. 6. Channel block was relieved by pyrophosphate (5 mM) and ATP (5 mM), two agents that interact with the nucleotide-binding domains (NBDs) of CFTR to greatly stimulate channel activity. 7. ATP (5 mM) prevented the genistein-induced decrease in Po, but was without effect on the genistein-induced decrease in i. 8. The genistein-induced decrease in i was voltage dependent, whereas the genistein-induced decrease in Po was voltage independent. 9. The data suggest that genistein may inhibit CFTR by two mechanisms. First, it may interact with NBD1 to potently inhibit channel opening. Second, it may bind within the CFTR pore to weakly block Cl- permeation.

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407 Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein. Login to comment
409 Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein. 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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87 Alterations within NBD1 (such as missense mutation G551D) may also affect CFTR regulation of other channels such as the outwardly rectifying chloride channel [21] or ROMK2 potassium channel [22]. Login to comment

[hide] Characterization of a novel 21-kb deletion, CFTRde... Hum Genet. 2000 Mar;106(3):259-68. Dork T, Macek M Jr, Mekus F, Tummler B, Tzountzouris J, Casals T, Krebsova A, Koudova M, Sakmaryova I, Macek M Sr, Vavrova V, Zemkova D, Ginter E, Petrova NV, Ivaschenko T, Baranov V, Witt M, Pogorzelski A, Bal J, Zekanowsky C, Wagner K, Stuhrmann M, Bauer I, Seydewitz HH, Neumann T, Jakubiczka S
Characterization of a novel 21-kb deletion, CFTRdele2,3(21 kb), in the CFTR gene: a cystic fibrosis mutation of Slavic origin common in Central and East Europe.
Hum Genet. 2000 Mar;106(3):259-68., [PMID:10798353]

Abstract [show]
We report a large genomic deletion of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, viz., a deletion that is frequently observed in Central and Eastern Europe. The mutation, termed CFTRdele2,3(21 kb), deletes 21,080 bp spanning introns 1-3 of the CFTR gene. Transcript analyses have revealed that this deletion results in the loss of exons 2 and 3 in epithelial CFTR mRNA, thereby producing a premature termination signal within exon 4. In order to develop a simple polymerase chain reaction assay for this allele, we defined the end-points of the deletion at the DNA sequence level. We next screened for this mutation in a representative set of European and European-derived populations. Some 197 CF patients, including seven homozygotes, bearing this mutation have been identified during the course of our study. Clinical evaluation of CFTRdele2,3(21 kb) homozygotes and a comparison of compound heterozygotes for deltaF508/CFTRdele2,3(21 kb) with pairwise-matched deltaF508 homozygotes indicate that this deletion represents a severe mutation associated with pancreatic insufficiency and early age at diagnosis. Current data show that the mutation is particularly common in Czech (6.4% of all CF chromosomes), Russian (5.2%), Belorussian (3.3%), Austrian (2.6%), German (1.5%), Polish (1.5%), Slovenian (1.5%), Ukrainian (1.2%), and Slovak patients (1.1%). It has also been found in Lithuania, Latvia, Macedonia and Greece and has sporadically been observed in Canada, USA, France, Spain, Turkey, and UK, but not in CF patients from Bulgaria, Croatia, Romania or Serbia. Haplotype analysis has identified the same extragenic CF-haplotype XV-2c/KM. 19 "A" and the same infrequent intragenic microsatellite haplotype 16-33-13 (IVS8CA-IVS 17bTA-IVS 17bCA) in all examined CFTRdele2,3(21 kb) chromosomes, suggesting a common origin for this deletion. We conclude that the 21-kb deletion is a frequent and severe CF mutation in populations of Eastern- and Western-Slavic descent.

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56 261 Second, a combined loss of these exons was found in about half of the nasal epithelial CFTR mRNA transcripts from a German CF patient who was compound heterozygous for G551D and the presumed deletion (Fig.1a). Login to comment
70 RT-PCR was performed on epithelial CFTR mRNA from the nasal polyp of a patient heterozygous for G551D and CFTRdele2,3(21 kb). Login to comment
145 Subsequently, in the region of the current Czech Republic and northern Austria, the Slavs assimilated with the remaining Celtic population, as objectively documented by an increased prevalence of the "Celtic" CF mutation, G551D, in Czech and Austrian CF populations (Macek et al. 1991). 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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28 R553X, G551D, S549N, R1162X, and 3120+1G→A were tested by PCR-based restriction analysis as previously described (Osborne et al. 1992; Jones et al. 1992; Picci et al. 1992; Shoshani et al. 1992). Login to comment
42 On the other hand, the R553X, G551D and 1924del7 mutations had a frequency <1% and 2869insG, R1162X, 3120+1G→A were not found in the population studied. 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
78 In contrast, the G551D and R553X mutations, which are among the first five most common worldwide, were only found in one chromosome each. Login to comment
79 In particular, mutation G551D has a high frequency in northern European and USA populations, while it is very rare in Hispanic and Latin American CF patients (CFGAC 1994). Login to comment

[hide] Role of CFTR in the colon. Annu Rev Physiol. 2000;62:467-91. Greger R
Role of CFTR in the colon.
Annu Rev Physiol. 2000;62:467-91., [PMID:10845099]

Abstract [show]
In contrast to the airways, the defects in colonic function in cystic fibrosis (CF) patients are closely related to the defect in CFTR. The gastrointestinal phenotype of CF transgenic mice closely resembles the phenotype in CF patients, which clearly indicates the crucial role of CFTR in colonic Cl- secretion and the absence of an effective compensation. In the colon, stimulation of CFTR Cl- channels involves cAMP- or cGMP-dependent phosphorylation. Exocytosis is not involved. Activation of CFTR leads to coactivation of basolateral KVLQT1-type K+ channels and inhibition of luminal Na+ channels (ENaC). In contrast to cultured cells, Ca2+ does not activate luminal Cl- channels in intact enterocytes. It activates basolateral SK4-type K+ channels and luminal K+ channels, which provide additional driving force for Cl- exit. The magnitude of Cl- secretion, however, completely depends on the presence of at least a residual CFTR function in the luminal membrane. These findings have been clearly demonstrated by Ussing chamber experiments in colon epithelium biopsies of CF and normal individuals: Colonic Cl- secretion in CF patients is variable and reflects the genotype; a complete defect of CFTR is paralleled by the absence of Cl- secretion and unmasks Ca(2+)-regulated K+ channels in the luminal membrane; overabsorption of Na+ in CF reflects the absence of ENaC inhibition by CFTR; and the functional status of CF colon can be mimicked by the complete suppression of cAMP stimulation in enterocytes of healthy individuals.

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250 Shortly thereafter it was observed that ENaC was downregulated by activated CFTR but not by mutated CFTR (DF508 CFTR or G551D CFTR) in Xenopus laevis oocytes overexpressing these membrane transport proteins (77). Login to comment

[hide] Control of epithelial Na+ conductance by the cysti... Pflugers Arch. 2000 Jun;440(2):193-201. Kunzelmann K, Schreiber R, Nitschke R, Mall M
Control of epithelial Na+ conductance by the cystic fibrosis transmembrane conductance regulator.
Pflugers Arch. 2000 Jun;440(2):193-201., [PMID:10898518]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl- channel expressed in luminal membranes of secretory and reabsorptive epithelia. CFTR plays a predominant role in both cAMP- and Ca2+-activated secretion of electrolytes. Although Ca2+-dependent Cl- channels exist independent of CFTR in the airway epithelium, their physiological significance remains to be determined. However, CFTR seems to be the only relevant Cl- conductance in the colonic epithelium. Apart from its secretory function, CFTR also has a task in regulating the reabsorption of electrolytes by controlling the activity of the epithelial Na+ channel, ENaC. Accordingly, defects in CFTR causing the disease cystic fibrosis (CF) lead to disturbances of both the secretion and absorption of electrolytes. Therefore, it is unclear what is pathophysiologically more important for the development of CF lung disease, the impaired secretion of Cl- or the enhanced reabsorption of Na+ and consecutive hyperabsorption of electrolytes. The mechanisms of how CFTR and ENaC interact are unknown. Previous work has given rise to several interesting working hypothesis, such as direct protein interaction or interaction via cytoskeletal proteins. Recent studies demonstrate the importance of the first nucleotide binding fold of CFTR, not only for the inhibition of ENaC but also for the interaction with other ion channels. Further studies are required to demonstrate whether regulation of other ion channels and membrane transport by CFTR occur by a common mechanism.

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78 ENaC is not inhibited by mutant CFTR, e.g. ∆F508-CFTR or G551D-CFTR, suggesting that the enhanced Na+ conductance in CF is caused by a lack of downregulation of ENaC by defective CFTR. Login to comment
114 We found that wtCFTR and the common CFTR mutant G551D, but not the most frequent CFTR mutant ∆F508, accumulate in the cell membrane of Xenopus oocytes during cAMP-dependent stimulation (Fig. 3). Login to comment
147 Summary of fluorescence measurements using confocal microscopy images of cell membranes of oocytes expressing wtCFTR, ∆F508-CFTR and G551D-CFTR as well as water-injected control oocytes. Login to comment

[hide] Role of protein phosphatases in the activation of ... Am J Physiol Cell Physiol. 2000 Jul;279(1):C108-19. Luo J, Zhu T, Evagelidis A, Pato MD, Hanrahan JW
Role of protein phosphatases in the activation of CFTR (ABCC7) by genistein and bromotetramisole.
Am J Physiol Cell Physiol. 2000 Jul;279(1):C108-19., [PMID:10898722]

Abstract [show]
Genistein and bromotetramisole (Br-t) strongly activate cystic fibrosis transmembrane conductance regulator (CFTR; ABCC7) chloride channels on Chinese hamster ovary cells and human airway epithelial cells. We have examined the possible role of phosphatases in stimulation by these drugs using patch-clamp and biochemical methods. Genistein inhibited the spontaneous rundown of channel activity that occurs after membrane patches are excised from cAMP-stimulated cells but had no effect on purified protein phosphatase type 1 (PP1), PP2A, PP2B, PP2C, or endogenous phosphatases when assayed as [(32)P]PO(4) release from prelabeled casein, recombinant GST-R domain fusion protein, or immunoprecipitated full-length CFTR. Br-t also slowed rundown of CFTR channels, but, in marked contrast to genistein, it did inhibit all four protein phosphatases tested. Half-maximal inhibition of PP2A and PP2C was observed with 0.5 and 1.5 mM Br-t, respectively. Protein phosphatases were also sensitive to (+)-p-Br-t, a stereoisomer of Br-t that does not inhibit alkaline phosphatases. Br-t appeared to act exclusively through phosphatases since it did not affect CFTR channels in patches that had low apparent endogenous phosphatase activity (i.e., those lacking spontaneous rundown). We conclude that genistein and Br-t act through different mechanisms. Genistein stimulates CFTR without inhibiting phosphatases, whereas Br-t acts by inhibiting a membrane-associated protein phosphatase (probably PP2C) that presumably allows basal phosphorylation to accumulate.

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198 Data are means Ϯ SE; n ϭ 4 experiments. C GENISTEIN, BROMOTETRAMISOLE, AND DEPHOSPHORYLATION OF CFTR a deletion of phenylalanine 508 (21), whereas Br-t activates several mutants, including G551D, when cells have only basal PKA activity (4). Login to comment
256 In previous work, we found that (-)-Br-t inhibits CFTR rundown in patches from CHO and human airway epithelial cells [NP34 (4, 5)] and also activates quiescent G551D mutant channels that are normally unresponsive to forskolin. Login to comment

[hide] Genistein activates CFTR-mediated Cl(-) secretion ... Am J Physiol Cell Physiol. 2000 Aug;279(2):C383-92. Goddard CA, Evans MJ, Colledge WH
Genistein activates CFTR-mediated Cl(-) secretion in the murine trachea and colon.
Am J Physiol Cell Physiol. 2000 Aug;279(2):C383-92., [PMID:10913005]

Abstract [show]
The action of the isoflavone genistein on the cystic fibrosis transmembrane conductance regulator (CFTR) has been studied in many cell systems but not in intact murine tissues. We have investigated the action of genistein on murine tissues from normal and cystic fibrosis (CF) mice. Genistein increased the short-circuit current (I(sc)) in tracheal (16.4 +/- 2.8 microA/cm(2)) and colonic (40.0 +/- 4.4 microA/cm(2)) epithelia of wild-type mice. This increase was inhibited by furosemide, diphenylamine-2-carboxylate, and glibenclamide, but not by DIDS. In contrast, genistein produced no significant change in the I(sc) of the tracheal epithelium (0.9 +/- 1.1 microA/cm(2)) and decreased the I(sc) of colons from CF null (-13.1 +/- 2.3 microA/cm(2)) and DeltaF508 mice (-10.3 +/- 1.3 microA/cm(2)). Delivery of a human CFTR cDNA-liposome complex to the airways of CF null mice restored the genistein response in the tracheas to wild-type levels. Tracheas from DeltaF508 mice were also studied: 46% of trachea showed no response to genistein, whereas 54% gave an increase in I(sc) similar to that in wild type. We conclude that genistein activates CFTR-mediated Cl(-) secretion in the murine trachea and distal colon.

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42 It has recently been demonstrated that genistein can also activate the G551D mutant CFTR channel in HeLa cells and in cystic fibrosis (CF) patients (19). 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] Role of the cystic fibrosis transmembrane conducta... Proc Natl Acad Sci U S A. 2000 Aug 1;97(16):8822-8. Pier GB
Role of the cystic fibrosis transmembrane conductance regulator in innate immunity to Pseudomonas aeruginosa infections.
Proc Natl Acad Sci U S A. 2000 Aug 1;97(16):8822-8., 2000-08-01 [PMID:10922041]

Abstract [show]
Chronic Pseudomonas aeruginosa infection occurs in 75-90% of patients with cystic fibrosis (CF). It is the foremost factor in pulmonary function decline and early mortality. A connection has been made between mutant or missing CF transmembrane conductance regulator (CFTR) in lung epithelial cell membranes and a failure in innate immunity leading to initiation of P. aeruginosa infection. Epithelial cells use CFTR as a receptor for internalization of P. aeruginosa via endocytosis and subsequent removal of bacteria from the airway. In the absence of functional CFTR, this interaction does not occur, allowing for increased bacterial loads in the lungs. Binding occurs between the outer core of the bacterial lipopolysaccharide and amino acids 108-117 in the first predicted extracellular domain of CFTR. In experimentally infected mice, inhibiting CFTR-mediated endocytosis of P. aeruginosa by inclusion in the bacterial inoculum of either free bacterial lipopolysaccharide or CFTR peptide 108-117 resulted in increased bacterial counts in the lungs. CFTR is also a receptor on gastrointestinal epithelial cells for Salmonella enterica serovar Typhi, the etiologic agent of typhoid fever. There was a significant decrease in translocation of this organism to the gastrointestinal submucosa in transgenic mice that are heterozygous carriers of a mutant DeltaF508 CFTR allele, suggesting heterozygous CFTR carriers may have increased resistance to typhoid fever. The identification of CFTR as a receptor for bacterial pathogens could underlie the biology of CF lung disease and be the basis for the heterozygote advantage for carriers of mutant alleles of CFTR.

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133 Recent studies in transgenic CF mice with a stop mutation in Cftr (48), a ⌬F508 Cftr (49) allele, or a G551D Cftr allele (50) have confirmed the association between decreased lung cell internalization of P. aeruginosa in CF mice and increased bacterial burdens in lung tissue (unpublished data). Login to comment
134 The fact that transgenic Cftr knockout and G551D mice, both representative of alleles in humans with severe CF, have the same phenotype as ⌬F508 Cftr mice indicates that the defect in epithelial cell uptake of P. aeruginosa is not restricted to one allelic variant of Cftr. 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] 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
29 CFTR mutants that move successfully through the cell to the plasma membrane but are defective in cAMP regulation of chloride transport (e.g. G551D) are called class III mutations. Login to comment
72 Laboratory investigations have demonstrated in vitro activity in the form of increased production of mature CFTR and chloride transport at the cell surface for both wild type, 'F508, and G551D [43-46]. Login to comment
86 Genistein has also been shown to restore function of the class III mutation G551D [46]. Login to comment
96 Milrinone appears to be more successful in mouse nasal mucosa than human mucosa with respect to 'F508 and G551D defects. Login to comment
100 Whereas G551D is severely defective, G551S is a milder mutant. Login to comment
106 G551D has been shown to respond to the flavonoid compounds as mentioned above. Login to comment
107 ATP binding to G551D is severely affected, but overcome by interaction with the flavonoids. Login to comment
109 CF subjects bearing at least one G551D allele underwent nasal potential difference testing in which genistein was superfused following isoproterenol activation of CFTR. Login to comment
111 This degree of chloride transport (17% of that observed in normal subjects) might be augmented overall by increasing the level of G551D expression in nasal mucosa. 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] Nucleotide-binding domain 1 of cystic fibrosis tra... Eur J Biochem. 2000 Sep;267(17):5306-12. Duffieux F, Annereau JP, Boucher J, Miclet E, Pamlard O, Schneider M, Stoven V, Lallemand JY
Nucleotide-binding domain 1 of cystic fibrosis transmembrane conductance regulator production of a suitable protein for structural studies.
Eur J Biochem. 2000 Sep;267(17):5306-12., [PMID:10951189]

Abstract [show]
Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). This protein belongs to the large ATP-binding cassette (ABC) family of transporters. Most patients with cystic fibrosis bear a mutation in the nucleotide-binding domain 1 (NBD1) of CFTR, which plays a key role in the activation of the channel function of CFTR. Determination of the three dimensional structure of NBD1 is essential to better understand its structure-function relationship, and relate it to the biological features of CFTR. In this paper, we report the first preparation of recombinant His-tagged NBD1, as a soluble, stable and isolated domain. The method avoids the use of renaturing processes or fusion constructs. ATPase activity assays show that the recombinant domain is functional. Using tryptophan intrinsic fluorescence, we point out that the local conformation, in the region of the most frequent mutation DeltaF508, could differ from that of the nucleotide-binding subunit of histidine permease, the only available ABC structure. We have undertaken three dimensional structure determination of NBD1, and the first two dimensional 15N-1H NMR spectra demonstrate that the domain is folded. The method should be applicable to the structural studies of NBD2 or of other NBDs from different ABC proteins of major biological interest, such as multidrug resistance protein 1 or multidrug resistance associated protein 1.

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340 & Cohn, J.A. (2000) ATP hydrolysis by a CFTR domain: pharmacology and effects of G551D mutation. 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
27 The following genotypes were identi®ed: DF508/ DF508 (n=3); DF508/G551D (n=1); DF508/7(n=2);7/7 (n=2) (7=mutation not identi®ed). 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
224 In the present study we examined the eect of genistein on tissues derived from CF patients carrying DF508 CFTR on at least one allele and some patients were shown to be compound heterozygous with a second severe mutation (R553X, N1303K, 3905insT, G551D). 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] Regulation of the epithelial sodium channel (ENaC)... Curr Opin Nephrol Hypertens. 2000 Sep;9(5):529-34. Rotin D
Regulation of the epithelial sodium channel (ENaC) by accessory proteins.
Curr Opin Nephrol Hypertens. 2000 Sep;9(5):529-34., [PMID:10990373]

Abstract [show]
The epithelial sodium channel (ENaC) plays a key role in the regulation of fluid absorption in the kidney, lung, colon and exocrine glands, and in the regulation of blood pressure. Abnormal functioning of ENaC is associated with several human diseases, including pseudohypoaldosteronism type I, Liddle's syndrome, pulmonary edema, and cystic fibrosis. ENaC is regulated by several hormones, ions and accessory proteins. This review focuses on the regulation of ENaC by recently described accessory proteins, mainly Nedd4, syntaxin 1A, CFTR, sgk, K-Ras2A and Cap-1.

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53 provided support for this theory, and, moreover, demonstrated a lack of inhibition of ENaC by mutant CFTR (DF508, G551D) [11,49]. 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
89 This consisted of the primer from one locus attached to the probe of another locus. We used the cystic fibrosis loci ∆F508 and G551D (12). Login to comment
90 The probe sequence for ∆F508 was attached to the G551D forwards primer (Table 2, 551F). Login to comment
94 (i) HS, G551D unlabelled lower primer (Table 2, 551R) and two unlabelled ∆F508 primers without a probe sequence attached (Table 2, 508F and 508R), were added to the PCR reactions (Fig. 2, test 1). Login to comment
95 In this case the probe is unable to bind to the extension of the Scorpion primer, which contains the G551D rather than ∆F508 target site. Login to comment
97 Therefore the Scorpion can only act as a Molecular Beacon although it will have the G551D PCR product attached. Login to comment
99 Again the Scorpion can only act as a Molecular Beacon but without the G551D PCR product tail attached (Fig. 2, test 2). Login to comment
151 The G551D locus (Fig. 6E) gave the worst discrimination of all five Scorpions. 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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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

[hide] Expression of nucleotide-regulated Cl(-) currents ... Am J Physiol Cell Physiol. 2000 Nov;279(5):C1578-86. Thomas EJ, Gabriel SE, Makhlina M, Hardy SP, Lethem MI
Expression of nucleotide-regulated Cl(-) currents in CF and normal mouse tracheal epithelial cell lines.
Am J Physiol Cell Physiol. 2000 Nov;279(5):C1578-86., [PMID:11029305]

Abstract [show]
The dominant route for Cl(-) secretion in mouse tracheal epithelium is via Cl(-) channels different from the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the channel that is defective in CF. It has been proposed that the use of purinergic agonists to activate these alternative channels in human airways may be beneficial in CF. In the present study, two conditionally immortal epithelial cell lines were established from the tracheae of mice possessing the tsA58 T antigen gene, one of which [MTE18-(-/-)] was homozygous for a knockout of CFTR and the other [MTE7b-(+/-)] heterozygous for CFTR expression. In Ussing chamber studies, amiloride (10(-4) M) and a cocktail of cAMP-activating agents (forskolin, IBMX, and dibutyryl cAMP) resulted in small changes in the short-circuit current (I(sc)) and resistance of both cell lines, with larger increases in I(sc) being elicited by ionomycin (10(-6) M). Both cell lines expressed P(2)Y(2) receptors and responded to the purinergic agonists ATP, UTP, and 5'-adenylylimidodiphosphate (10(-4) M) with an increase in I(sc). This response could be inhibited by DIDS and was abolished in the presence of Cl(-)-free Ringer solution. Reducing the mucosal Cl(-) concentration increased the response to UTP of both cell lines, with a significantly greater increase in MTE18-(-/-) cells. Pretreatment of these cells with thapsigargin caused a direct increase in I(sc) and inhibited the response to UTP. These data suggest that both cell lines express purinergic-regulated Cl(-) currents and may prove valuable tools in studying the properties of this pathway.

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20 More recently, several transgenic mouse models of CF have been generated incorporating a knockout of CFTR or one of the CFTR mutations more commonly found in human populations (i.e., ⌬F508 or G551D) (12, 14, 15, 29, 33, 37, 39). Login to comment

[hide] Role of cystic fibrosis transmembrane conductance ... J Immunol. 2000 Oct 1;165(7):3941-50. Chroneos ZC, Wert SE, Livingston JL, Hassett DJ, Whitsett JA
Role of cystic fibrosis transmembrane conductance regulator in pulmonary clearance of Pseudomonas aeruginosa in vivo.
J Immunol. 2000 Oct 1;165(7):3941-50., 2000-10-01 [PMID:11034402]

Abstract [show]
Cystic fibrosis (CF)2 is a fatal genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) that is commonly associated with chronic pulmonary infections with mucoid Pseudomonas aeruginosa (PA). To test the hypothesis that CFTR plays a direct role in PA adhesion and clearance, we have used mouse lines expressing varying levels of human (h) or mouse (m) CFTR. A subacute intratracheal dose of 3 x 10(6) bacteria was cleared with similar kinetics in control wild-type (WT) and transgenic mice overexpressing hCFTR in the lung from the surfactant protein C (SP-C) promoter (SP-C-hCFTR+/-). In a second series of experiments, the clearance of an acute intratracheal dose of 1.5 x 10(7) PA bacteria was also similar in WT, hemizygous SP-C-hCFTR+/-, and bitransgenic gut-corrected FABP-hCFTR+/+-mCFTR-/-, the latter lacking expression of mCFTR in the lung. However, a small but significant decrease in bacterial killing was observed in lungs of homozygote SP-C-hCFTR+/+ mice. Lung pathology in both WT and SP-C-hCFTR+/+ mice was marked by neutrophilic inflammation and bacterial invasion of perivascular and subepithelial compartments. Bacteria were associated primarily with leukocytes and were not associated with alveolar type II or bronchiolar epithelial cells, the cellular sites of SP-C-hCFTR+/+ transgene expression. The results indicate that there is no direct correlation between levels of CFTR expression and bacterial clearance or association of bacteria with epithelial cells in vivo.

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246 Similar to our findings with the FABP-hCFTRϩ/ϩ -mCFTR-/- mice, the study of Thomas et al. (55) demonstrated abnormal LPS-induced inflammatory responses by macrophages in the lungs of a mouse with the G551D CFTR mutation. Login to comment

[hide] Cystic fibrosis in infertility: screening before a... Hum Reprod. 2000 Nov;15(11):2415-7. Lewis-Jones DI, Gazvani MR, Mountford R
Cystic fibrosis in infertility: screening before assisted reproduction: opinion.
Hum Reprod. 2000 Nov;15(11):2415-7., [PMID:11056144]

Abstract [show]
Cystic fibrosis (CF) is the most common autosomal recessive disease in Caucasians. In 97-98% of men with CF, bilateral congenital absence of the vas deferens (CBAVD) blocks the transport of spermatozoa resulting in azoospermia. Abnormalities in sperm parameters have also been identified in males with CF. To date, over 800 disease-causing mutations of the CF transmembrane conductance regulator (CFTR) gene have been identified (also called ABCC7). Current legislation suggests that prior to intracytoplasmic sperm injection (ICSI) treatment, men with CBAVD or unexplained oligozoospermia should be considered for screening. If the male is negative with routine screening then the female partner is not screened. This is fundamentally wrong because if the female is screened and is found to be CF positive on routine testing, her partner would then need the fullest possible investigation of the CFTR gene. It is ideal to screen both partners in cases of oligozoospermia. However, if the resources are stretched, then only the female needs to be routinely screened because if she is negative, then the couple's residual risk of having a CF or CBAVD child will be reduced to 1:960. Only when the female is found to be a carrier does the male partner need routine screening followed by full testing for known mutations.

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68 (1996) Mutations in the cystic fibrosis gene G551D 621ϩ1G→T 2789ϩ5G→A in men with congenital bilateral absence of the vas deferens. Login to comment

[hide] Gene therapy for cystic fibrosis. Expert Opin Investig Drugs. 2000 Jul;9(7):1523-35. Alton E, Kitson C
Gene therapy for cystic fibrosis.
Expert Opin Investig Drugs. 2000 Jul;9(7):1523-35., [PMID:11060757]

Abstract [show]
The gene for cystic fibrosis was identified in 1989 and this together with the emerging technology of gene therapy heralded a new dawn for the treatment of genetic disease. The initial optimism however gave way to the realisation that gene therapy for cystic fibrosis was unlikely to be straightforward. The lung was considered an ideal organ to target due to ease of access, but subsequent research has shown that the airway surface provides an efficient barrier to topically applied gene transfer agents. A number of Phase I clinical safety trials were carried out through the 1990s and provided proof of concept evidence that delivery of DNA by either viral or non-viral means was safe though not clinically efficacious. Current research is now focusing more on the barriers faced by delivery agents, with the aim that more efficient gene delivery will lead to a gene therapeutic for cystic fibrosis.

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205 Finally, a CF mouse carrying a rare mutation, G551D, has been produced [56], where the CFTR protein traffics normally to the apical surface but is non-functional [57]. Login to comment
400 DELANEY SJ, ALTON EW, SMITH SN et al.: Cystic fibrosis mice carrying the missense mutation G551D replicate human genotype-phenotype correlations. Login to comment
402 ILLEK B, ZHANG L, LEWIS NC, MOSS RB, DONG JY, FISCHER H: Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein. 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

[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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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
89 G551D is localized at the cell surface but exhibits impaired ATP binding and does not conduct chloride in response to elevated cAMP [71,72]. Login to comment
90 G551D is typically associated with pancreatic insufficiency and a severe phenotype. Login to comment
97 In a small study of five CF patients carrying at least one G551D mutation, perfusion of the nasal mucosa with genistein stimulated chloride-dependent NPD to an average of 16.9% of the responses found in healthy subjects [77••]. Login to comment
113 Although a recent study failed to demonstrate a significant effect of milrinone on chloride secretion in both ∆F508 and G551D CF patients [83], phosphodiesterase inhibitors such as rolipram, papaverine, and IBMX (3-isobutyl-1- methyxanthine) may still be beneficial for less stringent mutations. Login to comment
117 Adenosine combined with rolipram or papaverine can also activate G551D CFTR measured by halide efflux to approximately 30% of wild-type activity [86]. 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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55 Chromosomes were first tested for the presence of the L375F/G551D). 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
78 12 G551D/- (TG)10T9/(TG)13T5 These three novel mutations have not been found in more 18 2183AA→G/- (TG)10T7/(TG)12T5 than 200 non-CF chromosomes and in a sample of 300 CF24 R1162X/- (TG)10T9/(TG)12T5 chromosomes from local classical CF patients, nor were theyOne mutation detected without T5 allele (3/47 ϭ 6.4%) reported by any other member of the CF Genetic Analysis30 ∆F508/- (TG)10T9/(TG)10T7 34 R117H/- (TG)12T7/(TG)10T7 Consortium. Login to comment
80 No mutation detected with one T5 allele (2/47 ϭ 4.3%) A rare missense mutation, L375F, first described elsewhere 41 -/- (TG)11T5/(TG)11T7 (Je´ze´quel et al., 1996) and located in exon 8 which codes for11 -/- (TG)11T5/(TG)11T7 a cytoplasmic region between the m6 transmembrane domainNo mutation detected (12/47 ϭ 25.5%) and NBD1, was found twice, once associated with G551D in4 -/- nd 44 -/- (TG)12T7/(TG)10T7 a CUAVD phenotype (no. 40) surgically explored and once 19 -/- (TG)11T7/(TG)11T7 with R117H in a CBAVD phenotype (no. 25) clinically45 -/- (TG)11T7/(TG)10T7 diagnosed in a 30-year-old man with a normal sweat test21 -/- (TG)11T7/(TG)11T7 27 -/- (TG)11T7/(TG)10T7 (17 mEq/l) and asthma. Login to comment

[hide] Genotype analysis and phenotypic manifestations of... Chest. 2000 Dec;118(6):1591-7. Desmarquest P, Feldmann D, Tamalat A, Boule M, Fauroux B, Tournier G, Clement A
Genotype analysis and phenotypic manifestations of children with intermediate sweat chloride test results.
Chest. 2000 Dec;118(6):1591-7., [PMID:11115444]

Abstract [show]
STUDY OBJECTIVES: Cystic fibrosis (CF) is one of the most common inherited diseases among whites. Since the cloning of the CF transmembrane conductance regulator (CFTR) gene, a number of studies have focused on associations between the genotype and phenotype in CF. This had led to the progressive identification of new groups of patients, including those who have mild lung disease and those who have normal sweat chloride values (< 60 mEq/L). The aim of the present work was to provide information on the genotype and the phenotypic characteristics of children with intermediate-range sweat chloride test results. PATIENTS AND RESULTS: We focused on children referred to the pulmonary department for various types of pulmonary disease and who had several sweat chloride test results with median values in the range of 40 to 60 mEq/L. Twenty-four patients over a 10-year period were enrolled (mean age, 4.8 years). Respiratory manifestations at initial evaluation included recurrent bronchitis, wheezing, chronic cough, and pneumonia. The duration of the follow-up ranged from 0.5 to 10.5 years. Sputum cultures revealed the presence of Haemophilus influenzae (10 children), Staphylococcus aureus (4 children), and Pseudomonas aeruginosa (3 children). Pancreatic insufficiency was found in two patients. Analysis of the entire coding sequence allowed identification of 16 known mutations in CFTR gene. Fifteen chromosomes (31.2%) carried a mutation in CFTR gene and one allele carried two mutations. Three patients were homozygous or double heterozygous (DeltaF508/DeltaF508, DeltaF508/3849 + 10 kb C-->T, S1235R/G551D). The 5-thymidine allele was identified in four children. CONCLUSION: These results indicate an higher frequency of CFTR gene mutations in patients with borderline sweat chloride test results, compared to data reported in the general population. They lead to the recommendations for complete pulmonary and GI investigations in this group of patients, as well as assiduous care and medical follow-up.

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16 Three patients were homozygous or double heterozygous (⌬F508/⌬F508, ⌬F508/3849 ؉ 10 kb C3T, S1235R/G551D). Login to comment
81 One patient carried homozygous ⌬F508, and two patients carried compound heterozygous (⌬F508/3849 ϩ 10 kb C3T, S1235R/G551D). Login to comment
92 Genotype Poly T 1 -/- 7T/7T 2 R117C/- 7T/7T 3 R75X-D1270H/- 7T/7T 4 -/- 7T/7T 5 G91R/- 7T/5T 6 ⌬F508/- 7T/9T 7 -/- 7T/7T 8 -/- 7T/7T 9 S1235R/G551D 5T/7T 10 ⌬F508/- 9T/9T 11 7T/7T 12 ⌬F508/⌬F508 9T/9T 13 ⌬F508/- 7T/9T 14 -/- 7T/7T 15 ⌬F508/- 7T/9T 16 -/- 7T/5T 17 -/- 7T/7T 18 -/- 7T/7T 19 -/- 7T/9T 20 ⌬F508/- 7T/9T 21 -/- 7T/7T 22 W1282X/- 7T/5T 23 -/- 7T/7T 24 ⌬F508/3849 ϩ 10 kb C 3 T 7T/7T 1594 Clinical Investigations reported in the general population (frequency of the 5T allele in the general population, 5.2%).26 Based on the results of DNA analysis and according to the consensus statement on the diagnosis of CF, three patients (patients 9, 12, and 24) met the criteria of both respiratory manifestations and identification of two CF mutations.21 For patient 6, there was a diagnostic dilemma. Login to comment
99 It is interesting to point out that two patients were compound heterozygous (⌬F508/ 3849 ϩ 10 kb C3T, S1235R/G551D), and one was homozygous ⌬F508. 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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125 Yang et al. found that high concentrations of IBMX (4 mM) could induce chloride secretion in cells with the G551D-CFTR mutation, but only in 20% of cells with the ∆F508 mutation [46]. Login to comment
182 On the other hand, genistein activates the trafficking-competent G551D-CFTR mutant without pretreatment with 4PBA [34]. Login to comment
194 Also, a variety of other phosphatase inhibitors, such as levamisole and bromotetramisole [98-100], cyclosporin A and deltamethrin [101] have been shown to activate wild-type and mutated (G551D and ∆F508) CFTR. Login to comment
196 It has also been claimed that class III phosphodiesterase inhibitors such as milrinone could activate ∆F508-CFTR [102], but according to a recent study on nasal epithelia of ∆F508 or G551D mice, as well as on nasal epithelia of non-CF controls and CF patients, milrinone is without significant effect on chloride efflux in these tissues [103]. Login to comment
410 34. ILLEK B, ZHANG L, LEWIS NC, MOSS RB, DONG JY, FISCHER H: Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein. Am. J. Physiol. Login to comment

[hide] Perturbation of the pore of the cystic fibrosis tr... J Biol Chem. 2001 Apr 13;276(15):11575-81. Epub 2000 Dec 21. Kogan I, Ramjeesingh M, Huan LJ, Wang Y, Bear CE
Perturbation of the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibits its atpase activity.
J Biol Chem. 2001 Apr 13;276(15):11575-81. Epub 2000 Dec 21., 2001-04-13 [PMID:11124965]

Abstract [show]
Mutations in the cystic fibrosis gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) lead to altered chloride (Cl(-)) flux in affected epithelial tissues. CFTR is a Cl(-) channel that is regulated by phosphorylation, nucleotide binding, and hydrolysis. However, the molecular basis for the functional regulation of wild type and mutant CFTR remains poorly understood. CFTR possesses two nucleotide binding domains, a phosphorylation-dependent regulatory domain, and two transmembrane domains that comprise the pore through which Cl(-) permeates. Mutations of residues lining the channel pore (e.g. R347D) are typically thought to cause disease by altering the interaction of Cl(-) with the pore. However, in the present study we show that the R347D mutation and diphenylamine-2-carboxylate (an open pore inhibitor) also inhibit CFTR ATPase activity, revealing a novel mechanism for cross-talk from the pore to the catalytic domains. In both cases, the reduction in ATPase correlates with a decrease in nucleotide turnover rather than affinity. Finally, we demonstrate that glutathione (GSH) inhibits CFTR ATPase and that this inhibition is altered in the CFTR-R347D variant. These findings suggest that cross-talk between the pore and nucleotide binding domains of CFTR may be important in the in vivo regulation of CFTR in health and disease.

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128 This reduced ATPase activity is comparable with that obtained for mutations of conserved residues thought to reside in the nucleotide binding pocket in the NBDs (e.g. G551D; (18)). Login to comment
20 Moreover, the disease-causing mutation in the conserved Walker C motif of the first NBD of CFTR, i.e. CFTR- G551D, impaired both ATPase activity and the rate of channel opening (18). 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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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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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
176 Stop mutations account for about 7% of the patients at this center, and some of these mutations may be amenable to correction by encouraging read-through with agents such as aminoglycosides.21,22 The mutation G551D, which interferes with efficient adenosine triphosphate gating of CFTR, accounts for ϳ4% of our patients, and experiments indicate that the activity of G551D-CFTR can be increased with the isoflavone genistein.23 Whereas these kinds of mutations are relatively rare, mutations that lead to improper folding of CFTR, such as ⌬F508, are responsible for the great majority of CF.24 The molecular basis for these processes is under intense investigation,25 and strategies for inducing an increased proportion of properly folded, functional molecules at the plasma membrane have achieved success in vitro26-28. Login to comment

[hide] Assessment of CFTR chloride channel openers in int... Br J Pharmacol. 2001 Feb;132(3):659-68. Cuthbert AW
Assessment of CFTR chloride channel openers in intact normal and cystic fibrosis murine epithelia.
Br J Pharmacol. 2001 Feb;132(3):659-68., [PMID:11159718]

Abstract [show]
1. A method is described for the detection of CFTR chloride channel openers (ClCOs) and blockers. Murine colonic epithelia were used throughout, but the method is applicable to other epithelia and biopsy material. 2. The principle was to render the epithelial basolateral membranes electrically transparent so that the apical membrane alone could be voltage clamped. This was achieved by potassium depolarization on the basolateral side. Imposition of an apical to basolateral chloride gradient allowed the effects of ClCOs on an outward chloride current and on apical membrane conductance to be measured. 3. 1-ethyl-2-benzimidazolone (EBIO), forskolin, chlorzoxazone, and genistein all showed ClCO activity. In cystic fibrosis (CF) epithelia, either from CF null or CF Delta F508 mice, EBIO showed only a minor effect, indicating that CFTR was the target in wild type tissues. 4. 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) was shown to block CFTR chloride channels. The blockade was pH and voltage-dependent and indicated that while the charged form was the active moiety, movement into the cell depended on the unionized drug. It is concluded NPPB blocks CFTR from the cytosolic side and that the agent preferentially blocks at potentials opposing the inflow of chloride ions. No significant blockade was seen with either N-phenylanthranilic acid (DPC) or with glibenclamide, under standard conditions. 5. The method described can be used to examine compounds reported to increase the trafficking of Delta F508 CFTR to the membrane or those capable of opening Delta F508 CFTR chloride channels and to differentiate between them. Further, the method distinguishes between chloride channel openers and those acting indirectly to increase the flux through CFTR chloride channels by indirect means, for example, hyperpolarization.

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37 Futhermore, agents which increase the tracking of DF508 CFTR or G551D CFTR to the membrane can be revealed by agents, such as genistein (Hwang et al., 1997), which activates the mutant channels. 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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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
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] Aberrant CFTR-dependent HCO3- transport in mutatio... Nature. 2001 Mar 1;410(6824):94-7. Choi JY, Muallem D, Kiselyov K, Lee MG, Thomas PJ, Muallem S
Aberrant CFTR-dependent HCO3- transport in mutations associated with cystic fibrosis.
Nature. 2001 Mar 1;410(6824):94-7., 2001-03-01 [PMID:11242048]

Abstract [show]
Cystic fibrosis (CF) is a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). Initially, Cl- conductance in the sweat duct was discovered to be impaired in CF, a finding that has been extended to all CFTR-expressing cells. Subsequent cloning of the gene showed that CFTR functions as a cyclic-AMP-regulated Cl- channel; and some CF-causing mutations inhibit CFTR Cl- channel activity. The identification of additional CF-causing mutants with normal Cl- channel activity indicates, however, that other CFTR-dependent processes contribute to the disease. Indeed, CFTR regulates other transporters, including Cl(-)-coupled HCO3- transport. Alkaline fluids are secreted by normal tissues, whereas acidic fluids are secreted by mutant CFTR-expressing tissues, indicating the importance of this activity. HCO3- and pH affect mucin viscosity and bacterial binding. We have examined Cl(-)-coupled HCO3- transport by CFTR mutants that retain substantial or normal Cl- channel activity. Here we show that mutants reported to be associated with CF with pancreatic insufficiency do not support HCO3- transport, and those associated with pancreatic sufficiency show reduced HCO3- transport. Our findings demonstrate the importance of HCO3- transport in the function of secretory epithelia and in CF.

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50 Two sets of particularly interesting mutants are G551D and G551S and H620Q and A800G. Login to comment
52 As reported previously17,18,26 , G551D reduced Cl-transport to 53% of the control value. Login to comment
66 Notably, although a few of these mutants exhibit altered letters to nature NATURE |VOL 410 |1 MARCH 2001 |www.nature.com 95 NO3 - Forskolin 5 µM NO3 - Forskolin 5 µM a G551S b G551D 10mMCl- 200 s NO3 - NO3 - Forskolin 5 µM Forskolin 5 µM f H620Q g A800G h H620Q Forskolin 5 µMCl- free Cl- freeCl- free Cl-free c G551S d G551D Forskolin 5 µM Forskolin 5 µM 0.25pHunits 250 s e A800G 0 0.25 0.50 0.75 1.00 H620Q A800G G551D G551S j WT Forskolin 5 µM 0 0.25 0.50 0.75 1.00 H620Q A800G G551D G551S WT i [Cl- ]change(mMs-1 )HCO3 -transport (∆pH+ min-1 ) Figure 2 cAMP-stimulated Cl- and HCO3 transport by CFTR mutants associated with a severe or a mild form of CF. Login to comment
155 Illek, B. et al. Defective function of the cystic ®brosis-causing missense mutation G551D is recovered by genistein. Login to comment
186 letters to nature 96 NATURE |VOL 410 |1 MARCH 2001 |www.nature.com HCO3 -/Cl- transportratio 0 0.25 0.50 0.75 1.00 WT I148T G178R R297Q G551D H620Q G970R A1067T G1244E S1255P G1349D E193K G551S A800G H949Y R1070Q Pancreatic insufficient Pancreatic sufficientD648V N CI148T G178R E193K R297Q R117H A1067T R1070Q G1244E S1255P G1349D NBD2 RD H949Y G970R CL4CL3CL2CL1 NBD1 G551D G551S H620Q D648V A800G Figure 3 The HCO3:Cl-transport ratio of CFTR mutants associated with CF. 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
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

[hide] Novel CFTR chloride channel activators identified ... J Biol Chem. 2001 Jun 8;276(23):19723-8. Epub 2001 Mar 21. Galietta LJ, Springsteel MF, Eda M, Niedzinski EJ, By K, Haddadin MJ, Kurth MJ, Nantz MH, Verkman AS
Novel CFTR chloride channel activators identified by screening of combinatorial libraries based on flavone and benzoquinolizinium lead compounds.
J Biol Chem. 2001 Jun 8;276(23):19723-8. Epub 2001 Mar 21., 2001-06-08 [PMID:11262417]

Abstract [show]
The flavonoid genistein and the benzo[c]quinolizinium MPB-07 have been shown to activate the cystic fibrosis transmembrane conductance regulator (CFTR), the protein that is defective in cystic fibrosis. Lead-based combinatorial and parallel synthesis yielded 223 flavonoid, quinolizinium, and related heterocyclic compounds. The compounds were screened for their ability to activate CFTR at 50 microm concentration by measurement of the kinetics of iodide influx in Fisher rat thyroid cells expressing wild-type or G551D CFTR together with the green fluorescent protein-based halide indicator YFP-H148Q. Duplicate screenings revealed that 204 compounds did not significantly affect CFTR function. Compounds of the 7,8-benzoflavone class, which are structurally intermediate between flavones and benzo[c]quinoliziniums, were effective CFTR activators with the most potent being 2-(4-pyridinium)benzo[h]4H-chromen-4-one bisulfate (UCcf-029). Compounds of the novel structural class of fused pyrazolo heterocycles were also strong CFTR activators with the most potent being 3-(3-butynyl)-5-methoxy-1-phenylpyrazole-4-carbaldehyde (UCcf-180). A CFTR inhibitor was also identified. The active compounds did not induce iodide influx in null cells deficient in CFTR. Short-circuit current measurements showed that the CFTR activators identified by screening induced strong anion currents in the transfected cell monolayers grown on porous supports. Compared with genistein, the most active compounds had up to 10 times greater potency in activating wild-type and/or G551D-CFTR. The activators had low cellular toxicity and did not elevate cellular cAMP concentration or inhibit phosphatase activity, suggesting that CFTR activation may involve a direct interaction. These results establish an efficient screening procedure to identify CFTR activators and inhibitors and have identified 7,8-benzoflavones and pyrazolo derivatives as novel classes of CFTR activators.

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2 The compounds were screened for their ability to activate CFTR at 50 ␮M concentration by measurement of the kinetics of iodide influx in Fisher rat thyroid cells expressing wild-type or G551D CFTR together with the green fluorescent protein-based halide indicator YFP-H148Q. Login to comment
9 Compared with genistein, the most active compounds had up to 10 times greater potency in activating wild-type and/or G551D-CFTR. Login to comment
36 23, Issue of June 8, pp. 19723-19728, 2001 (c) 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. This paper is available on line at http://www.jbc.org 19723 atUniversityofNorthCarolinaatChapelHill,onAugust8,2011www.jbc.orgDownloadedfrom http://www.jbc.org/content/suppl/2001/06/07/276.23.19723.DC1.html Supplemental Material can be found at: compounds for CFTR-activating potency by a cell-based halide transport assay that utilized fluorescent epithelial cells stably expressing wild-type or G551D CFTR together with the green fluorescent protein halide indicator YFP-H148Q. Login to comment
56 Cell Culture and Transfection-Fischer rat thyroid (FRT) cells expressing the human wild-type CFTR or CFTR-G551D were transfected with the plasmid pcDNA3.1 (Invitrogen) containing the cDNA encoding YFP-H148Q and selected in G418 (0.75 mg/ml). Login to comment
84 Short-circuit Current Measurements-FRT cells stably expressing human wild-type or G551D CFTR were cultured on Snapwell inserts (Costar) at a density of 500,000 cells/insert. Login to comment
105 FRT cells coexpressing human wild-type CFTR or G551D CFTR (which causes CF) and YFP-H148Q were cultured on 96-well plates for the monitoring of YFP fluorescence in a plate reader. Login to comment
110 Neither activation of G551D CFTR nor of null cells that do not express CFTR was observed under these conditions (data not shown). Login to comment
113 Screening for activators of wild-type and G551D CFTR. Login to comment
116 Right panel, for G551D CFTR, the addition of a test compound can have no effect (heavy line) or can activate forskolin-independent or -dependent I- transport. Login to comment
120 C, a screening of FRT cells expressing G551D CFTR showing I- transport after the addition of 5 ␮M forskolin. Login to comment
132 A full screen was also carried out in FRT cells expressing G551D CFTR. Login to comment
133 Halide transport by G551D CFTR is not increased by cAMP elevation alone. Login to comment
137 Many of the same compounds that activated wild-type CFTR also activated G551D CFTR, although relative activating potencies differed, and a few compounds activated preferentially wild-type CFTR (e.g. UCCF-180) or G551D CFTR (e.g. UCCF-023 and UCCF-030). Login to comment
145 Fig. 3B shows a similar comparison for the activation of G551D CFTR. Login to comment
147 Short-circuit current was measured in polarized monolayers of FRT cells expressing wild-type or G551D CFTR. Login to comment
159 Representative fluorescence curves are shown for FRT cells expressing wild-type CFTR (A) and G551D CFTR (B). Login to comment
164 Fig. 4B shows short-circuit current analysis of G551D CFTR activation by genistein versus UCCF-030. Login to comment
166 Interestingly, the activator of wild-type CFTR, UCCF-180, was ineffective in activating G551D CFTR. Login to comment
167 The compounds UCCF-023, UCCF-027, and UCCF-028 were also effective in inducing short-circuit current in cells expressing G551D CFTR (data not shown). Login to comment
170 All compounds with activating potency on wild-type and/or G551D CFTR were nontoxic (cell growth Ͼ90% of control) except for UCCF-027, which was similar to genistein. Login to comment
185 Short-circuit current (Isc) measurements FRT cells expressing wild-type CFTR (A) and G551D CFTR (B). Login to comment
196 The CFTR activators did not induce Cl- currents in null cells, and short-circuit current analysis showed that they had activating potencies for wild-type and G551D CFTR that were substantially better than the existing lead compounds. Login to comment
199 Interestingly, the order of activating potencies of the CFTR activators on wild-type versus G551D CFTR was different. Login to comment
200 For example, UCCF-029 was the most potent compound in activating wild-type CFTR but had less effect on G551D CFTR. Login to comment
201 In contrast, UCCF-023, UCCF-028, and UCCF-030, which were substantially less potent than UCCF-029 in activating wild-type CFTR, were very potent in activating G551D CFTR in synergy with forskolin. Login to comment

[hide] Disease-associated mutations in the extracytoplasm... J Biol Chem. 2001 May 4;276(18):14848-54. Epub 2001 Feb 6. Hammerle MM, Aleksandrov AA, Riordan JR
Disease-associated mutations in the extracytoplasmic loops of cystic fibrosis transmembrane conductance regulator do not impede biosynthetic processing but impair chloride channel stability.
J Biol Chem. 2001 May 4;276(18):14848-54. Epub 2001 Feb 6., 2001-05-04 [PMID:11278813]

Abstract [show]
Consistent with its function as a chloride channel regulated entirely from the cytoplasmic side of the plasma membrane, the cystic fibrosis transmembrane conductance regulator (CFTR) glycoprotein exposes little of its mass on the exterior surface of cells. The first and fourth extracytoplasmic loops (ELs) contain approximately 15 and 30 residues, respectively; the other four ELs are extremely short. To examine the influence of missense mutants in ELs detected in patients with cystic fibrosis, we have expressed them in mammalian (baby hamster kidney (BHK21)) cells and assessed their biosynthetic processing and chloride channel activity. In contrast to previous findings that 18 of 30 disease-associated missense mutations in cytoplasmic loops caused retention of the nascent polypeptides in the endoplasmic reticulum, all the EL mutants studied matured and were transported to the cell surface. This pronounced asymmetry is consistent with the notion that endoplasmic reticulum quality control of nascent CFTR is exerted primarily on the cytoplasmic side of the membrane. Although this set of EL mutations has little effect on CFTR maturation, most of them seriously compromise its chloride channel activity. Substitutions at six different positions in EL1 and single positions in EL2 and EL4 all destabilized the open state, some of them severely, indicating that the ELs contribute to the stability of the CFTR ion pore.

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171 For example a nucleotide binding domain mutation, G551D, precludes virtually all TABLE II Relative charge transport capacity of mutants Mutants S108F Y109C D110H P111L P111A E116K R117H R117C R117L R117P E217G T908N P1013L Imutant/Iwt 100% 11 15 27 173 105 12 80 27 5 11 10 48 170 FIG. 5. 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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12 Cos-7 cell transfection with plasmids expressing DF508 or G551D mutant CFTR protein resulted in increased activation of a p50-containing NF-kB complex, but IL-8 secretion was similar to wild-type cells. Login to comment
110 Transfections The 6.2 kb wild-type, DF508 and G551D CFTR complementary deoxyribonucleic acids (cDNAs) were cloned into the Not I site of the eukaryotic expression vector pCMVb (Clontech Laboratories UK Ltd, Basingstoke, UK), from which the b-galactosidase reporter gene was removed through Not I digestion. Login to comment
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
166 The effects of transfection with wild-type, DF508 and G551D cystic ®brosis transmembrane conductance regulator on nuclear factor-kB activity and interleukin-8 secretion Cos-7 cells transfected with either DF508 or G551D CFTR plasmids, exhibited a signi®cant increase in p50-containing complex II when compared to wild-type transfectants (n=10, p<0.01) (®g. 7). Login to comment
175 Overexpression of DF508 and G551D mutant CFTR increased p50 activity in Cos-7 cells when compared to overexpression of wild-type CFTR; however, this increase was not accompanied by an increase in IL-8 secretion. Login to comment
224 In an attempt to further determine the effect of ER overload, the present study transiently overexpressed wild-type, DF508 or G551D CFTR protein in Cos-7 cells. Login to comment
226 A signi®cant increase in a p50 containing protein/DNA complex (complex II) was detected after DF508 or G551D overexpression when compared to overexpression of the wild-type protein. 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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65 Non DF 508 alleles in the two groups were: 1237A group: G551D (3); N1303K (2); R117H (1); R560T (1); Unknown (3). Login to comment
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
70 For subjects heterozygous for DF508, the second allele was matched as closely as possible and included the following: G551D, N1303K, R117H and R560T. Login to comment
81 infective exacerbations over 2 years 4.7+0.7 2.8+0.6 0.03 over 4 yearsb 10.5+1.8 4.5+1.1 0.006 FEV1 % predicted 55.5+7.4 67.5+5.5 NS at reference visit 2 years post 53.1+8.5 68.4+5.5 (n=14) 0.09 (NS) reference visitb a Non DF 508 alleles in the two groups were: 1237A group: G551D (3); R117H (1); R560T (1); N1303K (2); Unknown (3); 1237G group: G551D (4); R117H (1); R560T (1); 621+G4T (1); Unknown (3). Login to comment

[hide] ATP hydrolysis-coupled gating of CFTR chloride cha... Biochemistry. 2001 May 15;40(19):5579-86. Zou X, Hwang TC
ATP hydrolysis-coupled gating of CFTR chloride channels: structure and function.
Biochemistry. 2001 May 15;40(19):5579-86., 2001-05-15 [PMID:11341822]

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200 Among them, the glycine-to-aspartate mutation (G551D) has a worldwide frequency of ~2%. Login to comment
201 It has been shown that the G551D CFTR has a diminished ATPase activity, and the open probability of this mutant CFTR is less than 10% of that of wild-type channels (11). Login to comment
202 By comparing ATP hydrolysis rates and gating parameters of wild-type and G551D CFTR channels, Bear et al. (50) were able to confirm the proposal that ATP hydrolysis is directly coupled to the individual gating transition (30, 38, 51; cf. ref 46). 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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125 It was unexpected that six of the next most common mutations after 3120 ϩ 1GϾA would be of Caucasian origin (R1158X, R117H, G551D, 1812-1GϾA, 1898 ϩ 1GϾA, and R1066C). Login to comment
128 By comparison, eight "African" mutations accounted for a similar percentage of the chromosomes analyzed (23%) in the study by Macek et al.6 In contrast, 11 of the 20 mutations detected in this study are considered to be "Caucasian" mutations and account for 10.5% of the chromosomes analyzed (R117H, 621 ϩ 1GϾT, R334W, Q493X, G551D, 1812-1GϾA, 1898 ϩ 1GϾA, R1066C, R1158X, R1162X, and 3905insT). Login to comment

[hide] Activation of NF-kappaB in airway epithelial cells... Am J Physiol Lung Cell Mol Physiol. 2001 Jul;281(1):L71-8. Weber AJ, Soong G, Bryan R, Saba S, Prince A
Activation of NF-kappaB in airway epithelial cells is dependent on CFTR trafficking and Cl- channel function.
Am J Physiol Lung Cell Mol Physiol. 2001 Jul;281(1):L71-8., [PMID:11404248]

Abstract [show]
Polymorphonuclear leukocyte-dominated airway inflammation is a major component of cystic fibrosis (CF) lung disease and may be associated with CF transmembrane conductance regulator (CFTR) dysfunction as well as infection. Mutant DeltaF508 CFTR is mistrafficked, accumulates in the endoplasmic reticulum (ER), and may cause "cell stress" and activation of nuclear factor (NF)-kappaB. G551D mutants also lack Cl- channel function, but CFTR is trafficked normally. We compared the effects of CFTR mutations on the endogenous activation of an NF-kappaB reporter construct. In transfected Chinese hamster ovary cells, the mistrafficked DeltaF508 allele caused a sevenfold activation of NF-kappaB compared with wild-type CFTR or the G551D mutant (P < 0.001). NF-kappaB was also activated in 9/HTEo-/pCep-R cells and in 16HBE/pcftr antisense cell lines, which lack CFTR Cl- channel function but do not accumulate mutant protein in the ER. This endogenous activation of NF-kappaB was associated with elevated interleukin-8 expression. Impaired CFTR Cl- channel activity as well as cell stress due to accumulation of mistrafficked CFTR in the ER contributes to the endogenous activation of NF-kappaB in cells with the CFTR mutation.

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11 Mutant ⌬F508 CFTR is mistrafficked, accumulates in the endoplasmic reticulum (ER), and may cause "cell stress" and activation of nuclear factor (NF)-␬B. G551D mutants also lack Cl-channel function, but CFTR is trafficked normally. Login to comment
13 In transfected Chinese hamster ovary cells, the mistrafficked ⌬F508 allele caused a sevenfold activation of NF-␬B compared with wild-type CFTR or the G551D mutant (P Ͻ 0.001). Login to comment
47 The G551D CFTR mutant, in which a conserved glycine in the ATP binding cassette is mutated, lacks Cl-channel function, but the protein is properly folded and trafficked (12). Login to comment
48 Patients with the G551D mutation have a clinical disease that is indistinguishable from that caused by the more common ⌬F508 mutation (10). Login to comment
60 Stably transfected Chinese hamster ovary (CHO)-K1 cells that express ⌬F508 CFTR or G551D CFTR were obtained from J. Riordan (Mayo Clinic Scottsdale, Scottsdale, AZ) and grown in ␣MEM plus 200 ␮M methotrexate plus 8% FCS. Login to comment
61 CHO-K1 cells were obtained from the American Type Culture Collection (ATCC) and lipofected with pCep plasmid constructs expressing either wild-type CFTR, G551D CFTR, ⌬F508 CFTR, or the vector alone, provided by M. Drumm (Case Western Reserve University, Cleveland OH). Login to comment
107 Activation of NF-␬B in CHO Cells Expressing Wild-Type and Mutant CFTR CHO cells that were transiently transfected with the pCep empty vector or pCep vector expressing wild-type CFTR had low levels of basal NF-␬B activation as did the cells transfected with the plasmid expressing the mutant G551D CFTR (Fig. 3A). Login to comment
109 Experiments done with stably transfected CHO cells yielded similar results (Fig. 3C); expression of ⌬F508 CFTR was associated with a sevenfold increase in reporter activation compared with that in the CHO cells expressing the G551D mutation (P Ͻ 0.001) or CHO cells transfected with the reporter construct alone (P Ͻ 0.001). Login to comment
111 In the CHO cells, expression of the ⌬F508 CFTR mutation was sufficient to activate NF-␬B, whereas the presence of comparable amounts of the mutant G551D CFTR was not. Login to comment
132 A: relative activation of an NF-␬B-luciferase reporter construct in CHO cells without CFTR (-) or transiently transfected with the pCep vector; wild-type (wt) CFTR, G551D CFTR, or ⌬F508 CFTR cloned in pCep (n ϭ 12 experiments). Login to comment
134 B: immunodetection of CFTR in the transiently transfected CHO cells: Lane A, control CHO cells; lane B, pCep-wild-type CFTR; lane C, pCep- G551D CFTR; lane D, pCep-⌬F508 CFTR. Login to comment
138 Lane A, without CFTR (control); lane B, G551D CFTR; lane C, ⌬F508 CFTR. Login to comment
179 The G551D CFTR, which does not function appropriately as a Cl-channel but is trafficked normally to the apical surface of the respiratory epithelial cell (10), did not stimulate NF-␬B. Login to comment
181 Although both the G551D and ⌬F508 CFTR mutations are associated with clinical disease and lack of Cl-secretion in response to cAMP (10), the major difference between these mutants is the mistrafficking and accumulation of ⌬F508 CFTR within the ER. Login to comment

[hide] Regulation and properties of KCNQ1 (K(V)LQT1) and ... J Membr Biol. 2001 Jul 1;182(1):39-47. Boucherot A, Schreiber R, Kunzelmann K
Regulation and properties of KCNQ1 (K(V)LQT1) and impact of the cystic fibrosis transmembrane conductance regulator.
J Membr Biol. 2001 Jul 1;182(1):39-47., 2001-07-01 [PMID:11426298]

Abstract [show]
The K+ channel KCNQ1 (K(V)LQT1) is a voltage-gated K+ channel, coexpressed with regulatory subunits such as KCNE1 (IsK, mink) or KCNE3, depending on the tissue examined. Here, we investigate regulation and properties of human and rat KCNQ1 and the impact of regulators such as KCNE1 and KCNE3. Because the cystic fibrosis transmembrane conductance regulator (CFTR) has also been suggested to regulate KCNQ1 channels we studied the effects of CFTR on KCNQ1 in Xenopus oocytes. Expression of both human and rat KCNQ1 induced time dependent K+ currents that were sensitive to Ba2+ and 293B. Coexpression with KCNE1 delayed voltage activation, while coexpression with KCNE3 accelerated current activation. KCNQ1 currents were activated by an increase in intracellular cAMP, independent of coexpression with KCNE1 or KCNE3. cAMP dependent activation was abolished in N-terminal truncated hKCNQ1 but was still detectable after deletion of a single PKA phosphorylation motif. In the presence but not in the absence of KCNE1 or KCNE3, K+ currents were activated by the Ca2+ ionophore ionomycin. Coexpression of CFTR with either human or rat KCNQ1 had no impact on regulation of KCNQ1 K+ currents by cAMP but slightly shifted the concentration response curve for 293B. Thus, KCNQ1 expressed in Xenopus oocytes is regulated by cAMP and Ca2+ but is not affected by CFTR.

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152 Moreover, coexpression and stimulation of G551D-CFTR had no further impact (data not shown). 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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54 This ®gure is much lower than in European populations (48.8%) where several mutations such as G551D, N1303K, and W1282X are relatively frequent and associated with haplotype B (EWGCFG, 1990; Castaldo et al., 1996]. 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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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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39 One Australian centre (South Australia) includes the mutations G551D, G452X, DI507, R553X and R117H as part of routine screening of infants with an elevated IRT [12]. Login to comment
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] A combined analysis of the cystic fibrosis transme... Mol Biol Evol. 2001 Sep;18(9):1771-88. Chen JM, Cutler C, Jacques C, Boeuf G, Denamur E, Lecointre G, Mercier B, Cramb G, Ferec C
A combined analysis of the cystic fibrosis transmembrane conductance regulator: implications for structure and disease models.
Mol Biol Evol. 2001 Sep;18(9):1771-88., [PMID:11504857]

Abstract [show]
Over the past decade, nearly 1,000 variants have been identified in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in classic and atypical cystic fibrosis (CF) patients worldwide, and an enormous wealth of information concerning the structure and function of the protein has also been accumulated. These data, if evaluated together in a sequence comparison of all currently available CFTR homologs, are likely to refine the global structure-function relationship of the protein, which will, in turn, facilitate interpretation of the identified mutations in the gene. Based on such a combined analysis, we had recently defined a "functional R domain" of the CFTR protein. First, presenting two full-length cDNA sequences (termed sCFTR-I and sCFTR-II) from the Atlantic salmon (Salmo salar) and an additional partial coding sequence from the eastern gray kangaroo (Macropus giganteus), this study went further to refine the boundaries of the two nucleotide-binding domains (NBDs) and the COOH-terminal tail (C-tail), wherein NBD1 was defined as going from P439 to G646, NBD2 as going from A1225 to E1417, and the C-tail as going from E1418 to L1480. This approach also provided further insights into the differential roles of the two halves of CFTR and highlighted several well-conserved motifs that may be involved in inter- or intramolecular interactions. Moreover, a serious concern that a certain fraction of missense mutations identified in the CFTR gene may not have functional consequences was raised. Finally, phylogenetic analysis of all the full-length CFTR amino acid sequences and an extended set of exon 13--coding nucleotide sequences reinforced the idea that the rabbit may represent a better CF model than the mouse and strengthened the assertion that a long-branch attraction artifact separates the murine rodents from the rabbit and the guinea pig, the other Glires.

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100 Moreover, all of the 11 most common missense mutations or single-amino-acid deletions (i.e., F508del, G551D, N1303K, R117H, R347P, I507del, G85E, R560T, A455E, R334W, and S549N) identified in classic and atypical CF patients worldwide (http://www.genet.sickkids.on.ca/cftr) occur in stringently conserved residues across the 15 CFTR sequences. 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

[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
85 Previously, it has been shown that pancreatic insufficiency was related to mutation G551D and pancreatic sufficiency to R347W and S549N [11]. Login to comment

[hide] A phase I study of aerosolized administration of t... Hum Gene Ther. 2001 Oct 10;12(15):1907-16. Aitken ML, Moss RB, Waltz DA, Dovey ME, Tonelli MR, McNamara SC, Gibson RL, Ramsey BW, Carter BJ, Reynolds TC
A phase I study of aerosolized administration of tgAAVCF to cystic fibrosis subjects with mild lung disease.
Hum Gene Ther. 2001 Oct 10;12(15):1907-16., 2001-10-10 [PMID:11589832]

Abstract [show]
Cystic fibrosis (CF) is one of the most common autosomal recessive disorders in North America, leading to significant morbidity and early mortality. The defect in the cystic fibrosis transmembrane conductance regulator protein (CFTR) function can be corrected in vitro by gene replacement with a wild-type gene. A Phase I, single administration, dose escalation trial was designed and executed to assess safety and delivery of tgAAVCF, an adeno-associated virus (AAV) vector encoding the human CFTR cDNA, by nebulization to the lungs of CF subjects. Four cohorts of three subjects each were administered increasing doses of the study agent, beginning with 10(10) DNase-resistant particles (DRP) and escalating in log increments up to 10(13) DRP. Sequential bronchoscopies were performed to gather analytical samples throughout the study. All 12 subjects completed the study. There were a total of 242 adverse events (AEs), six of which were defined as serious and three of which were defined as possibly being related to the study drug. A clear dose-response relationship was observed in vector gene transfer. A maximum of 0.6 and 0.1 vector copies per brushed cell were observed 14 days and 30 days, respectively, following nebulization of 10(13) DRP tgAAVCF, and this declined to nearly undetectable levels by day 90. Vector gene transfer was evenly distributed throughout the fourth airway generation following single-dose administration. RNA-specific PCR did not detect vector-derived mRNA. This Phase I trial shows that aerosolized tgAAVCF is safe and widely delivered to the proximal airways of CF subjects by nebulization.

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156 SUBJECT DEMOGRAPHICS Age Day (214) tgAAVCF Subject Gender (years) CF mutation FEV1% dose 101 M 29 F508/F508 71 1010 102 F 40 F508/unknown 90 1010 103 F 34 F508/unknown 79 1010 301 M 37 F508/F508 63 1011 104 F 28 F508/unknown 104 1011 105 F 38 F508/F508 79 1011 201 F 29 F508/unknown 99 1012 302 M 24 F508/R347P 65 1012 106 F 28 F508/R334W 69 1012 203 M 41 G551D/G551D 64 1013 303 M 19 F508/F508 82 1013 107 F 26 F508/unknown 75 1013 TABLE 2. Login to comment

[hide] Activation of G551D CFTR channel with MPB-91: regu... Am J Physiol Cell Physiol. 2001 Nov;281(5):C1657-66. Derand R, Bulteau-Pignoux L, Mettey Y, Zegarra-Moran O, Howell LD, Randak C, Galietta LJ, Cohn JA, Norez C, Romio L, Vierfond JM, Joffre M, Becq F
Activation of G551D CFTR channel with MPB-91: regulation by ATPase activity and phosphorylation.
Am J Physiol Cell Physiol. 2001 Nov;281(5):C1657-66., [PMID:11600430]

Abstract [show]
We have designed and synthesized benzo[c]quinolizinium derivatives and evaluated their effects on the activity of G551D cystic fibrosis transmembrane conductance regulator (CFTR) expressed in Chinese hamster ovary and Fisher rat thyroid cells. We demonstrated, using iodide efflux, whole cell patch clamp, and short-circuit recordings, that 5-butyl-6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-91) restored the activity of G551D CFTR (EC(50) = 85 microM) and activated CFTR in Calu-3 cells (EC(50) = 47 microM). MPB-91 has no effect on the ATPase activity of wild-type and G551D NBD1/R/GST fusion proteins or on the ATPase, GTPase, and adenylate kinase activities of purified NBD2. The activation of CFTR by MPB-91 is independent of phosphorylation because 1) kinase inhibitors have no effect and 2) the compound still activated CFTR having 10 mutated protein kinase A sites (10SA-CFTR). The new pharmacological agent MPB-91 may be an important candidate drug to ameliorate the ion transport defect associated with CF and to point out a new pathway to modulate CFTR activity.

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0 281:C1657-C1666, 2001.Am J Physiol Cell Physiol Becq Caroline Norez, Leila Romio, Jean-Michel Vierfond, Michel Joffre and Frédéric L. Daniel Howell, Christoph Randak, Luis J. V. Galietta, Jonathan A. Cohn, Renaud Dérand, Laurence Bulteau-Pignoux, Yvette Mettey, Olga Zegarra-Moran, regulation by ATPase activity and phosphorylation Activation of G551D CFTR channel with MPB-91: You might find this additional info useful... 25 articles, 15 of which can be accessed free at:This article cites http://ajpcell.physiology.org/content/281/5/C1657.full.html#ref-list-1 3 other HighWire hosted articlesThis article has been cited by [PDF][Full Text][Abstract] , September 27, 2002; 277 (39): 35999-36004.J. Login to comment
2 Renaud Dérand, Laurence Bulteau-Pignoux and Frédéric Becq the Inhibitory Genistein Binding Site Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel and Abolishes The Cystic Fibrosis Mutation G551D Alters the Non-Michaelis-Menten Behavior of the [PDF][Full Text][Abstract] , November 13, 2007; 37 (6): 631-639.AJRCMB Lusky, Frederic Becq, Pierre Boulanger, Joseph Zabner and Saw-See Hong Ophelia Granio, Caroline Norez, Katherine J. Login to comment
11 It is published 12 timesAJP - Cell Physiology Activation of G551D CFTR channel with MPB-91: regulation by ATPase activity and phosphorylation RENAUD DE´ RAND,1 LAURENCE BULTEAU-PIGNOUX,1 YVETTE METTEY,2 OLGA ZEGARRA-MORAN,3 L. DANIEL HOWELL,4 CHRISTOPH RANDAK,5 LUIS J. V. GALIETTA,3 JONATHAN A. COHN,4 CAROLINE NOREZ,1 LEILA ROMIO,3 JEAN-MICHEL VIERFOND,2 MICHEL JOFFRE,1 AND FRE´ DE´ RIC BECQ1 1 Laboratoire de Physiologie des Re´gulations Cellulaires, Unite´ Mixte de Recherche 6558, 86022 Poitiers, and 2 Laboratoire de Chimie Organique, Faculte´ de Me´decine et de Pharmacie, 86005 Poitiers, France; 3 Laboratorio di Genetica Molecolare, Istituto G. Gaslini, Genoa 16148, Italy; 4 Durham Veterans Affairs and Duke University Medical Center, Durham, North Carolina; and 5 Kinderklinik im Dr. von Haunerschen Kinderspital, Ludwig-Maximilians-Universita¨t, Munich, Germany Received 19 April 2001; accepted in final form 16 July 2001 De´rand, Renaud, Laurence Bulteau-Pignoux, Yvette Mettey, Olga Zegarra-Moran, L. Daniel Howell, Christoph Randak, Luis J. V. Galietta, Jonathan A. Cohn, Caroline Norez, Leila Romio, Jean-Michel Vierfond, Michel Joffre, and Fre´de´ric Becq. Login to comment
12 Activation of G551D CFTR channel with MPB-91: regulation by ATPase activity and phosphorylation. Login to comment
13 Am J Physiol Cell Physiol 281: C1657-C1666, 2001.-We have designed and synthesized benzo[c]quinolizinium derivatives and evaluated their effects on the activity of G551D cystic fibrosis transmembrane conductance regulator (CFTR) expressed in Chinese hamster ovary and Fisher rat thyroid cells. Login to comment
14 We demonstrated, using iodide efflux, whole cell patch clamp, and short-circuit recordings, that 5-butyl-6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-91) restored the activity of G551D CFTR (EC50 ϭ 85 ␮M) and activated CFTR in Calu-3 cells (EC50 ϭ 47 ␮M). Login to comment
15 MPB-91 has no effect on the ATPase activity of wild-type and G551D NBD1/R/GST fusion proteins or on the ATPase, GTPase, and adenylate kinase activities of purified NBD2. Login to comment
22 The main mutations are a deletion of the phenylalanine at position 508 (⌬F508, class II mutation), which represents ϳ66% of the mutated chromosomes, and a glycine-to-aspartic acid missense mutation at codon 551 (G551D, class III mutation) with a frequency of 2-5% (33). Login to comment
23 Both mutations are located in the first nucleotide binding domain (NBD1) (21), but, whereas ⌬F508-CFTR is not correctly addressed toward the membrane (8, 13), the processing, maturation, and assigned apical location of G551D CFTR are not affected (30, 33). Login to comment
24 However, in cells expressing the G551D mutant, chloride transport cannot be activated by cAMP-elevating agents (1, 11, 33). Login to comment
27 We have further performed a structure-activity study and identified 5-butyl-6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-91), a compound able to restore chloride secretion in G551D and wild-type CFTR-expressing cells. Login to comment
41 Chinese hamster ovary (CHO) cells stably transfected with pNUT vector alone (CHO pNUT) or containing wild-type CFTR [CFTR(ϩ) CHO], G551D CFTR (G551D CHO), or 10SA CFTR (10SA CHO) were provided by J. R. Riordan and X.-B. Chang (Scottsdale, AZ) (6, 31). Login to comment
57 Ussing chamber experiments in G551D Fisher rat thyroid cells. Login to comment
93 We found it necessary to study the additive stimulatory effect of forskolin and MPB-91 in CFTR(ϩ) CHO cells, since the effect on G551D CFTR had been tested in the same cell line. Login to comment
107 C1659MPB-91 STIMULATES G551D CFTR CHANNEL ACTIVITY AJP-Cell Physiol • VOL 281 • NOVEMBER 2001 • www.ajpcell.org 1.11 Ϯ 0.14 min-1 , n ϭ 8), even in the presence of forskolin (not shown). Login to comment
119 MPB-91 but not MPB-07 is an activator of G551D CFTR. Login to comment
120 Because little is known about the pharmacology of G551D CFTR, we determined the effects of MPB-07 and MPB-91 on the activity of G551D expressed in CHO cells using iodide efflux and whole cell patch-clamp methods. Login to comment
121 Control experiments confirmed that forskolin indeed failed to stimulate iodide efflux from G551D-CFTR-expressing cells (peak rate-to-basal: 0.10 Ϯ 0.01 min-1 , n ϭ 14; 10 ␮M forskolin: 0.10 Ϯ 0.01 min-1 , n ϭ 14). Login to comment
122 We found that MPB-07 (250 ␮M) either alone (peak rate: 0.09 Ϯ 0.01 min-1 , n ϭ 4) or in combination with 10 ␮M forskolin (peak rate: 0.09 Ϯ 0.01 min-1 , n ϭ 4) failed to stimulate the iodide efflux in G551D cells. Login to comment
134 These results indicate that the MPB-91-stimulated iodide efflux and whole cell chloride current in G551D cells are due to CFTR activation. Login to comment
135 Figure 4 shows short-circuit current measurements on FRT cells transfected with G551D CFTR. Login to comment
137 However, subsequent stimulation with MPB-91 produced a dose-dependent increase of transepithelial conductance in G551D CFTR cells (Fig. 4). Login to comment
142 The activation of G551D CFTR by MPB-91 occurred with an EC50 of 84.8 Ϯ 9.3 ␮M (n ϭ 3). Login to comment
145 Whole cell CFTR current activated by MPB-91 in CHO G551D cells. Login to comment
150 Transepithelial conductance (G) response to MPB-91 on G551D CFTR-transfected FRT cells. Login to comment
151 A: response of a G551D CFTR monolayer to 500 ␮M 8-(4-chlorophenylthio)adenosine-3Ј,5Ј-cyclic monophosphate (CPT-cAMP) followed by increasing concentrations of MPB-91 (in ␮M) in the apical solution. Login to comment
155 C1661MPB-91 STIMULATES G551D CFTR CHANNEL ACTIVITY AJP-Cell Physiol • VOL 281 • NOVEMBER 2001 • www.ajpcell.org both NBD1 and NBD2 ATPase activities. Login to comment
156 First, wild-type and G551D NBD1/R/GST ATPase activities were measured at their respective Michaelis-Menten constant (Km) values (70 and 250 ␮M; Ref. 14) and at 1 mM ATP. Login to comment
157 G551D NBD1 has a significantly reduced ATPase activity compared with wild-type NBD1 (Fig. 5A) as expected from previous studies (14, 19). Login to comment
158 MPB-91 does not affect the ATPase activity of wild-type or G551D NBD1 at any ATP and MPB-91 concentrations tested. Login to comment
164 To determine whether wild-type and G551D CFTR activation by MPB-91 involved phosphorylation of the channel, the effects of two kinase inhibitors were investigated. Login to comment
173 Wild-type (WT) NBD1/R/GST (50 ng) and G551D NBD1/R/GST (50 ng) were tested for ATPase activity at their respective Km values (70 and 250 ␮M) and at 1 mM ATP. Login to comment
185 C1662 MPB-91 STIMULATES G551D CFTR CHANNEL ACTIVITY AJP-Cell Physiol • VOL 281 • NOVEMBER 2001 • www.ajpcell.org onAugust,2011ajpcell.physiology.orgDownloadedfrom 91-stimulated iodide efflux in Calu-3 cells (P Ͼ 0.05, for both inhibitors). Login to comment
187 Finally, experiments were performed on G551D CFTR cells exposed to forskolin (10 ␮M) and MPB-91 (250 ␮M). Login to comment
193 C and D: MPB-91-mediated iodide efflux in Calu-3 (C) and in G551D CHO cells (D). Login to comment
196 C1663MPB-91 STIMULATES G551D CFTR CHANNEL ACTIVITY AJP-Cell Physiol • VOL 281 • NOVEMBER 2001 • www.ajpcell.org kolin ϩ MPB-91 (P Ͻ 0.01, n ϭ 8, Fig. 6D). Login to comment
204 Indeed, we found that MPB-07, which is able to activate wild-type CFTR (2), is not effective as a G551D CFTR activator. Login to comment
205 Modification of the chemical structure using SAR identified an important position within the MPB skeleton, leading to MPB-91, a compound that kept its ability to activate wild-type CFTR but more importantly acquired the property to activate G551D CFTR. Login to comment
208 We demonstrated that MPB-91 restores CFTR-mediated chloride secretion in cells expressing G551D CFTR. Login to comment
209 The inhibitory profile of G551D CFTR channel activity is similar to that described for wild-type CFTR (13, 26). Login to comment
214 Because ATP hydrolysis at NBD1 has been linked to CFTR activation, we initially hypothesized that MPB-91 could modulate wild-type ATPase and/or could restore normal G551D ATPase function (e.g., by reducing the Km value for ATP). Login to comment
215 To study this, we first measured the ATPase activity of wild-type NBD1 in the presence of MPB-91. Our results clearly demonstrated that MPB-91 did not compete with ATP binding, did not modulate ATPase activity, and had no significant effect on reduced ATPase activity of G551D NBD1. Login to comment
216 We then concluded that the mutation G551D interferes with the mechanism of channel opening that is dependent of ATP hydrolysis at NBD1. Login to comment
217 Because the compound MPB-91 was found to be able to activate wild-type CFTR and G551D CFTR without directly affecting NBD1 ATPase activity, these observations strongly suggest that an alternative pathway of activation exists that bypasses the defective ATPase activity of G551D CFTR proteins. Login to comment
223 For example, these two drugs are both able to activate wild-type and G551D CFTR, but probably through two distinct mechanisms. Login to comment
232 One major difference currently observed between wild-type and G551D CFTR is the role of phosphorylation. Login to comment
234 Although G551D CFTR could be normally phosphorylated (11) and despite the fact that phosphorylation by itself is not sufficient to activate G551D CFTR (11, 33), it is required for CFTR to be opened by MPB-91 and other compounds like genistein (17). Login to comment
237 In addition, MPB-91 does not alter the cAMP levels in both Calu-3 and G551D CHO cells (T. Me´taye´, R. De´rand, L. Bulteau, and F. Becq, unpublished data). Login to comment
240 In G551D CHO cells we found, however, that MPB-91 alone is not sufficient to stimulate the chloride current. Login to comment
242 However, once phosphorylated, G551D CFTR can be activated by MPB-91. Login to comment
244 These results suggest that phosphorylation of G551D CFTR may act only as a switch, making the mutated protein responsive or not to MPB-91. Login to comment
248 In conclusion, in this report we have presented the potential interest of MPB-91 as a putative drug in CF, since it not only modulates wild-type CFTR but also activates CFTR having the disease-causing mutation G551D. Login to comment

[hide] Cell-based assay for high-throughput quantitative ... Am J Physiol Cell Physiol. 2001 Nov;281(5):C1734-42. Galietta LV, Jayaraman S, Verkman AS
Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists.
Am J Physiol Cell Physiol. 2001 Nov;281(5):C1734-42., [PMID:11600438]

Abstract [show]
Drug discovery by high-throughput screening is a promising approach to develop new therapies for the most common lethal genetic disease, cystic fibrosis. Because disease-causing mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) protein produce epithelial cells with reduced or absent Cl(-) permeability, the goal of screening is to identify compounds that restore cell Cl(-) transport. We have developed a rapid, quantitative screening procedure for analysis of CFTR-mediated halide transport in cells with the use of a conventional fluorescence plate reader. Doubly transfected cell lines were generated that express wild-type or mutant CFTR together with a yellow fluorescent protein (YFP)-based halide sensor. CFTR function was assayed from the time course of cell fluorescence in response to extracellular addition of 100 mM I(-) followed by forskolin, resulting in decreased YFP fluorescence due to CFTR-mediated I(-) entry. Cell lines were chosen, and conditions were optimized to minimize basal halide transport to maximize assay sensitivity. In cells cultured on 96-well plastic dishes, the assay gave reproducible halide permeabilities from well to well and could reliably detect a 2% activation of CFTR-dependent halide transport produced by low concentrations of forskolin. Applications of the assay are shown, including comparative dose-dependent CFTR activation by genistein, apigenin, 8-cyclopentyl-1,3-dipropylxanthine, IBMX, 8-methoxypsoralen, and milrinone as well as activation of alternative Cl(-) channels. The fluorescence assay and cell lines should facilitate the screening of novel CFTR activators and the characterization of alternative Cl(-) channels and transporters.

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35 Fischer rat thyroid (FRT) cells were transfected with wild-type or G551D CFTR and selected in 0.6 mg/ml zeocin. Login to comment
40 In addition, a stably transfected Chinese hamster ovary (CHO) cell line was generated that expressed YFP-H148Q and was subsequently transfected with an episomal plasmid (pCEP4) containing the coding sequence for wild-type or G551D CFTR. Login to comment
53 FRT cells stably expressing wild-type or G551D CFTR were cultured on Snapwell inserts (Costar) for 8-10 days to form epithelial monolayers with high electrical resistance (3-4 k⍀⅐cm2 ). Login to comment
58 Cells stably expressing CFTR (wild type or G551D) and YFP-H148Q were plated in 96-well black microplates (Corning Costar) at a density of 20,000 cells/well using a Labsystems Multidrop apparatus for automated liquid delivery. Login to comment
86 Activation of G551D CFTR, a mutant that is processed normally but is relatively Cl- impermeable at the plasma membrane (17), was studied in stably transfected FRT cells. Login to comment
108 B: experiments as in A done in Fischer rat thyroid (FRT) cells expressing G551D CFTR. Login to comment
169 We evaluated with the plate reader assay the efficacy of genistein, a known activator of CFTR (8, 21, 23), to induce halide transport in cells expressing wild-type and G551D CFTR. Login to comment
171 Forskolin was added during the assay at the submaximal concentration of 250 nM for wild-type CFTR or the high concentration of 5 ␮M for G551D CFTR. Login to comment
176 In FRT cells stably expressing G551D CFTR, genistein alone did not activate halide transport, but subsequent addition of forskolin gave a small but significant activation of G551D CFTR at the higher genistein concentrations (Fig. 5B). Login to comment
195 B: same as in A, except on FRT cells expressing G551D CFTR. Login to comment

[hide] A cluster of negative charges at the amino termina... J Physiol. 2001 Oct 15;536(Pt 2):459-70. Fu J, Ji HL, Naren AP, Kirk KL
A cluster of negative charges at the amino terminal tail of CFTR regulates ATP-dependent channel gating.
J Physiol. 2001 Oct 15;536(Pt 2):459-70., 2001-10-15 [PMID:11600681]

Abstract [show]
1. The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is activated by protein kinase A (PKA) phosphorylation of its R domain and by ATP binding at its nucleotide-binding domains (NBDs). Here we investigated the functional role of a cluster of acidic residues in the amino terminal tail (N-tail) that also modulate CFTR channel gating by an unknown mechanism. 2. A disease-associated mutant that lacks one of these acidic residues (D58N CFTR) exhibited lower macroscopic currents in Xenopus oocytes and faster deactivation following washout of a cAMP -activating cocktail than wild-type CFTR. 3. In excised membrane patches D58N CFTR exhibited a two-fold reduction in single channel open probability due primarily to shortened open channel bursts. 4. Replacing this and two nearby acidic residues with alanines (D47A, E54A, D58A) also reduced channel activity, but had negligible effects on bulk PKA phosphorylation or on the ATP dependence of channel activation. 5. Conversely, the N-tail triple mutant exhibited a markedly inhibited response to AMP-PNP, a poorly hydrolysable ATP analogue that can nearly lock open the wild-type channel. The N-tail mutant had both a slower response to AMP-PNP (activation half-time of 140 +/- 20 s vs. 21 +/- 4 s for wild type) and a lower steady-state open probability following AMP-PNP addition (0.68 +/- 0.08 vs. 0.92 +/- 0.03 for wild type). 6. Introducing the N-tail mutations into K1250A CFTR, an NBD2 hydrolysis mutant that normally exhibits very long open channel bursts, destabilized the activity of this mutant as evidenced by decreased macroscopic currents and shortened open channel bursts. 7. We propose that this cluster of acidic residues modulates the stability of CFTR channel openings at a step that is downstream of ATP binding and upstream of ATP hydrolysis, probably at NBD2.

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5 Other mutations (e.g. G551D) exhibit normal protein trafficking but lower channel activity; many of these mutations are associated with less severe disease (Sheppard et al. 1993; Illek et al. 1999). Login to comment
323 Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein. 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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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] Correction of delF508-CFTR activity with benzo(c)q... J Cell Sci. 2001 Nov;114(Pt 22):4073-81. Dormer RL, Derand R, McNeilly CM, Mettey Y, Bulteau-Pignoux L, Metaye T, Vierfond JM, Gray MA, Galietta LJ, Morris MR, Pereira MM, Doull IJ, Becq F, McPherson MA
Correction of delF508-CFTR activity with benzo(c)quinolizinium compounds through facilitation of its processing in cystic fibrosis airway cells.
J Cell Sci. 2001 Nov;114(Pt 22):4073-81., [PMID:11739639]

Abstract [show]
A number of genetic diseases, including cystic fibrosis, have been identified as disorders of protein trafficking associated with retention of mutant protein within the endoplasmic reticulum. In the presence of the benzo(c)quinolizinium drugs, MPB-07 and its congener MPB-91, we show the activation of cystic fibrosis transmembrane conductance regulator (CFTR) delF508 channels in IB3-1 human cells, which express endogenous levels of delF508-CFTR. These drugs were without effect on the Ca(2+)-activated Cl- transport, whereas the swelling-activated Cl- transport was found altered in MPB-treated cells. Immunoprecipitation and in vitro phosphorylation shows a 20% increase of the band C form of delF508 after MPB treatment. We then investigated the effect of these drugs on the extent of mislocalisation of delF508-CFTR in native airway cells from cystic fibrosis patients. We first showed that delF508 CFTR was characteristically restricted to an endoplasmic reticulum location in approximately 80% of untreated cells from CF patients homozygous for the delF508-CFTR mutation. By contrast, 60-70% of cells from non-CF patients showed wild-type CFTR in an apical location. MPB-07 treatment caused dramatic relocation of delF508-CFTR to the apical region such that the majority of delF508/delF508 CF cells showed a similar CFTR location to that of wild-type. MPB-07 had no apparent effect on the distribution of wild-type CFTR, the apical membrane protein CD59 or the ER membrane Ca(2+),Mg-ATPase. We also showed a similar pharmacological effect in nasal cells freshly isolated from a delF508/G551D CF patient. The results demonstrate selective redirection of a mutant membrane protein using cell-permeant small molecules of the benzo(c)quinolizinium family and provide a major advance towards development of a targetted drug treatment for cystic fibrosis and other disorders of protein trafficking.

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15 We also showed a similar pharmacological effect in nasal cells freshly isolated from a delF508/G551D CF patient. Login to comment
50 Incubation and fixation of nasal epithelial cells Airway epithelial cells obtained by nasal brushing were from five non-CF individuals, four delF508/delF508 CF individuals and one delF508/G551D CF individual undergoing flexible bronchoscopy under sedation. Login to comment
164 Effect of MPB compounds on the location of CFTR from delF508/G551D cystic fibrosis individuals The technique allows us to investigate the effect of MPB compounds on other naturally occurring genotypes and we show CFTR distribution in cells obtained from nasal brushings from a CF individual with a delF508/G551D genotype. Login to comment
170 Thus MPB-91 dramatically changed the cellular distribution of mutant CFTR from a perinuclear towards an apical location in nasal cells from a delF508/G551D CF patient. Login to comment
180 Effect of MPB-91 on confocal immunofluorescent labelling of CFTR in ∆F508/G551D cystic fibrosis epithelial cells from nasal brushings. Images show CFTR immunofluorescence in green with the nucleus counterstained with propidium iodide (red). Login to comment
207 Their application to the majority of CF patients is highlighted by our results in the present report that nasal cells obtained from a compound heterozygote individual (delF508/G551D) have an altered CFTR location similar to delF508/delF508 cells. Login to comment
208 The data suggest that delF508-CFTR is more strongly expressed than G551D or that its restriction to the endoplasmic reticulum also retains G551D. Login to comment
209 Incubation of delF508/G551D cells with MPB-91 markedly altered the location of mutant CFTR towards a wild-type distribution, although we cannot distinguish whether the apically located CFTR is delF508 or G551D. Login to comment
210 Thus, MPB-91 provides a new class of compounds that not only activate G551D-CFTR (R.D., L.B.-P. and F.B., unpublished) but also traffic mutant CFTR to the apical membrane in CF compound heterozygotes with a delF508/G551D mutation. Login to comment
211 In conclusion, the present study demonstrates that benzoquinolizinium compounds, which directly activate wild-type (Becq et al., 1999) and G551D-CFTR Cl-channels (R.D., L.B.-P. and F.B., unpublished), have a dramatic effect in restoring defective chloride transport in CF cells by increasing delF508-CFTR at the apical membrane. This is a major step forward in the goal of devising a rational drug treatment aimed at repair or rescue of the activity of the mutant cystic fibrosis gene protein and has implications for other disorders of protein trafficking (Aridor and Balch, 1999). Login to comment

[hide] A(2) adenosine receptors regulate CFTR through PKA... Am J Physiol Lung Cell Mol Physiol. 2002 Jan;282(1):L12-25. Cobb BR, Ruiz F, King CM, Fortenberry J, Greer H, Kovacs T, Sorscher EJ, Clancy JP
A(2) adenosine receptors regulate CFTR through PKA and PLA(2).
Am J Physiol Lung Cell Mol Physiol. 2002 Jan;282(1):L12-25., [PMID:11741811]

Abstract [show]
We investigated adenosine (Ado) activation of the cystic fibrosis transmembrane conductance regulator (CFTR) in vitro and in vivo. A(2B) Ado receptors were identified in Calu-3, IB-3-1, COS-7, and primary human airway cells. Ado elevated cAMP in Calu-3, IB-3-1, and COS-7 cells and activated protein kinase A-dependent halide efflux in Calu-3 cells. Ado promoted arachidonic acid release from Calu-3 cells, and phospholipase A(2) (PLA(2)) inhibition blocked Ado-activated halide efflux in Calu-3 and COS-7 cells expressing CFTR. Forskolin- and beta(2)-adrenergic receptor-stimulated efflux were not affected by the same treatment. Cytoplasmic PLA(2) (cPLA(2)) was identified in Calu-3, IB-3-1, and COS-7 cells, but cPLA(2) inhibition did not affect Ado-stimulated cAMP concentrations. In cftr(+) and cftr(-/-) mice, Ado stimulated nasal Cl(-) secretion that was CFTR dependent and sensitive to A(2) receptor and PLA(2) blockade. In COS-7 cells transiently expressing DeltaF508 CFTR, Ado activated halide efflux. Ado also activated G551D CFTR-dependent halide efflux when combined with arachidonic acid and phosphodiesterase inhibition. In conclusion, PLA(2) and protein kinase A both contribute to A(2) receptor activation of CFTR, and components of this signaling pathway can augment wild-type and mutant CFTR activity.

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17 Ado also activated G551D CFTR-dependent halide efflux when combined with arachidonic acid and phosphodiesterase inhibition. Login to comment
51 Wild-type (WT), ⌬F508, or G551D CFTR under control of the T7 promoter in the pTM-1 vector was then introduced into the cells in complex with N-[1-(2,3-dioleoyloxy)propyl]- N,N,N-trimethylammonium (DOTAP)-propane-1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE; 20 ␮g DOTAP-DOPE and 5 ␮g pTM-1 CFTR/5 ϫ 105 cells) for 4 h. Login to comment
53 Cells were then washed in PBS, returned to DMEM plus 10% FBS, and studied 18-24 h postinfection (for WT CFTR and G551D CFTR) or after being grown at 29°C for 48 h (⌬F508 CFTR). Login to comment
55 To study CFTR activation, we measured halide efflux in COS-7 cells transiently expressing WT, ⌬F508, or G551D CFTR or in Calu-3 cells with the halide-quenched dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ; Molecular Probes, Eugene, OR) as previously described (16, 17). Login to comment
243 A2B-ARs activate ⌬F508 CFTR and G551D CFTR. Login to comment
245 To determine whether A2B receptor signaling could be used to improve the activity of common disease-causing CFTR mutations, we transiently expressed ⌬F508 CFTR (class II mutation) and G551D CFTR (class III mutation) in COS-7 cells. Login to comment
266 In Fig. 11, COS-7 cells expressing G551D CFTR were stimulated with forskolin, Ado, or AA. Login to comment
269 Two nonspecific PDE inhibitors (papaverine and theophylline) were studied at 200 ␮M. PDE inhibitors such as milrinone have been evaluated in clinical trials for their ability to activate Cl-conductance in normal subjects and in CF patients carrying the G551D CFTR mutation (68). Login to comment
270 At the concentrations used, none of the PDE inhibitors alone consistently elevated cAMP in COS-7 cells (10-min exposure; data not shown), and none of the stimuli (including Ado) activated G551D CFTR-specific halide efflux (Fig. 11A). Login to comment
272 In Fig. 11B, G551D CFTR-expressing COS-7 cells were ex- Fig. 6. Login to comment
330 Finally, we showed that the two most common disease-associated CFTR mutations can be activated by A2B receptors alone (⌬F508 CFTR) or by using messenger pathways stimulated by A2B receptors (G551D CFTR). Login to comment
355 Activation of halide efflux in G551D CFTR-expressing cells. Login to comment
356 A: G551D CFTR-expressing cells compared with wild-type (WT) CFTR-expressing cells. Login to comment
357 COS-7 cells transiently expressing WT or G551D CFTR were studied with 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) as described in METHODS; n 40 cells/condition. Login to comment
358 G551D CFTR-expressing cells failed to activate halide efflux when stimulated with Ado (200 ␮M) ϩ papaverine (PAP; 200 ␮M), Ado ϩ rolipram (ROL; 20 ␮M) in combination, or Forsk (20 ␮M; arrow). Login to comment
360 B: G551D CFTR-expressing COS-7 cells stimulated with Ado (200 ␮M) and AA (100 ␮M) combined with a series of phosphodiesterase inhibitors (PDEis). Login to comment
385 The effect would need to be quite dramatic, however, because G551D CFTR, which has previously been shown to be poorly responsive to powerful phosphorylating stimuli, appears to be activated by costimulation with AA (Fig. 11B, Refs. Login to comment

[hide] Genistein restores functional interactions between... J Biol Chem. 2002 Mar 15;277(11):8928-33. Epub 2001 Dec 28. Suaud L, Li J, Jiang Q, Rubenstein RC, Kleyman TR
Genistein restores functional interactions between Delta F508-CFTR and ENaC in Xenopus oocytes.
J Biol Chem. 2002 Mar 15;277(11):8928-33. Epub 2001 Dec 28., 2002-03-15 [PMID:11773060]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its Cl(-) channel properties, has regulatory interactions with other epithelial ion channels including the epithelial Na(+) channel (ENaC). Both the open probability and surface expression of wild type CFTR Cl(-) channels are increased significantly when CFTR is co-expressed in Xenopus oocytes with alphabetagamma-ENaC, and conversely, the activity of ENaC is inhibited following wild type CFTR activation. Using the Xenopus oocyte expression system, a lack of functional regulatory interactions between DeltaF508-CFTR and ENaC was observed following activation of DeltaF508-CFTR by forskolin and isobutylmethylxanthine (IBMX). Whole cell currents in oocytes expressing ENaC alone decreased in response to genistein but increased in response to a combination of forskolin and IBMX followed by genistein. In contrast, ENaC currents in oocytes co-expressing ENaC and DeltaF508-CFTR remained stable following stimulation with forskolin/IBMX/genistein. Furthermore, co-expression of DeltaF508-CFTR with ENaC enhanced the forskolin/IBMX/genistein-mediated activation of DeltaF508-CFTR. Our data suggest that genistein restores regulatory interactions between DeltaF508-CFTR and ENaC and that combinations of protein repair agents, such as 4-phenylbutyrate and genistein, may be necessary to restore DeltaF508-CFTR function in vivo.

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133 A similar NBD-1/R peptide fragment containing the G551D mutation did not demonstrate forskolin/IBMX-regulated inhibition of ENaC (45). 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

[hide] Spectrum of mutations in the CFTR gene of patients... Genet Test. 2001 Fall;5(3):235-42. Strandvik B, Bjorck E, Fallstrom M, Gronowitz E, Thountzouris J, Lindblad A, Markiewicz D, Wahlstrom J, Tsui LC, Zielenski J
Spectrum of mutations in the CFTR gene of patients with classical and atypical forms of cystic fibrosis from southwestern Sweden: identification of 12 novel mutations.
Genet Test. 2001 Fall;5(3):235-42., [PMID:11788090]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the CFTR gene. The spectrum of CFTR mutations varies between populations and depends on different factors, such as ethnic background and geographical location. The extensive CFTR mutation screening of 129 patients with classical or atypical CF from the south-western region of Sweden revealed the presence of 37 CFTR mutations, including 12 novel alleles. The overall mutation detection rate in this study population was 92%, the highest among all tested regions in Sweden. Eight mutations with a frequency above 1% (DeltaF508, 394delTT, R117C, 3659delC, E60X, 1112delT, R764X, and 621 + 1G --> T) accounted for 78% of CF chromosomes and have been recommended for inclusion in the CFTR mutation screening panel for molecular diagnosis of CF in this region. The multiple occurrence of specific CFTR alleles less common than the predominant DeltaF508 mutation (394delTT, R117C, 3659delC) allowed for genotype-phenotype comparisons and revealed consistent relationships between these mutations and disease severity.

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27 MUTATIONS IDENTIFIED IN 258 CHROMOSOMES IN THE CF POPULATION ATTENDING THE SOUTH-WESTERN SWEDISH CF CENTRE Location in the Frequency of Mutation gene, exon Number of mutations mutation (%) Homozygotes Heterozygotes DF508 10 161 62.4 56 49 394delTT 3 13 5.0 3 7 R117C 4 7 2.7 7 3659delC 19 5 1.9 5 E60X 3 4 1.6 4 1112delT 7 4 1.6 1 2 R764X 13 4 1.6 1 2 621 1 1G ® T 4 3 1.2 3 G551D 11 2 0.8 2 I506L 10 2 0.8 2 N1088D (R75Q) 17b 2 0.8 2 Q1238X 19 2 0.8 2 R117H (IVS8-5T) 4 2 0.8 2 V603F (IVS8-5T) 13 2 0.8 2 1716G ® A 10 2 0.8 2 R75Q 3 2 0.8 2 R533X 11 1 0.4 1 2329A ® G Promoter 1 0.4 1 297-3 C ® A 2 1 0.4 1 Y161D 4 1 0.4 1 994del9 Exon/intron 6b 1 0.4 1 1154insTC 7 1 0.4 1 W361R 7 1 0.4 1 T338I 7 1 0.4 1 1249-5A ® G Intron 7 1 0.4 1 1717-2A ® G Intron 10 1 0.4 1 R560T 11 1 0.4 1 E1401X 23 1 0.4 1 3126del4 17a 1 0.4 1 S945L 15 1 0.4 1 R668C 13 1 0.4 1 2622 1 2del6 Intron 13 1 0.4 1 R1162Q Exon 19 1 0.4 1 3849 1 10kbC ® T Intron 19 1 0.4 1 R74W Exon 3 1 0.4 1 2363C ® T Promoter 1 0.4 1 IVS8-5Ta Intron 8 1 0.4 1 Unidentified 20 7.8 Total 258 100 61 116 The new mutations are displayed in bold. 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] The severe G480C cystic fibrosis mutation, when re... Hum Mol Genet. 2002 Feb 1;11(3):243-51. Dickinson P, Smith SN, Webb S, Kilanowski FM, Campbell IJ, Taylor MS, Porteous DJ, Willemsen R, de Jonge HR, Farley R, Alton EW, Dorin JR
The severe G480C cystic fibrosis mutation, when replicated in the mouse, demonstrates mistrafficking, normal survival and organ-specific bioelectrics.
Hum Mol Genet. 2002 Feb 1;11(3):243-51., 2002-02-01 [PMID:11823443]

Abstract [show]
The majority of cystic fibrosis patients produce a mutant form of CFTR (DeltaF508) which has been shown to be mislocalized in both humans and mice. G480C, another clinically 'severe' mutation, has also been demonstrated to be defective in its intracellular processing, but when allowed to traffic in Xenopus oocytes showed similar channel characteristics to that of wild-type CFTR. We have replicated the G480C mutation in the murine Cftr gene using the 'hit and run' double recombination procedure. As expected, the G480C cystic fibrosis mouse model expresses the G480C mutant transcript at a level comparable to that of wild-type CFTR: The homozygous mutant mice were fertile, had normal survival, weight, tooth colour and no evidence of caecal blockage, despite mild goblet cell hypertrophy in the intestine. Analysis of the mutant protein revealed that the majority of G480C CFTR was abnormally processed and no G480C CFTR-specific immunostaining in the apical membranes of intestinal cells was detected. The bioelectric phenotype of these mice revealed organ-specific electrophysiological effects. In contrast to DeltaF508 'hit and run' homozygotes, the classic defect of forskolin-induced chloride ion transport is not replicated in the caecum, but the response to low chloride in the nose is clearly defective in the G480C mutant animals. The mild phenotype of these G480C mutant animals combined with the defective chloride transport in the nose uniquely provides a valuable resource to test novel pharmacological agents aimed at improving trafficking and correcting the electrophysiological defect in the respiratory tract.

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26 In such mutant mice (e.g. the ∆F508 Cftrtm2Cam and the G551D Cftrtm1G551D mice), it is therefore difficult to assess whether the phenotype is due to the precise mutation or principally related to a reduction in the level of the transcript. 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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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

[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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17 Class III mutations, of which G551D is an example, are regulatory mutations in which protein reaches the surface of the cell but fails to respond consistently to cyclic AMP activation signals. Login to comment
82 II ⌬F508 (36), W1282X (1) III G551D (10), N1303K (4), R560T (2), A559T (1) IV R117H (14), R347H (3) V 3849 ϩ 10KbC→T (7), 3120G→A (3) AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 165 2002 All patients were recruited from a single center, and the sample size of this study was large compared with previously published studies of exercise capacity in children with CF (21). 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] Characterization of LPS-induced lung inflammation ... J Appl Physiol. 2002 May;92(5):2169-76. Freedman SD, Weinstein D, Blanco PG, Martinez-Clark P, Urman S, Zaman M, Morrow JD, Alvarez JG
Characterization of LPS-induced lung inflammation in cftr-/- mice and the effect of docosahexaenoic acid.
J Appl Physiol. 2002 May;92(5):2169-76., [PMID:11960971]

Abstract [show]
The mechanism by which Pseudomonas causes excessive inflammation in the cystic fibrosis lung is unclear. We have reported that arachidonic acid is increased and docosahexaenoic acid (DHA) decreased in lung, pancreas, and ileum from cftr-/- mice. Oral DHA corrected this defect and reversed the pathology. To determine which mediators regulate inflammation in lungs from cftr-/- mice and whether inhibition occurs with DHA, cftr-/- and wild-type (WT) mice were exposed to aerosolized Pseudomonas lipopolysaccharide (LPS). After 2 days of LPS, tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2, and KC levels in bronchoalveolar lavage fluid were increased in cftr-/- compared with WT mice and not suppressed by pretreatment with oral DHA. Neutrophil levels were not different between cftr-/- and WT mice. After 3 days of aerosolized LPS, neutrophil concentration, TNF-alpha, and the eicosanoids 6-keto-PGF1alpha, PGF2alpha, PGE2, and thromboxane B2 were all increased in bronchoalveolar lavage fluid from cftr-/- mice compared with WT controls. Oral DHA had no significant effect on TNF-alpha levels in cftr-/- mice. In contrast, neutrophils and eicosanoids were decreased in cftr-/- but not in WT mice treated with DHA, indicating that the effects of DHA on these inflammatory parameters may be related to correction of the membrane lipid defect.

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110 Using a different approach, Thomas et al. (26a) have shown that intravenous administration of LPS leads to a qualitative increase in the number of neutrophils in the lung parenchyma of mice with the G551D mutation in CFTR compared Fig. 1. Login to comment
111 Using a different approach, Thomas et al. (26a) have shown that intravenous administration of LPS leads to a qualitative increase in the number of neutrophils in the lung parenchyma of mice with the G551D mutation in CFTR compared Fig. 1. 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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113 G551D G551S PI Defective chloride channel Regulation Reduced or absent cell surface chloride transport Genistein Pyrophosphate UTP INS36217 Moli1901 Class IV Mutations located within membrane spanning domain, implicated in forming the pore of the channel. Login to comment
174 In vitro investigations have demonstrated an increased production of mature CFTR and chloride transport at the cell surface for both wild type, ∆F508 and G551D, by a mechanism that involves upregulation at the transcriptional level and modulation of protein folding steps [47]. Login to comment
184 Genistein has also been shown to restore G551D function (class III mutation) [54]. Login to comment
186 ATP binding and phosphorylation to G551D are severely affected but can be overcome by interaction with flavonoids. Login to comment
445 Illek B, Zhang L, Lewis NC, Moss RB, Dong JY, Ficsher H: Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein. Login to comment

[hide] Gene complementation of airway epithelium in the c... Hum Mol Genet. 2002 May 1;11(9):1059-67. Oceandy D, McMorran BJ, Smith SN, Schreiber R, Kunzelmann K, Alton EW, Hume DA, Wainwright BJ
Gene complementation of airway epithelium in the cystic fibrosis mouse is necessary and sufficient to correct the pathogen clearance and inflammatory abnormalities.
Hum Mol Genet. 2002 May 1;11(9):1059-67., 2002-05-01 [PMID:11978765]

Abstract [show]
Increasingly, cystic fibrosis (CF) is regarded as an inflammatory disorder where the response of the lung to Pseudomonas aeruginosa is exaggerated as a consequence of processes mediated by the product of the CF gene, CFTR. Of importance to any gene-replacement strategy for treatment of CF is the identification of the cell type(s) within the lung milieu that need to be corrected and an indication whether this is sufficient to restore a normal inflammatory response and bacterial clearance. We generated G551D CF mice transgenically expressing the human CFTR gene in two tissue compartments previously demonstrated to mediate a CFTR-dependent inflammatory response: lung epithelium and alveolar macrophages. Following chronic pulmonary infection with P. aeruginosa, CF mice with epithelial-expressed but not macrophage-specific CFTR showed an improvement in pathogen clearance and inflammatory markers compared with control CF animals. Additionally, these data indicate the general role for epithelial cell-mediated events in the response of the lung to bacterial pathogens and the importance of CFTR in mediating these processes.

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2 We generated G551D CF mice transgenically expressing the human CFTR gene in two tissue compartments previously demonstrated to mediate a CFTR-dependent inflammatory response: lung epithelium and alveolar macrophages. Login to comment
25 Another CF mouse model, the G551D mouse (CftrG551D/G551D ), exhibits abnormal lung inflammation in response to bacterial lipopolysaccharide (LPS), and the bone-marrow-derived macrophages (BMM) from these animals demonstrate hypersensitivity to LPS-induced cytokine production (24). Login to comment
28 We generated G551D CF mice expressing human CFTR cDNA in airway epithelia, or in macrophages using lineage-specific promoters. Login to comment
29 The G551D homozygous mice have a reduced capacity to clear bacteria compared with normal mice after pulmonary infection with P. aeruginosa embedded in beads (27). Login to comment
32 RESULTS Establishment of the G551D-K18 and G551D-fms bitransgenic mice We used the human cytokeratin 18 (K18) promoter to direct CFTR cDNA expression selectively to respiratory epithelium. Login to comment
45 From three transgenic founders bearing the D6.7fms-CFTR transgene, two transmitted the transgene to their offspring. We then mated K18-CFTR mice and D6.7fms-CFTR mice with G551D CF mice (CftrG551D/G551D ). Login to comment
46 Then, by intercross breeding of F1 mice, we generated bitransgenic CftrG551D/ G551D K18-CFTR þ / 7 (G551D-K18), which are CF mice expressing human CFTR in the respiratory epithelial cells, and CftrG551D/G551D D6.7fms-CFTR þ / 7 (G551D-fms) which are CF mice expressing human CFTR in macrophages. Login to comment
47 Subsequent studies were focused on those mice together with G551D CF mice (CftrG551D/G551D ) and normal mice (CftrG551D/ þ and Cftr þ / þ ) that are the littermates of both G551D-K18 and G551D-fms as controls. Login to comment
62 The electrophysiological responses to IBMX and forskolin were examined in excised tracheal and caecal tissues from G551D, G551D-K18, G551D-fms and normal mice. Login to comment
63 In trachea, the change in short-circuit current (DISC) after IBMX/forskolin challenge was significantly reduced in G551D mice compared with normal animals (P < 0.05) (Fig. 3A). Login to comment
64 In the G551D-K18 mice, the DISC was significantly higher (P ¼ 0.01) and the average was increased by approximately 2.5 fold compared with G551D animals, indicating that the human transgene had restored function to the murine CF trachea. Login to comment
65 By contrast, the IBMX/ forskolin response in the trachea of G551D-fms animals was not different from that in G551D mice, suggesting that expression of the transgene in the macrophages did not restore the chloride channel activity in the trachea. Login to comment
67 The forskolin response in the cecum was markedly reduced in G551D compared with normal mice (P < 0.001), and in G551D-K18 mice the DISC was increased by approximately 40% of the value of normal mice and significantly higher (P < 0.05) than G551D animals (Fig. 3B). Login to comment
68 As in the trachea, the forskolin response in the cecum of G551D-fms mice was comparable to that in G551D mice. Login to comment
70 Expression of human CFTR in airway epithelial cells corrects P. aeruginosa clearance in the lung To test whether complementation of human CFTR in airway epithelial cells or in alveolar macrophages corrected the pathogen clearance defect in CF mice, we studied the effects of chronic lung infection in G551D-K18 and G551D-fms mice. Login to comment
72 After 3 days of infection, G551D mice had a significantly higher (P < 0.05) number of P. aeruginosa in the lung than normal mice (Fig. 4A and Table 1), indicating an impaired clearance capacity. Login to comment
74 The bacterial burden in the lung of G551D-K18 mice was comparable to that in normal mice and significantly (P ¼ 0.01) lower than that in G551D mice. However, there was no significant difference between G551D and G551D-fms animals. Login to comment
77 One out of 14 (7.1%) G551D mice and 2 out of 11 (18.2%) G551D-fms mice died by 3 days after infection, whereas none of the G551D-K18 and normal mice died (Table 1). Login to comment
78 Although it did not reach statistical significance, this result supports the quantitative bacterial clearance assay that G551D and G551D-fms mice are more susceptible to P. aeruginosa lung infection. Login to comment
80 Two days after infection, weight loss stabilized in normal and G551D-K18 mice, whereas it continued in G551D and G551D-fms mice (Fig. 4B). Login to comment
81 Thus, by day 3 after infection, G551D and G551D-fms mice lost more weight than G551D-K18 and normal mice, although this did not reach statistical significance (P < 0.2). Login to comment
84 (A) The response to IBMX and forskolin on in vitro short-circuit current change (DISC) in the trachea of normal (n ¼ 8), G551D (n ¼ 8), G551D-K18 (n ¼ 6) and G551D-fms (n ¼ 4) mice. Login to comment
85 (B) Change in ISC in response to forskolin in the cecum of normal (n ¼ 18), G551D (n ¼ 7), G551D-K18 (n ¼ 4) and G551D-fms (n ¼ 4) mice. Login to comment
87 (P < 0.05) elevated in the G551D mice compared with normal animals after 3 days of infection with P. aeruginosa agar beads (Fig. 5). Login to comment
88 In the G551D-K18 mice, the concentrations of these molecules were comparable to those in normal mice, and significantly lower (P < 0.01) than there in G551D mice. Login to comment
90 Bacterial burden in the lung and mortality after 3 days of P. aeruginosa beads infection Bacterial burden Mortality Mean Median Normal 6.23 Â 106 6.78 Â 103 0/11 (0%) G551D 7.74 Â 107 5.22 Â 105 1/14 (7.1%) G551D-K18 8.17 Â 104 1.56 Â 104 0/13 (0%) G551D-fms 2.88 Â 107 2.88 Â 104 2/11 (18.2%) Values for bacterial burden are expressed in CFU/ml lung homogenates. Login to comment
94 (A) Bacterial burden in lung homogenates after 3 days of P. aeruginosa infection in normal (squares, n ¼ 11), G551D (circles, n ¼ 13), G551D-K18 (triangles, n ¼ 13) and G551D-fms (crosses, n ¼ 10) mice. Login to comment
98 (B) Change in body weight during P. aeruginosa infection: squares normal mice; circles, G551D; triangles, G551D-K18; crosses, G551D-fms. Login to comment
105 Sample size are as follows: normal, n ¼ 11; G551D, n ¼ 13; G551D-K18, n ¼ 12; G551D-fms, n ¼ 9. Login to comment
107 If upregulation of the inflammatory response in CF mice is caused by altered CFTR function in macrophages, we anticipated correction in the G551D-fms mice. However, levels of these cytokines were not significantly different in these mice compared to G551D animals. Login to comment
110 Thus, the higher concentration of cytokine/chemokines in the BAL fluid of G551D and G551D-fms mice was most likely a consequence of ineffective bacterial clearance by the pulmonary epithelium, resulting in the accumulation of bacterial products and stimulation of macrophages. Login to comment
115 In keeping with these findings, G551D CF mice that carry a type III mutation in the Cftr gene also show reduced bacterial clearance after infection with P. aeruginosa-laden agar beads (27). Login to comment
118 On average, the G551D mice retained 12-fold more bacteria in the lung than normal mice after 3 days of P. aeruginosa infection (Table 1). Login to comment
119 In contrast, the bacterial clearance capacity of G551D-K18 mice was comparable to that of normal animals and significantly better than that of G551D mice. However, the bacterial clearance defect was not corrected in the G551D-fms mice - suggesting that expression of exogenous CFTR in macrophages had no effect in eliminating bacterial infection. Login to comment
121 Thomas and co-workers (24) reported that G551D CF mice have an abnormal inflammatory response in the lung as well as in isolated macrophages after bacterial LPS induction. Login to comment
125 However, G551D-fms mice exhibited an excessive inflammatory response after P. aeruginosa infection, as indicated by higher concentrations of TNF-a, mip-2 and KC in BAL fluid. Login to comment
126 In contrast, G551D-K18 mice showed reduced cytokine/chemokine production, comparable to normal levels. Login to comment
133 Each symbol represents an individual mouse: squares, normal mice; circles, G551D; triangles, G551D-K18; crosses, G551D-fms. Login to comment
145 Female transgenic offspring were mated with CftrG551D/G551D males (129Sv/CD1), which are homozygous for the G551D mutation in the murine Cftr gene (44). Login to comment
148 PCR followed by restriction enzyme digestion was used to identify the G551D mutation as described (44). Login to comment
157 Electrophysiological measurements Electrophysiological studies were performed in excised trachea and cecum from G551D, bitransgenic G551D-K18, G551D-fms and normal mice. 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

[hide] Predictors of deterioration of lung function in cy... Pediatr Pulmonol. 2002 Jun;33(6):483-91. Schaedel C, de Monestrol I, Hjelte L, Johannesson M, Kornfalt R, Lindblad A, Strandvik B, Wahlgren L, Holmberg L
Predictors of deterioration of lung function in cystic fibrosis.
Pediatr Pulmonol. 2002 Jun;33(6):483-91., [PMID:12001283]

Abstract [show]
The severity of lung disease in cystic fibrosis (CF) may be related to the type of mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and to environmental and immunological factors. Since pulmonary disease is the main determinant of morbidity and mortality in CF, it is important to identify factors that can explain and predict this variation. The aim of this longitudinal study of the whole Swedish CF population over age 7 years was to correlate genetic and clinical data with the rate of decline in pulmonary function. The statistical analysis was performed using the mixed model regression method, supplemented with calculation of relative risks for severe lung disease in age cohorts.The severity of pulmonary disease was to some extent predicted by CFTR genotype. Furthermore, the present investigation is the first long-term study showing a significantly more rapid deterioration of lung function in patients with concomitant diabetes mellitus. Besides diabetes mellitus, pancreatic insufficiency and chronic Pseudomonas colonization were found to be negative predictors of pulmonary function. In contrast to several other reports, we found no significant differences in lung function between genders. Patients with pancreatic sufficiency have no or only a slight decline of lung function with age once treatment is started, but an early diagnosis in this group is desirable.

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88 Furthermore, the inferred values for FEV1 and VC at age 5 years (the intercepts) were significantly lower TABLE 1- Allele Frequencies of 10 Most Common CFTR Mutations in Swedish CF Population Mutation Allele frequency (%) DF508 67.9 394delTT 7.1 3659delC 6.4 S945L 1.2 R117C 1.0 R117H 0.55 T338I 0.55 G551D 0.55 R553X 0.55 I506L 0.41 compared with those in the other CF patients (63.4% and 68.2% vs. 89% and 93.3%). 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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40 Some mutations can reach higher frequencies in particular populations due to founder effect (e.g., M1101K in Hutterites) [Zielenski et al., 1993] or genetic drift (e.g., G551D in the historic Celts), and other mutations are recurrent (e.g., R1162X in Zuni and Northwest Italian populations) which explains their presence in historically unrelated populations [Morral et al., in an attempt to quantify a pattern between the number of mutations present in a population and the CF incidence and/or the ∆F508 frequency of that population. 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
102 There are also a multitude of other interesting regions or populations such as Reunion Island, which shows its distinct ancestral founder effect by having both high rates of G551D and FIGURE 1. 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
170 Another mutation that seems to have a clear history is the G551D allele. Login to comment
179 The effects of this movement can be seen today by the nearly three times higher than expected frequency of G551D in the Czech Republic. 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] Glutathione levels and BAX activation during apopt... J Biol Chem. 2002 Aug 2;277(31):27912-8. Epub 2002 May 22. Jungas T, Motta I, Duffieux F, Fanen P, Stoven V, Ojcius DM
Glutathione levels and BAX activation during apoptosis due to oxidative stress in cells expressing wild-type and mutant cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 2002 Aug 2;277(31):27912-8. Epub 2002 May 22., 2002-08-02 [PMID:12023951]

Abstract [show]
Cystic fibrosis is characterized by chronic inflammation and an imbalance in the concentrations of alveolar and lung oxidants and antioxidants, which result in cell damage. Modifications in lung glutathione concentrations are recognized as a salient feature of inflammatory lung diseases such as cystic fibrosis, and glutathione plays a major role in protection against oxidative stress and is important in modulation of apoptosis. The cystic fibrosis transmembrane conductance regulator (CFTR) is permeable to Cl(-), larger organic ions, and reduced and oxidized forms of glutathione, and the DeltaF508 CFTR mutation found in cystic fibrosis patients has been correlated with impaired glutathione transport in cystic fibrosis airway epithelia. Because intracellular glutathione protects against oxidative stress-induced apoptosis, we studied the susceptibility of epithelial cells (HeLa and IB3-1) expressing normal and mutant CFTR to apoptosis triggered by H(2)O(2). We find that cells with normal CFTR are more sensitive to oxidative stress-induced apoptosis than cells expressing defective CFTR. In addition, sensitivity to apoptosis could be correlated with glutathione levels, because depletion of intracellular glutathione results in higher levels of apoptosis, and glutathione levels decreased faster in cells expressing normal CFTR than in cells with defective CFTR during incubation with H(2)O(2). The pro-apoptotic BCL-2 family member, BAX, is also activated faster in cells expressing normal CFTR than in those with mutant CFTR under these conditions, and artificial glutathione depletion increases the extent of BAX activation. These results suggest that glutathione-dependent BAX activation in cells with normal CFTR represents an early step in oxidative stress-induced apoptosis of these cells.

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42 HeLa cells stably transfected with the plasmid alone (pTracer), the wild-type CFTR construct (pTCFwt), the CFTR ⌬F508 mutation (pTCF⌬F508), and the CFTR G551D mutation (pTCFG551D) were prepared as previously described (30). Login to comment
124 The ability of H2O2 to activate BAX was then tested in pTracer, wild-type CFTR cells, ⌬F508, and cells expressing another CFTR mutation, G551D. Login to comment
125 The G551D mutation is less common than ⌬F508, but it traffics normally to the plasma membrane, unlike ⌬F508, most of which is processed improperly and remains within the cell (44). Login to comment
162 BAX activation in HeLa cells stably transfected with plasmid (black bars), wild-type CFTR (white bars), ⌬F508 CFTR (gray bars), or G551D CFTR (striped bars) was measured as a function of time after addition of 90 ␮M H2O2. Login to comment

[hide] Distinct Mg(2+)-dependent steps rate limit opening... J Gen Physiol. 2002 Jun;119(6):545-59. Dousmanis AG, Nairn AC, Gadsby DC
Distinct Mg(2+)-dependent steps rate limit opening and closing of a single CFTR Cl(-) channel.
J Gen Physiol. 2002 Jun;119(6):545-59., [PMID:12034762]

Abstract [show]
The roles played by ATP binding and hydrolysis in the complex mechanisms that open and close cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels remain controversial. In this work, the contributions made by ATP and Mg(2+) ions to the gating of phosphorylated cardiac CFTR channels were evaluated separately by measuring the rates of opening and closing of single channels in excised patches exposed to solutions in which [ATP] and [Mg(2+)] were varied independently. Channel opening was found to be rate-limited not by the binding of ATP alone, but by a Mg(2+)-dependent step that followed binding of both ATP and Mg(2+). Once a channel had opened, sudden withdrawal of all Mg(2+) and ATP could prevent it from closing for tens of seconds. But subsequent exposure of such an open channel to Mg(2+) ions alone could close it, and the closing rate increased with [Mg(2+)] over the micromolar range (half maximal at approximately 50 microM [Mg(2+)]). A simple interpretation is that channel closing is stoichiometrically coupled to hydrolysis of an ATP molecule that remains tightly associated with the open CFTR channel despite continuous washing. If correct, that ATP molecule appears able to reside for over a minute in the catalytic site that controls channel closing, implying that the site must entrap, or have an intrinsically high apparent affinity for, ATP, even without a Mg(2+) ion. Such stabilization of the open-channel conformation of CFTR by tight binding, or occlusion, of an ATP molecule echoes the stabilization of the active conformation of a G protein by GTP.

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372 ATP hydrolysis by a CFTR domain: pharmacology and effects of G551D mutation. 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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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] Relationship between genotype and phenotype for th... J Med Genet. 2002 Jun;39(6):E32. Dugueperoux I, Bellis G, Ferec C, Gillet D, Scotet V, De Braekeleer M
Relationship between genotype and phenotype for the CFTR gene W846X mutation.
J Med Genet. 2002 Jun;39(6):E32., [PMID:12070264]

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127 Western Brittany has one of the highest rates of CF in the world.1 2 Over 98% of the CF mutations in the Celtic population of Brittany have been identified.3 4 In this population, only five mutations account for 92.4% of the chromosomes: ∆F508 81.2%, 1078delT 4.9%, G551D 4.1%, 1717-1G→A 1.1%, and W846X2 1.1%. Login to comment

[hide] A novel natural product compound enhances cAMP-reg... Mol Med. 2002 Feb;8(2):75-87. deCarvalho AC, Ndi CP, Tsopmo A, Tane P, Ayafor J, Connolly JD, Teem JL
A novel natural product compound enhances cAMP-regulated chloride conductance of cells expressing CFTR[delta]F508.
Mol Med. 2002 Feb;8(2):75-87., [PMID:12080183]

Abstract [show]
BACKGROUND: Cystic fibrosis (CF) results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel localized at the plasma membrane of diverse epithelia. The most common mutation leading to CF, Delta F508, occurs in the first nucleotide-binding domain (NBD1) of CFTR. The Delta F508 mutation disrupts protein processing, leading to a decreased level of mutant channels at the plasma membrane and reduced transepithelial chloride permeability. Partial correction of the Delta F508 molecular defect in vitro is achieved by incubation of cells with several classes of chemical chaperones, indicating that further investigation of novel small molecules is warranted as a means for producing new therapies for CF. MATERIALS AND METHODS: The yeast two-hybrid assay was used to study the effect of CF-causing mutations on the ability of NBD1 to self-associate and form dimers. A yeast strain demonstrating defective growth as a result of impaired NBD1 dimerization due to Delta F508 was used as a drug discovery bioassay for the identification of plant natural product compounds restoring mutant NBD1 interaction. Active compounds were purified and the chemical structures determined. The purified compounds were tested in epithelial cells expressing CFTR Delta F508 and the resulting effect on transepithelial chloride permeability was assessed using short-circuit chloride current measurements. RESULTS: Wild-type NBD1 of CFTR forms homodimers in a yeast two-hybrid assay. CF-causing mutations within NBD1 that result in defective processing of CFTR (Delta F508, Delta I507, and S549R) disrupted NBD1 interaction in yeast. In contrast, a CF-causing mutation that does not impair CFTR processing (G551D) had no effect on NBD1 dimerization. Using the yeast-based assay, we identified a novel limonoid compound (TS3) that corrected the Delta F508 NBD1 dimerization defect in yeast and also increased the chloride permeability of Fisher Rat Thyroid (FRT) cells stably expressing CFTR Delta F508. CONCLUSION: The establishment of a phenotype for the Delta F508 mutation in the yeast two-hybrid system yielded a simple assay for the identification of small molecules that interact with the mutant NBD1 and restore dimerization. The natural product compound identified using the system (TS3) was found to increase chloride conductance in epithelial cells to an extent comparable to genistein, a known CFTR activator. The yeast system will thus be useful for further identification of compounds with potential for CF drug therapy.

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10 In contrast, a CF-causing mutation that does not impair CFTR processing (G551D) had no effect on NBD1 dimerization. Login to comment
37 Plasmids identical to pBDNBD1 and pADNBD1, but containing CF-causing mutations (⌬F508, ⌬I507, S549R, and G551D) were similarly constructed (producing pBD⌬F, pAD⌬F, pBD⌬I507, pAD⌬I507, pBDS549R, pADS549R, pBDG551D, pADG551D). Login to comment
109 To assess the effect of CF-causing mutations on NBD1 homodimerization, ⌬I507, ⌬F508, S549R, and G551D were each introduced into NBD1 in both pADNBD1 and pBDNBD1. Login to comment
111 The CF-causing mutation G551D, which impairs chloride channel function, but not processing of CFTR, did not result in defective NBD1 dimerization in yeast (Fig. 1). Login to comment
215 In contrast, the G551D mutation, which does not impair CFTR processing, had no effect on NBD1 association. 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
83 However, the presence of the R553X and S549N mutations precluded hybridization to the G551D wild-type probe (data not shown). Login to comment
84 Thus, although the assay correctly identified the R553X mutation in a heterozygous state, the lack of hybridization with the G551D wild-type probe (absence of signal in the wild-type allele) indicated the need for additional analysis to determine the genotype of the sample. 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] Mutations in the nucleotide binding domain 1 signa... J Biol Chem. 2002 Sep 27;277(39):35896-905. Epub 2002 Jul 10. DeCarvalho AC, Gansheroff LJ, Teem JL
Mutations in the nucleotide binding domain 1 signature motif region rescue processing and functional defects of cystic fibrosis transmembrane conductance regulator delta f508.
J Biol Chem. 2002 Sep 27;277(39):35896-905. Epub 2002 Jul 10., 2002-09-27 [PMID:12110684]

Abstract [show]
The gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP binding cassette (ABC) transporter that functions as a phosphorylation- and nucleotide-regulated chloride channel, is mutated in cystic fibrosis (CF) patients. Deletion of a phenylalanine at amino acid position 508 (DeltaF508) in the first nucleotide binding domain (NBD1) is the most prevalent CF-causing mutation and results in defective protein processing and reduced CFTR function, leading to chloride impermeability in CF epithelia and heterologous systems. Using a STE6/CFTRDeltaF508 chimera system in yeast, we isolated two novel DeltaF508 revertant mutations, I539T and G550E, proximal to and within the conserved ABC signature motif of NBD1, respectively. Western blot and functional analysis in mammalian cells indicate that mutations I539T and G550E each partially rescue the CFTRDeltaF508 defect. Furthermore, a combination of both revertant mutations resulted in a 38-fold increase in CFTRDeltaF508-mediated chloride current, representing 29% of wild type channel activity. The G550E mutation increased the sensitivity of CFTRDeltaF508 and wild type CFTR to activation by cAMP agonists and blocked the enhancement of CFTRDeltaF508 channel activity by 2 mm 3-isobutyl-1-methylxanthine. The data show that the DeltaF508 defect can be significantly rescued by second-site mutations in the nucleotide binding domain 1 region, that includes the LSGGQ consensus motif.

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36 Interestingly, the site of this mutation is flanked by two residues where CF-causing mutations have been identified that impair folding/trafficking of CFTR (S549R) or ATP-dependent channel gating (G551D) (37-41). Login to comment
143 The functional importance of the NBD1 signature motif is evident from the characterization of the CF, causing mutation G551D, which does not affect processing but results in decreased CFTR chloride channel function (41, 50) (Table II). Login to comment
167 CFTR variant Isc n % of CFTR wt CFTR S549R 1.70 Ϯ 0.11 4 CFTR G551D 1.17 Ϯ 0.12 4 CFTR ⌬F508 0.76 Ϯ 0.07 17 CFTR ⌬F/G550E 9.30 Ϯ 0.55 14 (*) CFTR ⌬F/G550D 6.06 Ϯ 0.63 12 (*) CFTR ⌬F/G550H 4.17 Ϯ 0.40 8 (*) Mutations in the ABC Signature Motif Region Rescue CFTR⌬F50835900 CFTR⌬F/G550E relative to FRT-CFTR ⌬F508 (Fig. 4B), although the mature band C was barely detectable, and the steady-state levels of band B were decreased for the revertant (Fig. 4A). Login to comment
206 CFTR variants bearing CF-causing mutations within the NBD1 signature motif, G551D and S549R, were included in the experiment and produced low Isc, as expected. Login to comment
284 The CF-causing mutation of the invariant Gly residue in the NBD1 signature motif of CFTR (LSGGQ), G551D, has been shown to reduce ATP binding (41, 77) and hydrolysis (18). 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]

Abstract [show]
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

[hide] The cystic fibrosis mutation G551D alters the non-... J Biol Chem. 2002 Sep 27;277(39):35999-6004. Epub 2002 Jul 17. Derand R, Bulteau-Pignoux L, Becq F
The cystic fibrosis mutation G551D alters the non-Michaelis-Menten behavior of the cystic fibrosis transmembrane conductance regulator (CFTR) channel and abolishes the inhibitory Genistein binding site.
J Biol Chem. 2002 Sep 27;277(39):35999-6004. Epub 2002 Jul 17., 2002-09-27 [PMID:12124395]

Abstract [show]
Loss of cystic fibrosis transmembrane conductance regulator (CFTR) channel activity explains most of the manifestations of the cystic fibrosis (CF) disease. To understand the consequences of CF mutations on CFTR channel activity, we compared the pharmacological properties of wild-type (wt) and G551D-CFTR. Dose-dependent relationships of wt-CFTR activated by genistein follows a non-Michaelis-Menten behavior consistent with the presence of two binding sites. With phosphorylated CFTR, a high affinity site for genistein is the activator (K(s) approximately 3 microm), whereas a second site of low affinity (K(i) approximately 75 microm) is the inhibitor. With non-phosphorylated CFTR, K(s) was increased (K(s) approximately 12 microm), but K(i) was not affected (K(i) approximately 70 microm). In G551D-CFTR cells, channel activity was recovered by co-application of forskolin and genistein in a dose-dependent manner. A further stimulation of G551D-CFTR channel activity was measured at concentrations from 30 microm to 1 mm. The dose response is described by a classical Michaelis-Menten kinetics with only a single apparent site (K(m) approximately 11 microm). Our results suggest glycine 551 in NBD1 as an important location within the low affinity inhibitory site for genistein and offers new evidence for pharmacological alteration caused by an NBD1 mutation of CFTR. This study also reveals how a mutation of an ion channel converts a non-Michaelis-Menten behavior (two binding sites) into a classical Michaelis-Menten model (one binding site).

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0 The Cystic Fibrosis Mutation G551D Alters the Non-Michaelis-Menten Behavior of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel and Abolishes the Inhibitory Genistein Binding Site* Received for publication, June 20, 2002 Published, JBC Papers in Press, July 17, 2002, DOI 10.1074/jbc.M206121200 Renaud De´rand, Laurence Bulteau-Pignoux, and Fre´de´ric Becq‡ From the From LBSC, CNRS UMR 6558, Universite´ de Poitiers, 40 Avenue du Recteur Pineau, 86022 Poitiers, France Loss of cystic fibrosis transmembrane conductance regulator (CFTR) channel activity explains most of the manifestations of the cystic fibrosis (CF) disease. Login to comment
1 To understand the consequences of CF mutations on CFTR channel activity, we compared the pharmacological properties of wild-type (wt) and G551D-CFTR. Login to comment
5 In G551D-CFTR cells, channel activity was recovered by co-application of forskolin and genistein in a dose-dependent manner. Login to comment
6 A further stimulation of G551D-CFTR channel activity was measured at concentrations from 30 ␮M to 1 mM. Login to comment
13 The glycine-to-aspartic acid missense mutation at codon 551 (G551D) is a class III mutation located within the nucleotide binding domain 1 (NBD1) (5). Login to comment
15 G551D is one of the five most frequent CF mutations with a frequency of 2-5% depending of the population of origin but is associated with a severe CF phenotype (5). Login to comment
17 The G551D mutated protein is fully glycosylated, correctly located at the apical membrane (i.e. normal biosynthesis, trafficking, and processing), and normally phosphorylated at the R domain by cAMP-dependent protein kinase (6). Login to comment
18 However, G551D proteins have a decreased nucleotide binding (7) and a reduced ATPase activity at NBD1 (8, 9). Login to comment
22 In this work, we compared the effect of genistein on both activation and inhibition of wt-CFTR and G551D-CFTR chloride channels. Login to comment
23 Analyzing dose-response relationships pointed to a novel type of alteration due to the disease-causing mutation G551D. Login to comment
26 EXPERIMENTAL PROCEDURES Cell Culture-Chinese hamster ovary (CHO) cells stably transfected with pNUT vector alone (pNUT CHO) or containing wild-type CFTR (CFTR(ϩ) CHO) or G551D (G551D CHO) mutation were provided by J. R. Riordan and X.-B. Chang, Scottsdale, AZ (2, 6). Login to comment
109 Perturbation of the non-Michaelis-Menten Behavior of CFTR by the G551D Mutation-The glycine-to-aspartic acid missense mutation at codon 551 (G551D) is a class III mutation located within the NBD1 domain (5). Login to comment
110 We used CHO cells stably expressing G551D-CFTR to analyze the effect of genistein. Login to comment
112 The addition of up to 100 ␮M genistein failed to activate chloride transport in G551D-CFTR-expressing cells. Login to comment
113 We also noticed that up to 10 ␮M forskolin failed to stimulate G551D-CFTR channels (not shown). Login to comment
115 To compare the effect of genistein on G551D-CFTR with our study with wt-CFTR cells, we generated dose-response relationships for genistein in the presence of 10 ␮M forskolin. Login to comment
117 Complete dose-response relationships for activation of G551D-CFTR using concentrations of genistein from 0.1 ␮M to 1000 ␮M were generated from six separate experiments. Login to comment
125 Second, at concentrations that normally induced an inhibition of wt-CFTR (i.e. Ͼ 30 ␮M), genistein did not inhibit G551D-CFTR chloride channel activity. Login to comment
139 Classical Michaelis-Menten kinetics of G551D-CFTR channel activated by genistein. Login to comment
140 A, example traces of iodide efflux showing the stimulation of G551D-CFTR genistein in the presence of 10 ␮M forskolin. Login to comment
151 Several inhibitors were also used to further characterize G551D-CFTR chloride channel activity. Login to comment
152 Fig. 6, C-E, shows that in the presence of 100 ␮M glibenclamide, the activation of G551D-CFTR currents is abolished. Login to comment
154 A summary of the effect of four different inhibitors on the rate of iodide efflux stimulated by FSK ϩ GST is also presented in Fig. 6F, showing the inhibition of G551D-CFTR channel activity by 100 ␮M glibenclamide and 250 ␮M DPC but not by 200 ␮M DIDS nor by 100 nM calixarene. Login to comment
155 DISCUSSION We described here the mechanism of action and kinetic parameters of wt-CFTR chloride channel activated by genistein and the profound alteration resulting from the G551D mutation. Login to comment
156 This study reveals two important results: (i) a non- Michaelis-Menten behavior describes the pharmacological effect of genistein on wt-CFTR, suggesting the presence of one activatory site and one inhibitory site; (ii) the non-Michaelis-Menten behavior of wt-CFTR is absent and transformed into a classical Michaelis-Menten behavior by the CF mutation G551D. Login to comment
163 Our results support a model with two binding sites for genistein on wt-CFTR but only one binding site on G551D-CFTR. Login to comment
172 Activation of G551D-CFTR chloride currents using whole cell patch clamp recording from G551D CHO cells. Login to comment
173 G551D-CFTR currents were stimulated with 10 ␮M FSK and 30 ␮M GST. Login to comment
180 F, histograms summarizing the inhibition of genistein-mediated 125 I efflux in G551D CHO cells. Login to comment
188 Indeed, one major difference concerning the phosphorylation process is that with G551D-CFTR, much higher concentrations of forskolin are required to achieve the stimulation by genistein of the CFTR channel activity. Login to comment
189 The G551D-CFTR protein is a class III mutation that produced a fully glycosylated protein correctly located at the apical membrane (i.e. normal biosynthesis, trafficking, and processing) and normally phosphorylated at the R domain by cAMP-dependent protein kinase (6). Login to comment
190 However, G551D proteins have decreased nucleotide binding (7) and a reduced ATPase activity at NBD1 (8, 9). Login to comment
191 The mutation G551D also disrupts activation and regulation of CFTR at the plasma membrane since phosphorylation alone is not able to achieve efficient stimulation of chloride transport in G551D cells (12, 29, 30). Login to comment
192 Our study of the activity of G551D-CFTR mutant demonstrates that the inhibitory component of the dual mechanism of the action of genistein on wt-CFTR is lacking. Login to comment
195 In addition, our study reveals that the G551D mutation profoundly alters the pharmacological behavior of CFTR. Login to comment
200 We have described here a similar effect with the class III CF mutation G551D and showed that it abolishes the inhibitory response to genistein. Login to comment
203 In conclusion, our results show that the high affinity stimulatory site for genistein, located in NBD2, is dependent on the phosphorylation status of CFTR and preserved by G551D mutation. Login to comment
204 The second site is lacking in G551D-CFTR cells. 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]

Abstract [show]
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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28 Class 3 mutations affect the regulatory domains of the CFTR protein and include G551D, which is frequent in Northern Europe. Login to comment
97 After gene cloning, Hamosh et al. [1992] found that G551D carried a decreased risk of meconium ileus in contrast to previous studies [Kerem et al., 1989c]. 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

[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
76 After the 5T allele, the relative frequent mutations with two to four alleles were: R117H (four alleles), W1282X (four alleles), G551D (three alleles), L206W (three alleles) and D1270 (two alleles). 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] High-affinity activators of cystic fibrosis transm... J Biol Chem. 2002 Oct 4;277(40):37235-41. Epub 2002 Aug 2. Ma T, Vetrivel L, Yang H, Pedemonte N, Zegarra-Moran O, Galietta LJ, Verkman AS
High-affinity activators of cystic fibrosis transmembrane conductance regulator (CFTR) chloride conductance identified by high-throughput screening.
J Biol Chem. 2002 Oct 4;277(40):37235-41. Epub 2002 Aug 2., 2002-10-04 [PMID:12161441]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein that reduce cAMP-stimulated Cl(-) conductance in airway and other epithelia. The purpose of this investigation was to identify new classes of potent CFTR activators. A collection of 60,000 diverse drug-like compounds was screened at 10 microm together with a low concentration of forskolin (0.5 microm) in Fisher rat thyroid epithelial cells co-expressing human CFTR and a green fluorescent protein-based Cl(-) sensor. Primary screening yielded 57 strong activators (greater activity than reference compound apigenin), most of which were unrelated in chemical structure to known CFTR activators, and 284 weaker activators. Secondary analysis of the strong activators included analysis of CFTR specificity, forskolin requirement, transepithelial short-circuit current, activation kinetics, dose response, toxicity, and activation mechanism. Three compounds, the most potent being a dihydroisoquinoline, activated CFTR by elevating cellular cAMP, probably by phosphodiesterase inhibition. Fourteen compounds activated CFTR without cAMP elevation or phosphatase inhibition, suggesting direct CFTR interaction. The most potent compounds had tetrahydrocarbazol, hydroxycoumarin, and thiazolidine core structures. These compounds induced CFTR Cl(-) currents rapidly (<5 min) with K(d) down to 200 nm and were CFTR-selective, reversible, and nontoxic. Several compounds, the most potent being a trifluoromethylphenylbenzamine, activated the CF-causing mutant G551D, but with much weaker affinity (K(d) > 10 microm). When added for 10 min, none of the compounds activated DeltaPhe(508)-CFTR in transfected cells grown at 37 degrees C (with DeltaPhe(508)-CFTR trapped in the endoplasmic reticulum). However, after correction of trafficking by 48 h of growth at 27 degrees C, tetrahydrocarbazol and N-phenyltriazine derivatives strongly stimulated Cl(-) conductance with K(d) < 1 microm. The new activators identified here may be useful in defining molecular mechanisms of CFTR activation and as lead compounds in CF drug development.

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10 Several compounds, the most potent being a trifluoromethyl- phenylbenzamine, activated the CF-causing mutant G551D, but with much weaker affinity (Kd > 10 ␮M). Login to comment
23 Such activators could provide information about CFTR activating mechanisms and might activate some CF-causing CFTR mutants, such as G551D-CFTR, that are processed normally but have reduced intrinsic Cl- permeability. Login to comment
40 Novel activators were identified that strongly activated CFTR Cl-conductance at concentrations well under 1 ␮M and without elevation of cAMP, as well as compounds that activated the CF-causing mutants G551D-CFTR and ⌬Phe508 -CFTR. Login to comment
41 MATERIALS AND METHODS Cell Culture-Fischer rat thyroid (FRT) cells stably co-expressing human CFTR (wild-type, or mutants G551D or ⌬Phe508 ) and the yellow fluorescent protein YFP-H148Q were cultured on plastic in Coon`s modified F12 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 ␮g/ml streptomycin as described previously (13). Login to comment
157 The activators of wild-type CFTR were screened for their ability to activate two CF-causing CFTR mutants: G551D, which is transported to the plasma membrane but is not active after cAMP elevation, and ⌬Phe508 , which is retained at the endoplasmic reticulum and has impaired function even after transport to the cell surface (by low temperature incubation or chemical chaperones). Login to comment
158 Fig. 5B, top, shows representative fluorescence assays of I- influx in FRT cells coexpressing G551D-CFTR and YFP-H148Q. Login to comment
159 Measurements were performed using 20 ␮M forskolin, which itself does not activate G551D-CFTR, and 50 ␮M of each test compound. Login to comment
160 Of the 57 activators of wild-type CFTR identified in the fluorescence assay, only three compounds activated G551D-CFTR; the most potent compound (structure in Fig. 5B, middle) was not one of the strongest activators of wild-type CFTR. Login to comment
161 The other G551D-CFTR activators (N-[6-methyl-2-(4-methylphenyl)-2H-benzotriazol-5-yl]-4- nitrobenzamide and ␣-methyl-4-oxo-5-(phenylmethylene)-2- thioxo-3-thiazolidineacetic acid) were also not strong activators of wild-type CFTR. Login to comment
162 The potencies of these compounds for activation of G551D-CFTR were relatively poor. Login to comment
204 B, activation of G551D-CFTR. Login to comment
205 Fluorescence assays of I- influx on cells co-expressing G551D-CFTR and YFP-H148Q. Login to comment
217 The compounds that strongly activated wild-type CFTR were screened for their efficacy in activating G551D-CFTR and ⌬Phe508 -CFTR, two mutant CFTRs that cause human CF. Login to comment
218 Of the 57 strong activators of wild-type CFTR, only three compounds activated G551D-CFTR significantly, indicating that CFTR activation is mutation-specific. Login to comment
219 Unfortunately, the compounds that activated G551D-CFTR did so weakly (Kd 10 ␮M or higher), no better than existing flavones. 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] Genistein modifies the activation kinetics and mag... J Membr Biol. 2002 Aug 1;188(3):175-82. Bulteau-Pignoux L, Derand R, Metaye T, Joffre M, Becq F
Genistein modifies the activation kinetics and magnitude of phosphorylated wild-type and G551D-CFTR chloride currents.
J Membr Biol. 2002 Aug 1;188(3):175-82., 2002-08-01 [PMID:12181609]

Abstract [show]
We have studied the mechanism by which genistein activates cystic fibrosis transmembrane conductance regulator (CFTR) in CHO cells expressing wild type or G551D-CFTR. In wild-type CHO cells, after exposure to 2.5 microM forskolin, 25 microM genistein induced a further 2-fold and rapid increase of the forskolin-activated CFTR current. In both types of cells, when forskolin was added after genistein preincubation, whole-cell current density was greatly reduced compared to that measured when genistein was added after phosphorylation of CFTR, and all activation kinetic parameters were significantly altered. Genistein had no effect on the adenylate cyclase activity. Our results suggest that the occupancy of a putative genistein binding site is critical for the gating mechanism of CFTR chloride channels, which, depending on the phosphorylation status of the R-domain, drives CFTR either into a refractory state or alternatively to a highly activated state.

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1 We have studied the mechanism by which genistein activates cystic ®brosis transmembrane conductance regulator (CFTR) in CHO cells expressing wild type or G551D-CFTR. Login to comment
8 Among the 900 mutations associated with a CF disease phenotype, the G551D mutation is the third most common one with a frequency of 2±5%, depending on the population of origin (Hamosh et al., 1992; Cashman et al., 1995). Login to comment
10 G551D, localized in NBD1 of CFTR, interferes with ATP binding (Logan et al., 1994) and hydrolysis (Li et al., 1996). Login to comment
28 In the present study, we further explored the mechanism by which genistein activates wild type and G551D-CFTR expressed in CHO cells. Login to comment
30 Materials and Methods CELL CULTURE Chinese Hamster Ovary cells, stably transfected with pNUT vector containing wild-type CFTR (CFTR(+) CHO) or G551D mutation (G551D CHO) were provided by J.R. Riordan and X.-B. Chang, Scottsdale, AZ, USA (Tabcharani et al., 1991). Login to comment
31 Cells cultured at 37°C in 0.5% CO2 were maintained in aMEM containing 7% fetal bovine serum, 0.5% L-glutamine, 0.5% antibiotics (50 IU/ml penicillin and 50 lg/ml streptomycin) and 100 or 20 lM methotrexate for, respectively, CFTR(+) or G551D CHO. Login to comment
60 Results We compared activation of wild type and G551D-CFTR by low concentrations of forskolin and genistein, with two protocols of activation, using the whole-cell and perforated patch-clamp techniques. Login to comment
61 In the ®rst protocol, cells were preincubated during 3 minutes with 2.5 lM (for CFTR(+) CHO) or 10 lM (for G551D CHO) forskolin, then 25 lM genistein was added in the presence of forskolin. Login to comment
83 Since one of the main dierences between wild-type and G551D-CFTR is the lack of responsiveness of the mutant to cAMP agonists, we then performed the same study in G551D CHO cells (Fig. 4). Login to comment
84 No current was activated in the presence of 10 lM fors- Fig. 4. Eect of genistein on forskolin-stimulated G551D-CFTR ClÀ channels. Representative current traces for CFTR. Login to comment
87 Fig. 5. Eect of forskolin on genistein-stimulated G551D-CFTR ClÀ channels. Representative current traces for CFTR for cell ®rst incubated with 25 lM genistein (A), then in the presence of 10 lM forskolin (B). Login to comment
92 In G551D CHO cells not treated with forskolin (Fig. 5), genistein failed to stimulate ClÀ current (Fig. 5A). Login to comment
97 When genistein was ®rst added to the bath, subsequent addition of forskolin led to a current density $3-fold (for wild type) and $4-fold (for G551D) lower than when CFTR was ®rst stimulated by forskolin. Login to comment
98 A time course of the activation of G551D-CFTR current is shown in Fig. 7. Login to comment
102 Activation for G551D-CFTR current (for both conditions) is signi®cantly slower than for wild-type CFTR (compare time constants in Tables 1 and 2). Login to comment
108 Here we show that low concentration of genistein (25 lM) dierentially activates wild type or G551D-CFTR channels depending on whether the protein is phosphorylated before or after genistein interaction with CFTR. Login to comment
110 When forskolin was added after genistein preincubation, (1) whole-cell current density was reduced by $ 3-fold (for wild type) and $4-fold (for G551D), (2) time to activation was greatly delayed, and (3) time constant of activation was reduced when compared with genistein activation after phosphorylation of CFTR. Login to comment
111 Direct interaction of genistein with CFTR has been shown by Randak et al. (1999) who demonstrated Table 2. Comparison of time constants determined at dierent conditions of activation in G551D CHO cells First activator Second activator Activation delay Time constant 1/K sec n P sec n P Fsk Gst 8 1 5 138 30 5 Gst Fsk 120 40 3 * 522 39 3 ** For methods of analysis, see Table 1. Login to comment
114 Fig. 6. Comparison of wild-type and G551D-CFTR current densities at +40 mV holding potential. Login to comment
126 We also found that the kinetic parameters of activation by genistein are similar in wild type and G551D CFTR cells. 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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10 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 Léon), 4005+1G→A (2.9% in Cornouaille) and W846X (1.5% in western Brittany). Login to comment
99 The main mutation, viz. ∆F508, was present in 75% of chromosomes, whereas five other mutations were found with a frequency greater than or equal to 1%: 1078delT (3.8%); G551D (3.7%), N1303 K (1.3%), W846X (1.1%) and 2789+5G→A (1.0%). Login to comment
103 Only three genotypes were responsible for two thirds of CF cases in the region: ∆F508/∆F508 (57.6%), ∆F508/1078delT (4.9%) and ∆F508/ G551D (4.7%). Login to comment
114 The G551D mutation appeared to be concentrated along the coasts of North Finistère, in Léon (7.7% of mutated alleles). Login to comment
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
123 Consequently, western Brittany showed a specific mutation spectrum containing the mutations 1078delT (9.4% of mutated alleles in the diocese of Cornouaille), G551D (7.7% in the diocese of Léon), 4005+1G→A (2.9% in Cornouaille) and W846X (1.5% in western Brittany). Login to comment
155 Some mutations that are rare in France have a high frequency of occurrence in the Breton population (1078delT: 3.8%; G551D: 3.7%; W846X: 1.1%). Login to comment
157 Indeed, 34 of the 49 cases of the 1078delT mutation observed in France (69.4%) have been found here, together with 33 of the 73 G551D alleles (45.2%) and 10 of the 20 W846X alleles (50%; Claustres et al. 2000). Login to comment

[hide] Electrolyte transport in the mouse trachea: no evi... J Membr Biol. 2002 Sep 15;189(2):143-51. Schreiber R, Murle B, Sun J, Kunzelmann K
Electrolyte transport in the mouse trachea: no evidence for a contribution of luminal K(+) conductance.
J Membr Biol. 2002 Sep 15;189(2):143-51., 2002-09-15 [PMID:12235489]

Abstract [show]
Recent studies on frog skin acini have challenged the question whether Cl(-) secretion or Na(+) absorption in the airways is driven by luminal K(+) channels in series to a basolateral K(+) conductance. We examined the possible role of luminal K(+) channels in electrolyte transport in mouse trachea in Ussing-chamber experiments. Tracheas of both normal and CFTR (-/-) mice showed a dominant amiloride-sensitive Na+ absorption under both, control conditions and after cAMP-dependent stimulation. The lumen-negative transepithelial voltage was enhanced after application of IBMX and forskolin and Cl(-) secretion was activated. Electrolyte secretion induced by IBMX and forskolin was inhibited by luminal glibenclamide and the blocker of basolateral Na(+2)Cl(-)K(+) cotransporter azosemide. Similarly, the compound 293B, a blocker of basolateral KCNQ1/KCNE3 K(+) channels effectively blocked Cl(-) secretion when applied to either the luminal or basolateral side of the epithelium. RT-PCR analysis suggested expression of additional K(+) channels in tracheal epithelial cells such as Slo1 and Kir6.2. However, we did not detect any functional evidence for expression of luminal K(+) channels in mouse airways, using luminal 293B, clotrimazole and Ba(2+) or different K(+) channel toxins such as charybdotoxin, apamin and a-dendrotoxin. Thus, the present study demonstrates Cl(-) secretion in mouse airways, which depends on basolateral Na(+2)Cl(-)K(+) cotransport and luminal CFTR and non-CFTR Cl(-) channels. Cl(-) secretion is maintained by the activity of basolateral K(+) channels, while no clear evidence was found for the presence of a luminal K(+) conductance.

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79 An inhibitory eect of glibenclamide on Isc-Amil was also detected in tracheas of cftr(G551/G551D) mice (Fig. 3A). Login to comment
134 Summary of the eects of glibenclamide (Glib) on short-circuit currents measured in tracheas of G551D (À/À) mice. Login to comment
205 We thank Prof. Dr. B. Wainwright for supplying the cftr(G551D/G551D) mice. 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
80 *Includes 1525-1G?T/1525-1G?T, Y569C/Y569D, G551D/G551D, 1525-1G?A/1525-1G?A, R1162X/R1162X, R01/ 07/R01/07, 2184insA/2184insA, Y568D/Y568D, 1VSB1-1/1VSB1-1, 1506M/1506M, 3849+10kbC?T/3849+10kbC?T and Q98X/ Q98X. 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
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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0 Correction of G551D-CFTR transport defect in epithelial monolayers by genistein but not by CPX or MPB-07 *,1 Olga Zegarra-Moran, 1 Leila Romio, 1 Chiara Folli, 1 Emanuela Caci, 2 Frederic Becq, 3 Jean-Michel Vierfond, 3 Yvette Mettey, 4 Giulio Cabrini, 5 Pascale Fanen & 1 Luis J.V. Galietta 1 Laboratorio di Genetica Molecolare, Istituto G. Gaslini, L.go G. Gaslini 5, Genova-16148, Italy; 2 Physiologie des Regulations Cellulaires, UniversiteÂ de Poitiers, 40 avenue du Recteur Pineau, 86022 Poitiers, France; 3 Laboratoire de Chimie Organique, FaculteÂ de MeÂ decine et de Pharmacie de Poitiers, 34 rue du Jardin des Plantes, 86005 Poitiers, France; 4 Laboratorio di Patologia Molecolare, Centro Regionale Fibrosi Cistica, Piazzale Stefani 1, Verona-37126, Italy and 5 Institut National de la Sante et la Recherche Medicale U.468, Service de Biochimie et Genetique, Hopital Henri Mondor, AH-HP, 94010 Creteil, France 1 This study compares the eect of three chemically unrelated cystic ®brosis transmembrane conductance regulator (CFTR) activators on epithelial cell monolayers expressing the G551D-CFTR mutant. Login to comment
2 The correction of G551D-CFTR defect was quanti®ed by comparison with maximal activity elicited in cells expressing wild type CFTR. Login to comment
3 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. Login to comment
5 After normalising for CFTR expression, the response of FRT-G551D epithelia was 1% that of wild type monolayers. Login to comment
6 4 Addition of genistein (10 ± 200 mM), but not of 8-cyclopentyl-1,3-dipropylxanthine (CPX, 1 ± 100 mM) or of the benzo[c]quinolizinium MPB-07 (10 ± 200 mM) 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. Login to comment
8 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. Login to comment
9 6 On human nasal epithelia the correction of G551D-CFTR defective conductance obtained with genistein was 20% that of wild type. Login to comment
10 The impressive eect of genistein suggests that it might correct the Cl7 transport defect on G551D patients. Login to comment
11 British Journal of Pharmacology (2002) 137, 504 ± 512. doi:10.1038/sj.bjp.0704882 Keywords: CFTR; CF-G551D; monolayers; nasal epithelia; CFTR openers; genistein; CPX; MPB-07 Abbreviations: CF, cystic ®brosis; CFTR, cystic ®brosis transmembrane conductance regulator; CPT-cAMP, 8-(4-chlorophenylthio) adenosine-3'-5'-cyclic monophosphate; CPX, 8-cyclopentyl-1,3-dipropylxanthine; GFP, green ¯uorescent protein; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; DIDS, 4,4'-diisothiocyanato- stilbene-2,2'-disulfonic acid; FRT, Fisher rat thyroid; NBD, nucleotide binding domain. Login to comment
16 Among the other mutations, the glycine-to-aspartic acid change at codon 551 (G551D) is the third commonest mutation with a worldwide frequency of 3.1% among CF chromosomes (Hamosh et al., 1992) although populations of Celtic descent display frequencies as high as 8% (Cashman et al., 1995). Login to comment
17 The phenotype of patients carrying one F508del and one G551D allele is characterized by a clinical course as severe as that of homozygous for F508del but with a reduced risk of meconium ileus (Hamosh et al., 1992). Login to comment
18 The G551D is a class III mutation that produces a protein that is well synthesized, processed and correctly inserted in the plasma membrane but with a strongly reduced channel activity (Welsh & Smith, 1993). Login to comment
19 G551D is localized in the ®rst nucleotide binding British Journal of Pharmacology (2002) 137, 504 ± 512 ã 2002 Nature Publishing Group All rights reserved 0007 ± 1188/02 $25.00 www.nature.com/bjp *Author for correspondence; E-mail: ozegarra@tin.it domain (NBD) of CFTR and interferes with ATP hydrolysis (Howell et al., 2000; Li et al., 1996; Logan et al., 1994). Login to comment
31 Recent data indicate that the iso¯avone genistein is particularly eective on the G551D-CFTR Cl7 channel (Illek et al., 1999) and we showed that also the benzo[c]quinolizinium MPB-91 does activate this mutation (Derand et al., 2001). Login to comment
33 This information is important for the development of improved CFTR activators eective on G551D and similar mutations. Login to comment
35 Our results, obtained on a heterologous expression system, show that the three compounds are similarly eective on wild type CFTR but that only genistein is active on the G551D mutant. Login to comment
36 This result is con®rmed on nasal monolayers obtained from a G551D CF patient, where the transport correction by genistein is as high as 20% that of wild type CFTR. Login to comment
62 It has been well established that G551D mutation yields an amount of correctly processed protein similar to that of wild type CFTR (Logan et al., 1994; Welsh & Smith, 1993) therefore normalisation by CFTR mRNA relative levels (CFTR/ G3PDH ratio) was considered a good estimation of CFTR expression. Login to comment
65 Two wild type (N8 and N10) and two G551D clones (6E and A1) were used in this study. Login to comment
69 CF patient clinical feature and genotype, and nasal polyp cell cultures The CF patient is a 12-year-old female carrying the G551D mutation on one chromosome. Login to comment
89 Results Northern blot and immunoprecipitation analysis of CFTR expression To assess the eect of dierent CFTR openers, we stably expressed wild type (wt) and G551D-CFTRs in Fisher rat thyroid (FRT) epithelial cells. Login to comment
92 We considered two clones with wild type CFTR, labelled as N8 and N10, and two clones with G551D, A1 and 6E. Login to comment
96 The G551D clones A1 and 6E showed a CFTR expression lower than N8, but considerably stronger than N10. Login to comment
98 The ratio of CFTR to G3PDH expression was 2.37, 0.12, 0.72 and 0.98 for wt-N8, wt-N10, G551D-6E and G551D-A1, respectively. Login to comment
101 Response of wtand G551D-CFTR monolayers to cAMP elevating agents Application of the cAMP analogue CPT ± cAMP to intact FRT monolayers of cells transfected with wtor G551D-CFTR in symmetrical Cl7 was unable to stimulate a measurable Isc (Figure 2). Login to comment
108 On monolayers expressing G551D-CFTR cells, CPT ± cAMP activated a Isc that was considerably lower. Login to comment
110 Despite the dierence in the amplitude of Isc, the concentration that gave half of the maximal eect in G551D clones was 68.6+13.4 mM, not statistically dierent from that measured in wt-CFTR monolayers. Login to comment
111 Response of wtand G551D-CFTR monolayers to genistein Apical application of genistein to wt-CFTR monolayers produced a sustained Isc increase (Figure 3). Login to comment
117 (a) Northern analysis of RNA obtained from non transfected (NT) FRT cells and from cells stably transfected with G551D-CFTR (clones A1 and 6E) or with wt-CFTR (clones N8 and N10) CFTR. Login to comment
124 Fully glycosylated (band C, 175 kDa) and core glycosylated (band B, 150 kDa) CFTR can be observed in wild type and G551D clones, but not in non-transfected FRT cells (lane NT). Login to comment
128 In contrast to wild type CFTR, the G551D mutant did not respond to genistein (10 ± 200 mM). Login to comment
135 The currents activated by CPT ± cAMP and/or genistein in wild type and G551D-CFTR cells were totally blocked by 500 mM glibenclamide. Login to comment
140 CPX alone was unable to activate G551D-CFTR. Login to comment
145 Representative traces depicting the response of wt-CFTR (a and c, clone N10) and of G551D-CFTR (b and d, clone 6E) monolayers to 500 mM CPT ± cAMP and the inhibition caused by 500 mM glibenclamide. Login to comment
148 (e) Dose-response relationships to CPT ± cAMP of wild type and G551D-CFTR monolayers. Login to comment
149 Each point is the mean of 8 (wt) or 9 (G551D) experiments and vertical bars are s.e. Figure 3 Isc response to genistein. Login to comment
150 Representative traces depicting the response of wt-CFTR (a and c, clone N10) and of G551D-CFTR (b and d, clone 6E) permeabilized monolayers to apical application of genistein. Login to comment
152 (e) Dose-response relationship to genistein of wild type and G551D-CFTR epithelia after preactivation with 30 mM or 500 mM CPT ± cAMP, respectively. Login to comment
154 Response of wtand G551D-CFTR to the benzo[c]quinolizinium MPB-07 Apical MPB-07 added to wt-CFTR monolayers after a low CPT ± cAMP concentration (30 mM) evoked a sustained Isc increase (126.3+17.4 mA cm72 , n=3, see Figure 5e). Login to comment
155 Nevertheless, when MPB-07 was added without prestimulation it had no signi®cant eect on wtor on G551D-CFTR monolayers. Login to comment
157 Relative efficacy of genistein, CPX and MPB-07 on G551D FRT cells To determine the relative eect of CFTR openers on the mutant CFTR, Isc activated on G551D cells in response to the dierent compounds in the presence of 500 mM CPT ± cAMP was compared with the response of wild type CFTR monolayers to CPT ± cAMP alone, which we assume as Figure 4 Isc response to CPX. Login to comment
158 Traces depict the response of wt-CFTR (a and c, clone N8) and of G551D-CFTR (b and d, clone A1) permeabilized monolayers to apical addition of 100 mM CPX. Login to comment
162 Traces depict the response of wt-CFTR (a and c, clone N10) and of G551D-CFTR (b and d, clone 6E) permeabilized monolayers to apical application of 200 mM MPB-07. Login to comment
166 Mean of wt-CFTR epithelia were obtained pooling data from clones N8 and N10, and mean of G551D-CFTR epithelia were obtaining pooling data from clones 6E and A1. Login to comment
170 Genistein (200 mM) increased the G551D-CFTR Isc from 0.8+0.3% to 32.2+4.4% of the wild type value (from 4.2+0.5 to 168.1+23.1 mA cm72 , see Figure 5e). Login to comment
174 Response to CFTR openers of nasal G551D and non-CF primary cultures To strengthen the results obtained with transfected FRT monolayers, we decided to test the openers on human airway epithelial cells. Login to comment
175 Primary cultures of nasal polyps were obtained from four non-CF subjects and from one G551D CF patient. Login to comment
176 Application of 500 mM CPT ± cAMP to amiloride treated G551D epithelia elicited a small Isc increase of 0.20+0.05 mA cm72 (n=13). Login to comment
179 In nasal G551D epithelia, genistein eect was also dose-dependent and the dose-response relationship was bell-shaped. Login to comment
182 The response of G551D nasal epithelia to genistein required the presence of the cAMP analogue (see Figure 6d). Login to comment
183 Neither CPX nor MPB07 elicited a signi®cant stimulatory eect on G551D epithelia despite a good eect on non-CF nasal monolayers (not shown). Login to comment
185 Figure 6f depicts mean responses of non-CF and G551D-CF epithelia to cAMP and the correction of G551D monolayers obtained with 200 mM genistein. Login to comment
186 Chronic stimulation with genistein To determine if chronic stimulation with genistein alters the electrophysiological characteristics and viability of epithelia, wtand G551D-FRT monolayers and CF and non-CF nasal epithelia were incubated for 48 h with genistein (50 or 200 mM). Login to comment
188 For example, resistance and PD were 4.388+0.192 and 4.200+0.236 kO cm2 and 76.4+0.5 and 75+0.7 mV for control and genistein treated G551D-CFTR FRT monolayers. Login to comment
189 For control and genistein treated G551D nasal epithelia, resistance and PD were 1.910+0.103 and 2.092+0.149 kO cm2 and 758+1 and 757+4 mV, respectively. Login to comment
197 In the present study we compare the pharmacological properties of three major CFTR openers, namely genistein, CPX, and MPB- Figure 6 G551D and non-CF nasal polyps. Login to comment
198 Representative traces showing the response of G551D nasal epithelia to CPT ± cAMP and genistein (a and c, respectively). Login to comment
207 G551D is a relatively common CF mutation with the characteristic of being correctly processed and inserted in apical membrane, therefore a good target for a comparative pharmacological study. Login to comment
213 We found that the maximal cAMP-dependent Isc in permeabilised G551D-CFTR was less than 1% of the response of wt monolayers. Login to comment
215 Indeed, while CPX only doubled the conductance of G551D monolayers, genistein potentiated it to values that correspond to 7, 15 and 30% of wild type monolayers (with 50, 100 and 200 mM genistein, respectively). Login to comment
217 Therefore, the results obtained in our FRT cell model indicate that genistein, but not CPX or MPB-07, might correct the G551D CF phenotype. Login to comment
218 We recently found that MPB-91, an improved derivative of MPB-07, is able to increase the conductance of G551D transfected FRT monolayers. Login to comment
222 Given that only one allele carries the G551D mutation in the nasal epithelia that we analysed, the recovery of 20% of CFTR function by 200 mM genistein is in good agreement with the 30% correction found in FRT cells which carry exclusively the G551D mutant. Login to comment
224 Nevertheless, the pattern of response to genistein, including the shift of the dose-response relationship to higher concentrations, and to CPX and MPB-07, is similar in G551D nasal polyp and in G551D-transfected FRT cells. This suggests that the response to openers in the nasal polyp is mainly due to stimulation of G551D-CFTR. Login to comment
226 The meaningful correction of G551D monolayers conductance with genistein supports recent data by Illek et al. (1999). Login to comment
227 These authors observed with genistein an increase of isoproterenol-dependent nasal PD of G551D CF patients and calculated that the hyperpolarization obtained corresponded to 17% of the response of normal subjects. Login to comment
228 The maximal eective genistein concentration on G551D-CFTR was far higher than that observed on wild type CFTR. Login to comment
229 Indeed, it was 200 ± 300 mM in our experiments on G551D FRT and nasal epithelia while 30 ± 50 mM genistein is the maximal eective concentration observed on wild type CFTR in our experiments and in single cell measurements by other authors (Illek et al., 1995; Wang et al., 1998). Login to comment
232 The most appealing explanation for the dierence between wild type and G551D-CFTR data, is that G551D mutant is displaying genistein-dependent activation, but the typical inhibition is shifted to much higher opener concentrations due to a dicult interaction with mutated NBD1. Login to comment
234 If genistein is inhibiting K+ channels on our airway epithelia, this action seems much less important than the stimulatory eect on G551D-CFTR since the net result is a substantial increase on Cl7 secretion. Login to comment
238 These measurements also con®rm that genistein alone does not activate G551D-CFTR in the absence of an intracellular cAMP increase (see Figure 3b) since CFTR activation would have reduced resistance of the monolayers. Login to comment
239 In conclusion, our study shows that genistein is a particularly potent CFTR agonist able to correct the G551D functional defect to therapeutic levels in polarized preparations. Login to comment
243 Our study suggests that ¯avonoid- based combinatorial libraries will be a promising source of potent and more selective derivatives that could show a signi®cant eect on G551D and similar CFTR mutants. Login to comment
123 (b) Immunoprecipitation of G551D- and wt-CFTR proteins was done using a monoclonal antibody that recognises the C-terminus of CFTR. Login to comment

[hide] Genistein improves regulatory interactions between... J Biol Chem. 2002 Dec 27;277(52):50341-7. Epub 2002 Oct 16. Suaud L, Carattino M, Kleyman TR, Rubenstein RC
Genistein improves regulatory interactions between G551D-cystic fibrosis transmembrane conductance regulator and the epithelial sodium channel in Xenopus oocytes.
J Biol Chem. 2002 Dec 27;277(52):50341-7. Epub 2002 Oct 16., 2002-12-27 [PMID:12386156]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) in addition to its well defined Cl(-) channel properties regulates other ion channels. CFTR inhibits epithelial Na(+) channel (ENaC) currents in many epithelial and non-epithelial cells, whereas the presence of ENaC increases CFTR functional expression. This interregulation is reproduced in Xenopus oocytes where both the open probability and surface expression of wild type CFTR Cl(-) channels are increased when CFTR is co-expressed with alphabetagamma-mouse ENaC (mENaC) and conversely when the activity of mENaC is inhibited after wild type CFTR activation. Using the Xenopus oocyte expression system, different functional regulatory interactions were observed between G551D-CFTR and alphabetagamma-mENaC. The co-expression of G551D-CFTR and alphabetagamma-mENaC resulted in a 5-fold increase in G551D-CFTR Cl(-) current compared with oocytes expressing G551D-CFTR alone. Oocytes co-injected with both G551D-CFTR and ENaC expressed an amiloride-sensitive whole cell current that was similar to that observed before and after G551D-CFTR activation with forskolin/isobutylmethylxanthine. Treatment with genistein both enhanced the functional expression of G551D-CFTR and improved regulatory interactions between G551D-CFTR and ENaC. These data suggest that genistein may be useful in patients with cystic fibrosis and the G551D-CFTR mutation.

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0 Genistein Improves Regulatory Interactions between G551D-Cystic Fibrosis Transmembrane Conductance Regulator and the Epithelial Sodium Channel in Xenopus Oocytes* Received for publication, September 19, 2002, and in revised form, October 11, 2002 Published, JBC Papers in Press, October 16, 2002, DOI 10.1074/jbc.M209641200 Laurence Suaud‡§, Marcelo Carattino§¶, Thomas R. Kleyman¶, and Ronald C. Rubenstein‡ʈ** From the ‡Division of Pulmonary Medicine, Children`s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, ʈDepartment of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, and ¶Department of Medicine, Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 The cystic fibrosis transmembrane conductance regulator (CFTR) in addition to its well defined Cl-channel properties regulates other ion channels. Login to comment
3 Using the Xenopus oocyte expression system, different functional regulatory interactions were observed between G551D-CFTR and ␣beta␥-mENaC. Login to comment
4 The co-expression of G551D-CFTR and ␣beta␥- mENaC resulted in a 5-fold increase in G551D-CFTR Cl- current compared with oocytes expressing G551D-CFTR alone. Login to comment
5 Oocytes co-injected with both G551D-CFTR and ENaC expressed an amiloride-sensitive whole cell current that was similar to that observed before and after G551D-CFTR activation with forskolin/isobutylmethylxanthine. Login to comment
6 Treatment with genistein both enhanced the functional expression of G551D-CFTR and improved regulatory interactions between G551D-CFTR and ENaC. Login to comment
7 These data suggest that genistein may be useful in patients with cystic fibrosis and the G551D-CFTR mutation. Login to comment
16 The G551D mutation in CFTR results in a channel that is appropriately localized to the apical membrane but does not normally conduct Clin response to cAMP stimulation (20). Login to comment
17 Genistein can improve G551D-CFTRs cAMP-dependent chloride transport (19). Login to comment
18 Others have shown that G551D-CFTR does not inhibit sodium transport by ENaC in Xenopus oocytes (6). Login to comment
19 We hypothesized that genistein would restore the functional interactions between G551D-CFTR and mENaC and examined this interaction in the Xenopus oocyte expression system. Login to comment
20 ENaC was able to enhance the functional expression of G551D-CFTR in the absence of genistein, in contrast to our previous results examining interactions between ⌬F508-CFTR and ENaC (11). Login to comment
21 In agreement with previous data, we observed that G551D-CFTR did not inhibit the functional expression of ENaC after activation with IBMX/forskolin (6). Login to comment
22 As we observed with ⌬F508-CFTR, genistein restored functional interactions between G551D-CFTR and ENaC. Login to comment
25 Expression of Human G551D-CFTR and Mouse ENaC in Xenopus Oocytes-Human G551D-CFTR and mouse ENaC were expressed in Xenopus oocytes as described previously (10, 11). Login to comment
26 Human G551D-CFTR and mouse ␣-, beta-, and ␥-ENaC cRNAs were prepared using a cRNA synthesis kit (mMESSAGE mMachine, Ambion Inc, Austin, TX) according to the manufacturer`s protocol. Login to comment
40 Each batch of oocytes obtained from an individual frog was injected (50 nl/oocyte) using a Nanoject II microinjector (Drummond Scientific, Broomall, PA) with ␣, beta, and ␥ subunits of mENaC (0.33 ng/subunit), G551D-CFTR (10 ng), or a combination of mENaC and G551D-CFTR cRNAs dissolved in RNase-free water. Login to comment
49 G551D-CFTR was activated by perfusion of the oocyte with buffer containing 10 ␮M forskolin and 100 ␮M IBMX for 25 min (10, 11). Login to comment
51 In all experiments, G551D-CFTR Cl- current was defined as the difference between amiloride-insensitive current measured before and after perfusion with forskolin/IBMX (or before and after perfusion with forskolin/IBMX/genistein). Login to comment
59 Expression of G551D-CFTR in Xenopus oocytes. Login to comment
60 G551D-CFTR was expressed in oocytes, and TEV was performed as described under "Experimental Procedures." Login to comment
64 C, G551D-CFTR was expressed in oocytes, and TEV was performed as described with the exception that the ND96 buffer was supplemented with 5 mM tetraethylamine chloride, 5 mM BaCl2, and 200 ␮M DIDS (4,4Ј-diisothiocyanatostilbene-2,2Ј-disulfonic acid). Login to comment
76 A two-tailed t test was used when comparing currents obtained from oocytes injected with a cRNA for a single transporter (i.e. ENaC or G551D-CFTR versus oocytes co-injected with a cRNAs for both ENaC and G551D-CFTR). Login to comment
78 RESULTS Functional Expression of G551D-CFTR in Xenopus Oocytes-We used the Xenopus oocyte expression system and two-electrode voltage clamp technique to examine the functional expression of G551D-CFTR. Login to comment
79 We measured whole cell currents at varying clamping potentials in oocytes expressing G551D-CFTR (Fig. 1) and generated I/V curves from data obtained prior to and after 20 min of incubation with 10 ␮M forskolin and 100 ␮M IBMX to activate endogenous protein kinase A (Fig. 1A). Login to comment
81 These data are consistent with previous observations that forskolin/IBMX-stimulated chloride current in G551D-CFTR-expressing oocytes is very low (14). Login to comment
82 As the isoflavone genistein is able to stimulate G551D-CFTR chloride conductance (19), whole cell currents in G551D-CFTR-expressing oocytes were assessed before and after additional stimulation with 50 ␮M genistein (in addition to IBMX and forskolin). Login to comment
83 Fig. 1B demonstrates the I/V relationship prior to and after activation of G551D-CFTR by 25 min of incubation with 10 ␮M forskolin and 100 ␮M IBMX followed by 15 min of incubation with 10 ␮M forskolin, 100 ␮M IBMX, and 50 ␮M genistein. Login to comment
85 These data are consistent with IBMX/forskolin-stimulated G551D-CFTR-mediated chloride current being enhanced by genistein in Xenopus oocytes. Login to comment
88 Forskolin, IBMX, and genistein did not alter whole cell currents in oocytes that had not been injected with G551D-CFTR (n ϭ 3). Login to comment
89 We also assessed the I/V relationship of G551D-CFTR-injected oocytes in the presence of inhibitors of putative endogenous channels (Fig. 1C). Login to comment
92 Co-expression of G551D-CFTR and ␣beta␥-ENaC-Several groups have reported (7, 9-11) that when WT CFTR and ENaC were co-expressed in Xenopus oocytes, ENaC-mediated Naϩ currents were inhibited in response to CFTR activation. Login to comment
94 Therefore, we assessed the interregulation of G551D-CFTR and mENaC. Login to comment
95 Whole cell currents measured at a holding potential of -100 mV in oocytes injected with G551D-CFTR (10 ng) or ␣beta␥-ENaC (0.33 ng/subunit) or both G551D-CFTR and ␣beta␥-ENaC are shown Fig. 2. Login to comment
97 Expression of G551D-CFTR and ENaC in Xenopus oocytes. Login to comment
98 G551D-CFTR and ␣beta␥-ENaC were expressed separately or together in oocytes, and TEV was performed as described under "Experimental Procedures." Login to comment
100 B, amiloride-sensitive whole cell currents (-100-mV holding potential) were determined in oocytes expressing ENaC or co-expressing ENaC and G551D-CFTR prior to (open bars) and following (gray bars) stimulation with 10 ␮M forskolin and 100 ␮M IBMX. Login to comment
101 Data obtained from the same G551D-CFTR/ ENaC co-injected oocytes are presented in panels A and B. Means Ϯ S.E. are illustrated. Login to comment
104 Oocytes co-injected with G551D-CFTR and ␣beta␥-ENaC had 5-fold increased forskolin/IBMX-stimulated, amiloride-insensitive current compared with oocytes injected with G551D-CFTR alone (Fig. 2A, ICl ϭ -0.1 Ϯ 0.0 ␮A (G551D-CFTR) versus -0.5 Ϯ 0.1 ␮A (G551D-CFTR/␣beta␥- ENaC), p ϭ 0.007). Login to comment
105 Thus, a co-expression of ENaC in oocytes resulted in enhanced forskolin/IBMX-stimulated G551D-CFTR Cl- current similar to its previously described enhancement of WT CFTR current (7, 9-11, 21). Login to comment
106 Oocytes co-injected with both G551D-CFTR and ENaC expressed amiloride-sensitive whole cell currents (-2.8 Ϯ 0.6 ␮A, n ϭ 25) in the absence of forskolin/IBMX that were significantly lower than the amiloride-sensitive whole cell currents recorded in the absence of forskolin/IBMX in oocytes injected with ENaC alone (-5.3 Ϯ 1.0 ␮A, n ϭ 22). Login to comment
107 However, similar levels of amiloride-sensitive whole cell currents were observed in oocytes co-injected with G551D-CFTR and ENaC prior to and after G551D-CFTR activation with forskolin/IBMX (-2.8 Ϯ 0.6 ␮A versus -2.5 Ϯ 0.6 ␮A, n ϭ 25, p ϭ n.s.) (Fig. 2B), which is similar to ⌬F508-CFTR behavior and differs from WT CFTR behavior in this system (10, 11). Login to comment
108 G551D-CFTR/ENaC Interregulation with IBMX/Forskolin Does Not Require Sodium Transport-When Naϩ in the ND96 bath buffer was replaced by the impermeant cation NMDGϩ , oocytes co-injected with G551D-CFTR and ␣beta␥-ENaC had a 6-fold increased forskolin/IBMX-stimulated whole cell current compared with oocytes injected with G551D-CFTR alone (ICl ϭ -5.1 Ϯ 1.4-fold stimulation (ND96), n ϭ 25, versus -6.3 Ϯ 1.7-fold stimulation (NMDG-CL96), n ϭ 18, p ϭ n.s.) (Fig. 3). Login to comment
109 These data suggest that ENaC enhancement of forskolin/ IBMX-mediated activation of G551D-CFTR is not dependent on extracellular Naϩ , Naϩ influx, or inward Naϩ conductance. Login to comment
110 Interregulation of G551D-CFTR and ␣beta␥-mENaC after IBMX/Forskolin/Genistein Stimulation-G551D-CFTR exhibits a poor Cl-conductance in response to cAMP. Login to comment
111 As this might reflect the presence of alterations in other functional properties of this mutant, we examined whether genistein, which activates G551D-CFTR Cl-conductance, would restore functional interactions between G551D-CFTR and ENaC. Login to comment
112 As shown in Fig. 4A, the amiloride-insensitive component of the forskolin/ IBMX/genistein-stimulated whole cell current in oocytes coexpressing G551D-CFTR and ␣beta␥-ENaC was 4-fold greater than the forskolin/IBMX/genistein-stimulated current meas- FIG. 4. Login to comment
113 Genistein restores regulation interactions between G551D-CFTR and ␣beta␥-ENaC. Login to comment
114 G551D-CFTR and ␣beta␥-ENaC were expressed separately or together in oocytes, and TEV was performed as described under "Experimental Procedures." Login to comment
116 B, amiloride-sensitive whole cell currents (-100-mV holding potential) were determined in oocytes expressing ENaC or co-expressing ENaC and G551D-CFTR prior to (open bars) and following (dark gray bars) stimulation with 10 ␮M forskolin, 100 ␮M IBMX, and 50 ␮M genistein. Login to comment
117 Data obtained from the same G551D-CFTR/ENaC-co-injected oocytes are presented in panels A and B. Means Ϯ S.E. are illustrated. FIG. 3. Login to comment
118 G551D-CFTR/ENaC regulatory interactions restored by genistein do not require Na؉ transport. Whole cell currents (-100-mV holding potential) were determined in oocytes expressing G551D-CFTR alone (open bars) or co-expressing G551D-CFTR and ␣beta␥- ENaC (closed bars). Login to comment
119 ND96, changes in whole cell currents that were not inhibited by 10 ␮M amiloride (presumed to be G551D-CFTR-mediated) were determined following stimulation with 10 ␮M forskolin and 100 ␮M IBMX in standard ND96 (NaCl) buffer. NMDG-Cl96, changes in whole cell currents were determined following stimulation with 10 ␮M forskolin and 100 ␮M IBMX in oocytes bathed in a Naϩ -free solution (Naϩ was replaced with NMDG). Login to comment
120 Data (means Ϯ S.E.) are expressed relative to the mean change in whole cell current of oocytes injected with G551D-CFTR alone. Login to comment
121 ured in oocytes expressing G551D-CFTR alone (-0.3 Ϯ 0.0 ␮A (G551D-CFTR, n ϭ 21) versus -1.2 Ϯ 0.4 ␮A (G551D-CFTR/ ENaC, n ϭ 20), p ϭ 0.03). Login to comment
123 Amiloride-sensitive whole cell currents recorded before and after stimulation by forskolin/ IBMX/genistein in oocytes expressing ENaC and G551D-CFTR were significantly lower than the amiloride-sensitive currents obtained both before and after stimulation by forskolin/IBMX/ genistein in oocytes expressing ENaC alone (Fig. 4B). Login to comment
124 Although amiloride-sensitive whole cell currents significantly increased in oocytes expressing ENaC alone following stimulation by forskolin/IBMX/genistein (Fig. 4B), amiloride-sensitive currents recorded in oocytes co-expressing ENaC and G551D-CFTR remained stable following stimulation with forskolin/ IBMX/genistein (-2.1 Ϯ 0.7 ␮A (-forskolin/IBMX/genistein) versus -2.0 Ϯ 0.3 (ϩforskolin/IBMX/genistein), n ϭ 20, p ϭ n.s.). Login to comment
125 These data suggest that the activation of G551D-CFTR by forskolin/IBMX in the presence of genistein partially restored the inhibition of ENaC activity by activated G551D-CFTR and are similar to our observations for ⌬F508-CFTR (11). Login to comment
126 We again assessed whether the enhanced forskolin/IBMX/ genistein-stimulated amiloride-insensitive whole cell currents recorded in oocytes co-injected with G551D-CFTR and ENaC compared with oocytes injected with G551D-CFTR alone depended on ENaC-mediated sodium transport. Login to comment
127 Oocytes co-injected with G551D-CFTR and ␣beta␥-ENaC had a 3.75-fold increased forskolin/IBMX/genistein-stimulated G551D-CFTR Cl- current compared with oocytes injected with G551D-CFTR alone both in ND96 (NaCl) buffer and in NMDG-Cl buffer (Fig. 5). Login to comment
128 These data suggest that ENaC enhancement of forskolin/ IBMX/genistein-mediated activation of G551D-CFTR is not dependent on extracellular Naϩ , Naϩ influx, or inward Naϩ conductance. Login to comment
129 Synergistic Activation of G551D-CFTR by Genistein and ENaC-In Fig. 6, we have replotted the whole oocyte forskolin/ IBMX-stimulated (Fig. 3, NMDG-Cl- ) or forskolin/IBMX/genistein-stimulated (Fig. 5, NMDG-Cl- ) currents and expressed these relative to that of oocytes injected with G551D-CFTR alone and stimulated with IBMX/forskolin. Login to comment
131 These data demonstrate synergistic activation of G551D-CFTR function by genistein and ENaC in the presence of forskolin/IBMX and suggest that genistein and ENaC enhance G551D-CFTR function in oocytes by different and complementary mechanisms. Login to comment
132 Influence of ␣beta␥-mENaC on G551D-CFTR Single Channel Activity in Oocytes-We next sought to assess the mechanism by which ␣beta␥-mENaC increased G551D-CFTR-mediated current in co-injected oocytes. Login to comment
133 Oocytes were injected with G551D-CFTR alone or co-injected with ENaC, and G551D-CFTR was stimulated with forskolin/IBMX/genistein prior to single channel recordings of chloride transport (Fig. 7). Login to comment
134 At a pipette potential of -80 mV in the cell-attached mode, single channel currents in G551D-CFTR-injected oocytes (1.08 Ϯ 0.13 pA, n ϭ 6) FIG. 6. Login to comment
135 Synergistic activation of G551D-CFTR by genistein and ENaC. Login to comment
136 Whole oocyte IBMX/forskolin-stimulated (Fig. 3, NMDG-Cl- ) or IBMX/forskolin/genistein-stimulated (Fig. 5, NMDG-Cl- ) currents are replotted and expressed relative to that of oocytes injected with G551D-CFTR alone and stimulated with IBMX/forskolin. Login to comment
138 G551D-CFTR/ENaC regulatory interactions restored by genistein do not require Na؉ transport. Whole cell currents (-100-mV holding potential) were determined in oocytes expressing G551D-CFTR alone (open bars) or co-expressing G551D-CFTR and ␣beta␥- ENaC (closed bars). Login to comment
139 ND96, changes in whole cell currents that were not inhibited by 10 ␮M amiloride (presumed to be G551D-CFTR-mediated) were determined following stimulation with 10 ␮M forskolin, 100 ␮M IBMX, and 50 ␮M genistein in standard ND96 (NaCl) buffer. NMDG-Cl96, changes in whole cell currents were determined following stimulation with 10 ␮M forskolin, 100 ␮M IBMX, and 50 ␮M genistein in oocytes bathed in a Naϩ -free solution (Naϩ was replaced with NMDG). Login to comment
140 Data (means Ϯ S.E.) are expressed relative to the mean change in whole cell current of oocytes injected with G551D-CFTR alone. Login to comment
141 were similar in magnitude to the currents recorded in oocytes co-expressing G551D-CFTR and ENaC (0.92 Ϯ 0.14 pA, n ϭ 6, p ϭ n.s.). Login to comment
142 The Po for G551D-CFTR in oocytes co-injected with ENaC (0.24 Ϯ 0.6, mean Ϯ S.E., n ϭ 6) was not significantly different from that determined in oocytes injected with G551D-CFTR alone (0.29 Ϯ 0.2, n ϭ 6, p ϭ n.s.). Login to comment
143 We did not observe a difference in number of G551D-CFTR channels/patch from oocytes injected with G551D-CFTR alone (n ϭ 2, 3, 1, 2, 1, and 4 for n ϭ 6 patches) or co-injected with G551D-CFTR and ENaC (n ϭ 3, 1, 1, 4, 2, and 2 for n ϭ 6 patches). Login to comment
144 Given the difficulties in estimating small changes in channel density from single channel analyses and the lack of a difference in G551D-CFTR open probability following stimulation with forskolin/IBMX/ genistein in oocytes expressing G551D-CFTR alone or co-expressing G551D-CFTR and ENaC, our results are consistent with the increase in forskolin/IBMX/genistein-stimulated G551D-CFTR currents in the presence of ENaC, reflecting an increase in the number of G551D-CFTR channels at the plasma membrane. Login to comment
145 As genistein increases the Po for G551D-CFTR (22), these data are consistent with genistein and ENaC enhancing G551D-CFTR function in oocytes by different and complementary mechanisms. Login to comment
150 The first nucleotide binding fold (nucleotide binding domain (NBD-1)) of CFTR in which G551D mutation is located seems important for this interaction. Login to comment
152 A similar NBD1/R peptide fragment containing the G551D mutation did not demonstrate forskolin/IBMX-regulated inhibition of ENaC (14). Login to comment
153 G551D-CFTR when co-expressed with ␣beta␥-ENaC and activated by IBMX/forskolin was unable to inhibit ENaC in our experiments. Login to comment
158 Genistein activates CFTR (WT, ⌬F508, and G551D) (16, 22) by increasing channel open time and decreasing channel closing, thereby increasing channel Po. Login to comment
161 Co-expression of ␣beta␥-ENaC does not alter the open probability of forskolin/IBMX/genistein-stimulated G551D-CFTR. Login to comment
163 The upper tracings show 60 s of a continuous recording performed in the cell-attached mode performed as described under "Experimental Procedures" in an oocyte expressing G551D-CFTR alone or an oocyte co-expressing G551D-CFTR and ␣beta␥-ENaC. Login to comment
165 B, G551D-CFTR open probability (mean Ϯ S.E.) determined in forskolin/IBMX/genistein-treated oocytes expressing either G551D-CFTR alone or co-expressing G551D-CFTR and ENaC. Login to comment
166 being able to activate both ⌬F508- and G551D-CFTR as these two mutants have intact NBD-2s and further suggests that in some way an interaction of NBD-2 influences NBD-1 form and/or function. Login to comment
168 In oocyte co-injected with G551D-CFTR and ␣beta␥-mENaC, the enhancement of G551D-CFTR Cl- current most probably correlates with an increase in the N or the number of Cl-channels present at the oocyte surface as the measured open probability of forskolin/IBMX/genistein-stimulated G551D-CFTR was the same in the presence or absence of ENaC. Login to comment
169 We also observed that amiloride-inhibited whole oocyte currents decreased with co-expression of G551D-CFTR (in these studies) or with WT CFTR in our previous studies (11). Login to comment
171 One potential explanation for these observations is that co-expression of ENaC increases G551D-CFTR N, whereas co-expression of G551D-CFTR could decrease either ENaC N or open probability. Login to comment
172 Previous studies in a number of systems have suggested that co-expression of WT CFTR decreases ENaC open probability (21, 27); however, such an effect on open probability was not noted for G551D-CFTR in lipid bilayers (8). Login to comment
173 Our data do not directly address this issue, but if G551D-CFTR does not decrease ENaC open probability in oocytes (similar to its behavior in lipid bilayers), we can speculate that G551D-CFTR inhibits ENaC functional expression via an N effect. Login to comment
177 The co-expression of an appropriately folded and trafficked CFTR (G551D-CFTR as shown in Figs. Login to comment
180 Another potential site of interaction/competition between CFTR and ENaC is at the plasma membrane where cAMP-activated WT CFTR but not G551D-CFTR and ⌬F508-CFTR inhibited ENaC function. Login to comment
181 One possibility is that both G551D-CFTR and ⌬F508-CFTR undergo more rapid removal from the plasma membrane when compared with WT CFTR as has been suggested for ⌬F508-CFTR (25). Login to comment
182 Another possibility is that G551D-CFTR and ⌬F508-CFTR, perhaps because of their aberrant NBD-1s, are not able to interact or interact differently with either ENaC or proteins important in transducing cAMP-activated CFTR signal to inhibit ENaC function. 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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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] Airways in cystic fibrosis are acidified: detectio... Thorax. 2002 Nov;57(11):926-9. Tate S, MacGregor G, Davis M, Innes JA, Greening AP
Airways in cystic fibrosis are acidified: detection by exhaled breath condensate.
Thorax. 2002 Nov;57(11):926-9., [PMID:12403872]

Abstract [show]
BACKGROUND: The loss of cystic fibrosis transmembrane conductance regulator (CFTR) mediated chloride conductance does not fully explain the diverse pathologies evident in patients with cystic fibrosis (CF). Bicarbonate (HCO(3)(-)) secretion is also impaired in CFTR expressing tissues and CFTR is thought to regulate HCO(3)(-) secretion at the apical membrane of epithelial cells. We hypothesised that the epithelial lining fluid (ELF) of patients with CF would be acidified and that this may be worsened during an infective exacerbation due to the increased inflammatory burden. METHODS: pH and nitrite levels in exhaled breath condensate (EBC) from 12 healthy non-smoking controls and 30 patients with CF (11 of whom were in an infective exacerbation) were measured. A further nine patients were studied before and after intravenous antibiotic treatment for an exacerbation of CF. RESULTS: The pH of EBC was significantly lower in patients with stable CF than in controls (5.88 (0.32) v 6.15 (0.16), p=0.017), and was further reduced in CF patients with an exacerbation (5.32 (0.38), p=0.001) compared with stable CF patients. EBC pH increased significantly following antibiotic treatment from 5.27 (0.42) to 5.71 (0.42), p=0.049). Nitrite levels in EBC were increased in CF patients with an exacerbation compared with control subjects (4.4 (4.0) micro m v 1.6 (1.6) micro m p=0.047). No correlation was found between EBC pH and nitrite levels. CONCLUSIONS: These findings support the hypothesis that airway acidification occurs in CF. This acidity is in part a function of inflammation as the pH of the EBC of patients increased significantly with treatment of an exacerbation, although not to control levels. Acidic pH of the ELF may play a role in the pathophysiology of CF lung disease and requires further investigation.

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240 Choi et al11 showed that cultured cells expressing CFTR mutations known to be associated with pancreatic sufficiency (class 4-5 mutations such as R117H, A455E) displayed significantly greater bicarbonate conductance than mutations associated with pancreatic insufficiency (class 1-3 mutations, G452X, ∆F508, G551D). Login to comment

[hide] A molecular mechanism for aberrant CFTR-dependent ... EMBO J. 2002 Nov 1;21(21):5662-72. Ko SB, Shcheynikov N, Choi JY, Luo X, Ishibashi K, Thomas PJ, Kim JY, Kim KH, Lee MG, Naruse S, Muallem S
A molecular mechanism for aberrant CFTR-dependent HCO(3)(-) transport in cystic fibrosis.
EMBO J. 2002 Nov 1;21(21):5662-72., 2002-11-01 [PMID:12411484]

Abstract [show]
Aberrant HCO(3)(-) transport is a hallmark of cystic fibrosis (CF) and is associated with aberrant Cl(-)-dependent HCO(3)(-) transport by the cystic fibrosis transmembrane conductance regulator (CFTR). We show here that HCO(3)(-) current by CFTR cannot account for CFTR-activated HCO(3)(-) transport and that CFTR does not activate AE1-AE4. In contrast, CFTR markedly activates Cl(-) and OH(-)/HCO(3)(-) transport by members of the SLC26 family DRA, SLC26A6 and pendrin. Most notably, the SLC26s are electrogenic transporters with isoform-specific stoichiometries. DRA activity occurred at a Cl(-)/HCO(3)(-) ratio > or =2. SLC26A6 activity is voltage regulated and occurred at HCO(3)(-)/Cl(-) > or =2. The physiological significance of these findings is demonstrated by interaction of CFTR and DRA in the mouse pancreas and an altered activation of DRA by the R117H and G551D mutants of CFTR. These findings provide a molecular mechanism for epithelial HCO(3)(-) transport (one SLC26 transporter-electrogenic transport; two SLC26 transporters with opposite stoichiometry in the same membrane domain-electroneutral transport), the CF-associated aberrant HCO(3)(-) transport, and reveal a new function of CFTR with clinical implications for CF and congenital chloride diarrhea.

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6 The physiological signi®- cance of these ®ndings is demonstrated by interaction of CFTR and DRA in the mouse pancreas and an altered activation of DRA by the R117H and G551D mutants of CFTR. Login to comment
220 In (A) and (B), the cells were transfected with 0.5 mg of CFTR, in (C) with 0.5 mg of CFTR(R117H) and in (D) with 0.5 mg of CFTR(G551D). Login to comment
249 We focused on the R117H and the G551D mutants since these mutants are well characterized clinically and R117H is associated with a mild form and G551D with a severe form of CF (Wilschanski et al., 1995). Login to comment
252 Figure 8C and E shows that the R117H mutant activated DRA by only 2.5-fold (26% of that by the wild-type CFTR), whereas the G551D mutant was unable to activate DRA. Login to comment
283 A signi®cant ®nding was the modi®ed activation of DRA by the R117H and G551D mutants of CFTR. Login to comment
292 The R117H and G551D mutants were prepared as described (Choi et al., 2001). 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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160 Some of them are always responsible for a unique phenotype that can be either CF-PI (for instance, the case of N1303K in class II, G551D in class III, and R1066C in class IV), or CF-PS (for instance, the case of G551S in class III) or CBVAD for D1152H (class IV). Login to comment

[hide] Chloride channels in the kidney: lessons learned f... Am J Physiol Renal Physiol. 2002 Dec;283(6):F1176-91. Devuyst O, Guggino WB
Chloride channels in the kidney: lessons learned from knockout animals.
Am J Physiol Renal Physiol. 2002 Dec;283(6):F1176-91., [PMID:12426234]

Abstract [show]
Cl- channels are involved in a range of functions, including regulation of cell volume and/or intracellular pH, acidification of intracellular vesicles, and vectorial transport of NaCl across many epithelia. Numerous Cl- channels have been identified in the kidney, based on single-channel properties such as conductance, anion selectivity, gating, and response to inhibitors. The molecular counterpart of many of these Cl- channels is still not known. This review will focus on gene-targeted mouse models disrupting two structural classes of Cl- channels that are relevant for the kidney: the CLC family of voltage-gated Cl- channels and the CFTR. Disruption of several members of the CLC family in the mouse provided useful models for various inherited diseases of the kidney, including Dent's disease and diabetes insipidus. Mice with disrupted CFTR are valuable models for cystic fibrosis (CF), the most common autosomal recessive, lethal disease in Caucasians. Although CFTR is expressed in various nephron segments, there is no overt renal phenotype in CF. Analysis of CF mice has been useful to identify the role and potential interactions of CFTR in the kidney. Furthermore, observations made in CF mice are potentially relevant to all other models of Cl- channel knockouts because they emphasize the importance of alternative Cl- pathways in such models.

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283 In one additional model, the glycine at position 551 has been changed to an aspartate (G551D) (39). Login to comment
284 The G551D mutation is relatively common in patients with CF and results in a mutated CFTR protein that is processed normally but shows a significant reduction in cAMP-regulated Cl-channel activity (187). Login to comment
285 The G551D mutation is relatively common in patients with CF and results in a mutated CFTR protein that is processed normally but shows a significant reduction in cAMP-regulated Cl- channel activity (187). 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
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
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
117 In the case of the G551D/WT R553X/WT genotype, the Roche test needs careful interpretation (and ought to be improved) because the hybridisation at the one locus destabilised the hybridisation at the other locus which is only 4 bp apart. Login to comment
118 Accordingly, the line assay showed a G551D/G551D and R553X/R553X (two homozygote mutations) genotype instead of the correct G551D/WT and R553X/WT (compound heterozygote) genotype. 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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130 The prominent differences were that 1677delTA was relatively common in our patients, whereas G551D, the 5th common mutation in Europeans, was not present at all. Login to comment

[hide] Down-regulation of volume-sensitive Cl- channels b... Pflugers Arch. 2002 Nov;445(2):177-86. Epub 2002 Sep 7. Ando-Akatsuka Y, Abdullaev IF, Lee EL, Okada Y, Sabirov RZ
Down-regulation of volume-sensitive Cl- channels by CFTR is mediated by the second nucleotide-binding domain.
Pflugers Arch. 2002 Nov;445(2):177-86. Epub 2002 Sep 7., [PMID:12457238]

Abstract [show]
Transient expression of wild-type human cystic fibrosis transmembrane conductance regulator (CFTR) in HEK293T cells resulted in a profound decrease in the amplitude of volume-sensitive outwardly rectifying Cl- channel (VSOR) current without changing the single-channel amplitude. This effect was not mimicked by expression of the DeltaF508 mutant of CFTR, which did not reach the plasma membrane. The VSOR regulation by CFTR was not affected by G551D mutation at first nucleotide-binding domain (NBD1), which is known to impair CFTR interaction with the outwardly rectifying chloride channel, ORCC, epithelial amiloride-sensitive Na-channel, ENaC, and renal potassium channel, ROMK2. The CFTR-VSOR interaction was insensitive to the deletion mutation, DeltaTRL, which is known to impair CFTR-PDZ domain binding. In contrast, the G1349D mutant, which impairs ATP binding at NBD2, effectively abolished the down-regulatory effect of CFTR. Furthermore, the K1250M mutation at the Walker A motif and the D1370N mutation at the Walker B motif, both known to impair ATP hydrolysis at NBD2, completely abolished the VSOR regulation by CFTR. Thus, we conclude that an ATP-hydrolysable conformation of NBD2 is essential for the regulation of the VSOR by the CFTR protein, and that VSOR is a first channel regulated by CFTR through its NBD2.

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3 The VSOR regulation by CFTR was not affected by G551D mutation at first nucleotide-binding domain (NBD1), which is known to impair CFTR interaction with the outwardly rectifying chloride channel, ORCC, epithelial amiloride-sensitive Na-channel, ENaC, and renal potassium channel, ROMK2. Login to comment
94 Crucial role of NBD2 in the CFTR-VSOR interaction To clarify the molecular basis for the CFTR-VSOR interaction, we first introduced mutations into NBD1 (G551D), NBD2 (G1349D) and the C-terminal PDZ-binding domain (DTRL). Login to comment
98 Quantitative densitometry (Fig. 4Bb) revealed that the G551D, G1349D and DTRL mutants were expressed to a comparable extent in the plasma membrane. Login to comment
116 Gly551 fiAsp and Gly1349 fiAsp (G551D and G1349D) are naturally occurring mutations in NBD1 and NBD2, known to cause a severe CF phenotype by decreasing nucleotide binding at NBDs [25]. Login to comment
117 The G551D mutation has been shown to impair CFTR regulation of ORCC [7, 17] and ENaC [20]. Login to comment
118 In our experiments, however, the G551D mutant was an effective down-regulator of VSOR activity with an efficiency close to that of WT CFTR (Fig. 5). Login to comment
120 In contrast to the mutation in NBD1, we found that the G1349D mutation failed to affect VSOR currents (Fig. 5), although it was expressed in the plasma membrane to an extent comparable to that of the G551D and DTRL mutants (Fig. 4). Login to comment
127 Fig. 5A, B Effects of expression of WT CFTR and CFTR proteins mutated at NBD1, NBD2 or the PDZ-binding domain on VSOR current densities in HEK293T cells. A Time-course of VSOR current activation by hypotonic stimulation of cells transfected with DTRL (top), G551D (middle) and G1349D (bottom) mutants, taken during application of alternating pulses from 0 to €40 mV every 15 s. B Peak VSOR current densities from HEK293T cells transfected with vector alone (Mock), WT CFTR or one of the three mutants, recorded at +40 mV after reaching a steady-state level. Login to comment
137 Since VSOR-non-regulating NBD2 mutants (G1349D, K1250M and D1370N) were expressed to approximately the same level as VSOR-regulating WT and G551D CFTR (as seen from immunostaining and Western blotting data), we may exclude the possibility that the down-regulation is simply a side-effect of overexpression of a foreign protein. Login to comment
144 The G551D mutant of CFTR, which produces severe lung disease in 3% of all CF patients, is normally transported to the plasma membrane, but does not generate a functional Cl-conductance due to impaired ATP binding at NBD1 [58]. Login to comment
152 As was the case for ORCC (but not for VSOR in the present study), the point mutation at NBD1, G551D, abolished the CFTR-mediated regulation of the epithelial sodium channel in both oocytes [20] and planar lipid membranes [15]. Login to comment
156 Asterisk, P<0.05 versus mock data which is identical to that presented in Fig. 5B hybrid analysis in which the interaction was sensitive to the G551D mutation [20]. Login to comment
159 The G551D mutation at NBD1, however, abolished the CFTR-dependent component of glibenclamide-induced inhibition. Login to comment
162 Although the features of CFTR-mediated regulation are different in all three cases (activation for ORCC, inhibition for ENaC and glibenclamide sensitivity for ROMK2), the modulation itself is due to a direct or indirect interaction involving NBD1 and is sensitive to the G551D mutation. Login to comment
163 In contrast, the results obtained in the present study show that VSOR differs markedly from these three channels in an important point: CFTR-mediated regulation was insensitive to the CF-causing mutation G551D in NBD1. Login to comment

[hide] Thiazolidinone CFTR inhibitor identified by high-t... J Clin Invest. 2002 Dec;110(11):1651-8. Ma T, Thiagarajah JR, Yang H, Sonawane ND, Folli C, Galietta LJ, Verkman AS
Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion.
J Clin Invest. 2002 Dec;110(11):1651-8., [PMID:12464670]

Abstract [show]
Secretory diarrhea is the leading cause of infant death in developing countries and a major cause of morbidity in adults. The cystic fibrosis transmembrane conductance regulator (CFTR) protein is required for fluid secretion in the intestine and airways and, when defective, causes the lethal genetic disease cystic fibrosis. We screened 50,000 chemically diverse compounds for inhibition of cAMP/flavone-stimulated Cl(-) transport in epithelial cells expressing CFTR. Six CFTR inhibitors of the 2-thioxo-4-thiazolidinone chemical class were identified. The most potent compound discovered by screening of structural analogs, CFTR(inh)-172, reversibly inhibited CFTR short-circuit current in less than 2 minutes in a voltage-independent manner with K(I) approximately 300 nM. CFTR(inh)-172 was nontoxic at high concentrations in cell culture and mouse models. At concentrations fully inhibiting CFTR, CFTR(inh)-172 did not prevent elevation of cellular cAMP or inhibit non-CFTR Cl(-) channels, multidrug resistance protein-1 (MDR-1), ATP-sensitive K(+) channels, or a series of other transporters. A single intraperitoneal injection of CFTR(inh)-172 (250 micro g/kg) in mice reduced by more than 90% cholera toxin-induced fluid secretion in the small intestine over 6 hours. Thiazolidinone CFTR inhibitors may be useful in developing large-animal models of cystic fibrosis and in reducing intestinal fluid loss in cholera and other secretory diarrheas.

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121 Similar inhibitory potencies were found in cells that natively express wild-type CFTR, including T84 cells and primary cultures of human bronchial epithelial cells, as well as in transfected FRT cells expressing G551D-CFTR and ∆F508-CFTR (after low temperature correction) (not shown). Login to comment

[hide] 2nd international symposium: Frontiers in pancreat... Pancreas. 2003 Jan;26(1):e1-11. Hayakawa T, Naruse S, Kim KH, Go VL
2nd international symposium: Frontiers in pancreatic research-from basics to clinic and exocrine glands, Japan-Korea.
Pancreas. 2003 Jan;26(1):e1-11., [PMID:12499931]

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191 Notably, the mutant CFTR(R117H), associated with a mild form of CF, showed reduced activation of DRA/CLD, whereas the mutant CFTR(G551D), associated with a severe form of CF, was unable to activate Cl- /HCO3 - exchange by DRA/CLD. 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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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
101 The results of studies by Illek et al. [1999] suggested correction of defective function of the G551D mutation by using genistein. Login to comment

[hide] Plasma membrane CFTR regulates RANTES expression v... Mol Cell Biol. 2003 Jan;23(2):594-606. Estell K, Braunstein G, Tucker T, Varga K, Collawn JF, Schwiebert LM
Plasma membrane CFTR regulates RANTES expression via its C-terminal PDZ-interacting motif.
Mol Cell Biol. 2003 Jan;23(2):594-606., [PMID:12509457]

Abstract [show]
Despite the identification of 1,000 mutations in the cystic fibrosis gene product CFTR, there remains discordance between CFTR genotype and lung disease phenotype. The study of CFTR, therefore, has expanded beyond its chloride channel activity into other possible functions, such as its role as a regulator of gene expression. Findings indicate that CFTR plays a role in the expression of RANTES in airway epithelia. RANTES is a chemokine that has been implicated in the regulation of mucosal immunity and the pathogenesis of airway inflammatory diseases. Results demonstrate that CFTR triggers RANTES expression via a mechanism that is independent of CFTR's chloride channel activity. Neither pharmacological inhibition of CFTR nor activation of alternative chloride channels, including hClC-2, modulated RANTES expression. Through the use of CFTR disease-associated and truncation mutants, experiments suggest that CFTR-mediated transcription factor activation and RANTES expression require (i) insertion of CFTR into the plasma membrane and (ii) an intact CFTR C-terminal PDZ-interacting domain. Expression of constructs encoding wild-type or dominant-negative forms of the PDZ-binding protein EBP50 suggests that EBP50 may be involved in CFTR-dependent RANTES expression. Together, these data suggest that CFTR modulates gene expression in airway epithelial cells while located in a macromolecular signaling complex at the plasma membrane.

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57 The CFTR-encoding constructs utilized were pRSV-WT-CFTR (9) (a gift from Erik M. Schwiebert, University of Alabama at Birmingham), pGFP-WT-CFTR, pGFP-⌬F508, pGFP-G551D, and pGFP-⌬TRL (18) (all pGFP constructs were generous gifts from Bruce A. Stanton, Dartmouth Medical School). Login to comment
147 For these experiments, we took advantage of the ⌬F508, G551D, and ⌬TRL CFTR mutations, which differ in their levels of chloride transport activity. Login to comment
165 In contrast, G551D is a missense mutation within NBD-1 resulting in the conversion of a glycine residue to an aspartic acid residue. Login to comment
166 Unlike ⌬F508, the G551D mutation traffics normally to the plasma membrane; however, it is unable to function as a chloride channel (43). Login to comment
169 In these experiments, IB3-1 cells were transfected with constructs encoding WT-CFTR, ⌬F508-CFTR, G551D-CFTR, ⌬TRL-CFTR, or a mock control. Login to comment
173 In sharp contrast, cells expressing ⌬F508-CFTR, G551D-CFTR, or the mock control were not responsive to the effects of cAMP agonists. Login to comment
176 In sharp contrast, G551D restored RANTES expression to a level slightly less than but comparable to that observed with WT-CFTR (Fig. 5). Login to comment
182 As shown in Fig. 6, biotinylation analysis revealed that WT-CFTR, G551D-CFTR, and ⌬TRL-CFTR were each expressed at the cell surface; WTand ⌬TRL-CFTR surface expression levels were comparable while G551D-CFTR expression was detectable yet significantly less than that of the others. Login to comment
184 Because WTand G551D-CFTR, but neither ⌬TRL- nor ⌬F508-CFTR, were able to restore RANTES expression, these results suggest that insertion of an intact CFTR molecule into the plasma membrane is necessary for CFTR-dependent RANTES expression. Login to comment
190 IB3-1 cells were cotransfected with constructs encoding beta-galactosidase (pSV-beta-galactosidase; 0.5 ␮g/well) and mock control (pEGFP-C1; open circles) or WT-CFTR (pGFP-WT-CFTR; closed circles) (A) or G551D-CFTR (pGFP-G551D; gray circles), ⌬F508-CFTR (pGFP-⌬F508; open circles), or ⌬TRL-CFTR (pGFP-⌬TRL; closed circles) (B) (each at 1.0 ␮g/well). Login to comment
216 For these experiments, IB3-1 cells were transfected with constructs encoding WT-CFTR, G551D-CFTR, ⌬TRL-CFTR, or a mock control and then cultured in the presence and absence of TNF-␣ and IFN-␥. Login to comment
220 IB3-1 cells were cotransfected with constructs encoding beta-galactosidase and mock control, WT-CFTR, G551D-CFTR, ⌬F508-CFTR, or ⌬TRL-CFTR as described in the Fig. 4 legend and then cultured with or without TNF-␣ and IFN-␥ as described in the Fig. 1 legend. Login to comment
226 As shown in Fig. 9C, cells transfected with constructs encoding G551D-CFTR displayed a pattern of enhanced NF-␬B binding that resembled the pattern of NF-␬B binding observed for cells transfected with constructs encoding WT-CFTR; in contrast, ⌬TRL-CFTR did not promote the binding of NF-␬B either in the presence or in the absence of TNF-␣ and IFN-␥ (Fig. 9C). Login to comment
229 IB3-1 cells were cotransfected with constructs encoding beta-galactosidase and mock control (M) or WT-CFTR (A), G551D-CFTR or ⌬TRL-CFTR (B), or ⌬F508-CFTR (C) as described in the Fig. 4 legend. Login to comment
270 (C) IB3-1 cells were transfected with constructs encoding beta-galactosidase, WT-CFTR, G551D-CFTR, or ⌬TRL-CFTR; stimulated for 30 min with and without TNF-␣-IFN-␥ (denoted by ϩ and -, respectively); and then prepared for EMSA. Login to comment
281 For these experiments, IB3-1 cells were transfected transiently with constructs encoding WT-CFTR, G551D-CFTR, ⌬TRL-CFTR, or a mock control; cultured in the presence and absence of TNF-␣ and IFN-␥; and then prepared for EMSA. Login to comment
285 As shown in Fig. 11C, cells transfected with constructs encoding G551D-CFTR displayed a pattern of enhanced NF-␬B binding that resembled the pattern of NF-␬B binding observed for cells transfected with constructs encoding WT-CFTR; in contrast, ⌬TRL-CFTR did not promote the binding of NF-␬B either in the presence or in the absence of TNF-␣ and IFN-␥ (Fig. 11C). Login to comment
300 Data obtained through the use of CFTR disease-associated (G551D and ⌬F508) and truncation (⌬TRL) mutations presented herein FIG. 11. Login to comment
306 (C) IB3-1 cells were transfected with constructs encoding beta-galactosidase, WT-CFTR, G551D-CFTR, or ⌬TRL-CFTR; stimulated with and without TNF-␣-IFN-␥ (denoted by ϩ and -, respectively); and then prepared for EMSA as described in the Fig. 9 legend. Login to comment
310 First, the ability of G551D, but not ⌬F508, to restore RANTES expression to a level comparable with that observed with WT-CFTR suggests that CFTR molecules inserted into the plasma membrane restore RANTES expression. Login to comment
314 It should be noted that, although ⌬F508- and G551D-CFTR differ in their ability to restore RANTES expression, both of these CFTR mutations are associated with severe forms of CF. Login to comment
315 If the lack of RANTES expression is an important contributor to CF-associated airway inflammation, then it would be expected that neither ⌬F508 nor G551D would support RANTES expression; however, this was not the response that was observed. Login to comment
316 With regard to the correlation of ⌬F508- and G551D-CFTR mutations, RANTES expression, and severe CF pulmonary disease, we note that Mickle and Cutting have reported that, despite its being a monogenic disorder, the genotype-phenotype relationship in CF is complex (14). Login to comment
321 Specifically, reports by Prince and coworkers indicate that cells expressing the CFTR mutation ⌬F508 or G551D as well as non-CFTR-expressing cells display increased NF-␬B activation in response to a cytokine or P. aeruginosa stimulus compared with non-CF and "CFTR-corrected" cells; assessments were determined via measurements of NF-␬B binding, NF-␬B reporter construct activity, and NF-␬B nuclear translocation (42). Login to comment
326 In contrast, results presented herein suggest that human airway epithelial cells expressing WT-CFTR or G551D-CFTR, but not ⌬TRL-CFTR, exhibit enhanced NF-␬B activation in the presence of TNF-␣ and IFN-␥ compared with mock controls. Login to comment
297 Analyses of the CFTR mutants G551D- and ⌬TRL-CFTR, which differ in their ability to transport chloride and restore RANTES expression, support these observations. 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] ENaC is inhibited by an increase in the intracellu... Pflugers Arch. 2003 Jan;445(4):504-12. Epub 2002 Nov 20. Kunzelmann K
ENaC is inhibited by an increase in the intracellular Cl(-) concentration mediated through activation of Cl(-) channels.
Pflugers Arch. 2003 Jan;445(4):504-12. Epub 2002 Nov 20., [PMID:12548397]

Abstract [show]
Activation of the CFTR Cl(-) channel inhibits epithelial Na(+) absorption, according to studies on native epithelia derived from airways, colon and kidney, and can also be demonstrated in overexpressing cells. However, Na(+) absorption is not inhibited by CFTR in the native sweat duct epithelium. The mechanism for the inhibition of epithelial sodium channels (ENaC) has been examined in most detail in Xenopus oocytes coexpressing CFTR and ENaC. It was shown that ENaC is inhibited during stimulation of CFTR in Xenopus oocytes, independent of the experimental setup and the magnitude of the whole-cell current. However, a minimal Cl(-) conductance is required for inhibition of ENaC, and inhibition is augmented at higher CFTR-to-ENaC currents ratios. Low-CFTR-to-ENaC conductance ratios may be the reason for the absence of ENaC inhibition, as described recently. Similar to CFTR, ClC-0 Cl(-) currents also inhibit ENaC, as well as high extracellular Na(+) and Cl(-) in partially permeabilized oocytes. Thus, inhibition of ENaC is not specific to CFTR and could be mediated by Cl(-) flow and/or changes in the intracellular Cl(-) concentration. These results are reminiscent of the Cl(-) feedback regulation observed in mouse mandibular duct cells. Current results obtained with ENaC mutants examined in Xenopus oocytes suggest a charge interaction of Cl(-) ions with the epithelial sodium channel.

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36 Amiloride-sensitive short-circuit currents are inhibited by cAMP in normal mouse colon, but not in transgenic G551D-CFTR mice [55]. Login to comment

[hide] Effects of oral genistein in mice. Pediatr Pathol Mol Med. 2003 Mar-Apr;22(2):131-41. Bhandari A, Crawford SE, Huang L, Reenstra WW
Effects of oral genistein in mice.
Pediatr Pathol Mol Med. 2003 Mar-Apr;22(2):131-41., [PMID:12556293]

Abstract [show]
In cell culture systems, genistein, a soy-derived isoflavone with chemopreventive and estrogenic effects, enhances cAMP-dependent activation of the most common cystic fibrosis-causing mutation, deltaF508-CFTR, by as much as 20-fold. DeltaF508-CFTR is present in the apical membrane at far lower levels than wild-type CFTR. If genistein can enhance cyclic AMP-dependent activity in vivo, the presence of deltaF508-CFTR, at even a few percent of wild-type levels, might permit genistein to be of therapeutic benefit to cystic fibrosis patients with this mutation. Before determining if oral genistein would be of benefit in mice with a deltaF508 mutation in the murine CFTR gene, a maximal dose of oral genistein with minimal side effects needed to be established. Accordingly, C57Bl/6 mice pups were randomly weaned onto soy-free diet, AIN-76, containing between 0 and 1.0 g/kg genistein and allowed to feed ad libitum for 3 weeks. Genistein had no significant effects on growth rates of either male or female mice. Histology of the lung, heart, kidney, liver, and intestine revealed no significant genistein-dependent changes in morphology. When mice on a 1.0 g/kg of genistein diet were sacrificed in the morning, the mean level of serum genistein was 1.4+/-0.2 micro moles/L. Serum genistein increased during the daylight hours reaching a maximum of 7.5+/-0.6 micro moles/L in the early evening. Our results demonstrate that dietary genistein is not inhibitory to growth or caloric intake and up to 1.0 g/kg ad libitum genistein causes no significant organ specific abnormalities.

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18 Genistein also has been shown to potentiate PKA-dependent activation of the most common CFTR mutant DF508 and another common CF causing mutation G551D [3,6]. Login to comment
105 A similar e¡ect hasbeen observed with G551D, another common CFmutationthat is more e/ciently targetedtothe plasma membrane [6]. Login to comment
106 If similar e¡ects can be achieved in vivo, and with the proviso that DF508-CFTR and G551D-CFTR are present in the apical membrane, genistein could be of considerable therapeutic bene't to CF patients with these mutations. Login to comment
120 However, since its utility as a treatment for CF would require continuous treatment, we chose to study the e¡ects of relatively long-term ad libitum consumption of genistein before assessing the ability of genistein to improve the morbidity and mortality of knockin mice with DF508- or G551D-CFTR. 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
71 The G551D mutation (7.6%) was found at a higher frequency than the UK (15) (3.08%) and Northern Ireland (13) (5.1%) and was found to be similar to that reported in a previous Irish study (11) (6.9%). Login to comment

[hide] Chronic pancreatitis and cystic fibrosis. Gut. 2003 May;52 Suppl 2:ii31-41. Witt H
Chronic pancreatitis and cystic fibrosis.
Gut. 2003 May;52 Suppl 2:ii31-41., [PMID:12651880]

Abstract [show]
Recent discoveries of trypsinogen and trypsin inhibitor mutations in patients with chronic pancreatitis (CP) support the hypothesis that an inappropriate activation of pancreatic zymogens to active enzymes within the pancreatic parenchyma starts the inflammatory process. Current data suggest that CP may be inherited dominant, recessive, or complex as a result of mutations in the above mentioned or yet unidentified genes. Evaluation of patients with CP should include genetic testing. Cystic fibrosis (CF) is an autosomal recessive inherited disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene and is characterised by pancreatic insufficiency and chronic bronchopulmonary infection. The progression and severity of pulmonary disease differs considerably between people with identical CFTR mutations and does not seem to correlate with the type or class of the CFTR mutation. The identification of further disease modifying genetic factors will increase the pathophysiological understanding and may help to identify new therapeutic targets.

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430 Nucleus Class I defective protein synthesis (R553X, W1282X, 3950delT) Class II abnormal processing/trafficking (del508, N1303K) Class VI defective regulation of other ion channels (del508, G551D) Class V reduced synthesis (3849+10kbC>T) Class IV decreased conductance (R117H, R347P, D1152H) Class III defective activation (G551D) I II VI V III IV RD ATP Endoplasmic reticulum NBD NBD Golgi mutations result in a decreased amount of functional protein by abnormal splicing or reduced trafficking. Login to comment
433 It is worthwhile to note that a specific mutation can be associated with more than one of the above mentioned mechanisms: the G551D mutation affects activation (class III) as well as the regulatory property of CFTR on other ion channels ("class VI").89 Genotype phenotype correlation and modifier genes The course of disease differs between individuals with identical CFTR mutations even in the same family suggesting that environmental and inherited factors may modify the disease phenotype. Pancreatic function is strongly determined by the CFTR genotype, whereas the course of pulmonary disease seems to be largely dependent on secondary factors. Login to comment

[hide] Development of cystic fibrosis and noncystic fibro... Am J Physiol Lung Cell Mol Physiol. 2003 May;284(5):L844-54. Epub 2003 Jan 10. Zabner J, Karp P, Seiler M, Phillips SL, Mitchell CJ, Saavedra M, Welsh M, Klingelhutz AJ
Development of cystic fibrosis and noncystic fibrosis airway cell lines.
Am J Physiol Lung Cell Mol Physiol. 2003 May;284(5):L844-54. Epub 2003 Jan 10., [PMID:12676769]

Abstract [show]
In this study, we utilized the reverse transcriptase component of telomerase, hTERT, and human papillomavirus type 16 (HPV-16) E6 and E7 genes to transform normal and cystic fibrosis (CF) human airway epithelial (HAE) cells. One cell line, designated NuLi-1 (normal lung, University of Iowa), was derived from HAE of normal genotype; three cell lines, designated CuFi (cystic fibrosis, University of Iowa)-1, CuFi-3, and CuFi-4, were derived from HAE of various CF genotypes. When grown at the air-liquid interface, the cell lines were capable of forming polarized differentiated epithelia that exhibited transepithelial resistance and maintained the ion channel physiology expected for the genotypes. The CF transmembrane conductance regulator defect in the CuFi cell lines could be corrected by infecting from the basolateral surface using adenoviral vectors. Using nuclear factor-kappaB promoter reporter constructs, we also demonstrated that the NuLi and CuFi cell lines retained nuclear factor-kappaB responses to lipopolysaccharide. These cell lines should therefore be useful as models for studying ion physiology, therapeutic intervention for CF, and innate immunity.

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140 Immortal normal and CF HAE cell lines Cell Line CF Genotype Rt, ⍀⅐cm2 NuLi-1 WT Ͼ450 NuLi-2 WT Ͻ50 CuFi-1 ⌬F508/⌬F508 Ͼ450 CuFi-2 ⌬F508/⌬F508 Ͻ50 CuFi-3 ⌬F508/R553X Ͼ450 CuFi-4 ⌬F508/G551D Ͼ450 Cells were transduced with hTERT and human papilloma virus type 16 E6/E7 retroviruses. Login to comment
190 Primary lung epithelial cells from CF lung transplant recipients were infected with hTERT and HPV-16 E6/E7 to generate four different immortalized CF cells: CuFi-1 (⌬508/⌬508), CuFi-2 (⌬508/⌬508), CuFi-3 (⌬508/R553X), and CuFi-4 (⌬508/G551D). Login to comment
241 Differentiated primary cultures of airway epithelial cells from 3 different immortalized CF cells: CuFi-1 (⌬508/⌬508, A), CuFi-3 (⌬508/R553X, B), and CuFi-4 (⌬508/G551D, C) at passage 18 were individually thawed and seeded onto semipermeable filters and compared with their primary source. Login to comment
258 Several groups have screened compounds that may affect ⌬F508 or G551D CFTR-expressing cells to restore Cl-channel function (3, 8, 22, 34, 36, 47). 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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21 They reported a signi®cantly higher prevalence of the DF508/DF508 and DF508/G551D genotypes in patients with nasal polyps requiring surgery compared with the control group: 57.5% versus 49.9% and 12% versus 8% respectively. Login to comment
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
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] Effects of nitric oxide on Pseudomonas aeruginosa ... Infect Immun. 2003 May;71(5):2341-9. Darling KE, Evans TJ
Effects of nitric oxide on Pseudomonas aeruginosa infection of epithelial cells from a human respiratory cell line derived from a patient with cystic fibrosis.
Infect Immun. 2003 May;71(5):2341-9., [PMID:12704103]

Abstract [show]
Cystic fibrosis (CF) is characterized by airway inflammation and chronic bacterial lung infection, most commonly with Pseudomonas aeruginosa, an opportunistic human pathogen. Despite the persistent airway inflammation observed in patients with CF, although phagocyte inducible nitric oxide synthase (iNOS) production is upregulated, expression of iNOS in the respiratory epithelium is markedly reduced. Given the antimicrobial action of NO, this may contribute to the chronic airway infection of this disease. To define the role of epithelium-derived NO in airway defense against P. aeruginosa, we infected differentiated human bronchial epithelial cells derived from a patient with CF (CFBE41o- cells) with different strains of this pathogen at low multiplicities of infection. Using cells transfected with human iNOS cDNA, we studied the effect of NO on P. aeruginosa replication, adherence, and internalization. P. aeruginosa adherence to iNOS-expressing cells was reduced by 44 to 72% (P = 0.02) compared with control values. Absolute P. aeruginosa uptake into these cells was reduced by 44%, but uptake expressed as a percentage of adherent bacteria did not differ from the control uptake. Survival of P. aeruginosa within iNOS-expressing cells was reduced at late times postinfection (P = 0.034). NO production did not alter host cell viability. NO production reduced P. aeruginosa adherence to human bronchial epithelial cells and enhanced killing of internalized bacteria, suggesting that a lack of epithelial iNOS in patients with CF may contribute to P. aeruginosa infection and colonization.

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278 However, some patients with CF express a nonfunctional apical CFTR (e.g., G551D) that has the necessary domain to bind and internalize P. aeruginosa, and yet these patients have chronic colonization of the airways with P. aeruginosa as severe as that found in patients with no surface CFTR (e.g., ⌬F508) (52). Login to comment

[hide] Adenosine receptors and phosphodiesterase inhibito... Am J Respir Cell Mol Biol. 2003 Sep;29(3 Pt 1):410-8. Epub 2003 Apr 24. Cobb BR, Fan L, Kovacs TE, Sorscher EJ, Clancy JP
Adenosine receptors and phosphodiesterase inhibitors stimulate Cl- secretion in Calu-3 cells.
Am J Respir Cell Mol Biol. 2003 Sep;29(3 Pt 1):410-8. Epub 2003 Apr 24., [PMID:12714375]

Abstract [show]
We investigated cystic fibrosis transmembrane conductance regulator (CFTR) activation by clinically used phosphodiesterase inhibitors (PDEis) in Calu-3 cell monolayers alone and in combination with A2B adenosine receptor stimulation. This receptor pathway has previously been shown to activate wild-type and mutant CFTR molecules. Several PDEis, including milrinone, cilostazol (Pletal), papaverine, rolipram, and sildenafil (Viagra), produced a short circuit current (Isc) that was glibenclamide-sensitive, achieving 20-85% of forskolin-stimulated Isc. Papaverine, cilostazol, and rolipram also augmented both the magnitude and the duration of Isc following low dose stimulation of adenosine receptors with Ado (0.1-1.0 microM, P < 0.01). Subsequent studies demonstrated that very low concentrations of cilostazol or papaverine (approximately 1/2 peak serum concentrations) were sufficient to activate Isc, and both agents markedly augmented Ado-stimulated Isc (1 microM, P < 0.01). Our results provide evidence that select PDEis, at concentrations achieved as part of systemic therapies, can activate CFTR-dependent Isc in Calu-3 cell monolayers. These studies also indicate that PDEis have the capacity to augment an endogenous CFTR-activating pathway in an "in vivo"-like model system, and supports future investigations of these agents relevant to cystic fibrosis.

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14 29, pp. 410-418, 2003 Originally Published in Press as DOI: 10.1165/rcmb.2002-0247OC on April 24, 2003 Internet address: www.atsjournals.org inhibitors could theoretically potentiate low level activity of mutant CFTR molecules that spontaneously localize to the cell surface (e.g., R117H CFTR, G551D CFTR) or can be localized to the cell surface at low levels by corrective molecules (e.g., butyrate compounds for rescue of ⌬F508 CFTR, aminogylcosides to suppress premature stop mutations) (12-15). 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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35 Geographical patterns A geographical description of allele frequency patterns was obtained by drawing maps of gene frequencies for the most common CF mutations (namely F508del, G452X, G551D, N1303 K, and W1285X, which are the ones found at average frequencies 41%), as well as for genetic diversity estimates, by using Surfer 7.0 (http://www.goldensoftware. Login to comment
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
94 In this process, selection will pull up the frequency of any mutant allele as long as it confers a selective advantage to the heterozygote, that is, as discussed above, any CF causing mutation. Thus, the initial spectrum of CF causing mutations is that found when the selection process started; selection will increase the frequency of those mutations already present in the different populations, and, in the end, the total frequency of the mutant class may be similar Figure 3 Geographical distribution (a) and spatial autocorrelogram (b) of the G551D 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 4 Geographical distribution (a) and spatial autocor- relograms (b) of the N1303 K 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. across populations (as long as the selection pressure is similar), but the actual mutations that fill up this frequency determined by selection may be different across populations. Login to comment

[hide] The role of regulated CFTR trafficking in epitheli... Am J Physiol Cell Physiol. 2003 Jul;285(1):C1-18. Bertrand CA, Frizzell RA
The role of regulated CFTR trafficking in epithelial secretion.
Am J Physiol Cell Physiol. 2003 Jul;285(1):C1-18., [PMID:12777252]

Abstract [show]
The focus of this review is the regulated trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) in distal compartments of the protein secretory pathway and the question of how changes in CFTR cellular distribution may impact on the functions of polarized epithelial cells. We summarize data concerning the cellular localization and activity of CFTR and attempt to synthesize often conflicting results from functional studies of regulated endocytosis and exocytosis in CFTR-expressing cells. In some instances, findings that are inconsistent with regulated CFTR trafficking may result from the use of overexpression systems or nonphysiological experimental conditions. Nevertheless, judging from data on other transporters, an appropriate cellular context is necessary to support regulated CFTR trafficking, even in epithelial cells. The discovery that disease mutations can influence CFTR trafficking in distal secretory and recycling compartments provides support for the concept that regulated CFTR recycling contributes to normal epithelial function, including the control of apical CFTR channel density and epithelial protein secretion. Finally, we propose molecular mechanisms for regulated CFTR endocytosis and exocytosis that are based on CFTR interactions with other proteins, particularly those whose primary function is membrane trafficking. These models provide testable hypotheses that may lead to elucidation of CFTR trafficking mechanisms and permit their experimental manipulation in polarized epithelial cells.

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171 They also showed that the internalization rate of G551D CFTR was similar to that of the wild-type protein but that, interestingly, its endocytosis was not affected by cAMP. Login to comment
172 This finding suggested that either the channel activity of CFTR is related to its ability to be retrieved from the cell surface or the altered structure of the G551D mutant precludes regulation of both gating and trafficking. 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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5 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. Login to comment
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
50 Dublin Centre 1262 CF alleles 35 mutations F508del 76.5% G551D 6.5% R117H 3.0% R560T 2.4% 621+1G>T 1.7% Cork Area 278 CF alleles 10 mutations F508del 81.3% G551D 9.7% R117H 1.4% Brittany 778 CF alleles 62 mutations F508del 74.8% G551D 3.7% 1078delT 3.6% N1303K 1.4% W846X2 1.0% 1717-1G>A 1.0% Statistical analysis We determined the spectrum of the CFTR mutations identified in the three cohorts of patients and compared their respective frequencies by a Chi square test. Login to comment
59 Besides the F508del deletion, the G551D mutation was also observed with an elevated frequency in each cohort: it was found in 9.7% of the CF alleles in Cork, in 6.5% in Dublin and in 3.7% in Brittany. Login to comment
61 The G551D mutation was the most frequent molecular anomaly after the deletion F508del in the two Irish cohorts. 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
71 Despite a similar high frequency of the F508del and G551D mutations, the other most common mutations observed in each region were not the same (Table 1, Figure 1). Login to comment
74 In the cohort from Cork, only one mutation other than F508del and G551D reached a frequency of 1%: R117H (1.4%). 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
81 Just three genotypes were responsible for two thirds of CF cases in this region: F508del/F508del (57.6%), F508del/G551D (4.9%) and F508del/1078delT (4.6%). Login to comment
83 The other most frequent genotypes were: F508del/G551D (9.5%), F508del/R117H (3.5%) and F508del/R560T (2.7%). Login to comment
84 Finally, in the Cork area, about 70.0% of patients were homozygous for the main mutation, 13.7% were F508del/G551D and 2.9% were F508del/R117H. Login to comment
85 The frequency of the F508del/G551D genotype differed significantly between the cohorts (χ²=12.2 - p=0.0022). Login to comment
89 This study highlights, on one hand, the common background of these populations with a common origin, through the high frequency of the "Celtic" mutation (G551D) resulting from the expansion of the Celts which was at its height between the IVth and IIIth centuries BC, and, on the other hand, the specificities of each cohort, reflecting the influence of their own history. 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
101 We, in collaboration with others, have shown by studying the haplotypes for three intragenic microsatellites markers (IVS8CA, IVS17bTA and IVS17bCA) that the G551D alleles of Irish, Scottish, English, Breton and Czech subjects were associated with a unique haplotype (marker names 16-7-17). Login to comment
104 Besides the F508del and G551D mutations, disparities appeared in the spectrum of mutations between the three cohorts, reflecting the influence of the past of each population, with their own populations` movements, genetic drifts, founder effects, … (Tables 2 and 3). Login to comment
115 We concluded that western Brittany presented a specific mutation spectrum (1078delT, G551D, 4005+1G>A, W846X2), whereas the eastern part of the region showed a spectrum of mutations more similar to that generally observed in France (N1303K, Y1092X, 2789+5G>A, etc). Login to comment
124 The spectrums of CFTR mutations show disparities, even if G551D is the most frequent mutation after the deletion F508del in each of the studied cohorts. Login to comment

[hide] GFP-tagged CFTR transgene is functional in the G55... J Membr Biol. 2003 Apr 1;192(3):159-67. Oceandy D, McMorran B, Schreiber R, Wainwright BJ, Kunzelmann K
GFP-tagged CFTR transgene is functional in the G551D cystic fibrosis mouse colon.
J Membr Biol. 2003 Apr 1;192(3):159-67., 2003-04-01 [PMID:12820661]

Abstract [show]
Trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is central to its function, with the most common mutation, deltaF508, resulting in abnormal processing and trafficking. Therefore, there is a significant need to develop tools, which enable the trafficking of CFTR to be studied in vitro and in vivo. In previous studies it has been demonstrated that fusion of the green fluorescent protein (GFP) to the N-terminus of CFTR does lead to functional expression of CFTR chloride channels in epithelial cell lines. The aim of the present study was to examine whether it is possible to express GFP-tagged CFTR as a transgene in colonic and airway epithelial cells of cystic fibrosis (CF) mice and to correct the CF defect. Using the epithelial-specific human cytokeratin promoter K18, we generated bitransgenic mice cftr(G551D/G551) K18-GFP-CFTR(+/-), designated GFP mice. Transcripts for GFP-CFTR could be detected in bitransgenic mice by use of RT-PCR techniques. Expression of GFP-CFTR protein was detected specifically in the colonic epithelium by both direct GFP fluorescence and the use of an anti-GFP antibody. Ussing chamber studies showed that the ion transport defect in colon and airways observed in cftr(G551D/G551D) mice was partially corrected in the bitransgenic animals. Thus, K18-GFP-CFTR is functionally expressed in transgenic mice, which will be a valuable tool in studies on CFTR synthesis, processing and ion transport in native epithelial tissues.

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1 2003 GFP-Tagged CFTR Transgene is Functional in the G551D Cystic Fibrosis Mouse Colon D. Oceandy l, B. McMorran l, R. Schreiber2, B.J. Wainwright1, K. Kunzelmann2 IInstitute for Molecular Biosciences, The University of Queensland, St. Lucia QLD 4072, Australia 2Institut ffir Physiologic, Universit~it Regensburg, UniversitfitstraBe 31, D-93053 Regensburg Received: 25 September 2002/Revised: 13 December 2003 Abstract. Login to comment
52 The transgenic founder was bred with CD1 mice and the transgenic offspring were crossed with G551D CF mice [5] to generate bitransgenic animals carrying the K18-GFP-CFTR transgene and the cftr c'SsjD allele. Login to comment
54 The four strains used in this study, ~;[?r+/+ (wt), ~JtrG551D/G551D (CF), QQrG5slD/G551D K18-CFTR ~ (K18) [30], and ~ftr ~ K18-GFP-CFTR +/ (GFP) have equivalent genetic backgrounds (a mixture of CDI, C57-B6 and 129). Login to comment
80 Results PHENOTYPIC ASPECT OF cftrG551D/G551D K18-GFP-CFTR +/- MICE Typically cftrGSslD/Gs51D have a significant perinatal mortality primarily due to meconium ileus and sur- Fig. 1. Login to comment
85 Bitransgenic cftr G551D/G551D (CF) mice carrying the K18-GFP-CFTR transgene showed in general a good viability, which exceeded that of the cftr G551D/G551D mice. Login to comment
87 The bitransgenic CF mice were also significantly heavier than the cftr G551D/G551D mice and similar to non-transgenic cftr +/+ mice. Login to comment
88 Mean weights (+ standard deviation) of males at 10 weeks of age were 33.0-4-2.5 g, 24.8 + 3.0 g and 34.7 • 2.9 g for bitransgenic cftr c551D/C55~I~j, cftr G551D/G551I~ and cftr +/+ mice respectively (n > 5), indicating that the transgene was complementing the gastrointestinal defect at the broad phenotypic level. Login to comment
89 EXPRESSIONOF THE GFP-CFTR TRANSGENEIN THE COLON Expression of the human CFTR transgene could be readily detected in the colon of bitransgenic cftr G551D/G551D K18-GFP-CFTR +! Login to comment
107 TRANSPORT PROPERTIES OF THE COLONIC EPITHELIUM AND IN THE TRACHEA OF cftr G551D/G551D K18-GFP-CFTR +/- MICE We examined the ion transport in the colonic epithelium of GFP mice and compared the results with those obtained in the colon of cftr cSslD/G551D mice, wt mice and bitransgenic cftr ~SslD/~Ssm K18- Fig. 4. Login to comment
132 After stimulation with IBMX and forskolin and activation of CFTR, a negative voltage deflection is induced in wt, GFP and K18 mice but not in G551D animals (Figs. 5 and 6). Login to comment
152 We were interested in studying expression of a GFP-CFTR G551D/G551D transgene in cftr mice. Login to comment
154 The present results indicate expression of transcripts and protein of GFP-CFTR in G55 ID/G551D bitransgenic cftr K18-GFP-CFTR mice. Login to comment
180 Although multiple and also reciprocal interactions between CFTR and other ion channels such as ENaC have been described in several previous studies [reviewed in 20, 35], the reason and functional mechanism for the decreased Na + absorption in the colon of G551D animals, and the increase in Na+ transport after expression of K18-GFP-CFTR remain obscure. Login to comment
181 However, it seems important to note that the colon of G551D (CF) mice is smaller than that of the other animals, that it demonstrates an altered quality and is difficult to prepare for Ussing chamber recordings. Login to comment

[hide] Nanomolar affinity small molecule correctors of de... J Biol Chem. 2003 Sep 12;278(37):35079-85. Epub 2003 Jun 27. Yang H, Shelat AA, Guy RK, Gopinath VS, Ma T, Du K, Lukacs GL, Taddei A, Folli C, Pedemonte N, Galietta LJ, Verkman AS
Nanomolar affinity small molecule correctors of defective Delta F508-CFTR chloride channel gating.
J Biol Chem. 2003 Sep 12;278(37):35079-85. Epub 2003 Jun 27., 2003-09-12 [PMID:12832418]

Abstract [show]
Deletion of Phe-508 (Delta F508) is the most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) causing cystic fibrosis. Delta F508-CFTR has defects in both channel gating and endoplasmic reticulum-to-plasma membrane processing. We identified six novel classes of high affinity potentiators of defective Delta F508-CFTR Cl- channel gating by screening 100,000 diverse small molecules. Compounds were added 15 min prior to assay of iodide uptake in epithelial cells co-expressing Delta F508-CFTR and a high sensitivity halide indicator (YFP-H148Q/I152L) in which Delta F508-CFTR was targeted to the plasma membrane by culture at 27 degrees C for 24 h. Thirty-two compounds with submicromolar activating potency were identified; most had tetrahydrobenzothiophene, benzofuran, pyramidinetrione, dihydropyridine, and anthraquinone core structures (360-480 daltons). Further screening of >1000 structural analogs revealed tetrahydrobenzothiophenes that activated DeltaF508-CFTR Cl- conductance reversibly with Kd < 100 nm. Single-cell voltage clamp analysis showed characteristic CFTR currents after Delta F508-CFTR activation. Activation required low concentrations of a cAMP agonist, thus mimicking the normal physiological response. A Bayesian computational model was developed using tetrahydrobenzothiophene structure-activity data, yielding insight into the physical character and structural features of active and inactive potentiators and successfully predicting the activity of structural analogs. Efficient potentiation of defective Delta F508-CFTR gating was also demonstrated in human bronchial epithelial cells from a Delta F508 cystic fibrosis subject after 27 degrees C temperature rescue. In conjunction with correctors of defective Delta F508-CFTR processing, small molecule potentiators of defective Delta F508-CFTR gating may be useful for therapy of cystic fibrosis caused by the Delta F508 mutation.

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51 Some measurements were done with stably transfected FRT cells expressing YFP-H148Q and wild-type or G551D-CFTR (18). Login to comment
134 The six ⌬F508-CFTR potentiators shown in Fig. 3A were tested for activation of wild-type and G551D-CFTR in transfected FRT cells. Login to comment
135 None of the compounds gave measurable G551D-CFTR activation at 10 ␮M in the presence of 20 ␮M FIG. 1. Login to comment
238 None of the compounds activated G551D-CFTR even in the presence of a high concentration of a cAMP agonist nor did they cause endoplasmic reticulum-to-plasma membrane transport of ⌬F508-CFTR, as assessed functionally and biochemically. 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
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] Pseudomonas aeruginosa-induced apoptosis is defect... Am J Respir Cell Mol Biol. 2003 Aug;29(2):188-97. Cannon CL, Kowalski MP, Stopak KS, Pier GB
Pseudomonas aeruginosa-induced apoptosis is defective in respiratory epithelial cells expressing mutant cystic fibrosis transmembrane conductance regulator.
Am J Respir Cell Mol Biol. 2003 Aug;29(2):188-97., [PMID:12878584]

Abstract [show]
Chronic lung infection with Pseudomonas aeruginosa constitutes the most severe manifestation of cystic fibrosis, a scenario that results from defects in early clearance of the microbe. Early clearance involves epithelial cell ingestion of bacteria, rapid activation of nuclear factor-kappa B and cellular desquamation within minutes of P. aeruginosa infection, processes that are deficient in cells with mutant alleles of Cftr. Analyzing the effect of Cftr genotype on the apoptotic response of airway epithelial cells to P. aeruginosa, we found that human bronchial epithelial cells expressing Delta F508 cystic fibrosis transmembrane conductance regulator (CFTR) underwent significantly delayed apoptosis compared with cells expressing wild-type (WT) CFTR. Mice with a WT Cftr allele had apoptotic cells in their lungs after P. aeruginosa infections, whereas mice homozygous for the Delta F508 or G551D Cftr alleles showed little apoptosis in response to acute infection. Pseudomonal infection induced expression of CD95 and CD95 ligand, a response that was also delayed in cells homozygous for mutant Cftr alleles. Thus, WT CFTR expression promotes a rapid expression of CD95/CD95 ligand and apoptotic response to P. aeruginosa infection. Prompt apoptosis of infected epithelial cells may be critical for clearance of P. aeruginosa, and CFTR-associated defects in apoptosis may contribute to the pathogenesis of the lung disease in cystic fibrosis.

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3 Mice with a WT Cftr allele had apoptotic cells in their lungs after P. aeruginosa infections, whereas mice homozygous for the ⌬F508 or G551D Cftr alleles showed little apoptosis in response to acute infection. Login to comment
110 Equivalent areas examined for TUNEL-positive cells showed a striking absence in mice homozygous for either the ⌬F508 or G551D Cftr alleles (Figures 4A and 4C), whereas heterozygous littermates had TUNEL-positive cells both in the lumen of bronchi (Figures 4B and 4D, arrows) or scattered among the columnar epithelial cells lining the bronchi (Figure 4D). Login to comment
123 D and E show the lungs of an infected mouse heterozygous for WT and G551D cftr, and F depicts the lungs of a homozygous G551D littermate. Login to comment
248 They also thank Gloria Meulini and Fadie Coleman for help in breeding the ⌬F508 and G551D mice and Ervin Meulini for sectioning the mouse lungs. 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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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
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
256 This pooling is more noticeable in the diminution of the signal for the mutations of exon 11 relative to the wild-type analyte (e.g., mutants G551D and R560T in Fig. 3). 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] Phase I trial of intranasal and endobronchial admi... Hum Gene Ther. 2003 Jul 20;14(11):1079-88. Flotte TR, Zeitlin PL, Reynolds TC, Heald AE, Pedersen P, Beck S, Conrad CK, Brass-Ernst L, Humphries M, Sullivan K, Wetzel R, Taylor G, Carter BJ, Guggino WB
Phase I trial of intranasal and endobronchial administration of a recombinant adeno-associated virus serotype 2 (rAAV2)-CFTR vector in adult cystic fibrosis patients: a two-part clinical study.
Hum Gene Ther. 2003 Jul 20;14(11):1079-88., 2003-07-20 [PMID:12885347]

Abstract [show]
Recombinant adeno-associated serotype 2-based vectors (rAAV2) possess a number of theoretical advantages for cystic fibrosis (CF) gene therapy because they elicit little or no inflammatory response and generally result in stable expression. rAAV2 vectors expressing the cystic fibrosis transmembrane conductance regulator (CFTR) gene have previously been shown to mediate stable correction of the CF defect in CF bronchial epithelial cells and stable expression of CFTR in rabbit and nonhuman primate models. Here we report the results of the first trial initiated with rAAV in humans, a phase I study in 25 adult and adolescent CF patients with mild to moderate lung disease. Doses of the rAAV-CFTR vector (tgAAVCF) ranging from 3 x 10(1) to 1 x 10(9) replication units (RU), which is equivalent to approximately 6 x 10(4) to 2 x 10(12) DNase resistant particles (DRP), were administered to one side of the nose and to the superior segment of the lower lobe of the right lung. Several adverse events were noted prior to and/or after vector delivery, but most of them appeared to be related to the endogenous CF lung disease or a result of the bronchoscopic procedures. Only one of the serious events was judged to be possibly vector-related (based on temporal association), and this event was a pulmonary exacerbation very similar to several others experienced by the same subject in the three months preceding vector delivery. Vector shedding was minimal throughout the study, and serum-neutralizing antibodies were detected after vector delivery to subjects in the highest dosage cohorts. Gene transfer as measured by DNA polymerase chain reaction (PCR) was not observed until cohort 10 in nasal and bronchial epithelia. Sporadic low-level copy numbers suggested gene transfer of anywhere from 0.002 copies per cell up to 0.5 copies per cell was possible; however, DNA PCR was positive in lungs prior to direct dosing suggesting aspiration from the nasal dosing. These data indicate the need for continued evaluation of rAAV-CFTR vectors in additional clinical trials.

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62 Genotypes: homozygous or compound transfusion within 1 year or coughing of heterozygous DF508, G551D, . Login to comment
145 DEMOGRAPHICS AND BASELINE CHARACTERISTICS Low dose Medium High dose part 1 dose part 1 part 1 Part 2 (cohorts 1, (cohorts 4, (cohorts 7, (cohorts 10 2, and 3) 5, and 6) 8, and 9) and 11) Total Characteristic n 5 6 n 5 7 n 5 6 n 5 6 n 5 25 Age (years) 28.4 6 10.6 26.6 6 6.70 28.9 6 9.20 24.3 6 10.1 27.0 6 8.80 (range, 15 to 43) Height (cm) 168.0 6 11.40 171.1 6 13.30 171.5 6 10.70 157.5 6 44.80 170.4 6 11.00 Weight (kg) 60.5 6 9.90 71.2 6 19.9 63.7 6 8.80 66.8 6 11.6 65.8 6 13.4 Gender Male 3 (50.0) 5 (71.4) 4 (66.7) 5 (83.3) 17 (68.0) Female 3 (50.0) 2 (28.6) 2 (33.3) 1 (16.7) 08 (32.0) Race Caucasian 6 (100.0) 07 (100.0) 06 (100.0) 06 (100.0) 25 (100.0) CF Genotype DF508 homozygous 4 (66.7) 6 (85.7) 3 (50.0) 5 (83.3) 18 (72.0) DF508 heterozygous 2 (33.3) 1 (14.3) 2 (33.3) 1 (16.7) 06 (24.0) G551D heterozygous 0 (0.0)0 0 (0.0)0 1 (16.7) 0 (0.0)0 01 (4.0)0 Baseline PFT (Day 0) FEV1 (% predicted) 71.3 6 11.1 52.4 6 22.1 67.3 6 23.1 73.4 6 23.1 65.6 6 21.1 FVC (% predicted) 83.0 6 9.30 71.5 6 12.9 78.5 6 16.5 86.3 6 16. Login to comment

[hide] Prolonged nonhydrolytic interaction of nucleotide ... J Gen Physiol. 2003 Sep;122(3):333-48. Basso C, Vergani P, Nairn AC, Gadsby DC
Prolonged nonhydrolytic interaction of nucleotide with CFTR's NH2-terminal nucleotide binding domain and its role in channel gating.
J Gen Physiol. 2003 Sep;122(3):333-48., [PMID:12939393]

Abstract [show]
CFTR, the protein defective in cystic fibrosis, functions as a Cl- channel regulated by cAMP-dependent protein kinase (PKA). CFTR is also an ATPase, comprising two nucleotide-binding domains (NBDs) thought to bind and hydrolyze ATP. In hydrolyzable nucleoside triphosphates, PKA-phosphorylated CFTR channels open into bursts, lasting on the order of a second, from closed (interburst) intervals of a second or more. To investigate nucleotide interactions underlying channel gating, we examined photolabeling by [alpha32P]8-N3ATP or [gamma32P]8-N3ATP of intact CFTR channels expressed in HEK293T cells or Xenopus oocytes. We also exploited split CFTR channels to distinguish photolabeling at NBD1 from that at NBD2. To examine simple binding of nucleotide in the absence of hydrolysis and gating reactions, we photolabeled after incubation at 0 degrees C with no washing. Nucleotide interactions under gating conditions were probed by photolabeling after incubation at 30 degrees C, with extensive washing, also at 30 degrees C. Phosphorylation of CFTR by PKA only slightly influenced photolabeling after either protocol. Strikingly, at 30 degrees C nucleotide remained tightly bound at NBD1 for many minutes, in the form of nonhydrolyzed nucleoside triphosphate. As nucleotide-dependent gating of CFTR channels occurred on the time scale of seconds under comparable conditions, this suggests that the nucleotide interactions, including hydrolysis, that time CFTR channel opening and closing occur predominantly at NBD2. Vanadate also appeared to act at NBD2, presumably interrupting its hydrolytic cycle, and markedly delayed termination of channel open bursts. Vanadate somewhat increased the magnitude, but did not alter the rate, of the slow loss of nucleotide tightly bound at NBD1. Kinetic analysis of channel gating in Mg8-N3ATP or MgATP reveals that the rate-limiting step for CFTR channel opening at saturating [nucleotide] follows nucleotide binding to both NBDs. We propose that ATP remains tightly bound or occluded at CFTR's NBD1 for long periods, that binding of ATP at NBD2 leads to channel opening wherupon its hydrolysis prompts channel closing, and that phosphorylation acts like an automobile clutch that engages the NBD events to drive gating of the transmembrane ion pore.

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421 ATP hydrolysis by a CFTR domain: pharmacology and effects of G551D mutation. 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

[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
70 The missense mutation G551D is an example of a class III mutation. 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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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

[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] Comparative pharmacology of the activity of wild-t... J Membr Biol. 2003 Jul 15;194(2):109-17. Derand R, Bulteau-Pignoux L, Becq F
Comparative pharmacology of the activity of wild-type and G551D mutated CFTR chloride channel: effect of the benzimidazolone derivative NS004.
J Membr Biol. 2003 Jul 15;194(2):109-17., 2003-07-15 [PMID:14502435]

Abstract [show]
The pharmacological activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel mutated at glycine 551 (G551D-CFTR) was studied in the presence of the benzimidazolone derivative NS004 and compared to that of wild-type (wt) CFTR. Using iodide ((125)I) efflux and whole-cell patch-clamp techniques we found dose-dependent stimulation of phosphorylated wt-CFTR channels by NS004 with an EC(50) approximately 11 microM. With non-phosphorylated CFTR, the effect of NS004 was apparent only at concentration >100 microM. In G551D-CFTR-expressing CHO cells, neither forskolin (from 0.1 to 10 microM) nor NS004 (from 0.1 to 200 microM) added separately were able to stimulate channel activity. However, in the presence of 10 microM forskolin, NS004 stimulated G551D-CFTR activity in a dose-dependent manner with an EC(50) approximately 1.5 microM. We also determined the half-maximal effective concentration of forskolin ( EC(50) approximately 3.2 microM) required to stimulate G551D channel activity in presence of 1.5 micro M NS004. No inhibitory effect was observed at high concentration of NS004 with both wt- and G551D-CFTR. Whole-cell recordings of CFTR chloride currents from cells expressing wild-type or G551D-CFTR in the presence of NS004 were linear, time- and voltage-independent. The inhibitory profile of G551D-CFTR channel activity was similar to that of wild type, i.e., inhibition by glibenclamide (100 microM) and DPC (250 microM) but not by DIDS (200 microM) nor calixarene (100 nM). These results show that NS004 activates wt-CFTR channel and restores G551D-CFTR channel activity, the potency of which depends on both the concentration of NS004 and the phosphorylation status of CFTR.

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0 Comparative Pharmacology of the Activity of Wild-type and G551D Mutated CFTR Chloride Channel: Effect of the Benzimidazolone Derivative NS004 R. De´ rand, L. Bulteau-Pignoux, F. Becq Laboratoire des Biomembranes et Signalisation Cellulaire, UMR 6558 CNRS, Universite´ de Poitiers, 40 Avenue du Recteur Pineau, 86022 Poitiers, France Received: 2 December 2002/Revised: 10 April 2003 Abstract. Login to comment
1 The pharmacological activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel mutated at glycine 551 (G551D-CFTR) was studied in the presence of the benzimidazolone derivative NS004 and compared to that of wild-type (wt) CFTR. Login to comment
4 In G551D-CFTR-expressing CHO cells, neither forskolin (from 0.1 to 10 lM) nor NS004 (from 0.1 to 200 lM) added separately were able to stimulate channel activity. Login to comment
5 However, in the presence of 10 lM forskolin, NS004 stimulated G551D-CFTR activity in a dose-dependent manner with an EC50 » 1.5 lM. Login to comment
6 We also determined the half-maximal effective concentration of forskolin (EC50 » 3.2 lM) required to stimulate G551D channel activity in presence of 1.5 lM NS004. Login to comment
7 No inhibitory effect was observed at high concentration of NS004 with both wtand G551D-CFTR. Login to comment
8 Whole-cell recordings of CFTR chloride currents from cells expressing wild-type or G551D-CFTR in the presence of NS004 were linear, time-and voltage-independent. Login to comment
9 The inhibitory profile of G551D-CFTR channel activity was similar to that of wild type, i.e., inhibition by glibenclamide (100 lM) and DPC (250 lM) but not by DIDS (200 lM) nor calixarene (100 nM). Login to comment
10 These results show that NS004 activates wt-CFTR channel and restores G551D-CFTR channel activity, the potency of which depends on both the concentration of NS004 and the phosphorylation status of CFTR. Login to comment
11 Key words: Cystic Fibrosis - NS004 - G551D mutant - Pharmacology - Phosphorylation Introduction Cystic fibrosis (CF), the most common lethal autosomal recessive genetic disease among Caucasians, results from mutations of the gene encoding the Cystic Fibrosis Transmembrane conductance Regulator (CFTR), a chloride channel that normally mediates ClÀ transepithelial transport in epithelia (Riordan et al., 1989; Tabcharani et al., 1991; Quinton, 1999). Login to comment
14 The class III mutation glycine-to-aspartic acid at codon 551 (G551D) is located within the NBD1 domain (Cutting et al., 1990). Login to comment
16 With a frequency of 2-5%, depending on the population of origin, G551D is one of the five most frequent CF mutations and is always associated with a severe CF phenotype (Cutting et al., 1990), pulmonary dysfunction and pancreatic insufficiency. Login to comment
17 G551D-mutated protein is fully glycosylated, correctly located at the apical membrane (i.e., normal biosynthesis, trafficking and processing) (Smit et al., 1993; Welsh & Smith, 1993) and normally phosphorylated at the R J. Membrane Biol. 194, 109-117 (2003) DOI: 10.1007/s00232-003-2030-z Correspondence to: F. Becq; email: frederic.becq@univ-poitiers.fr domain by cAMP-dependent protein kinase (Chang et al., 1993). Login to comment
19 G551D mutation confers a decreased nucleotide binding (Logan et al., 1994) and a reduced ATPase activity at NBD1 (Li et al., 1996; Howell, Borchardt & Cohn, 2000). Login to comment
20 The pharmacological modulation of G551D-CFTR chloride-channel activity has not been clearly and systematically characterized and compared to that of wild-type CFTR channel. Login to comment
21 The ability of high doses of IBMX to activate mutated CFTR, including G551D, has, however, been reported (Drumm et al., 1991; Becq et al., 1994) and the phenylimidazothiazoles bromotetramisole and levamisole (Becq et al., 1994; 1996) as well as genistein (Illek et al., 1999) have been shown to activate the G551D-CFTR channel. Login to comment
22 Recently, we demonstrated that benzoquinolizinium derivatives activate wt-, G551D-CFTR chloride channels and modulate the trafficking of delF508-CFTR (Becq et al., 1999, De´ rand et al., 2001, Dormer et al., 2001). Login to comment
23 In this report, we have investigated and compared the effect of the benzimidazolone NS004 on the activation of both wild-type (wt) and G551D-CFTR chloride channels stably expressed in CHO cells, using iodide (125 I) efflux and whole-cell patch-clamp techniques. Login to comment
24 Materials and Methods CELL CULTURE Chinese Hamster Ovary (CHO) cells stably transfected with pNUT vector alone (pNUT CHO) or containing wild-type CFTR (CFTR(+) CHO) or G551D (G551D CHO) mutation were provided by J.R. Riordan and X.-B. Chang, Scottsdale, AZ, USA (Tabcharani et al., 1991; Chang et al., 1993). Login to comment
25 Cells cultured at 37°C in 5% CO2 were maintained in aMEM containing 7% fetal bovine serum, 0.5% antibiotics (50 IU/ml penicillin and 50 lg/ml streptomycin) and 100 lM or 20 lM methotrexate for CFTR(+), G551D and pNUT CHO cells, respectively. Login to comment
94 STIMULATION OF G551D-CFTR-MEDIATED 125 I EFFLUX BY NS004 The pharmacological properties of CFTR chloride channel having the glycine-to-aspartate mutation at codon 551 (G551D) were then studied. Login to comment
96 In G551D-CFTR cells, in the absence of Fsk, the peak rate of 125 I efflux was 0.09 ± 0.01 minÀ1 (n = 14, not shown) and did not differ significantly from experiments in which 10 lM Fsk was added (0.10 ± 0.01 minÀ1 , n = 14, Fig. 3A). Login to comment
97 We then studied the effect of NS004 on G551D-CFTR activity in the presence of 10 lM Fsk. Login to comment
99 In G551D-CFTR cells, 125 I efflux rates were 1.35 ± 0.17 (n = 12), 1.13 ± 0.04 (n = 8), and 3.41 ± 0.15 (n = 28) with 10 lM Fsk, 20 lM NS004 and 10 lM Fsk + 20 lM NS004, respectively. Login to comment
100 Figure 3A also shows that the addition of 20 lM NS004, without Fsk, failed to activate any significant 125 I efflux in G551D-CFTR cells. Login to comment
102 In contrast, in the presence of 10 lM Fsk, the stimulation of G551D-CFTR activity by NS004 increased dose-dependently with an EC50 = 1.47 ± 0.07 lM (Fig. 3B). Login to comment
103 We also searched for the minimal concentration of forskolin required to stimulate G551D in the presence of a low concentration of NS004. Login to comment
104 For this protocol, G551D-CFTR cells were simultaneously exposed to 1.5 lM NS004 and to increasing concentrations of Fsk. Login to comment
106 The half-maximal effective concentration of forskolin needed to stimulate G551D-CFTR with 1.5 lM NS004 was EC50 = 3.15 ± 0.2 lM (n = 4, Fig. 4B). Login to comment
107 These data indicate a synergistic effect of NS004 and forskolin, with the level of stimulation of G551D-CFTR activity being dependent on both NS004 and channel phosphorylation. Login to comment
108 ACTIVATION OF G551D-CFTR CHLORIDE CURRENT BY NS004 Whole-cell patch-clamp experiments were next performed, using G551D-CFTR cells. Login to comment
110 As expected from the iodide efflux experiments, G551D-CFTR cells are not responsive to up to 10 lM Fsk in whole-cell patch-clamp experiments, as previously reported (De´ rand et al., 2001; Illek et al., 1999). Login to comment
114 This lack of responsiveness of G551D-CFTR to cAMP agonists constitutes a hallmark of the mutant and a major difference with wild-type CFTR. Login to comment
115 To determine the effect of NS004 on G551D-CFTR current, cells were first exposed to 10 lM Fsk and then to 20 lM NS004. Login to comment
121 Iodide efflux experiments in G551D-CFTR CHO cells. Login to comment
124 (B) Dose-response relationship of the effect of NS004 on G551D-CFTR in the presence of 10 lM forskolin. Login to comment
129 INHIBITORY PROFILE OF G551D-CFTR CHLORIDE CHANNEL ACTIVITY STIMULATED BY NS004 Several inhibitors were then used to further characterize G551D-CFTR channel activated by NS004. Login to comment
131 G551D-CFTR currents were first activated by Fsk+NS004 (Fig. 5E) and then inhibited in the presence of 100 lM glibenclamide (Figs. Login to comment
136 From these observations, we concluded that the inhibitory profile of G551D-CFTR activated by NS004 did not differ from that of wild-type CFTR, and that G551D mutation did not affect these pharmacological properties of CFTR. Login to comment
137 Discussion The present work reports the characteristics of activation of G551D-CFTR channels by the benzimidazolone derivative NS004 and offers a comparison with wt-CFTR channel activity. Login to comment
139 First, NS004 acts on phosphorylated wt and G551D-CFTR. Login to comment
141 Third, the inhibitory profile of G551D-CFTR channel activity appears similar to that of wt-CFTR. Login to comment
149 Indeed, one major difference concerning the phosphorylation process is that higher concentrations of forskolin are required to achieve stimulation of the G551D-CFTR channel activity by NS004. Login to comment
150 Based on these observations, we could then speculate that NS004 activates G551D- Fig. 4. Login to comment
151 Minimum concentration of forskolin to stimulate G551D-CFTR activity. Login to comment
154 (B) Dose-response relationship of forskolin on G551D-CFTR in the presence of 1.5 lM NS004. Login to comment
160 In this study, we found that NS004 was able to stimulate forskolin-dependent phosphorylated G551D-CFTR activity. Login to comment
161 Interestingly, we also showed that, although up to 10 lM forskolin did not activate G551D-CFTR, only 1 lM was sufficient when 1.5 lM NS004 was also present. Login to comment
162 The maximum level of stimulation of G551D-CFTR was, however, achieved with higher concentrations of both agonists (see Fig. 3). Login to comment
165 The glycine-to-aspartic acid missense mutation at codon 551 (G551D) is a class III mutation located within NBD1, which disrupts activation and regulation of CFTR at the plasma membrane (Cutting et al., 1990). Login to comment
166 The G551D mutated protein is, however, fully glycosylated and correctly located at the apical membrane. Login to comment
167 Importantly, G551D proteins have a decreased nucleotide binding and a reduced ATPase activity at NBD1 (Logan et al., 1994; Li et al., 1996; Howell Fig. 5. Login to comment
168 Electrophysiological characteristics of G551D-CFTR chloride current. Login to comment
169 Whole-cell recordings using G551D-CFTR cells stimulated by 10 lM Forskolin (Fsk) and 20 lM NS004. Login to comment
172 (A-C) Representative currents showing the stimulation of G551D-CFTR chloride currents only with forskolin +NS004. Login to comment
173 Cell capacitance was 17 pF. (D-F) Effect of 100 lM glibenclamide on NS004-activated chloride conductance in G551D-CHO cells. Login to comment
176 Despite this apparent normal property, the channel activity of G551D mutant can not be stimulated by the classical pathway (Gregory et al., 1991). Login to comment
177 The importance of phosphorylation in the activation process of G551D-CFTR channel came from various studies. Login to comment
178 For example, it was found that phosphatase inhibitors activated G551D-CFTR as well as wt-CFTR (Becq et al., 1994). Login to comment
180 Thus, phosphorylation of the G551D-CFTR protein appears to be essential before opening the channel by other means. Login to comment
181 Since the ATPase activity at NBD1 is abnormal for G551D, it can be hypothesized that both the altered ATPase activity and the unresponsiveness to PKA stimulation are linked. Login to comment
190 Genistein has been proposed as an activator of the trafficking-competent G551D mutant after forskolin exposure (Illek et al., 1999; Bulteau-Pignoux et al., 2002; De´ - rand et al., 2002). Login to comment
191 Previous work from our laboratory showed that benzo[c]quinolizinium derivatives (MPB-91 in particular) are also potent activators of G551D-CFTR (Derand et al., 2001). Login to comment
194 Current-voltage relationships of G551D-CFTR chloride currents. Login to comment
200 Effect of calixarene and glibenclamide on NS004-activated chloride conductance in G551D-CHO cells. Login to comment
201 Representative experiments showing the time-dependent activation of G551D-CFTR chloride current in the presence of 10 lM forskolin and 20 lM NS004. Login to comment
209 When compared to wt-CFTR, the inhibitory profile of G551D was not altered, suggesting that the G551D mutation did not interfere with the pharmacological properties of the channel as we previously observed with genistein (Bulteau-Pignoux et al., 2002; De´ rand et al., 2002). Login to comment
213 We provided evidence in this report that NS004 and phosphorylation have a synergistic effect, which allows the stimulation of G551D-CFTR chloride channel activity. Login to comment

[hide] Pharmacologic approaches to correcting the basic d... N Engl J Med. 2003 Oct 9;349(15):1401-4. Lukacs GL, Durie PR
Pharmacologic approaches to correcting the basic defect in cystic fibrosis.
N Engl J Med. 2003 Oct 9;349(15):1401-4., 2003-10-09 [PMID:14534332]

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33 Depending on the location and the nature of the mu- tatedaminoacidresidue,missensemutationshave pleiotropicfunctionalconsequences.Someincrease the disposal and degradation of CFTR at the cell surface or interfere with its constitutive recycling to the cell membrane.Othersselectivelyimpairthe activationofionconductanceofCFTR(e.g.,G551D) attheplasmamembrane.Althoughindividualmis- sense mutations are rare, they collectively constitute 30 to 35 percent of all CFTR mutations. Login to comment
49 For personal use only. No other uses without permission. Copyright (c) 2003 Massachusetts Medical Society. All rights reserved. , 20031404 PERSPECTIVE Finally, high-throughput screening of large libraries of compounds with the use of a cell-based functional assay has recently led to the identifica- tionofsmallmoleculesthatcancorrecttheplasma- membrane channel activity of G551D and ∆F508 CFTR at very low concentrations.2 Although the clinical applications of these exciting studies will require substantial investments of time, work, and money, they demonstrate the potential value of this approach for identifying pharmacologic agents that will correct the cellular phenotype in cystic fibrosis and other diseases that are caused by misfolding. Login to comment

[hide] Long-acting bronchodilators in cystic fibrosis. Curr Opin Pulm Med. 2003 Nov;9(6):504-8. Colombo JL
Long-acting bronchodilators in cystic fibrosis.
Curr Opin Pulm Med. 2003 Nov;9(6):504-8., [PMID:14534403]

Abstract [show]
PURPOSE OF REVIEW: Over 80% of patients with cystic fibrosis (CF) have bronchodilator therapy prescribed, yet bronchodilator use in CF remains controversial. The development of long-acting beta-agonist drugs for clinical use has provided additional rationale for considering bronchodilator therapy in CF. This paper will review recent developments in bronchodilator use in CF patients, with emphasis on the long-acting beta agonists. RECENT FINDINGS: It is reported that 50 to 60% of CF patients demonstrate significant intermittent airway hyperreactivity in response to bronchodilators or challenges. The beta-agonist drugs are the most commonly prescribed bronchodilators. Several mechanisms may be implicated in therapeutic response of CF patients to bronchodilators including direct smooth muscle relaxation, increased mucociliary clearance, direct effects on inflammatory cells and bacterial adherence, and possible direct effects on CF transmembrane conductance regulator (CFTR) function. Several recent studies have shown improved outcomes with long-acting bronchodilators. SUMMARY: In spite of the widespread use of bronchodilators, there are very few long-term studies of their effects in CF patients. However, there are clearly clinical benefits in certain situations. Further research into the most appropriate utilization of these medications to improve outcomes in patients with CF would be helpful.

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110 However, the study by Hordvik et al. [15•] attempted to look at this, and reported a trend in the direction of greater improvement with salmeterol in patients with so-called trafficking-competent mutations, such as G551D [15•]. Login to comment
112 It should be noted, however, that other data suggest that CFTR protein with G551D does not respond to standard concentrations of forskolin or beta-agonists in vitro [19]. Login to comment

[hide] Airway inflammation and infection in congenital bi... Am J Respir Crit Care Med. 2004 Jan 15;169(2):174-9. Epub 2003 Oct 9. Gilljam M, Moltyaner Y, Downey GP, Devlin R, Durie P, Cantin AM, Zielenski J, Tullis DE
Airway inflammation and infection in congenital bilateral absence of the vas deferens.
Am J Respir Crit Care Med. 2004 Jan 15;169(2):174-9. Epub 2003 Oct 9., 2004-01-15 [PMID:14551163]

Abstract [show]
In cystic fibrosis (CF), airway disease begins early in life. Bacteria and elevated levels of neutrophils and inflammatory mediators have been detected in bronchoalveolar lavage (BAL) fluid from infants with CF. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) are common in men with congenital bilateral absence of the vas deferens (CBAVD) and it has been suggested that this syndrome represents a mild form of CF. We hypothesized that men with CBAVD also have subclinical pulmonary disease. Bronchoscopy with BAL, viral and quantitative bacterial cultures, and analyses of total and differential cell count, cytokines, and free neutrophil elastase was performed in eight men with CBAVD, who had mutations in the CFTR and intermediate or elevated sweat chloride levels, and in four healthy control subjects. There was light growth of Staphylococcus aureus in one of eight men with CBAVD, and small numbers of opportunistic gram-negative bacteria in six of eight men with CBAVD and in one control subject. BAL cell counts and neutrophil elastase were within the normal range. Interleukin-8 and tumor necrosis factor-alpha levels were higher for men with CBAVD than for control subjects. These data suggest that mutations in the CFTR in men with CBAVD, in addition to causing infertility, lead to subclinical bacterial pulmonary infection and inflammation consistent with mild CF.

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57 DEMOGRAPHIC DATA FOR MEN WITH CONGENITAL BILATERAL ABSENCE OF THE VAS DEFERENS Sweat Chlorides Nasal PD FEV1 Patient Age (yr) Genotype (mmol/l) (mean/⌬, mV) (%pred)‡ 1 41 ⌬F508/R117H, 7T 50 -34/13 106 2† 31 R117H, 7T/R117H, 7T 31, 40 -31/5 107 3† 36 G551D/R117H, 7T 66, 64 -29/8 118 4† 44 ⌬F508/R117H, 7T 54, 67 -23/6 109 5† 35 ⌬F508/4016insT 74, 88 -46/9 110 6 41 ⌬F508/unidentified, 5T 23, 24 -32/7 118 7 37 ⌬F508/M952T 41, 44 -20/20 122 8† 45 ⌬F508/P67L 47, 59 -36/6 78 Definition of abbreviations: mean/⌬ ϭ mean basal potential difference/change in response to perfusion with a chloride-free solution plus isoproterenol; PD ϭ potential difference. 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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38 The other 21 common mutations are often found in higher frequency in particular ethnic groups, such as the W1282X mutation in Askenazi Jewish populations (16), G551D in French Canadians (17), and 3,120 ϩ 1G → A in African/Mediterranean populations (18). Login to comment
79 Class 3 mutations, such as G551D, reach the cell membrane but the channel is not properly activated. 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] Protein kinase A regulates ATP hydrolysis and dime... Biochem J. 2004 Feb 15;378(Pt 1):151-9. Howell LD, Borchardt R, Kole J, Kaz AM, Randak C, Cohn JA
Protein kinase A regulates ATP hydrolysis and dimerization by a CFTR (cystic fibrosis transmembrane conductance regulator) domain.
Biochem J. 2004 Feb 15;378(Pt 1):151-9., 2004-02-15 [PMID:14602047]

Abstract [show]
Gating of the CFTR Cl- channel is associated with ATP hydrolysis at the nucleotide-binding domains (NBD1, NBD2) and requires PKA (protein kinase A) phosphorylation of the R domain. The manner in which the NBD1, NBD2 and R domains of CFTR (cystic fibrosis transmembrane conductance regulator) interact to achieve a properly regulated ion channel is largely unknown. In this study we used bacterially expressed recombinant proteins to examine interactions between these soluble domains of CFTR in vitro. PKA phosphorylated a fusion protein containing NBD1 and R (NBD1-R-GST) on CFTR residues Ser-660, Ser-700, Ser-712, Ser-737, Ser-768, Ser-795 and Ser-813. Phosphorylation of these serine residues regulated ATP hydrolysis by NBD1-R-GST by increasing the apparent K(m) for ATP (from 70 to 250 microM) and the Hill coefficient (from 1 to 1.7) without changing the V(max). When fusion proteins were photolabelled with 8-azido-[alpha-32P]ATP, PKA phosphorylation increased the apparent k(d) for nucleotide binding and it caused binding to become co-operative. PKA phosphorylation also resulted in dimerization of NBD1-R-GST but not of R-GST, a related fusion protein lacking the NBD1 domain. Finally, an MBP (maltose-binding protein) fusion protein containing the NBD2 domain (NBD2-MBP) associated with and regulated the ATPase activity of PKA-phosphorylated NBD1-R-GST. Thus when the R domain in NBD1-R-GST is phosphorylated by PKA, ATP binding and hydrolysis becomes co-operative and NBD dimerization occurs. These findings suggest that during the activation of native CFTR, phosphorylation of the R domain by PKA can control the ability of the NBD1 domain to hydrolyse ATP and to interact with other NBD domains.

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173 First, kinetic studies showed that the G551D mutation changed the Km but not the Vmax of the NBD1-R-GST preparation [14]. Login to comment
175 Thus even though a contaminating ATPase could have led to differences in the total measured ATPase activity of the G551D versus wild-type fusion protein preparations (e.g. if the G551D mutation affected the tendency of contaminants to associate with NBD1-R-GST during its purification), these findings indicate that NBD1-R-GST is the protein responsible for the observed ATP binding and ATP hydrolysis properties of the preparation. Login to comment

[hide] Progress toward generating a ferret model of cysti... Reprod Biol Endocrinol. 2003 Nov 7;1:83. Li Z, Engelhardt JF
Progress toward generating a ferret model of cystic fibrosis by somatic cell nuclear transfer.
Reprod Biol Endocrinol. 2003 Nov 7;1:83., 2003-11-07 [PMID:14613541]

Abstract [show]
Mammalian cloning by nuclear transfer from somatic cells has created new opportunities to generate animal models of genetic diseases in species other than mice. Although genetic mouse models play a critical role in basic and applied research for numerous diseases, often mouse models do not adequately reproduce the human disease phenotype. Cystic fibrosis (CF) is one such disease. Targeted ablation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in mice does not adequately replicate spontaneous bacterial infections observed in the human CF lung. Hence, several laboratories are pursuing alternative animal models of CF in larger species such as the pig, sheep, rabbits, and ferrets. Our laboratory has focused on developing the ferret as a CF animal model. Over the past few years, we have investigated several experimental parameters required for gene targeting and nuclear transfer (NT) cloning in the ferret using somatic cells. In this review, we will discuss our progress and the hurdles to NT cloning and gene-targeting that accompany efforts to generate animal models of genetic diseases in species such as the ferret.

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106 Since chimeraplasts are most efficient at introducing single base-pair alterations, we have evaluated the ability of chimeraplasts to target the G551D mutation to the CFTR gene that requires only a single-base alteration. Login to comment
107 With the ultimate goal of generating ferret fibroblast cell lines heterozygous for the G551D mutation, we first sought to develop targeting and screening methodologies in Hela cells. Login to comment
123 The sequences for the wild-type and mutant ASO primers were as follows: 1) wild-type, GAGT- GGAGGTCAACGAG, and 2) G551D mutant, GAGTGGA- GATCAACGAG. Login to comment
133 ASO screening of 44 single-cell clones generated from chimeraplast-transfected Hela cells resulted in 3 positive clones that strongly hybridized to the G551D mutant oligonucleotide (Figure 4b and 4c). Login to comment
144 Recent reports have suggested that the polarity of the DNA segment in the Chimeraplast targeting of the G551D mutation to the humanCFTR gene in Hela cellsFigure 4 Chimeraplast targeting of the G551D mutation to the human CFTR gene in Hela cells. Login to comment
148 (B, C) Cell lysates prepared with 44 targeted Hela cell clones after G551D chimeric oligonucleotide transfection were amplified by 2 rounds of PCR and analyzed by ASO hybridization against (B) wild-type CFTR and (C) G551D mutant oligonucleotide probes. Login to comment
149 Three of these cell clones (2b, 3c, and 6a) were highly positive for both the wild-type and the G551D genotype, as indicated by the asterisks. Login to comment
150 Positive plasmid cDNA controls for the wild-type (1e) and the G551D (1f) CFTR sequences were also run as standards. 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
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
80 Two other mutations, R553X and G551D, are common worldwide, but are rare in Brazil. 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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60 This group includes G551D and G551S. Login to comment
61 A lead compound has already been identified that is capable of activating the G551D channel to near wild-type levels, offering the greatest hope for pharmacologic correction. 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
117 Genistein, a tyrosine kinase inhibitor, not only stimulates ∆F508 chloride conductance, but is potent at stimulating the G551D mutant [66-70]. Login to comment
206 Fischer rat thyroid cells stably coexpressing the green fluorescent protein and either wild-type, ∆F508 or G551D CFTR were used to screen 60,000 chemically diverse compounds (at 10 µM) for activation of forskolin-mediated chloride efflux [144,145]. Login to comment
416 69. ILLEK B, ZHANG L, LEWIS NC, MOSS RB, DONG JY, FISCHER H: Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein. Am. J. Physiol. 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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20 Excessive cytokine production may reflect a cell stress response caused by mutant, misfolded CFTR accumulation in the endoplasmic reticulum, but cells with the G551D mutation, which express and traffic inactive CFTR to the cell surface, also had enhanced Ca2ϩ signaling and greater NF-␬B activity (17). Login to comment
51 5 25 NTD 67 F 4 25 41 F ⌬F508/G551D 5 26 NTD 57 M 5 27 NTD 48 M 5 28 NTD 62 F 5 29 NTD 44 F 5 30 NTD 48 M 5 31 NTD 55 F 5 32 NTD 26 M 5 33 NTD 49 M 5 Definition of abbreviations: CF ϭ cystic fibrosis; COPD ϭ chronic obstructive pulmonary disease; NTD ϭ nontransplant donor, lung unsuitable because of acute injury, age, etc.; PF ϭ pulmonary fibrosis; TD ϭ excess airway from lung transplant donor; ? Login to comment

[hide] Structure of nucleotide-binding domain 1 of the cy... EMBO J. 2004 Jan 28;23(2):282-93. Epub 2003 Dec 18. Lewis HA, Buchanan SG, Burley SK, Conners K, Dickey M, Dorwart M, Fowler R, Gao X, Guggino WB, Hendrickson WA, Hunt JF, Kearins MC, Lorimer D, Maloney PC, Post KW, Rajashankar KR, Rutter ME, Sauder JM, Shriver S, Thibodeau PH, Thomas PJ, Zhang M, Zhao X, Emtage S
Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator.
EMBO J. 2004 Jan 28;23(2):282-93. Epub 2003 Dec 18., 2004-01-28 [PMID:14685259]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a chloride channel. Nucleotide-binding domain 1 (NBD1), one of two ABC domains in CFTR, also contains sites for the predominant CF-causing mutation and, potentially, for regulatory phosphorylation. We have determined crystal structures for mouse NBD1 in unliganded, ADP- and ATP-bound states, with and without phosphorylation. This NBD1 differs from typical ABC domains in having added regulatory segments, a foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated NBD1 has undetectable ATPase activity and its structure is essentially the same independent of ligand state. Phe508, which is commonly deleted in CF, is exposed at a putative NBD1-transmembrane interface. Our results are consistent with a CFTR mechanism, whereby channel gating occurs through ATP binding in an NBD1-NBD2 nucleotide sandwich that forms upon displacement of NBD1 regulatory segments.

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216 CF mutations in NBD1 The majority of sites of CF-causing missense mutations occur in NBD1, primarily in its a-subdomain, and the locations in the mNBD1 structure of the most common of these (A455E, G480C, I506T, DI507, DF508, S549N, S549R, G551D, A559T, R560T, Y569D, and D648V; Bobadilla et al, 2002) are shown in Figure 3D. Login to comment

[hide] Localization of cystic fibrosis transmembrane cond... J Immunol. 2004 Jan 1;172(1):418-25. Kowalski MP, Pier GB
Localization of cystic fibrosis transmembrane conductance regulator to lipid rafts of epithelial cells is required for Pseudomonas aeruginosa-induced cellular activation.
J Immunol. 2004 Jan 1;172(1):418-25., 2004-01-01 [PMID:14688350]

Abstract [show]
The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein is an epithelial cell receptor for the outer core oligosaccharide of the Pseudomonas aeruginosa LPS. Bacterial binding leads to CFTR-dependent bacterial internalization, initiation of NF-kappaB nuclear translocation, cellular desquamation, and eventual apoptosis of the infected cells, all of which are critical for innate immune resistance to infection with this pathogen. Lack of this reaction in CF patients underlies their hypersusceptibility to chronic P. aeruginosa infection. In this study we tested whether these epithelial cell responses are dependent upon the localization of CFTR to lipid rafts. Confocal microscopy showed that green fluorescent protein-tagged CFTR (GFP-CFTR) and the lipid raft marker ganglioside GM1 colocalized at sites of P. aeruginosa contact and internalization. GFP-CFTR localized to low density Triton X-100-insoluble fractions in lysates of Madin-Darby canine kidney GFP-CFTR cells, and P. aeruginosa infection increased the levels of GFP-CFTR in these fractions as determined by Western blot. Cells expressing GFP-DeltaF508-CFTR did not have rafts with detectable CFTR protein. Extraction of cell surface cholesterol via cyclodextrin treatment of the cells inhibited CFTR entry into rafts. In addition, cyclodextrin treatment of both human and canine epithelial cells inhibited cellular ingestion of P. aeruginosa, NF-kappaB nuclear translocation, and apoptosis. These results indicate that lipid raft localization of CFTR is required for signaling in response to P. aeruginosa infection. Such signaling is needed for the coordination of innate immunity to P. aeruginosa lung infection, a process that is defective in CF.

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243 There are even some CFTR mutant proteins that are expressed on the cell surface, such as the G551D protein (41, 42), but are still associated with a high susceptibility of patients to P. aeruginosa infection. These non-⌬F508 CFTR mutant alleles may not properly respond to P. aeruginosa, as we recently showed that transgenic mice homozygous for the G551D Cftr allele are unable to produce a lung epithelial cell apoptotic reaction in response to P. aeruginosa infection (22). Login to comment
245 That it is CFTR in epithelial cells that is key to resistance to P. aeruginosa lung infection was shown in an elegant experiment by Wainwright and colleagues (43), who expressed wild-type CFTR selectively in either the lung epithelial cells or alveolar macrophages of mice homozygous for the G551D allele of Cftr and found that recovery of resistance to P. aeruginosa infection approaching that of wild-type mice was only achieved in the mice expressing wild-type CFTR in the epithelium. Login to comment

[hide] Association between serum oncofetal antigens CA 19... Acta Paediatr. 2003 Nov;92(11):1267-71. Gronowitz E, Pitkanen S, Kjellmer I, Heikinheimo M, Strandvik B
Association between serum oncofetal antigens CA 19-9 and CA 125 and clinical status in patients with cystic fibrosis.
Acta Paediatr. 2003 Nov;92(11):1267-71., [PMID:14696845]

Abstract [show]
In cystic fibrosis (CF), mucus plugging in the airways and in the gastrointestinal tract leads to severe morbidity and mortality. The mucin-associated antigens CA 19-9 and CA 125 are markers of gastrointestinal malignancy, and CA 19-9 has also been reported in association with pulmonary function in CF. AIM: To test whether these antigens might serve as markers for the severity of pulmonary and gastrointestinal disease in CF. METHODS: In 99 patients, aged 1 to 48 y, serum levels of CA 19-9 and CA 125 were measured by RIA and ELISA and related to clinical data. RESULTS: Patients with severe mutations had significantly increased serum levels of CA 125, indicating an association with a more severe CF phenotype. This was further supported by the association with lung function, chronic pulmonary colonization of Pseudomonas aeruginosa and pancreatic insufficiency. CA 19-9 was also shown to be associated with lung function and Ps. aeruginosa colonization. No gastrointestinal malignancy was found in our patients despite very high values of CA 19-9 in some patients. During a 5-y follow-up, the very high serum levels of CA 19-9 decreased along with improved general condition of the patients. CONCLUSION: Increased serum levels of CA 125 in CF patients were associated with severe cystic fibrosis transmembrane conductance regulator mutations and a severe phenotype. Both antigens were associated with pseudomonas colonization and lung function and CA 125 also with pancreatic insufficiency. The estimates of CA 19-9 are hampered by the influence of the Lewis histo-blood group system on the synthesis of CA 19-9.

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45 The remaining 23 patients had at least one mild (I506L, R117C, S945L, T338I, W301R, 3849 10KBC → T, 1249-5 → G, R117H, R75Q), moderate (G551D, R560T, V603F) or unknown mutation. 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] Nucleotide-binding domains of human cystic fibrosi... Cell Mol Life Sci. 2004 Jan;61(2):230-42. Callebaut I, Eudes R, Mornon JP, Lehn P
Nucleotide-binding domains of human cystic fibrosis transmembrane conductance regulator: detailed sequence analysis and three-dimensional modeling of the heterodimer.
Cell Mol Life Sci. 2004 Jan;61(2):230-42., [PMID:14745501]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) protein is encoded by the gene that is defective in cystic fibrosis, the most common lethal inherited disease among the Caucasian population. CFTR belongs to the ABC transporter superfamily, whose members form macromolecular architectures composed of two membrane-spanning domains and two nucleotide-binding domains (NBDs). The experimental structures of NBDs from several ABC transporters have recently been solved, opening new avenues for understanding the structure/function relationships and the consequences of some disease-causing mutations of CFTR. Based on a detailed sequence/structure analysis, we propose here a three-dimensional model of the human CFTR NBD heterodimer. This model, which is in agreement with recent experimental data, highlights the specific features of the CFTR asymmetric active sites located at the interface between the two NBDs. Moreover, additional CFTR-specific features can be identified at the subunit interface, which may play critical roles in active site interdependence and are uncommon in other NBD dimers.

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250 For example G551D, a class III mutation affecting the Gly residue located within the NBD1 ABC signature and participating in the 'conventional` active site B, should directly impair the ATP-binding properties of that site, a finding which has been demonstrated experimentally [65]. Login to comment

[hide] Synthesis, SAR, crystal structure, and biological ... J Med Chem. 2004 Feb 12;47(4):962-72. Marivingt-Mounir C, Norez C, Derand R, Bulteau-Pignoux L, Nguyen-Huy D, Viossat B, Morgant G, Becq F, Vierfond JM, Mettey Y
Synthesis, SAR, crystal structure, and biological evaluation of benzoquinoliziniums as activators of wild-type and mutant cystic fibrosis transmembrane conductance regulator channels.
J Med Chem. 2004 Feb 12;47(4):962-72., 2004-02-12 [PMID:14761197]

Abstract [show]
Chloride channels play important roles in homeostasis and regulate cell volume, transepithelial transport, and electrical excitability. Despite recent progress made in the genetic and molecular aspect of chloride channels, their pharmacology is still poorly understood. The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated epithelial chloride channel for which mutations cause cystic fibrosis. Here we have synthesized benzo[c]quinolizinium and benzo[f]indolo[2,3-a]quinolizinium salts (MPB) and performed a SAR to identify the structural basis for activation of the CFTR chloride channel. Synthesized compounds were evaluated on wild-type CFTR and on CFTR having the glycine-to-aspartic acid missense mutation at codon 551 (G551D-CFTR), using a robot and cell-based assay. The presence of an hydroxyl group at position 6 of the benzo[c]quinolizinium skeleton associated with a chlorine atom at position 10 or 7 and an alkyl chain at position 5 determined the highest activity. The most potent product is 5-butyl-7-chloro-6-hydroxybenzo[c]quinolizinium chloride (8u, MPB-104). 8u is 100 times more potent than the parent compound 8a (MPB-07).

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5 Synthesized compounds were evaluated on wild-type CFTR and on CFTR having the glycine-to-aspartic acid missense mutation at codon 551 (G551D-CFTR), using a robot and cell-based assay. Login to comment
11 The class III mutation glycine-to-aspartic acid at codon 551 (G551D) is found with a frequency of 2-5% in chromosomal analysis, depending on the population of origin. Login to comment
12 G551D is indeed one of the five most frequent CF mutations being always associated with a severe CF phenotype, pulmonary dysfunction, and pancreatic insufficiency.7 The G551D mutation is located within the first nucleotide binding domain of CFTR.7 Despite the fact that G551D mutated protein is fully glycosylated6,8 and normally phosphorylated at the R domain by cAMP-dependent protein kinases,9 its chloride channel activity cannot be stimulated pharmacologically by cAMP-elevating agents.6,7,10 G551D mutation also confers a decreased nucleotide binding11 and a reduced ATPase activity at NBD1.12,13 Searching for potent and specific small molecules able to modulate normal and mutated CFTR is crucial for both our understanding of the physiological role of CFTR in epithelial cell function and for the development of molecules of therapeutic interest to cure CF. In this regard, the pharmacology of G551D-CFTR chloride channel activity has not been clearly and systematically characterized and compared to that of wild-type CFTR channel. Login to comment
13 Recently, we have demonstrated that the benzo[c]quinolizinium derivatives MPB-07 (8a), MPB-27 (8d), and MPB-91 (8t) are activators of wild-type (wt) CFTR.14,15 Among these, only MPB-9115 but not MPB- 0715,16 was found capable to activate G551D-CFTR chloride channels. Login to comment
21 || Laboratoire de Cristallographie et RMN Biologiques, Universite´ de Paris V. 962 J. Med. Chem. 2004, 47, 962-972 10.1021/jm0308848 CCC: $27.50 (c) 2004 American Chemical Society Published on Web 01/17/2004 salts and generated many derivatives that have been tested on both wtand G551D-CFTR chloride channel activity. Login to comment
39 Functional Analysis of the Effect of Benzo[c]- quinolizinium and Benzo[f]indolo[2,3-a]quinolizinium Salts on the Chloride Channel Activities of Wtand G551D-CFTR. Login to comment
41 Chinese hamster ovary (CHO) cell lines stably expressing wt-CFTR14 and G551D-CFTR15 were used. Login to comment
67 The pharmacological characteristics of CFTR chloride channel having the glycine-to-aspartate mutation at codon 551 (G551D) were then studied. Login to comment
69 G551D-CFTR channels expressed in CHO cells are not responsive to up to 10 µM Fsk, as previously reported.15,21 This lack of responsiveness of G551D-CFTR to cAMP agonists constitutes a hallmark of the mutant and a major difference with wild-type CFTR. Login to comment
70 To determine the effect of compounds 8 on G551D-CFTR activity, cells were first exposed to 10 µM Fsk and then to these derivatives. Login to comment
71 In G551D-CFTR cells, in the absence of Fsk, the peak rate of 125I efflux was 0.09 ( 0.01 min-1 (n ) 14) and did not significantly differ from experiments in which 10 µM Fsk was added (0.10 ( 0.01 min-1, n ) 14, Figure 2B). Login to comment
72 We then studied the effect of compounds 8 on G551D-CFTR activity in the presence of 10 µM Fsk. Login to comment
74 The stimulation of G551D-CFTR activity by 8u increased dose-dependently with an EC50 ) 0.75 ( 0.15 µM (Figure 2B and Table 4). Login to comment
77 For each compound, the respective EC50 was determined for both wt-CFTR and G551D-CFTR channels. Login to comment
78 As can be seen from Table 4, compounds 8a and 8d were considered inactive on G551D-CFTR, because we could not determine the EC50, which must be above 200 µM. Login to comment
79 In contrast, the remaining six compounds 8u, 8v, 8t, 8w, 8s, and 8j were potent activators of both wt-CFTR and G551D-CFTR. Login to comment
97 Half-Maximal Effective Concentration (EC50) Determined for the Eight Most Potent Benzo[c]quinolizinium Derivativesa EC50 (µM) compound wt G551D 8u MPB-104 1.70 ( 0.15 0.75 ( 0.15 8v MPB-97 21.0 ( 1.5 20.0 ( 1.5 8t MPB-91 23.5 ( 1.5 34.0 ( 2 8w MPB-95 26.0 ( 1.5 32.5 ( 1.5 8s MPB-96 54.2 ( 1.2 48.0 ( 1.3 8j MPB-77 70.5 ( 1.5 95.0 ( 1.5 8a MPB-07 141 ( 15 >200 8d MPB-27 146 ( 14 >200 a Data are given as mean ( SEM for n ) 6 for each drug tested for wt-CFTR and G551D-CFTR. Login to comment
98 For the effect of 8a and 8d on G551D-CFTR, the dose-response relationship was not completed and the EC50 was estimated to be above 200 µM, a concentration too high compared to the other congeners. Login to comment
99 Experiments were performed in the presence of 1 µM Fsk (wt-CFTR) or 10 µM Fsk (G551D-CFTR). Login to comment
100 trafficking-competent G551D mutant after forskolin exposure.21,35 In our first report concerning the activity of benzo[c]- quinoliziniums on wt-CFTR chloride channel expressed in CHO cells,14 we studied only four compounds. Login to comment
111 Effect of MPB-104 (8u) on wt-CFTR (A) and G551D-CFTR (B) activity. Login to comment
113 On the right are presented the dose-response relationships for MPB-104 in the presence of 1 µM Fsk (wt-CFTR, A) or 10 µM Fsk (G551D-CFTR, B). Login to comment
115 The half-maximal effective concentration EC50 was 1.70 ( 0.15 µM and 0.75 ( 0.15 µM for wt-CFTR and G551D-CFTR, respectively. Login to comment
132 MPB-104, being the most potent drug of the series, has the same efficacy on both wtand G551D-CFTR. Login to comment
136 These compounds were tested on wild-type CFTR and on G551D-CFTR, a class III CF mutation, using a robot and cell-based assay. Login to comment
240 Chinese hamster ovary (CHO) cells stably transfected with pNUT vector alone (pNUT CHO) or containing wild-type CFTR (CFTR(+) CHO) or G551D (G551D CHO) mutation were provided by J. R. Riordan and X.-B. Chang, Scottsdale, AZ.2,9 Cells cultured at 37 °C in 5% CO2 were maintained in MEM containing 7% fetal bovine serum, 0.5% antibiotics (50 IU/mL penicillin and 50 µg/mL streptomycin) and 100 or 20 µM methotrexate for CFTR(+) and G551D or pNUT CHO cells, respectively. 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
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

[hide] Pharmacogenomics of cystic fibrosis. Mol Interv. 2001 Apr;1(1):54-63. Pollard HB, Eidelman O, Jacobson KA, Srivastava M
Pharmacogenomics of cystic fibrosis.
Mol Interv. 2001 Apr;1(1):54-63., [PMID:14993338]

Abstract [show]
Pharmacogenomics is becoming a frontline instrument of drug discovery, where the drug-dependent patterns of global gene expression are employed as biologically relevant end points. In the case of cystic fibrosis (CF), cells and tissues from CF patients provide the starting points of genomic analysis. The end points for drug discovery are proposed to reside in gene expression patterns of CF cells that have been corrected by gene therapy. A case is made here that successful drug therapy and gene therapy should, hypothetically, converge at a common end point. In response to a virtual tidal wave of genomic data, bioinformatics algorithms are needed to identify those genes that truly reveal drug efficacy. As examples, we describe the hierarchical clustering, GRASP, and GENESAVER algorithms, particularly within a hypothesis-driven context that focuses on data for a CF candidate drug. Pharmacogenomic approaches to CF, and other similar diseases, may eventually give us the opportunity to create drugs that work in a patient- or mutation-specific manner.

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305 Illek, B., Zhang, L., Lewis, N.C., Moss, R.B., Dong, J.Y., and Fisher, H. Defective function of the cystic fibrosis causing missense mutation G551D is recovered by genistein. 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
98 Three additional mutations in three female subjects were identi®ed by two other test methods: two mutations (DF508 and G551D) by the common mutation panel and one mutation (R74W) by CSGE. Login to comment
144 With this Table V. Description of female partners of CAVD patients and genetic results Subject no.a Ancestry Common mutation panel Sequence method CSGE method Interpretation Mutation panel/ CSGE/DNA sequence concordance 1F N.E. Cauc. Het. DF508 * * Mutation N/Ab 2F Asian Negative * Het. R74W Mutation No 3F Asian * Negative * No mutation detected Yes 4F Asian-Indian * Negative * No mutation detected Yes 5F Asian * Negative * No mutation detected Yes 6F N.E. Cauc./S.E.Cauc./ Ashkenazi Het. G551D * * Mutation N/Ab 7F Asian-Indian Negative Negative * No mutation detected Yes 8F Asian * Negative * No mutation detected Yes 9F Asian * Negative * No mutation detected Yes 10F S.E. Cauc./Ashkenazi * Negative * No mutation detected Yes 11F Hispanic * Negative * No mutation detected Yes 12F Asian * Negativec * No mutation detected Yes 13F Hispanic ² ² ² N/A N/A 14F S.E. Cauc./Asian * Het. L997F * Mutation Nod 15F Asian-Indian * Het. I807M * Mutation Nod 16F N.E. Cauc. 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] Activation of airway cl- secretion in human subjec... Am J Respir Cell Mol Biol. 2004 Aug;31(2):140-6. Epub 2004 Mar 23. Hentchel-Franks K, Lozano D, Eubanks-Tarn V, Cobb B, Fan L, Oster R, Sorscher E, Clancy JP
Activation of airway cl- secretion in human subjects by adenosine.
Am J Respir Cell Mol Biol. 2004 Aug;31(2):140-6. Epub 2004 Mar 23., [PMID:15039139]

Abstract [show]
We investigated cystic fibrosis (CF) transmembrane conductance regulator (CFTR) regulation by A2 adenosine (Ado) receptors and beta2 adrenergic receptors in CFTR-corrected CFBE41o- airway cells and human subjects. CFBE41o- cells stimulated with Ado (10 microM), isoproterenol (Iso, 10 microM), or Ado + Iso (10 microM each) elevated cyclic AMP (cAMP) above control conditions (P < 0.001), with the Iso conditions increasing cAMP approximately 10-fold above that produced by Ado alone (P < 0.001). All agonist conditions had similar effects on short circuit current at 10 and 25 microM, with no further currents produced by subsequent stimulation with forskolin (20 microM). CFTR dependence was demonstrated by glybenclamide block of agonist-stimulated currents. Nasal potential difference studies in normal (n = 50) subjects demonstrated that Ado (10 microM) and Ado + Iso (10 microM each) produced more polarization compared with Iso (10 microM Ado increase = 44%, 10 microM Ado + Iso increase = 52%, P < 0.05 for each condition compared with Iso alone). Studies completed in patients with CF (n = 10, "severe" genotypes) confirmed that Ado-stimulated polarization was CFTR-dependent. Together, these results indicate that Ado is a potent Cl- secretagogue in vivo, with relatively small effects on cAMP levels despite strong effects on CFTR-dependent short circuit current and nasal Cl- transport. These findings support growing evidence indicating a role for Ado regulation of CFTR-dependent Cl- secretion in vivo.

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124 Demographic information in normal subjects and in subjects with CF Condition Normal Subjects Subjects with CF Mean age, yr 30.9 22.9 Age range, yr 17-47 15-29 Sex Female 35 Female 6 Male 15 Male 4 Ethnicity African-American 11 African-American 0 Asian 0 Asian 0 White 39 White 10 Genotype (CF) N/A ⌬F508/⌬F508 9 ⌬F508/G551D 1 mean age than the CF group. Login to comment
186 The nature of this effect is unknown, but previous studies from our laboratory have shown that Ado stimulation of A2B ARs can activate surface localized mutant CFTR molecules, including ⌬F508 CFTR after growth at permissive temperatures, G551D CFTR, and R117H CFTR (15, 32). Login to comment

[hide] Syntaxin 8 impairs trafficking of cystic fibrosis ... J Cell Sci. 2004 Apr 15;117(Pt 10):1923-35. Epub 2004 Mar 23. Bilan F, Thoreau V, Nacfer M, Derand R, Norez C, Cantereau A, Garcia M, Becq F, Kitzis A
Syntaxin 8 impairs trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) and inhibits its channel activity.
J Cell Sci. 2004 Apr 15;117(Pt 10):1923-35. Epub 2004 Mar 23., 2004-04-15 [PMID:15039462]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-dependent chloride channel that mediates electrolyte transport across the luminal surface of epithelial cells. In this paper, we describe the CFTR regulation by syntaxin 8, a t-SNARE protein (target soluble N-ethylmaleimide-sensitive factor attachment protein receptor) involved in the SNARE endosomal complex. Syntaxin family members are key molecules implicated in diverse vesicle docking and membrane fusion events. We found that syntaxin 8 physically interacts with CFTR: recombinant syntaxin 8 binds CFTR in vitro and both proteins co-immunoprecipitate in HT29 cells. Syntaxin 8 regulates CFTR-mediated currents in chinese hamster ovary (CHO) cells stably expressing CFTR and syntaxin 8. Iodide efflux and whole-cell patch-clamp experiments on these cells indicate a strong inhibition of CFTR chloride current by syntaxin 8 overexpression. At the cellular level, we observed that syntaxin 8 overexpression disturbs CFTR trafficking. Confocal microscopy shows a dramatic decrease in green fluorescent protein-tagged CFTR plasma membrane staining, when syntaxin 8 is coexpressed in COS-7 cells. Using antibodies against Lamp-1, TfR or Rab11 we determined by immunofluorescence assays that both proteins are mainly accumulated in recycling endosomes. Our results evidence that syntaxin 8 contributes to the regulation of CFTR trafficking and chloride channel activity by the SNARE machinery.

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188 Moreover, we have observed, in COS-7 cells, that two cystic fibrosis widespread mutants, GFP-(F508del)CFTR and GFP- (G551D)CFTR, exhibited a pharmacological response and a trafficking consistent with the physiological data (data not shown). 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
20 The first reaction detects the G551D, R117H, and F508del mutations (87.2% frequency in Ireland, 67.9% 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
53 Melting curve profile of a R117H/G551D compound heterozygote. Login to comment
55 The F3 channel (bottom plot) detects the G551D ARMS products with a peak seen at 55 °C. 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
246 Three of the subjects had mutations of the most common CFTR mutation (DF 508) which is responsible for 67.5% of CFTR mutations in the local population and the other subject had the second most common mutation (G551D, 4.7%). Login to comment
297 Diagnostic value of tests that discriminate between exudative Table 1 CFTR mutations/sweat tests in 100 adults with bronchiectasis Age Sex Allele 1 Allele 2 5T variant Sweat test (chloride levels) 54 F DF 508 2ve +ve 38 mmol/l 70 F DF 508 2ve 2ve 34 mmol/l 72 F DF 508 2ve 2ve 36 mmol/l 69 M G551D 2ve 2ve Not done doi: 10.1136/thx.2003.020263 This work was supported by a grant from the National Health & Medical Research Council of Australia (to PK). Login to comment

[hide] Acidic duodenal pH alters gene expression in the c... Am J Physiol Gastrointest Liver Physiol. 2004 Aug;287(2):G480-90. Epub 2004 Apr 2. Kaur S, Norkina O, Ziemer D, Samuelson LC, De Lisle RC
Acidic duodenal pH alters gene expression in the cystic fibrosis mouse pancreas.
Am J Physiol Gastrointest Liver Physiol. 2004 Aug;287(2):G480-90. Epub 2004 Apr 2., [PMID:15064229]

Abstract [show]
The duodenum is abnormally acidic in cystic fibrosis (CF) due to decreased bicarbonate ion secretion that is dependent on the CF gene product CFTR. In the CFTR null mouse, the acidic duodenum results in increased signaling from the intestine to the exocrine pancreas in an attempt to stimulate pancreatic bicarbonate ion secretion. Excess stimulation is proposed to add to the stress/inflammation of the pancreas in CF. DNA microarray analysis of the CF mouse revealed altered pancreatic gene expression characteristic of stress/inflammation. When the duodenal pH was corrected genetically (crossing CFTR null with gastrin null mice) or pharmacologically (use of the proton pump inhibitor omeprazole), expression levels of genes measured by quantitative RT-PCR were significantly normalized. It is concluded that the acidic duodenal pH in CF contributes to the stress on the exocrine pancreas and that normalizing duodenal pH reduces this stress.

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39 A second mutant CFTR protein, G551D, which traffics correctly but does not have Cl-channel activity, did not induce NF-␬B activation. 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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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] Instability of the insertional mutation in CftrTgH... BMC Genet. 2004 Apr 21;5:6. Charizopoulou N, Jansen S, Dorsch M, Stanke F, Dorin JR, Hedrich HJ, Tummler B
Instability of the insertional mutation in CftrTgH(neoim)Hgu cystic fibrosis mouse model.
BMC Genet. 2004 Apr 21;5:6., 2004-04-21 [PMID:15102331]

Abstract [show]
BACKGROUND: A major boost to the cystic fibrosis disease research was given by the generation of various mouse models using gene targeting in embryonal stem cells. Moreover, the introduction of the same mutation on different inbred strains generating congenic strains facilitated the search for modifier genes. From the original CftrTgH(neoim)Hgu CF mouse model we have generated using strict brother x sister mating two inbred CftrTgH(neoim)Hgu mouse lines (CF/1 and CF/3). Thereafter, the insertional mutation was introgressed from CF/3 into three inbred backgrounds (C57BL/6, BALB/c, DBA/2J) generating congenic animals. In every backcross cycle germline transmission of the insertional mutation was monitored by direct probing the insertion via Southern RFLP. In order to bypass this time consuming procedure we devised an alternative PCR based protocol whereby mouse strains are differentiated at the Cftr locus by Cftr intragenic microsatellite genotypes that are tightly linked to the disrupted locus. RESULTS: Using this method we were able to identify animals carrying the insertional mutation based upon the differential haplotypic backgrounds of the three inbred strains and the mutant CftrTgH(neoim)Hgu at the Cftr locus. Moreover, this method facilitated the identification of the precise vector excision from the disrupted Cftr locus in two out of 57 typed animals. This reversion to wild type status took place without any loss of sequence revealing the instability of insertional mutations during the production of congenic animals. CONCLUSIONS: We present intragenic microsatellite markers as a tool for fast and efficient identification of the introgressed locus of interest in the recipient strain during congenic animal breeding. Moreover, the same genotyping method allowed the identification of a vector excision event, posing questions on the stability of insertional mutations in mice.

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13 These fall broadly into two different categories; those designed to mimic clinical human mutations such as the F508del [2-4], G551D [5] and G480C [6], and those with a disrupted Cftr gene resulting in either no or reduced production of CFTR. Login to comment

[hide] Role of the cystic fibrosis transmembrane conducta... Cell Microbiol. 2004 Jun;6(6):521-33. Darling KE, Dewar A, Evans TJ
Role of the cystic fibrosis transmembrane conductance regulator in internalization of Pseudomonas aeruginosa by polarized respiratory epithelial cells.
Cell Microbiol. 2004 Jun;6(6):521-33., [PMID:15104594]

Abstract [show]
Pseudomonas aeruginosa is an important human pathogen, producing lung infection in individuals with cystic fibrosis (CF), patients who are ventilated and those who are neutropenic. The respiratory epithelium provides the initial barrier to infection. Pseudomonas aeruginosa can enter epithelial cells, although the mechanism of entry and the role of intracellular organisms in its life cycle are unclear. We devised a model of infection of polarized human respiratory epithelial cells with P. aeruginosa and investigated the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in adherence, uptake and IL-8 production by human respiratory epithelial cells. We found that a number of P. aeruginosa strains could invade and replicate within cells derived from a patient with CF. Intracellular bacteria did not produce host cell cytotoxicity over a period of 24 h. When these cells were transfected with wild-type CFTR, uptake of bacteria was significantly reduced and release of IL-8 following infection enhanced. We propose that internalized P. aeruginosa may play an important role in the pathogenesis of infection and that, by allowing greater internalization into epithelial cells, mutant CFTR results in an increased susceptibility of bronchial infection with this microbe.

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40 Moreover, the mutant CFTR allele G551D is expressed normally at the apex of respiratory epithelial cells with the domain described by Pier and co-workers for P. aeruginosa uptake intact. 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] Capsaicin potentiates wild-type and mutant cystic ... Mol Pharmacol. 2004 Jun;65(6):1415-26. Ai T, Bompadre SG, Wang X, Hu S, Li M, Hwang TC
Capsaicin potentiates wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride-channel currents.
Mol Pharmacol. 2004 Jun;65(6):1415-26., [PMID:15155835]

Abstract [show]
To examine the effects of capsaicin on cystic fibrosis transmembrane conductance regulator (CFTR), we recorded wild-type and mutant CFTR chloride-channel currents using patch-clamp methods. The effects of capsaicin were compared with those of genistein, a well-characterized CFTR activator. In whole-cell experiments, capsaicin potentiates cAMP-stimulated wild-type CFTR currents expressed in NIH 3T3 cells or Chinese hamster ovary cells in a dose-dependent manner with a maximal response approximately 60% of that with genistein and an apparent Kd of 48.4 +/- 6.8 microM. In cell-attached recordings, capsaicin alone fails to activate CFTR in cells that show negligible basal CFTR activity, indicating that capsaicin does not stimulate the cAMP cascade. The magnitude of potentiation with capsaicin depends on the channel activity before drug application; the lower the prestimulated Po, the higher the potentiation. Single-channel kinetic analysis shows that capsaicin potentiates CFTR by increasing the opening rate and decreasing the closing rate of the channel. Capsaicin may act as a partial agonist of genistein because the maximally enhanced wild-type CFTR currents with genistein are partially inhibited by capsaicin. Capsaicin increases DeltaR-CFTR, a protein kinase A (PKA)-independent, constitutively active channel, in cell-attached patches. In excised inside-out patches, capsaicin potentiates the PKA-phosphorylated, ATP-dependent CFTR activity. Both capsaicin and genistein potentiate the cAMP-stimulated G551D-CFTR, DeltaF508-CFTR, and 8SA mutant channel currents. The binding site for capsaicin is probably located at the cytoplasmic domain of CFTR, because pipette application of capsaicin fails to potentiate CFTR activity. In conclusion, capsaicin is a partial agonist of genistein in activation of the CFTR chloride channel. Both compounds affect ATP-dependent gating of CFTR.

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9 Both capsaicin and genistein potentiate the cAMP-stimulated G551D-CFTR, ⌬F508-CFTR, and 8SA mutant channel currents. Login to comment
54 The 2.7-kb BspEI-PflMI fragments containing 8SA and G551D from the plasmids described above were exchanged with the corresponding ones in the pcDNA3.1 wild-type CFTR (Powe et al., 2002) to obtain the 8SA and G551D pcDNA3.1, respectively. Login to comment
62 The plasmid pBudCE4.1 split ⌬R-CFTR and pcDNA3.1 constructs containing wild-type, 8SA, or G551D CFTR cDNA were cotransfected with pEGFP-C3 (BD Biosciences Clontech, Palo Alto, CA) encoding green fluorescent protein using SuperFect transfection reagent (QIAGEN, Valencia, CA) according to manufacturer`s protocols. Login to comment
203 Figure 9A shows that 100 ␮M capsaicin increased the cAMP-dependent chloride currents by ϳ6 fold in NIH 3T3 cells transiently expressing G551D-CFTR. Login to comment
205 On average, the enhancement of G551D-CFTR with capsaicin was ϳ50% of that with genistein (n ϭ 6) (Fig. 9B). Login to comment
237 Fifth, both compounds potentiate several different mutant CFTR channels including 8SA, G551D, ⌬F508, and ⌬R-CFTR. Login to comment
279 Potentiation of G551D-CFTR and ⌬F508-CFTR by capsaicin and genistein. Login to comment
280 A, a whole-cell G551D-CFTR activity in the presence of various agonists. B, the I-V relationships of net G551D-CFTR-channel currents in different conditions as indicated in A. C, summary of fold increase in cAMP-stimulated G551D-CFTR activity on different agonists. Error bars represent S.E.M. **, P Ͻ 0.01 versus capsaicin. 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]

Abstract [show]
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
150 112 CF-Affected MA Infants Who Were Screened: Details of CF Newborn Screening Results and Diagnostic Follow-up Sweat Test Result (mEq Cl-/L) CF-Screen Positive CF-Screen Negative Total 2 Mutations 1 Mutation 0 Mutations Positive (Ն60) 62 19 3 2 86 Borderline (Ն30 and Ͻ60) Within expectations for specific CF genotype* 5 3 8¶ Monozygotic twin sweat test positive† 1 1 Negative (Ͻ30) Within expectations for specific CF genotype‡ 4 1 5 Incomplete (not done or QNS) 2 CFTR mutations identified and clinical symptoms§ 6 1 7 2 CFTR mutations identified without clinical symptoms࿣ 5 5 Total 82 25 3 2 112 * ⌬F508/R117H;7T (3), ⌬F508/3849 ϩ 10kb (2), ⌬F508/L206W (1), G551D/R117C (1), and G85E/R117C (1). Login to comment
154 ‡ ⌬F508/D1152H, ⌬F508/R117H (2), G85E/R117H, and G551D/R117H. 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] Risk of pancreatitis with mutation of the cystic f... Am J Gastroenterol. 2004 Jul;99(7):1358-63. Choudari CP, Imperiale TF, Sherman S, Fogel E, Lehman GA
Risk of pancreatitis with mutation of the cystic fibrosis gene.
Am J Gastroenterol. 2004 Jul;99(7):1358-63., [PMID:15233679]

Abstract [show]
BACKGROUND: Between 5% and 15% of patients with recurrent pancreatitis have no identified etiology after routine investigation and advanced endoscopic evaluation. OBJECTIVE: To determine whether mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is a risk factor for idiopathic pancreatitis. METHODS: We compared the frequency of CFTR mutations as measured by DNA probe analysis in a case group of persons with idiopathic pancreatitis and a control group without pancreatitis, all of whom underwent endoscopic retrograde cholangiopancreatography. A separate analysis compared the prevalence of CFTR mutations between the case group and controls with pancreatitis of known etiology. A subgroup comparison was made between cases of pancreas divisum with pancreatitis and controls with pancreas divisum and no pancreatitis. RESULTS: CFTR mutations were present in 19 (19%) of 96 cases and 7 (3.5%) of 198 controls without pancreatitis (odds ratio, OR = 6.7; 95% CI, 2.8-16.3; p < 0.00001). Compared to the controls with a known cause of pancreatitis (N = 78), cases had a higher prevalence of CFTR mutations (19% vs 2.6%, OR = 9.4; CI, 2.1-41.7; p= 0.0005). Among subjects with pancreas divisum, CFTR mutations were present in 8 (22%) of 37 cases compared to 0 (0%) of 20 controls (OR = 11.8; CI, 8.9-14.7; p= 0.02). CONCLUSION: The risk of idiopathic pancreatitis is greater among persons with CFTR mutations as compared to persons without CFTR mutations. Among persons with pancreas divisum, CFTR mutations appear to increase the risk for pancreatitis.

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45 ( F508, G551D, R553X, W1282X, N1303K, R117H, Delta I507, 621+1G- >T, R560T, 1717-1G->A, 711+1G->T, and R1162X; Nichols Institute, Nichols Institute Reference Laboratories, California). 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]

Abstract [show]
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] CFTR involvement in nasal potential differences in... Am J Physiol Lung Cell Mol Physiol. 2004 Nov;287(5):L936-43. Epub 2004 Jul 9. Salinas DB, Pedemonte N, Muanprasat C, Finkbeiner WF, Nielson DW, Verkman AS
CFTR involvement in nasal potential differences in mice and pigs studied using a thiazolidinone CFTR inhibitor.
Am J Physiol Lung Cell Mol Physiol. 2004 Nov;287(5):L936-43. Epub 2004 Jul 9., [PMID:15246976]

Abstract [show]
Nasal potential difference (PD) measurements have been used to demonstrate defective CFTR function in cystic fibrosis (CF) and to evaluate potential CF therapies. We used the selective thiazolidinone CFTR inhibitor CFTR(inh)-172 to define the involvement of CFTR in nasal PD changes in mice and pigs. In normal mice infused intranasally with a physiological saline solution containing amiloride, nasal PD was -4.7 +/- 0.7 mV, hyperpolarizing by 15 +/- 1 mV after a low-Cl- solution, and a further 3.9 +/- 0.5 mV after forskolin. CFTR(inh)-172 produced 1.1 +/- 0.9- and 4.3 +/- 0.7-mV depolarizations when added after low Cl- and forskolin, respectively. Systemically administered CFTR(inh)-172 reduced the forskolin-induced hyperpolarization from 4.7 +/- 0.4 to 0.9 +/- 0.1 mV but did not reduce the low Cl(-)-induced hyperpolarization. Nasal PD was -12 +/- 1 mV in CF mice after amiloride, changing by <0.5 mV after low Cl- or forskolin. In pigs, nasal PD was -14 +/- 3 mV after amiloride, hyperpolarizing by 13 +/- 2 mV after low Cl- and a further 9 +/- 1 mV after forskolin. CFTR(inh)-172 and glibenclamide did not affect nasal PD in pigs. Our results suggest that cAMP-dependent nasal PDs in mice primarily involve CFTR-mediated Cl- conductance, whereas cAMP-independent PDs are produced by a different, but CFTR-dependent, Cl- channel. In pigs, CFTR may not be responsible for Cl- channel-dependent nasal PDs. These results have important implications for interpreting nasal PDs in terms of CFTR function in animal models of CFTR activation and inhibition.

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53 Wild-type mice and CF mice (homozygous ‚F508 or G551D) were studied. Login to comment
55 Heterozygous mice were bred to generate homozygous mutant ‚F508 or G551D mutant mice. Login to comment
150 Inset: PD measurement done on a cystic fibrosis (CF; G551D mutant) mouse. Login to comment
151 Data are representative of studies on 19 wild-type mice (forskolin protocol), 11 wild-type mice (forskolin/genistein protocol), and 9 CF mice (6 G551D, 3 ‚F508). Login to comment
216 However, this conclusion must be reconciled with the observation here and from several laboratories that the low-Cl- induced hyperpolarization is absent in CF mice (CFTR null or ‚F508 or G551D) (4, 14). Login to comment

[hide] Role of Cftr genotype in the response to chronic P... Am J Physiol Lung Cell Mol Physiol. 2004 Nov;287(5):L944-52. Epub 2004 Jul 9. van Heeckeren AM, Schluchter MD, Drumm ML, Davis PB
Role of Cftr genotype in the response to chronic Pseudomonas aeruginosa lung infection in mice.
Am J Physiol Lung Cell Mol Physiol. 2004 Nov;287(5):L944-52. Epub 2004 Jul 9., [PMID:15246977]

Abstract [show]
Patients with cystic fibrosis have a lesion in the cystic fibrosis transmembrane conductance regulator gene (CFTR), which is associated with abnormal regulation of other ion channels, abnormal glycosylation of secreted and cell surface molecules, and vulnerability to bacterial infection and inflammation in the lung usually leading to the death of these patients. The exact mechanism(s) by which mutation in CFTR leads to lung infection and inflammation is not clear. Mice bearing different mutations in the murine homolog to CFTR (Cftr) (R117H, S489X, Y122X, and DeltaF508, all backcrossed to the C57BL/6J background) were compared with respect to growth and in their ability to respond to lung infection elicited with Pseudomonas aeruginosa-laden agarose beads. Body weights of mice bearing mutations in Cftr were significantly smaller than wild-type mice at most ages. The inflammatory responses to P. aeruginosa-laden agarose beads were comparable in mice of all four Cftr mutant genotypes with respect to absolute and relative cell counts in bronchoalveolar lavage fluid, and cytokine levels (TNF-alpha, IL-1beta, IL-6, macrophage inflammatory protein-2, and keratinocyte chemoattractant) and eicosanoid levels (PGE2 and LTB4) in epithelial lining fluid: the few small differences observed occurred only between cystic fibrosis mice bearing the S489X mutation and those bearing the knockout mutation Y122X. Thus we cannot implicate either misprocessing of CFTR or failure of CFTR to reach the plasma membrane in the genesis of the excess inflammatory response of CF mice. Therefore, it appears that any functional defect in CFTR produces comparable inflammatory responses to lung infections with P. aeruginosa.

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203 This has been demonstrated for cystic fibrosis mice bearing the S489X mutation both backcrossed to the C57BL/6 background (9) and on a mixed genetic background of 129P2 and C57BL/6 (10) and for cystic fibrosis mice bearing the G551D mice on a mixed genetic background of CD-1 and 129/Sv (17), in three different laboratories, each with one of two mucoid clinical strains of P. aeruginosa. Login to comment
236 These data are concordant with those of McMorran et al. (17), who studied mice bearing the G551D allele, which reaches the plasma membrane but fails to be activated to open the channel. Login to comment
248 However, together with clinical observations that patients with severe mutations all seem to have similar lung disease, whether the mutations are stop codons, processing mutants, or activation mutants that reach the membrane, such as G551D, our data suggest that lack of function of CFTR, and not the absence of CFTR at the membrane or the presence of misprocessed CFTR in the cells, is the predominant factor that accounts for the excess airway inflammation. Login to comment
200 This has been demonstrated for cystic fibrosis mice bearing the S489X mutation both backcrossed to the C57BL/6 background (9) and on a mixed genetic background of 129P2 and C57BL/6 (10) and for cystic fibrosis mice bearing the G551D mice on a mixed genetic background of CD-1 and 129/Sv (17), in three different laboratories, each with one of two mucoid clinical strains of P. aeruginosa. Login to comment
233 These data are concordant with those of McMorran et al. (17), who studied mice bearing the G551D allele, which reaches the plasma membrane but fails to be activated to open the channel. Login to comment
245 However, together with clinical observations that patients with severe mutations all seem to have similar lung disease, whether the mutations are stop codons, processing mutants, or activation mutants that reach the membrane, such as G551D, our data suggest that lack of function of CFTR, and not the absence of CFTR at the membrane or the presence of misprocessed CFTR in the cells, is the predominant factor that accounts for the excess airway inflammation. 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]

Abstract [show]
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] 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] Preconception and prenatal cystic fibrosis carrier... Genet Med. 2004 May-Jun;6(3):141-4. Monaghan KG, Bluhm D, Phillips M, Feldman GL
Preconception and prenatal cystic fibrosis carrier screening of African Americans reveals unanticipated frequencies for specific mutations.
Genet Med. 2004 May-Jun;6(3):141-4., [PMID:15354332]

Abstract [show]
PURPOSE: It is recommended that cystic fibrosis (CF) carrier screening be made available to African Americans who are either pregnant or planning a pregnancy. We analyzed the carrier and mutant allele frequencies for African Americans undergoing CF carrier screening in our laboratories. METHODS: Between December 2001 and September 2003, we performed carrier screening for 2189 African Americans, testing for at least the 25 recommended mutations. RESULTS: A total of 33 CF carriers were identified. The most common mutations detected were deltaF508, G622D, R117H/7T, and G551D. The G622D allele frequency among African Americans was 0.18%. We did not detect any 3120 + 1G --> A carriers, although 4 were expected (P < 0.05). CONCLUSIONS: When considering only the 25 recommended CF mutations, 1 in 75 African Americans screened in our laboratories were carriers (within the expected range, given a 69% mutation detection rate). The addition of 2 mutations, G622D and Q98R (incidentally identified while screening for ACOG/ACMG mutations), increased the observed carrier frequency to 1 in 66, which is not significantly different from the known African American carrier frequency of 1 in 65. The frequencies of several specific mutations detected were unanticipated, as was the absence of 3120 + 1G --> A carriers. Further studies on African American patients with classic CF are needed to examine the incidence of CF mutations that are not part of the current panel, such as G622D.

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41 The next most common mutations detected were G622D (19%), R117H/7T (12%), which was increased compared to previous estimates of this mutant allele frequency (P Ͻ 0.001), and G551D (6%). Login to comment
50 ⌬F508 was the most common CF mutation identified followed by G622D, R117H/7T, and G551D. Login to comment
51 R117H has been previously reported at an increased frequency among individuals undergoing carrier screening compared to those with a diagnosis of cystic fibrosis.8 An unexpected result was the lack of 3120ϩ1G3A carriers, although 4 were expected given that this mutation accounts for Ϸ12% of the CF muta- Table 1 Summary of carrier screening results using various methods employed between December 2001 and September 2003 OLA v2.0, heteroduplex analysis (exons 4 and 13) and RFLP analysis (3120ϩ1G3A) OLA v3.0 INNO-LiPA Total screened 818 1274 97 No. of carriers identified 16 14 3 Observed carrier frequency 1/51 1/81 Mutations identified ⌬F508 (6), G622D (3), R117H/7T (3), I148T (3199del6 negative), Q98R, 1898ϩ1G3A, and G551Da ⌬F508 (14), R117H/7T, R553X, and G551D a In addition, 2 persons were positive for F693L (TTG) and 1 was positive for P140S (C3T at 550); both are variants of unknown clinical significance. Login to comment
80 However, laboratories, which receive a large proportion of specimens from individuals with a lower Table 2 Summary of ACOG/ACMG CF mutations identified among 2189 African Americans (4378 chromosomes) undergoing carrier screening Mutation Carriers identified Mutation frequency (%) (this study) Published mutation frequency (%)6,7 ⌬F508 20/33 61 29a -48 R117H/7T 4/33 12 1 G551D 2/33 6 1 I148T (3199del6 negative) 1/33 3 0 1898ϩ1G3A 1/33 3 1 R553X 1/33 3 0.5 3120ϩ1G3A 0 0 12-14 a The lower frequency of ⌬F508 reported by Heim et al. was most likely due to ascertainment bias. Login to comment

[hide] Interleukin 8 secretion from monocytes of subjects... Clin Diagn Lab Immunol. 2004 Sep;11(5):819-24. Zaman MM, Gelrud A, Junaidi O, Regan MM, Warny M, Shea JC, Kelly C, O'Sullivan BP, Freedman SD
Interleukin 8 secretion from monocytes of subjects heterozygous for the deltaF508 cystic fibrosis transmembrane conductance regulator gene mutation is altered.
Clin Diagn Lab Immunol. 2004 Sep;11(5):819-24., [PMID:15358638]

Abstract [show]
Patients with cystic fibrosis (CF) exhibit an excessive host inflammatory response. The aim of this study was to determine (i) whether interleukin 8 (IL-8) secretion is increased from monocytes from subjects heterozygous as well as homozygous for cystic fibrosis transmembrane conductance regulator (CFTR) mutations and (ii) whether this is due to increased cell surface lipopolysaccharide (LPS) receptors or, alternatively, increased activation of mitogen-activated protein kinases (MAPK). The basal level of IL-8 secretion was higher from monocytes from CF patients than from monocytes from healthy controls (P = 0.02) and obligate heterozygotes (parents of the CF patients). The 50% effective concentrations for LPS-induced IL-8 production for monocytes from both CF patients and obligate heterozygotes were 100-fold lower than those for monocytes from healthy controls (P < 0.05). No differences in the levels of IL-1beta production were seen between these groups. Expression of the LPS surface receptors CD14 and Toll-like receptor 4 were not different between CF patients and healthy controls. In contrast, phosphorylation of the MAPKs p38 and ERK occurred at lower doses of LPS in monocytes from patients heterozygous and homozygous for CFTR mutations. These results indicate that a single allelic CFTR mutation is sufficient to augment IL-8 secretion in response to LPS. This is not a result of increased LPS receptor expression but, rather, is associated with alterations in MAPK signaling.

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25 A similar hypersensitivity to LPS-induced induction of TNF-␣ mRNA has been shown in cultured bone marrow-derived macrophages from G551D CF mice (22). Login to comment

[hide] Identification of novel and rare mutations in Cali... Hum Mutat. 2004 Oct;24(4):353. Alper OM, Wong LJ, Young S, Pearl M, Graham S, Sherwin J, Nussbaum E, Nielson D, Platzker A, Davies Z, Lieberthal A, Chin T, Shay G, Hardy K, Kharrazi M
Identification of novel and rare mutations in California Hispanic and African American cystic fibrosis patients.
Hum Mutat. 2004 Oct;24(4):353., [PMID:15365999]

Abstract [show]
In ethnic heterogeneous California, complete genetic information is currently lacking to build solid population-based cystic fibrosis (CF) screening programs because a large proportion of mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR/ABCC7) are still unknown, especially in non-Caucasian patients. A total of 402 [46 African American+356 Hispanic] Hispanic and African American patients from California CF patient registry were included in this study. Patients with at least one unidentified mutant allele were asked to donate blood samples for further analysis, first by Genzyme Genetics for a panel of 87 known mutations, followed by temporal temperature gradient gel electrophoresis (TTGE) scanning of the entire coding exons of CFTR gene. A total of eight novel mutations; one missense mutation, one splice-site mutation and six frame-shift mutations were identified. In addition to the eight novel mutations, 20 [corrected] distinct rare mutations that are not in the current available commercial mutation panels were identified by TTGE. The overall detection rate was raised to 95.7% for African American and 94.5% for Hispanic. The discovery of recurrent rare and novel mutations improves the diagnosis and care of persons with CF and improves our ability to adequately and equitably provide screening and genetic counseling services to non-Caucasians.

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14 In a worldwide study of more than 43,000 CF chromosomes, 1652_1654del (c.1520_1522del, p.Phe508del) accounted for 66% of the mutations, while the next four most common mutations 1756G>T(c.1624G>T, p. Gly542Ter), 4041C>G(c.3909C>G, p.Asn1303Lys), 1784G>A(c.1652G>A, p. Gly551Asp), 3978G>A(c.3846G>A, p.Trp1282Ter) had relative frequencies of 1.2-2.4 %. 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
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

[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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54 Among the over 2,300 AJ screenees tested, one screenee was identified who carried R117H, one carried G551D and two carried I148T. Login to comment
62 The additional mutations that were detected were R117H (n ϭ 7), I148T (6), A455E (2), R334W (2), G551D (1), and R553X (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
94 When MSSM and DY testing laboratories introduced expanded screening panels, several other CF mutations were found among AJ screenees; these were R117H, G551D, I148T, and 1898ϩ1GϾA. Login to comment
95 Of interest, R117H and G551D represented 0.8 and 2.5% of alleles in the non-Hispanic Caucasian CF patients, respectively,8 but were rare in the AJ population (each seen only once in over 2,300 screenees). 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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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

[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
85 G551D and R334W required a heterozygous allelic ratio with an upper limit set at 3.00. Login to comment
130 A G551D/R553X compound heterozygote was reported as G551D homozygote, R553X heterozygote. Login to comment
135 allele was not able to elongate from the G551 normal oligonucleotide, but the G551D mutant allele was able to elongate from the R553 normal oligonucleotide. Login to comment
137 There is no mismatch between the G551D mutant allele and the R553 normal oligonucleotide because the mutation is outside the region of complementarity. Login to comment
140 However, the reported genotype is unlikely and the sample would have been reflexed to another method for the analysis of the G551D and R553X mutations. Login to comment
162 j Proficiency sample ϩs: delF508/G551D, R117H/delF508, R553X, delF508/delF508, 621ϩ1GϾT/delF508, delI507, delF508/3659delC, delF508/G551D k Control samples were not included in calculation of failure rates. Login to comment
163 One control sample (G551D/R553X) failed in this study and is discussed in the text. Login to comment
175 The problem encountered with the G551D/R553X control sample was the result of the R553X mutant strand failing to elongate from the G551 normal probe due to reduced annealing efficiency. 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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190 Within this class, genistein and forskolin were able to increase chloride conductance of CFTR in G551D HeLa cells but not homozygous F508del cells. 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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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

[hide] CFTR: more than just a chloride channel. Pediatr Pulmonol. 2005 Apr;39(4):292-8. Mehta A
CFTR: more than just a chloride channel.
Pediatr Pulmonol. 2005 Apr;39(4):292-8., [PMID:15573386]

Abstract [show]
This review examines the cystic fibrosis transmembrane conductance regulator (CFTR) protein. After summarizing the ion channels regulated by CFTR, the review focuses on the functions of CFTR that do not relate directly to a disease mechanism based on a channelopathy. The key concept is that newly synthesized CFTR has to enter lipid vesicles which bud from the endoplasmic reticulum. This is abnormally low in DeltaF508 CFTR. Normal wild type vesicular CFTR enters a recycling pool of lipid vesicles which transiently dock with the apical membrane only for CFTR to be retrieved shortly after into a sub-apical recycling compartment. This retrieval is abnormally fast in DeltaF508 CFTR. The review discusses the relationship between this process and the difficult topic of fat metabolism and then explores the possible links between abnormal fatty acid turnover and inflammatory cascades that are abnormal in cystic fibrosis. Finally the review concentrates on the emerging functions of a protein kinase (AMP-activated kinase) which is bound near the C terminus of the CFTR protein whose functions could intergrate some of the abnormalities in lipid metabolism that result from mislocalization of CFTR in clinical disease.

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53 This number is different for unstimulated wild-type CFTR, stimulated wild-type CFTR, and the mutants DF508/G551D CFTR (see below). 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] Sildenafil (Viagra) corrects DeltaF508-CFTR locati... Thorax. 2005 Jan;60(1):55-9. Dormer RL, Harris CM, Clark Z, Pereira MM, Doull IJ, Norez C, Becq F, McPherson MA
Sildenafil (Viagra) corrects DeltaF508-CFTR location in nasal epithelial cells from patients with cystic fibrosis.
Thorax. 2005 Jan;60(1):55-9., [PMID:15618584]

Abstract [show]
BACKGROUND: Most patients with cystic fibrosis (CF) have a DeltaF508 mutation resulting in abnormal retention of mutant gene protein (DeltaF508-CFTR) within the cell. This study was undertaken to investigate DeltaF508-CFTR trafficking in native cells from patients with CF with the aim of discovering pharmacological agents that can move DeltaF508-CFTR to its correct location in the apical cell membrane. METHOD: Nasal epithelial cells were obtained by brushing from individuals with CF. CFTR location was determined using immunofluorescence and confocal imaging in untreated cells and cells treated with sildenafil. The effect of sildenafil treatment on CFTR chloride transport function was measured in CF15 cells using an iodide efflux assay. RESULTS: In most untreated CF cells DeltaF508-CFTR was mislocalised within the cell at a site close to the nucleus. Exposure of cells to sildenafil (2 hours at 37 degrees C) resulted in recruitment of DeltaF508-CFTR to the apical membrane and the appearance of chloride transport activity. Sildenafil also increased DeltaF508-CFTR trafficking in cells from individuals with CF with a single copy DeltaF508 (DeltaF508/4016ins) or with a newly described CF trafficking mutation (R1283M). CONCLUSIONS: The findings provide proof of principle for sildenafil as a DeltaF508-CFTR trafficking drug and give encouragement for future testing of sildenafil and related PDE5 inhibitors in patients with CF.

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213 This is in agreement with our previous data on the location of CFTR in wild type, DF508/DF508 and DF508/ G551D cells.8 Actions of sildenafil on DF508-CFTR location In DF508/DF508 cells treated with sildenafil (150 mM for 2 h at 37˚C), a marked change in DF508-CFTR location was observed (fig 1C and F) with a significant decrease (p,0.05) in the percentage of cells having a near nuclear location and a significant increase (from 10% to 32%, p,0.05) in cells having DF508-CFTR at the apical membrane. 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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3 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. Login to comment
81 Several mutations frequent in northern and central Europe, i.e., 621+1G>T, I507del, G551D, were not detected in our population and are also rare in other Mediterranean regions (Rendine et al. 1997; Casals et al. Login to comment
96 For example, the study promoted by the European Concerted Action for Quality Control on CF that involved 136 European laboratories (Dequeker & Cassiman, 1998, 2000) included samples bearing mutations G551D and 621+1G>T, which are virtually absent in southern Europe and Italy (Rendine et al. 1997). 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] Reversible silencing of CFTR chloride channels by ... J Gen Physiol. 2005 Feb;125(2):127-41. Epub 2005 Jan 18. Wang W, Oliva C, Li G, Holmgren A, Lillig CH, Kirk KL
Reversible silencing of CFTR chloride channels by glutathionylation.
J Gen Physiol. 2005 Feb;125(2):127-41. Epub 2005 Jan 18., [PMID:15657297]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation- and ATP-dependent chloride channel that modulates salt and water transport across lung and gut epithelia. The relationship between CFTR and oxidized forms of glutathione is of potential interest because reactive glutathione species are produced in inflamed epithelia where they may be modulators or substrates of CFTR. Here we show that CFTR channel activity in excised membrane patches is markedly inhibited by several oxidized forms of glutathione (i.e., GSSG, GSNO, and glutathione treated with diamide, a strong thiol oxidizer). Three lines of evidence indicate that the likely mechanism for this inhibitory effect is glutathionylation of a CFTR cysteine (i.e., formation of a mixed disulfide with glutathione): (a) channels could be protected from inhibition by pretreating the patch with NEM (a thiol alkylating agent) or by lowering the bath pH; (b) inhibited channels could be rescued by reducing agents (e.g., DTT) or by purified glutaredoxins (Grxs; thiol disulfide oxidoreductases) including a mutant Grx that specifically reduces mixed disulfides between glutathione and cysteines within proteins; and (c) reversible glutathionylation of CFTR polypeptides in microsomes could be detected biochemically under the same conditions. At the single channel level, the primary effect of reactive glutathione species was to markedly inhibit the opening rates of individual CFTR channels. CFTR channel inhibition was not obviously dependent on phosphorylation state but was markedly slowed when channels were first "locked open" by a poorly hydrolyzable ATP analogue (AMP-PNP). Consistent with the latter finding, we show that the major site of inhibition is cys-1344, a poorly conserved cysteine that lies proximal to the signature sequence in the second nucleotide binding domain (NBD2) of human CFTR. This region is predicted to participate in ATP-dependent channel opening and to be occluded in the nucleotide-bound state of the channel based on structural comparisons to related ATP binding cassette transporters. Our results demonstrate that human CFTR channels are reversibly inhibited by reactive glutathione species, and support an important role of the region proximal to the NBD2 signature sequence in ATP-dependent channel opening.

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253 For example, disease-associated mutations within the NBD1 signature sequence (e.g., G551D) dramatically inhibit the rate of CFTR channel opening (Li et al., 1996). Login to comment

[hide] Pathophysiologic consequences following inhibition... BMC Dev Biol. 2005 Feb 4;5:2. Cohen JC, Larson JE
Pathophysiologic consequences following inhibition of a CFTR-dependent developmental cascade in the lung.
BMC Dev Biol. 2005 Feb 4;5:2., [PMID:15694001]

Abstract [show]
BACKGROUND: Examination of late gestation developmental genes in vivo may be limited by early embryonic lethality and compensatory mechanisms. This problem is particularly apparent in evaluating the developmental role of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in the cystic fibrosis (CF) phenotype. A previously described transient in utero knockout (TIUKO) technology was used to address the developmental role of CFTR in the rat lung. RESULTS: Rat fetuses transiently treated with antisense cftr in utero developed pathology that replicated aspects of the human CF phenotype. The TIUKO CF rat developed lung fibrosis, chronic inflammation, reactive airway disease, and the CF Antigen (MRP8/14), a marker for CF in human patients, was expressed. CONCLUSIONS: The transient in utero antisense technology can be used to evaluate genes that exhibit either early lethality or compensating gene phenotypes. In the lung CFTR is part of a developmental cascade for normal secretory cell differentiation. Absence of CFTR results in a constitutive inflammatory process that is involved in some aspects of CF pathophysiology.

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202 Airway thickness was determined Table 2: Comparison of cystic fibrosis disease phenotypes between human and animal models HUMAN DISEASE PHENOTYPE CFTR KNOCKOUT MOUSE MODEL PHENOTYPE TIUKO CF RAT PHENOTYPE LUNG FIBROSIS FIBROSIS AND INFLAMMATION PRESENT BY 100 DAYS OF AGE MRP8/14 ELEVATED DETECTED IN G551D MOUSE INCREASED PRENATALLY AIRWAY REACTIVITY NOT DETECTED INCREASED WITH AGE CHRONIC INFLAMMATION DETECTED IN CONGENIC MICE PRESENT BY 100 DAYS OF AGE following staining with hematoxylin and eosin in 100 day old rats. 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] Binding site of activators of the cystic fibrosis ... Cell Mol Life Sci. 2005 Feb;62(4):446-60. Moran O, Galietta LJ, Zegarra-Moran O
Binding site of activators of the cystic fibrosis transmembrane conductance regulator in the nucleotide binding domains.
Cell Mol Life Sci. 2005 Feb;62(4):446-60., [PMID:15719171]

Abstract [show]
The use of substances that could activate the defective chloride channels of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) has been suggested as possible therapy for cystic fibrosis. Using epithelia formed by cells stably transfected with wildtype or mutant (G551D, G1349D) CFTR, we estimated the apparent dissociation constant, K(D), of a series of CFTR activators by measuring the increase in the apical membrane current. Modification of apparent K(D) of CFTR activators by mutations of the nucleotide-binding domains (NBDs) suggests that the binding site might be in these regions. The human NBD structure was predicted by homology with murine NBD1. An NBD1-NBD2 complex was constructed by overlying monomers to a bacterial ABC transporter NBD dimer in the "head-to-tail" conformation. Binding sites for CFTR activators were predicted by molecular docking. Comparison of theoretical binding free energy estimated in the model to free energy estimated from the apparent dissociation constants, K(D), resulted in a remarkably good correlation coefficient for one of the putative binding sites, located in the interface between NBD1 and NBD2.

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2 Using epithelia formed by cells stably transfected with wild-type or mutant (G551D, G1349D) CFTR, we estimated the apparent dissociation constant, KD, of a series of CFTR activators by measuring the increase in the apical membrane current. Login to comment
32 Materials and methods Cell cultures Fisher rat thyroid (FRT) cells expressing WT, G551D or G1349D CFTR were cultured on 60-mm petri dishes with Coon`s modified F12 containing 5% fetal bovine serum, 2 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin and 600 µg/ml zeocin, as previously described [18]. Login to comment
63 Compound WT G551D G1349D Apigenin(a) 3. Login to comment
64 1 ± 0.6 (4) 19.6 ± 6.1 (3) - Genistein(a) 19.6 ± 2.5 (15) 114.2 ± 12 (3) 34.38 ± 2.21 (3) UCCF-023(b) 0.5 ± 0.2 (3) - - UCCF-029(b) 1.5 ± 0.5 (4) 31.3 ± 3.5 (3) 11.7 ± 3.1 (5) UCCF-030(b) 0.5 ± 0.1 (3) 4.7 ± 1.3 (2) - UCCF-853(b) 1.17 (1) - - C02(b) 3.2 ± 0.3 (2) - - C03(b) 1.2 (1) 7.2 ± 3 (2) - Act01(c) 0.4 ± 0.04 (5) - 0.6 ± 0.2 (3) Act03(c) 0.07 ± 0.02 (2) - - Act04(c) 0.4 ± 0.08 (4) not active up to 20 µM - Act05(c) 0.13 ± 0.01 (4) not active up to 5 µM - Act09(d) 1.95 ± 0.7 (3) not active up to 50 µM - Act11(d) 0.3 ± 0.08 (3) not active up to 20 µM - Act12(d) 1.9 ± 1.3 (3) - - Act13(d) 0.2 ± 0.03 (3) not active up to 20 µM - Act14(d) 0.2 ± 0.04 (2) - - Act17(d) 1.1 ± 0.5 (6) - - Data were obtained from measurements of Isc on FRT cell monolayers expressing WT or mutant (G551D or G1349D) CFTR. Login to comment
84 Results Electrophysiology We compared the effect of different CFTR activators on the WT and on mutations of the signature sequences of CFTR, the NBD1 mutant G551D, and the mirror NBD2 mutant G1349D. Login to comment
90 Our data indicated, as already observed in previous experiments [18], that the genistein dose-response curve on G551D epithelia was shifted to the right with respect to WT protein (fig. 3A, B). Login to comment
92 The dose-response curve for the G1349D mutant was shifted to the right with respect to WT cells, but to a lesser extent than that of G551D (fig. 3B). Login to comment
94 In all cases, the affinities for the G551D mutant were significantly shifted to the right with respect to the WT protein, and the affinities for G1349D were in-between (fig. 3C, D). Login to comment
114 Effect of different CFTR activators on polarized epithelial preparations expressing WT or CFTR mutants (G1349D and G551D). Login to comment
117 Note that the lowest concentration that inhibited CFTR currents was 100 µM for WT and 200 µM for G1349D, while 200 µM still stimulated G551D. Login to comment
118 (B-D) Normalized dose-response relationships of FRT cells expressing WT (closed circles), G1349D (open triangles) and G551D (open squares) CFTR to genistein (B), UCCF-029 (C), and apigenin (D). Login to comment
170 The two mutations studied, G551D and G1349D, are located in the LSGGQ signature of NBD1 and NBD2, respectively. Login to comment
177 Hydrophobic (DGHB) and electrostatic (DGelec) contributions to interaction between NBD1 and NBD2 from WT and the mutants G551D and G1349D. Login to comment
178 WT G551D G1349D NBD1 + NBD2 ∆GHB (kJ/mol) -66.1 -68.4 -69.3 ∆Gelec (kJ/mol) -17.2 -12.5 -13.1 NBD1 + NBD2 + 2 ATP ∆GHB (kJ/mol) -98.4 -98.44 -101.05 ∆Gelec (kJ/mol) -28.9 -23.3 -24.3 Data were calculated from the interaction of NBDs in the absence of ATP, NBD1-NBD2, and from NBDs interacting in the presence of ATP, NBD1-NBD2-2 ATP. Login to comment
180 The hydrophobic contribution to the interaction energy is slightly improved in the mutants (DGHB = -68.4 kJ/mol for G551D, and DGHB = -69.3 kJ/mol for G1349D), but electrostatic terms of energy are significantly increased (DGElec = -12.5 kJ/mol for mutant G551D, and DGElec = -13.1 kJ/mol for mutant G1349D), with a net increase in the interaction energy of 2.9 kJ/mol and 1.4 kJ/mol for G551D and G1349D, respectively (table 2). Login to comment
183 For mutant G551D, there is a reduction in the affinity for ATP in site 2 (DDGbind = 13 kJ/mol), but ATP-binding site 1 is almost unaffected (DDGbind = 1.83 kJ/mol). Login to comment
205 Comparison of the binding free energy differences, DGbind estimated for ATP-binding sites 1 and 2, for WT and the mutants G551D and G1349D of human CFTR. Login to comment
206 WT WT-G551D WT-G1349D DDGbind (kJ/mol) DDGbind (kJ/mol) Site 1 1.83 11.06 Site 2 12.98 3.3 Site1-site 2 -2.78 13.92 -4.99 Changes are expressed as differences in DGbind (DDGbind), calculated at different conditions. Login to comment
234 Similarly, we estimated the KD for some of the compounds for which the affinity for the mutant G551D was too low to be measured experimentally (see table 1). Login to comment
242 Filled symbols represent data obtained docking the CFTR-activator to the WT model, open triangles are from G551D, and open circles are from G1349D. Login to comment
246 Discussion We recently studied the effect of some CFTR activators on cells expressing either the WT or G551D mutant CFTR [18]. Login to comment
247 We found that the genistein dose-response curve in G551D cells was shifted with respect to WT CFTR so that higher concentrations were required to observe activation. Login to comment
248 This result strongly indicated that the G551D mutation is near a binding site for genistein. Login to comment
259 Similar results have already been reported for genistein on the mutation G551D [18] and for genistein and two benzimidazolone analogs with the mutation dF508 [19]. Login to comment
260 Now, we extend these observation to five substances tested on mutant G551D and three tested on mutant G1349D. Login to comment
261 Interestingly, there is a decrease of affinity for every CFTR activator tested on the mutants, the reduction being more marked for mutant G551D than for mutant G1349D (table 1). Login to comment
292 Indeed, comparing the empirical estimations of the binding free energy difference, we found that DDGbind for mutant G551D is consistent with a strong reduction inATP affinity at site 2, where the mutated residue is more directly involved, and a smaller reduction in ATP affinity for site 1. Login to comment
294 These results are in agreement with direct measurements of ATP binding done in NBDs of multidrug resistance protein-1 [31], where mutations G771D and G1433D of LSGGQ signatures, equivalent to G551D and G1349D in CFTR NBDs, produced similar effects on ATP-NBD interactions. Login to comment
371 Acta 217: 23-28 18 Zegarra-Moran O., Romio L., Folli C., Caci E., Becq F., Vierfond J. M. et al. (2002) Correction of G551D-CFTR transport defect in epithelial monolayers by genistein but not by CPX or MPB-07. Login to comment

[hide] Phenylglycine and sulfonamide correctors of defect... Mol Pharmacol. 2005 May;67(5):1797-807. Epub 2005 Feb 18. Pedemonte N, Sonawane ND, Taddei A, Hu J, Zegarra-Moran O, Suen YF, Robins LI, Dicus CW, Willenbring D, Nantz MH, Kurth MJ, Galietta LJ, Verkman AS
Phenylglycine and sulfonamide correctors of defective delta F508 and G551D cystic fibrosis transmembrane conductance regulator chloride-channel gating.
Mol Pharmacol. 2005 May;67(5):1797-807. Epub 2005 Feb 18., [PMID:15722457]

Abstract [show]
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel cause cystic fibrosis. The delta F508 mutation produces defects in channel gating and cellular processing, whereas the G551D mutation produces primarily a gating defect. To identify correctors of gating, 50,000 diverse small molecules were screened at 2.5 microM (with forskolin, 20 microM) by an iodide uptake assay in epithelial cells coexpressing delta F508-CFTR and a fluorescent halide indicator (yellow fluorescent protein-H148Q/I152L) after delta F508-CFTR rescue by 24-h culture at 27 degrees C. Secondary analysis and testing of >1000 structural analogs yielded two novel classes of correctors of defective delta F508-CFTR gating ("potentiators") with nanomolar potency that were active in human delta F508 and G551D cells. The most potent compound of the phenylglycine class, 2-[(2-1H-indol-3-yl-acetyl)-methylamino]-N-(4-isopropylphenyl)-2-phenylace tamide, reversibly activated delta F508-CFTR in the presence of forskolin with K(a) approximately 70 nM and also activated the CFTR gating mutants G551D and G1349D with K(a) values of approximately 1100 and 40 nM, respectively. The most potent sulfonamide, 6-(ethylphenylsulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid cycloheptylamide, had K(a) approximately 20 nM for activation of delta F508-CFTR. In cell-attached patch-clamp experiments, phenylglycine-01 (PG-01) and sulfonamide-01 (SF-01) increased channel open probability >5-fold by the reduction of interburst closed time. An interesting property of these compounds was their ability to act in synergy with cAMP agonists. Microsome metabolism studies and rat pharmacokinetic analysis suggested significantly more rapid metabolism of PG-01 than SF-03. Phenylglycine and sulfonamide compounds may be useful for monotherapy of cystic fibrosis caused by gating mutants and possibly for a subset of delta F508 subjects with significant delta F508-CFTR plasma-membrane expression.

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3 The ⌬F508 mutation produces defects in channel gating and cellular processing, whereas the G551D mutation produces primarily a gating defect. Login to comment
5 Secondary analysis and testing of Ͼ1000 structural analogs yielded two novel classes of correctors of defective ⌬F508-CFTR gating ("potentiators") with nanomolar potency that were active in human ⌬F508 and G551D cells. Login to comment
6 The most potent compound of the phenylglycine class, 2-[(2-1H-indol-3-yl-acetyl)-methylamino]-N-(4-isopro- pylphenyl)-2-phenylacetamide, reversibly activated ⌬F508-CFTR in the presence of forskolin with Ka ϳ 70 nM and also activated the CFTR gating mutants G551D and G1349D with Ka values of ϳ1100 and 40 nM, respectively. Login to comment
28 The most common of the CFTR gating mutants is G551D, with a worldwide frequency of 3.1% among CF chromosomes (Hamosh et al., 1992), although people of Celtic descent have frequencies as high as 8% (Cashman et al., 1995). Login to comment
32 Potentiators may also be useful as monotherapy for CF caused by gating mutants of CFTR such as G551D. Login to comment
35 Flavones at high concentrations also are able to correct defective gating in G551D-CFTR (Illek et al., 1999; Zegarra-Moran et al., 2002). Login to comment
38 However, activation required high concentrations of cAMP agonists, and the benzothiophenes did not activate CFTR gating mutants such as G551D. Login to comment
41 These compounds were potent in ⌬F508-CFTR-transfected and natively expressing human cells, active in the presence of relatively low concentrations of cAMP agonists, and active against multiple CFTR gating mutants, including G551D. Login to comment
50 Some measurements were done using stably transfected FRT cells expressing YFP-H148Q and wild-type or G551D-CFTR (Galietta et al., 2001b). Login to comment
188 Activation of G551Dand G1349D-CFTR mutants. Login to comment
189 A and B, stimulation of apical membrane Cl- current by genistein (top) and PG-01 (bottom) in G551Dand G1349D-CFTR-expressing FRT cells. Login to comment
191 C and D, dose-responses for the PG-01 and genistein for activation of G551Dand G1349D-CFTR (S.E., n ϭ 4). Login to comment
217 Measurements were done in the "class III" mutants G551D and G1349D, which produce a severe gating defect without impairment in protein trafficking (Gregory et al., 1991). Login to comment
222 B, G551D-CFTR cells. Login to comment
225 The G551D and G1349D mutant CFTRs produced little Cl- current after the addition of maximal forskolin (Fig. 6, A and B). Login to comment
226 Genistein, a known activator of G551Dand G1349D-CFTR, increased Cl- cur- rent substantially, albeit at high micromolar concentrations (Fig. 6, A and B, top curves). Login to comment
227 PG-01 produced large currents in both G551Dand G1349D-CFTR-expressing cells as shown in Fig. 6, A and B (bottom curves) and summarized in Fig. 6, C and D. Login to comment
229 In contrast, PG-01, SF-01, and the benzothiophene ⌬F508act-02 did not increase Cl- currents in G551Dand G1349D-CFTR-expressing cells (data not shown). Login to comment
240 Nasal epithelial cells from a subject with the G551D mutation (Zegarra-Moran et al., 2002) showed a large response to PG-01 after forskolin stimulation (Fig. 7B). Login to comment
283 The phenyglycines corrected defective gating in a number of CF-causing CFTR mutants including ⌬F508, G551D, G1349D, and D1152H. Login to comment
284 G551D and G1349D affect critical glycine residues in nucleotide binding domains 1 and 2 of CFTR, respectively (Hyde et al., 1990), producing a severe gating defect (Gregory et al., 1991; Logan et al., 1994; Derand et al., 2002; Zegarra-Moran et al., 2002). Login to comment
286 The apparent Kd for PG-01 for G551D-CFTR activation was ϳ1 ␮M, approximately 100-fold better than that of genistein. Login to comment
294 The best phenylglycine was also effective on cells cultured from subjects with CF having G551D and D1152H CFTR mutations, supporting the possible use of this class of compounds for monotherapy of CF caused by some mutations. 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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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

[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
96 McGill and colleagues found common mutations G551D and R117H in one copy of CFTR in 2 (10.5%) of 19 PSC patients, a frequency not significantly different from that of carriers of CF mutations in the general population (21). Login to comment
106 of Classic CF Nonclassic CFTR Mutations Reference PSC Patients Mutations CF Mutations IVS8-5T of Unknown Effect McGill (1996) (21) 19 1 (G551D) 1 (R117H) NA NA Girodon (2002)(19) 29 0 3 (L997F, S1235R, D1270N) 2 1 (N782K) Sheth (2003)* (18) 19 0 3 (2752-26A→G, 3849 + 10kbC→T, I1139V) 1 3 (S686Y, I1366F, R75Q) Gallegos-Orozco (2004) 59 1 ( F508) 0 2 NA Total, no. 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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141 and Class III mutations such as W1282X, F508, and G551D result in little or no functional CFTR channel activity, and are usually associated with the classic CF phenotype of pulmonary disease, elevated sweat chlorides, pancreatic insufficiency, and male infertility. Login to comment
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] CFTR-regulated chloride transport at the ocular su... Invest Ophthalmol Vis Sci. 2005 Apr;46(4):1428-34. Levin MH, Verkman AS
CFTR-regulated chloride transport at the ocular surface in living mice measured by potential differences.
Invest Ophthalmol Vis Sci. 2005 Apr;46(4):1428-34., [PMID:15790911]

Abstract [show]
PURPOSE: To define the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in Cl(-) secretion at the mouse ocular surface in vivo. METHODS: Open-circuit potential differences (PDs) across the fluid-bathed ocular surface were measured in anesthetized wild-type and cystic fibrosis (CF) mice in response to Cl(-) ion substitution and transport agonists and inhibitors. RESULTS: Basal ocular surface PD was -23 +/- 1 mV (SE; 20 wild-type mice), depolarizing to -16 +/- 2 mV after amiloride, then hyperpolarizing to -34 +/- 3 mV after low Cl(-). CFTR activation by forskolin or a selective activator caused further sustained hyperpolarization to -50 to -60 mV. UTP produced a comparable but transient hyperpolarization. The CFTR inhibitors CFTR(inh)-172 and GlyH-101 largely reversed agonist- but not low Cl(-)-induced hyperpolarizations. PD in CF mice hyperpolarized by 2.1 mV after low Cl(-) and was insensitive to CFTR activators or inhibitors. CONCLUSIONS: CFTR provides a major pathway for mouse ocular surface Cl(-) secretion, suggesting the application of CFTR activators as therapy for dry eye. Amiloride-sensitive Na(+) transporters facilitate Na(+) absorption. PD measurements provide a robust and reproducible means of assessing ocular surface ion transporting mechanisms.

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39 MATERIALS AND METHODS Mice Wild-type mice and CF mice (homozygous G551D-CFTR mutant mice31 ) in a CD1 genetic background were bred at the University of California, San Francisco Animal Facility. Login to comment
40 G551D heterozygous mice were bred to generate homozygous CF mice. Login to comment

[hide] Genetic factors in pancreatitis. Rom J Gastroenterol. 2005 Mar;14(1):53-61. Grigorescu M, Grigorescu MD
Genetic factors in pancreatitis.
Rom J Gastroenterol. 2005 Mar;14(1):53-61., [PMID:15800694]

Abstract [show]
The understanding of pathogenesis of acute and chronic pancreatitis has benefited from the progress made in genetic investigations. The discoveries of the gain of function mutations of cationic trypsinogen gene (PRSS1) and the loss of function mutations of pancreatic secretory trypsin inhibitor (SPINK 1) or other potential defects in genes that regulate pancreatic secretory function or modulate inflammatory response to pancreatic injury has changed our current concepts on the pathogenesis of pancreatitis. Genetic factors play an important role in the susceptibility to pancreatic injury, severity and evolution of inflammatory process, leading in some cases to chronic inflammation and/or fibrosis. Acute pancreatitis is viewed as an event and chronic pancreatitis as a process, sequentially linked, reflecting a complex interaction between genetic and environmental factors.

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98 More than 1200 CFTR gene polymorphism have been reported which can be divided into six classes, based on the functional consequences of the polymorphisms on channel function: - class I-III mutations are severe (CFTRsev ), comprising: class I: defective protein synthesis (R553X, W1282X, 3950 del T); class II: abnormal processing trafficking (del 508, N1303K); class III: defective activation (G551D) and all result in functional loss of CFTR from the epithelial cell surface; - class IV mutations (R117H, R347P, D1152H) are mild-variable mutations (CFTRm-v ) and result in reduction but not absence of channel ion conductance; - class V mutations (3849+10KbC >T) diminish protein synthesis or stability and - class VI mutations may affect the regulatory function of CFTR on other ion channels (71-73). Login to comment

[hide] Parallel single nucleotide polymorphism genotyping... Anal Chem. 2005 Apr 15;77(8):2400-5. Chen Y, Shortreed MR, Olivier M, Smith LM
Parallel single nucleotide polymorphism genotyping by surface invasive cleavage with universal detection.
Anal Chem. 2005 Apr 15;77(8):2400-5., 2005-04-15 [PMID:15828773]

Abstract [show]
Large-scale investigations of sequence variation within the human species will provide information about the basis of heritable variation in disease susceptibility and human migration. The surface invader assay (an adaptation of the invasive cleavage reaction to an array format) is capable of exquisitely sensitive and specific detection of genetic variation. It is shown here that this genotyping technology can be multiplexed in a DNA array format, permitting the parallel analysis of a panel of single nucleotide polymorphisms (SNPs) directly from an unamplified genomic DNA target. In addition, a "universal" mode of detection was developed that makes use of a mixture of degenerate templates for DNA ligation to the surface-bound cleaved oligonucleotides and thereby makes this strategy amenable to any desired SNP site or combination of SNP sites, without regard to their particular DNA sequences. This approach was demonstrated on a proof-of-principle scale using small DNA arrays to genotype 6 SNP markers in the PTPN1 gene and 10 mutations in the cystic fibrosis transmembrane conductance regulator gene. This ability to analyze many different genetic variations in parallel, directly from unamplified human genomic DNA samples, lays the groundwork for the development of high-density arrays able to analyze hundreds of thousands or even millions of SNPs.

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170 508 of the protein product.23 The CF mutations chosen in this study, ∆F508, G551D, W1282X, N1303K, R117H, R560T, 3849+10kbCT, V520F, R334W, and I148T, are a subset of the standard panel. Login to comment
174 Among the six unrelated CF carriers, one was found to be homozygous for 3849+10kbCT, one homozygous for ∆F508, one heterozygous for both R117H and ∆F508, one heterozygous for W1282X/WT, one heterozygous for V520F/WT, and one heterozygous for G551D/WT. Login to comment

[hide] Activating cystic fibrosis transmembrane conductan... J Biol Chem. 2005 Jun 24;280(25):23622-30. Epub 2005 Apr 27. Wang W, Li G, Clancy JP, Kirk KL
Activating cystic fibrosis transmembrane conductance regulator channels with pore blocker analogs.
J Biol Chem. 2005 Jun 24;280(25):23622-30. Epub 2005 Apr 27., 2005-06-24 [PMID:15857825]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations that disrupt the surface localization and/or gating of the CF transmembrane conductance regulator (CFTR) chloride channel. The most common CF mutant is deltaF508-CFTR, which inefficiently traffics to the surfaces of most cells. The deltaF508 mutation may also disrupt the opening of CFTR channels once they reach the cell surface, but the extent of this gating defect is unclear. Here, we describe potent activators of wild-type and deltaF508-CFTR channels that are structurally related to 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), a negatively charged pore blocker that we show to have mixed agonistic activity (channel activation plus voltage-dependent pore block). These CFTR agonists include 1) an uncharged NPPB analog that stimulates channel opening at submicromolar concentrations without blocking the pore and 2) curcumin, a dietary compound recently reported to augment deltaF508-CFTR function in mice by an unknown mechanism. The uncharged NPPB analog enhanced the activities of wild-type and deltaF508-CFTR channels both in excised membrane patches and in intact epithelial monolayers. This compound increased the open probabilities of deltaF508-CFTR channels in excised membrane patches by 10-15-fold under conditions in which wild-type channels were already maximally active. Our results support the emerging view that CFTR channel activity is substantially reduced by the deltaF508 mutation and that effective CF therapies may require the use of channel openers to activate mutant CFTR channels at the cell surface.

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109 NPPB-AM Markedly Stimulates the Opening of ⌬F508-CFTR Channels under Conditions in Which Wild-type Channels Are Maximally Activated-We next tested the effects of NPPB and derivatives on the two most common CF mutants: G551D-CFTR and ⌬F508-CFTR. Login to comment
110 G551D-CFTR is a gating mutant (24) that, unlike ⌬F508-CFTR, is trafficked to the cell surface with efficiency similar to that of wild-type CFTR. Login to comment
111 The G551D mutation maps to a region in NBD1 that likely plays a role in MgATP binding or the conformational coupling between ATP binding and the opening of the pore within the transmembrane domains (ATP-binding cassette transporter signature sequence) (25). Login to comment
113 However, G551D-CFTR activity was markedly stimulated by high doses of the charged parent compound (NPPB), doses that were impossible to achieve for the less soluble uncharged derivative (Fig. 3E). Login to comment
114 In NPPB titration experiments, we observed an appreciable shift toward higher concentrations of NPPB for G551D-CFTR activation compared with wild-type channel activation (Fig. 3F). Login to comment
115 This result implies that the G551D mutation in NBD1 reduces the apparent affinity of NPPB (and presumably of NPPB-AM) for its activation site. Login to comment
168 E, NPPB-AM weakly activates G551D-CFTR, whereas high doses of NPPB markedly stimulate this mutant at high PKA concentrations in excised HEK-293T patches. Login to comment
170 See mean data in F. F, NPPB stimulates wild-type CFTR currents (WT; E) at lower doses than G551D-CFTR currents (●) in excised HEK-293T patches. Login to comment
172 The conditions for G551D-CFTR were as described for E. Login to comment
173 Shown are mean data at Ϯ80 mV obtained from four and seven experiments for the wild-type and G551D-CFTR channels, respectively. Data were normalized to the peak current induced by NPPB at each voltage. 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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50 Class 3 alleles (fourth panel) encode proteins that reach the cell surface, but contain missense mutations that render them incapable of being activated; replacement of glycine by aspartic acid at codon 551 (G551D allele) exemplifies this class. 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

[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

[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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58 C-D565G II DF508 D1507 S549R S549I S549N S549R S945D S945L H1054D G1061R L1065P R1066C R1066M L1077P H1085R N1303K G85E III G551D S492F V520F R553G R560T R560S Y569D IV R117H, R117C, R117P, R117L D1152H, L88S, G91R, E92K, Q98R, P205S, L206W, L227R, F311L, G314E, R334W, R334Q, I336K, T338I, L346P, R347C, R347H, R347L, R347P, L927P, R1070W, R1070Q V 3849 þ 10 kb C ! Login to comment
165 An example of this class is the glycine-to-aspartic acid change at codon 551 (G551D). Login to comment
167 The phenotype of patients carrying one DF508 and one G551D allele is characterized by a clinical course as severe as those who are homozygous for DF508, but with a reduced risk of meconium ileus.47 G551D is a class III mutation that produces a protein that is well- synthesized, processed, and correctly inserted in the plasma membrane, but with strongly reduced channel activity.48,49 G551D is localized in the first NBD of CFTR and interferes with ATP hydrolysis.48-50 CFTR Activators Several compounds were found to directly activate both wild-type and mutant CFTR. Login to comment
168 Among them, the most important are alkylxanthines, such as 8-cyclopentyl-1,3-dipropylxanthine (CPX) and genistein.51 The flavonoid genistein is a potent activator of CFTR and can overcome the affected ATP binding to G551D. Login to comment
171 Genistein restored cAMP-dependent G551D-CFTR activity in vitro and in vivo.52-56 CF subjects bearing at least one G551D allele underwent nasal potential difference testing, in which genistein was superfused following isoproterenol activation of CFTR.57 A modest repolarization of several millivolts on average was observed. Login to comment
172 This degree of chloride transport (17% of that observed in normal subjects) might be augmented overall by increasing the level of G551D expression in nasal mucosa. Login to comment
173 Perfusion of the nasal mucosa with genistein significantly hyperpolarized nasal PD both in G551D CF patients and in healthy subjects, indicative of genistein-induced ClÀ conductance in both groups. Login to comment
211 IBMX produced a small, CFTR-related secretory response in the jejunum, cecum, and rectum of G551D mice but had no effect in the nasal epithelium.78 NS-004 restored near-normal channel activity from P574H-CFTR (a mild, trafficking-impaired mutationinNBD1).79 Theseresultssuggestthat,inaddition to their direct effects on CFTR, these drugs may also have other effects that increase the number of channel proteins found in the membrane. Login to comment
252 Willumsen and Boucher108 found that responses to genistein of nasal PD in G551D CF patients corresponded to 13.6% of normal in terms of Cl current, which may have therapeutic significance. 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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123 For example, the G551D mutation (class III) is believed to possess little or no chloride-channel function in vivo because of abnormal function of a nucleotide-bindingdomain,resultingindisordered regulation. 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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68 These mutations are typically missense changes in regulatory regions of CFTR [46], with the most common being G551D (;2.2% of mutant alleles). Login to comment
69 G551D is within CFTR`s first nucleotide binding domain and is associated with a severe CF phenotype [3]. Login to comment
70 Genistein, an isoflavone that is present in milligram per kilogram quantities in tofu and soy, enhances chloride channel activity of wild-type and mutant CFTRs [47], including G551D [48]. Login to comment
71 Perfusion of genistein onto the nasal epithelia increased chloride transport, as assessed by NPD, in non-CF subjects, as well as in subjects with the G551D mutation [48]. Login to comment
95 DF508 [70,77,78•] and G551D [65,79] do not regulate ENaC appropriately. Login to comment
96 We hypothesized that augmentation of DF508 and G551D function with a potentiator would improve regulatory interactions between ENaC and these mutant CFTRs, and observed that genistein significantly improved the regulatory interactions of ENaC with DF508 [70] and G551D [80] in Xenopus oocytes. 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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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

[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] 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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272 DF508 mutation was not detected in DNA from 400 individuals, while none of the exon 11 mutations G551D, R553X, or S549 were identified in DNA from 200 of these patients. Login to comment
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
272 Only the mutations D1270N and G551D were analysed and they were associated with a more favourable outcome of pulmonary function. Nevertheless, previous studies have pointed out that, among functional class III mutations, there may exist a wide variability in their phenotypes that depends principally on the CFTR protein site for which they code.33 34 In summary, the results of this study suggest that the genotype, based on functional class mutation on the two chromosomes, seems to be one of the most decisive factors for pulmonary phenotype and for survival in relation to pulmonary damage. 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
24 Previously, it has been suggested [9, 19] that nasal polyps may be associated with the specific genotypes of CF patients, those homozygous for mutation DF508 and DF508/G551D, in particular. 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
73 As reported previously by Kingdom and co-workers [19], CF patients who had undergone surgery for nasal polyps were characterized by a higher prevalence of the DF508/DF508 and DF508/ G551D genotypes compared with the control population (57.5 vs. 49.9%, P =0.01, and 12 vs. 8%, P =0.05, respectively). 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
85 It has been previously suggested that individuals without CF, but with nasal polyps, tend to develop the CF mutation G551D [8]. Login to comment
94 As an example, a case report of a 35-year-old man suffering from nasal polyps, sinusitis, bronchial asthma and intolerance of acetysal- icylic acid for 19 years revealed the CFTR mutation G551D [22]. Login to comment
99 Currently, we are not aware of any further CFTR mutations in patients with nasal polyps, including G551D. 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
57 Alterations within NBF1, such as the missense mutation G551D, may additionally prevent the regulation of other channels associated with CFTR.19 Class IV: Decreased Conductance The fourth class of mutations involves amino acids located within the membrane-spanning domain, which is implicated in forming the pore of the channel and results in a CFTR channel with defective conductive properties. 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] Revisiting cystic fibrosis transmembrane conductan... Proc Am Thorac Soc. 2004;1(1):17-21. Hanrahan JW, Wioland MA
Revisiting cystic fibrosis transmembrane conductance regulator structure and function.
Proc Am Thorac Soc. 2004;1(1):17-21., [PMID:16113406]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a channel/enzyme which mediates passive diffusion of chloride and bicarbonate through epithelial cell membranes. It is expressed in many cell types throughout the body, but in the airways it is found mainly in secretory serous cells of the submucosal glands. CFTR belongs to a large super-family of ATP binding cassette transporters that have two nucleotide binding domains with characteristic sequences or "motifs". Although most other ATP binding cassette transporters consume ATP to actively transport various substrates, in CFTR the interactions of ATP with nucleotide binding domains control opening and closing of the channel pore (i.e., channel gating). Recent high resolution structures of bacterial nucleotide binding domains combined with new biochemical and electrophysiological studies of CFTR itself have led to major advances in our understanding of CFTR gating. For example, it is now clear that the ATPase activity of CFTR is not strictly required for its channel activity. CFTR has at least two distinct gating modes; one dependent on hydrolysis and the other requiring only stable ATP binding. In this article we discuss a working hypothesis for CFTR that incorporates these recent findings and discuss some interesting implications of the paradigm shift for other aspects of CFTR function and dysfunction.

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33 One of these is G551D, the second most common CF mutation. 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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134 However, G551D, a CFTR mutation that is almost exclusively associated with pancreatic insufficiency, confers a lower risk of meconium ileus (44, 57). Login to comment
318 Cystic fibrosis mice carrying the missense mutation G551D replicate human genotype-phenotype correlations. Login to comment
345 Genotype analysis for delta F508, G551D and R553X mutations in children and young adults with cystic fibrosis with and without chronic liver disease. Login to comment
400 Compound heterozygotes for G551D/deltaF508 are clinically indistinguishable from deltaF508 homozygotes. Login to comment
544 Two cystic fibrosis patients with the genotype G542X/G551D. Login to comment

[hide] Small-molecule correctors of defective DeltaF508-C... J Clin Invest. 2005 Sep;115(9):2564-71. Epub 2005 Aug 25. Pedemonte N, Lukacs GL, Du K, Caci E, Zegarra-Moran O, Galietta LJ, Verkman AS
Small-molecule correctors of defective DeltaF508-CFTR cellular processing identified by high-throughput screening.
J Clin Invest. 2005 Sep;115(9):2564-71. Epub 2005 Aug 25., [PMID:16127463]

Abstract [show]
The most common cause of cystic fibrosis (CF) is deletion of phenylalanine 508 (DeltaF508) in the CF transmembrane conductance regulator (CFTR) chloride channel. The DeltaF508 mutation produces defects in folding, stability, and channel gating. To identify small-molecule correctors of defective cellular processing, we assayed iodide flux in DeltaF508-CFTR-transfected epithelial cells using a fluorescent halide indicator. Screening of 150,000 chemically diverse compounds and more than 1,500 analogs of active compounds yielded several classes of DeltaF508-CFTR correctors (aminoarylthiazoles, quinazolinylaminopyrimidinones, and bisaminomethylbithiazoles) with micromolar potency that produced greater apical membrane chloride current than did low-temperature rescue. Correction was seen within 3-6 hours and persisted for more than 12 hours after washout. Functional correction was correlated with plasma membrane expression of complex-glycosylated DeltaF508-CFTR protein. Biochemical studies suggested a mechanism of action involving improved DeltaF508-CFTR folding at the ER and stability at the cell surface. The bisaminomethylbithiazoles corrected DeltaF508-CFTR in DeltaF508/DeltaF508 human bronchial epithelia but did not correct a different temperature-sensitive CFTR mutant (P574H-CFTR) or a dopamine receptor mutant. Small-molecule correctors may be useful in the treatment of CF caused by the DeltaF508 mutation.

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334 Pedemonte,N.,etal.2005.Phenylglycineandsulfon- amide correctors of defective ∆F508- and G551D-CFTR chloride channel gating. Login to comment
328 Pedemonte,N.,etal.2005.Phenylglycineandsulfon- amide correctors of defective ∆F508- and G551D-CFTR chloride channel gating. Login to comment

[hide] Nucleotide binding domains of human CFTR: a struct... Cell Mol Life Sci. 2005 Sep;62(18):2112-23. Eudes R, Lehn P, Ferec C, Mornon JP, Callebaut I
Nucleotide binding domains of human CFTR: a structural classification of critical residues and disease-causing mutations.
Cell Mol Life Sci. 2005 Sep;62(18):2112-23., [PMID:16132229]

Abstract [show]
Defective function of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) causes CF, the most frequent lethal inherited disease among the Caucasian population. The structure of this chloride ion channel includes two nucleotide-binding domains (NBDs), whose ATPase activity controls channel gating. Recently, the experimental structures of mouse and human CFTR NBD1 and our model of the human CFTR NBD1/NBD2 heterodimer have provided new insights into specific structural features of the CFTR NBD dimer. In the present work, we provide a structural classification of CF-causing mutations which may complement the existing functional classification. Our analysis also identified amino acid residues which may play a critical role in interdomain interaction and are located at the NBD1-NBD2 interface or on the surface of the dimer. In particular, a cluster of aromatic amino acids, which includes F508 and straddles the two NBDs, might be directly involved in the interaction of the NBD1/NBD2 heterodimer with the channel-forming membrane-spanning domains.

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51 Most of the class III mutants (affecting Cl-channel gating) are located within the ATP-binding sites, as exemplified here by G551D (salmon). Login to comment
161 The most striking example is the G551D missense mutation (labeled N in fig. 2) which involves a residue located in the LSGGQ motif of the NBD1 ABC signature of the conventional ATP-binding site B (fig. 1). Login to comment
163 Interestingly, in CF patients, the defective function of the appropriately localized G551D protein could be rescued by genistein [49]. Login to comment
348 J. Cyst. Fibros. 3: 183-190 48 Delaney S. J., Alton E. W., Smith S. N., Lunn D. P., Farley R., Lovelock P. K. et al. (1996) Cystic fibrosis mice carrying the missense mutation G551D replicate human genotype-phenotype correlations. Login to comment
350 B., Dong J. Y. and Fischer H. (1999) Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein. Login to comment

[hide] DCEBIO stimulates Cl- secretion in the mouse jejun... Am J Physiol Cell Physiol. 2006 Jan;290(1):C152-64. Epub 2005 Aug 31. Hamilton KL, Kiessling M
DCEBIO stimulates Cl- secretion in the mouse jejunum.
Am J Physiol Cell Physiol. 2006 Jan;290(1):C152-64. Epub 2005 Aug 31., [PMID:16135545]

Abstract [show]
We investigated the effects of 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one(DCEBIO) on the Cl- secretory response of the mouse jejunum using the Ussing short-circuit current (Isc) technique. DCEBIO stimulated a concentration-dependent, sustained increase in Isc (EC50 41 +/- 1 microM). Pretreating tissues with 0.25 microM forskolin reduced the concentration-dependent increase in Isc by DCEBIO and increased the EC50 (53 +/- 5 microM). Bumetanide blocked (82 +/- 5%) the DCEBIO-stimulated Isc consistent with Cl- secretion. DCEBIO was a more potent stimulator of Cl- secretion than its parent molecule, 1-ethyl-2-benzimidazolinone. Glibenclamide or NPPB reduced the DCEBIO-stimulated Isc by >80% indicating the participation of CFTR in the DCEBIO-stimulated Isc response. Clotrimazole reduced DCEBIO-stimulated Isc by 67 +/- 15%, suggesting the participation of the intermediate conductance Ca2+-activated K+ channel (IKCa) in the DCEBIO-activated Isc response. In the presence of maximum forskolin (10 microM), the DCEBIO response was reduced and biphasic, reaching a peak response of the change in Isc of 43 +/- 5 microA/cm2 and then falling to a steady-state response of 17 +/- 10 microA/cm2 compared with DCEBIO control tissues (61 +/- 6 microA/cm2). The forskolin-stimulated Isc in the presence of DCEBIO was reduced compared with forskolin control tissues. Similar results were observed with DCEBIO and 8-BrcAMP where adenylate cyclase was bypassed. H89, a PKA inhibitor, reduced the DCEBIO-activated Isc, providing evidence that DCEBIO increased Cl- secretion via a cAMP/PKA-dependent manner. These data suggest that DCEBIO stimulates Cl- secretion of the mouse jejunum and that DCEBIO targets components of the Cl- secretory mechanism.

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42 In addition, Becq and colleagues (12) have reported that NS004 activates phosphorylated (via forskolin) wild-type CFTR and G551D-CFTR expressed in Chinese hamster ovary cells. Login to comment

[hide] Antihypertensive 1,4-dihydropyridines as corrector... Mol Pharmacol. 2005 Dec;68(6):1736-46. Epub 2005 Sep 8. Pedemonte N, Diena T, Caci E, Nieddu E, Mazzei M, Ravazzolo R, Zegarra-Moran O, Galietta LJ
Antihypertensive 1,4-dihydropyridines as correctors of the cystic fibrosis transmembrane conductance regulator channel gating defect caused by cystic fibrosis mutations.
Mol Pharmacol. 2005 Dec;68(6):1736-46. Epub 2005 Sep 8., [PMID:16150931]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel gene. CF mutations like deltaF508 cause both a mistrafficking of the protein and a gating defect. Other mutations, like G551D, cause only a gating defect. Our aim was to find chemical compounds able to stimulate the activity of CFTR mutant proteins by screening a library containing approved drugs. Two thousand compounds were tested on Fischer rat thyroid cells coexpressing deltaF508-CFTR and a halide-sensitive yellow fluorescent protein (YFP) after correction of the trafficking defect by low-temperature incubation. The YFP-based screening allowed the identification of the antihypertensive 1,4-dihydropyridines (DHPs) nifedipine, nicardipine, nimodipine, isradipine, nitrendipine, felodipine, and niguldipine as compounds able to activate deltaF508-CFTR. This effect was not derived from the inhibition of voltage-dependent Ca2+ channels, the pharmacological target of antihypertensive DHPs. Indeed, methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-2(trifluoromethylphenyl)pyridine -5-carboxylate (BayK-8644), a DHP that is effective as an activator of such channels, also stimulated CFTR activity. DHPs were also effective on the G551D-CFTR mutant by inducing a 16- to 45-fold increase of the CFTR Cl- currents. DHP activity was confirmed in airway epithelial cells from patients with CF. DHPs may represent a novel class of therapeutic agents able to correct the defect caused by a set of CF mutations.

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2 Other mutations, like G551D, cause only a gating defect. Login to comment
8 DHPs were also effective on the G551D-CFTR mutant by inducing a 16- to 45-fold increase of the CFTR Cl- currents. Login to comment
25 6 Copyright (c) 2005 The American Society for Pharmacology and Experimental Therapeutics 15149/3064372 Mol Pharmacol 68:1736-1746, 2005 Printed in U.S.A. 1736 example of pure class III mutation is instead represented by G551D (glycine-to-aspartic acid change at position 551), which causes a severe impairment in CFTR channel activity (Gregory et al., 1991; Logan et al., 1994; Zegarra-Moran et al., 2002). Login to comment
27 For example, genistein and other flavonoids, at high micromolar concentrations, strongly stimulate the activity of G551D and of ⌬F508 (Hwang et al., 1997; Illek et al., 1999; Zegarra-Moran et al., 2002). Login to comment
34 We have found that the 1,4-dihydropyridines (DHPs) used to treat hypertension are able to stimulate the activity of ⌬F508- and G551D-CFTR mutants. Login to comment
39 Fischer rat thyroid (FRT) cells, stably transfected with ⌬F508- or G551D-CFTR (Galietta et al., 2001b; Zegarra-Moran et al., 2002), were retransfected with the YFP-H148Q/I152L fluorescent protein, which allows optimal sensitivity for the detection of mutant CFTR activity (Galietta et al., 2001a). Login to comment
168 DHPs were also tested as possible activators of the G551D mutant. Login to comment
170 With forskolin alone, no increase of halide transport was detected in G551D cells with the fluorescence assay. Login to comment
187 Dose-response relationships (Fig. 4B and Table 2) revealed that G551D activation required DHP concentrations higher than ⌬F508. Login to comment
188 The potency order for the G551D mutant was the following: felodipine ϭ nicardipine ϭ nitrendipine Ͼ nifedipine ϭ nimodipine ϭ isradipine Ͼ niguldipine. Login to comment
189 In Ussing chamber experiments, very small currents were activated after forskolin stimulation of G551D-CFTR cells. Login to comment
192 It is interesting that the maximal response to DHPs like felodipine or isradipine was at least 2-fold larger than that to genistein, a known activator of the G551D mutant (Illek et al., 1999; Zegarra-Moran et al., 2002). Login to comment
204 The S-(ϩ)-niguldipine had a modest activity also on G551D-CFTR, whereas the other stereoisomer was completely inactive. Login to comment
206 Both compounds were effective on ⌬F508- and G551D-CFTR, the apparent affinity being lower for the latter mutant (Fig. 5). Login to comment
207 BayK-8644 showed a modest but significant stereoselectivity for ⌬F508 (2-fold difference in Ka; p Ͻ 0.05) and no stereoselectivity at all for G551D. Login to comment
221 The two different isradipine enantiomers showed different activity on the G551D mutant (Fig. 5). Login to comment
223 Membrane patches from FRT cells expressing G551D-CFTR were excised in the inside-out configuration. Login to comment
227 Under these conditions, G551D-CFTR channels had a very low activity (Po ϭ 0.021 Ϯ 0.012; n ϭ 3), despite the presence of the catalytic subunit of the protein kinase A to induce maximal phosphorylation (Fig. 6A). Login to comment
232 Nasal epithelial cells from a patient carrying the G551D mutation (Fig. 7A) were largely unresponsive to maximal forskolin (20 ␮M), with an effect of only 0.05 to 0.2 ␮A/cm2 , in agreement with previous observations (Zegarra-Moran et al., 2002). Login to comment
246 Activation of G551D-CFTR by DHPs. Login to comment
247 A, representative traces from microplate reader experiments showing G551D-CFTR activity with saline alone, or forskolin (20 ␮M) with and without genistein (100 ␮M) or DHPs (20 ␮M). Login to comment
249 C, representative Ussing chamber traces showing G551D-CFTR currents upon stimulation with forskolin (20 ␮M) and the indicated concentrations of genistein or DHPs. Login to comment
274 An evidence in favor of this conclusion is represented by the differences of DHP potency for ⌬F508 and G551D mutants. Login to comment
276 Conversely, for G551D, the Ka values of these three DHPs were comparable and significantly higher than that for ⌬F508. Login to comment
287 The decreased channel activity of ⌬F508 and G551D mutants is considered to be the result of an intrinsic defect in CFTR protein function. Login to comment
289 To this respect, it is interesting to TABLE 2 Activation properties of 1,4-dihydropyridines measured in FRT cells expressing G551D-CFTR Values for Vmax and Ka were obtained from experiments of the type shown in Fig. 4B and are the mean Ϯ S.E.M. of 5 to 10 experiments. Login to comment
323 One classic example is G551D, which is a class III mutation characterized by a severe channel-open- Fig. 5. Login to comment
326 Data (mean Ϯ S.E.M. of 5-10 experiments) were obtained from ⌬F508 (left) and G551D (right) using the YFP assay. Login to comment
330 A, traces showing continuous recordings of currents from and inside-out membrane patch excised from a FRT cell expressing G551D-CFTR. Login to comment
335 B, traces from the same experiment showing activation of G551D channels by the addition of 100 ␮M felodipine to the bath solution. Login to comment
338 We have shown previously that genistein is an effective activator of the G551D mutant, with a level of functional correction close to 20% in FRT and nasal epithelial cells (Zegarra-Moran et al., 2002). Login to comment
339 Our present results show that the best DHP, felodipine, also stimulated Cl-secretion in G551D airway epithelial cells up to a level close to 20% of that measured in non-CF cells. Login to comment
342 The different sensitivity of ⌬F508 and G551D mutants suggests that the DHP binding site resides in the NBDs. Login to comment

[hide] Cystic fibrosis, disease severity, and a macrophag... Am J Respir Crit Care Med. 2005 Dec 1;172(11):1412-5. Epub 2005 Sep 22. Plant BJ, Gallagher CG, Bucala R, Baugh JA, Chappell S, Morgan L, O'Connor CM, Morgan K, Donnelly SC
Cystic fibrosis, disease severity, and a macrophage migration inhibitory factor polymorphism.
Am J Respir Crit Care Med. 2005 Dec 1;172(11):1412-5. Epub 2005 Sep 22., 2005-12-01 [PMID:16179637]

Abstract [show]
RATIONALE: Macrophage migration inhibitory factor (MIF) is a key proinflammatory mediator. It contributes toward an exaggerated gram-negative inflammatory response via its ability to induce Toll-like receptor-4 expression. Studies have shown that MIF knockout mice have less aggressive Pseudomonas infection (compared with wild-type). OBJECTIVES: To assess whether a novel functional MIF polymorphism was associated with clinical prognosis in a patient cohort with chronic gram-negative infection, namely cystic fibrosis (CF). METHODS: Collected genomic DNA was analyzed via polymerase chain reaction amplification for the polymorphic region for the CATT repeat polymorphism. Individuals may have a 5-, 6-, 7-, or 8-CATT tetranucleotide repeat unit on each allele. The 5-CATT repeat allele exhibits the lowest MIF promoter activity. MEASUREMENTS AND MAIN RESULTS: Patients with stable CF (n = 167) and a matched control group (n = 166) were enrolled. In patients with CF, the MIF5(+) group had a decreased incidence of Pseudomonas aeruginosa colonization (odds ratio, 0.25; 95% confidence interval, 0.09-0.65; p = 0.004) and a significant reduction in the risk of pancreatic insufficiency (odds ratio, 0.27; 95% confidence interval, 0.07-1.0; p = 0.05). A trend toward milder disease activity in the MIF5(+) group was seen with all other parameters. CONCLUSIONS: The results support the concept of a regulatory role for MIF in CF.

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85 After DF508, the commonest variant alleles were G551D (18 alleles) and R117H (10 alleles). Login to comment

[hide] Extensive sequencing of the CFTR gene: lessons lea... Hum Genet. 2005 Dec;118(3-4):331-8. Epub 2005 Sep 28. McGinniss MJ, Chen C, Redman JB, Buller A, Quan F, Peng M, Giusti R, Hantash FM, Huang D, Sun W, Strom CM
Extensive sequencing of the CFTR gene: lessons learned from the first 157 patient samples.
Hum Genet. 2005 Dec;118(3-4):331-8. Epub 2005 Sep 28., [PMID:16189704]

Abstract [show]
Cystic fibrosis (CF) is one of the most common monogenic diseases affecting Caucasians and has an incidence of approximately 1:3,300 births. Currently recommended screening panels for mutations in the responsible gene (CF transmembrane regulator gene, CFTR) do not detect all disease-associated mutations. Our laboratory offers extensive sequencing of the CFTR (ABCC7) gene (including the promoter, all exons and splice junction sites, and regions of selected introns) as a clinical test to detect mutations which are not found with conventional screening. The objective of this report is to summarize the findings of extensive CFTR sequencing from our first 157 consecutive patient samples. In most patients with classic CF symptoms (18/24, 75%), extensive CFTR sequencing confirmed the diagnosis by finding two disease-associated mutations. In contrast, only 5 of 75 (7%) patients with atypical CF had been identified with two CFTR mutations. A diagnosis of CF was confirmed in 10 of 17 (58%) newborns with either positive sweat chloride readings or positive immunoreactive trypsinogen (IRT) screen results. We ascertained ten novel sequence variants that are potentially disease-associated: two deletions (c.1641AG>T, c.2949_2853delTACTC), seven missense mutations (p.S158T, p.G451V, p.K481E, p.C491S, p.H949L, p.T1036N, p.F1099L), and one complex allele ([p.356_A357del; p.358I]). We ascertained three other apparently novel complex alleles. Finally, several patients were found to carry partial CFTR gene deletions. In summary, extensive CFTR gene sequencing can detect rare mutations which are not found with other screening and diagnostic tests, and can thus establish a definitive diagnosis in symptomatic patients with previously negative results. This enables carrier detection and prenatal diagnosis in additional family members.

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72 The two deletions Table 2 Atypical CF or nonclassic patients in whom extensive sequencing revealed two CFTR mutations Patient Genotype Phenotype Sex Age (years) Sweat chloride concentration (mmol/l) 1 p.S912L/DF508 Chronic lung and sinus disease F 52 Not done 2 p.R1070W/p.N1303K Recurrent respiratory infections F 4.5 2X intermediate 3 p.G551D/c.2789+2 InsA Pancreatic insufficiency, little lung involvement F 50 92, 96 4 c.3849+10kb C>T/p.L732X Failure to thrive, chronic cough, chronic sinusitis M 5.5 70,73 5 p.W1282X/p.R170H Chronic pancreatitis, CBVAD M 44 Borderline (c.1641 AG>T, and c.2949-2953 del TACTC) are expected to be severe disease-associated mutations, since they change the CFTR reading frame; the two patients harboring these novel deletions had a diagnosis of CF with elevated sweat chloride levels and carried a second, previously described, CF mutation. Login to comment
74 DF508/c.546insCTA CF; lung symptoms; PS; 2 sibs with CF NG Pos p.R1066C/c.3272-26 A>G Mild CF 40 115 [p.V562I;p.A1006E]b /p.R1158X CF, FTT 6 Not done DF508/c.1716G>A Classic CF 21 Not done p.R785X/c.2732insA Classic CF, PI 4 Not done DF508/p.R117C Classic CF 2 Not done DF508/p.R75X CF 19 Pos DF508/p.G451Va Mild CF 23 Pos DF508/p.L206W Classic CF 9 150s DF508/p.G542Xc Classic CF 15 Pos p.T1036N/p.T1036Na CF, PS 9 Pos DF508/c.3272-26 A>G Classic CF 33 Not done DF508/p.R117Hc Classic CF 35 Not done DF508/p.A455Ec CF 3 Pos p.G551D/p.Y275X a Novel CFTR variant b Complex CFTR allele c Both mutations are on the ACMG/ACOG panel Table 5 Diagnosis of CF in infants/newborns with abnormal newborn screening results Patient number Genotype Age at sequencing Sex Newborn screen result Sweat chloride concentration (mmol/l)a Phenotype 1 DF508/c.2789+2insA 3 months F Positive sweat test 88,96,89,84 Dx of CF, being treated prophylactically 2 DF508/c.2949del5b 3 months F IRT positive 105 Dx of CF 3 p.G551D/c.1259insA 14 months M Positive sweat test ?

In reference to DF508 and 1716G>A. Does this mean these two mutation have resulted in "classic CF"? Does this mean 1716G>A is disease causing?
Gibson75 on 2013-08-12 07:00:25

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[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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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
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

[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

[hide] Diagnostic dilemmas resulting from the immunoreact... J Pediatr. 2005 Sep;147(3 Suppl):S78-82. Parad RB, Comeau AM
Diagnostic dilemmas resulting from the immunoreactive trypsinogen/DNA cystic fibrosis newborn screening algorithm.
J Pediatr. 2005 Sep;147(3 Suppl):S78-82., [PMID:16202789]

Abstract [show]
OBJECTIVE: To quantitate the proportion of infants identified through cystic fibrosis (CF) newborn screening (NBS) by an immunoreactive trypsinogen (IRT)/DNA screening algorithm who have an unclear diagnosis as defined by the findings of an elevated IRT level and either 1) 2 CF gene (CFTR) mutations detected and sweat chloride level <60 mEq/L; or 2) 0 or 1 CFTR mutations and a "borderline" sweat chloride level >or=30 and <60 mEq/L. STUDY DESIGN: Using the 4-year cohort of CF-affected infants recently described by the Massachusetts CF NBS program, we identified and described the number of infants with the diagnostic characteristics (diagnostic dilemmas) aforementioned. RESULTS: Of infants with positive results on CF NBS who had 1 CFTR mutation detected and a borderline sweat chloride concentration, nearly 20% displayed a second CFTR mutation on further evaluation. Of all infants with positive CF NBS results considered affected with CF, 11% had a diagnosis that fell into 1 of the diagnostic dilemma categories aforementioned. CONCLUSIONS: Four problematic diagnostic categories generated by CF NBS are defined. In the absence of data on the natural history of such infants, careful follow-up is recommended for infants in whom a definitive diagnosis is elusive.

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65 Two infants with DF508/5T and borderline sweat chloride values were not included in the count of the true positive cohort, however follow-up continues Group IRT (mg/ml) IRT % CFTR Allele 1 CFTR Allele 2 [Cl2 ] mEq/L Sex I 64 97 DF508 R117H-7T 34 F 179 100 DF508 R117H-7T 33 F 79 99 DF508 R117H-7T 49 M 97 99 W1282X 3849110kb 54 M II 176 99.8 DF508 R117H-7T 24 F 129 99.7 G85E R117H 21 F 84 99 G551D R117H-7T 27 M III 94 99.1 DF508 unknown 58 M* 142 100 G85E R117C 33 F 72 98 G551D R117C 46 F 100 99.2 DF508 L206W 35 M IV 141 100 G85Ey R117C 41 M *Identified twin sibling has [Cl2 ] > 60 mEq/L. 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] 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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53 A, G551D, R553X, R560T, Q552X (i) 10, 11 2183AA . 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

[hide] Differential sensitivity of the cystic fibrosis (C... J Biol Chem. 2006 Jan 27;281(4):1970-7. Epub 2005 Nov 25. Cai Z, Taddei A, Sheppard DN
Differential sensitivity of the cystic fibrosis (CF)-associated mutants G551D and G1349D to potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel.
J Biol Chem. 2006 Jan 27;281(4):1970-7. Epub 2005 Nov 25., 2006-01-27 [PMID:16311240]

Abstract [show]
The genetic disease cystic fibrosis (CF) is caused by loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Two CF mutants, G551D and G1349D, affect equivalent residues in the highly conserved LSGGQ motifs that are essential components of the ATP-binding sites of CFTR. Both mutants severely disrupt CFTR channel gating by decreasing mean burst duration (MBD) and prolonging greatly the interburst interval (IBI). To identify small molecules that rescue the gating defects of G551D- and G1349D-CFTR and understand better how these agents work, we used the patch clamp technique to study the effects on G551D- and G1349D-CFTR of phloxine B, pyrophosphate (PP(i)), and 2'-deoxy ATP (2'-dATP), three agents that strongly enhance CFTR channel gating. Phloxine B (5 microm) potentiated robustly G551D-CFTR Cl- channels by altering both MBD and IBI. In contrast, phloxine B (5 microm) decreased the IBI of G1349D-CFTR, but this effect was insufficient to rescue G1349D-CFTR channel gating. PP(i) (5 mm) potentiated weakly G551D-CFTR and was without effect on the G1349D-CFTR Cl- channel. However, by altering both MBD and IBI, albeit with different efficacies, 2'-dATP (1 mm) potentiated both G551D- and G1349D-CFTR Cl- channels. Using the ATP-driven nucleotide-binding domain dimerization model of CFTR channel gating, we suggest that phloxine B, PP(i) and 2'-dATP alter channel gating by distinct mechanisms. We conclude that G551D- and G1349D-CFTR have distinct pharmacological profiles and speculate that drug therapy for CF is likely to be mutation-specific.

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1 Two CF mutants, G551D and G1349D, affect equivalent residues in the highly conserved LSGGQ motifs that are essential components of the ATP-binding sites of CFTR. Login to comment
3 To identify small molecules that rescue the gating defects of G551Dand G1349D-CFTR and understand better how these agents work, we used the patch clamp technique to study the effects on G551Dand G1349D-CFTR of phloxine B, pyrophosphate (PPi), and 2؅-deoxy ATP (2؅-dATP), three agents that strongly enhance CFTR channel gating. Login to comment
4 Phloxine B (5 ␮M) potentiated robustly G551D-CFTR Cl-channels by altering both MBD and IBI. Login to comment
6 PPi (5 mM) potentiated weakly G551D-CFTR and was without effect on the G1349D-CFTR Cl-channel. Login to comment
7 However, by altering both MBD and IBI, albeit with different efficacies, 2؅-dATP (1 mM) potentiated both G551Dand G1349D-CFTR Cl-channels. Login to comment
9 We conclude that G551Dand G1349D-CFTR have distinct pharmacological profiles and speculate that drug therapy for CF is likely to be mutation-specific. Login to comment
26 To explore the mutation specificity of CFTR potentiators and to understand better their mechanism of action, the aim of the present study was to investigate the effects of the CFTR potentiators phloxine B, pyrophosphate (PPi), and 2Ј-deoxy-ATP (2Ј- dATP) on the CF mutants G551D and G1349D. Login to comment
28 Conversely, we selected the CF mutants G551D and G1349D because these mutants profoundly disrupt channel gating by affecting equivalent residues in the LSGGQ motifs of the two ATP-binding sites of CFTR (G551D, site 2, and G1349D, site 1) (4-6; 15-19) and because G551D is a common CF mutation (2). Login to comment
29 To quantify the efficacy with which the different CFTR potentiators restore normal channel gating to G551Dand G1349D-CFTR, we employed high resolution single-channel recording and * This work was supported by the Cystic Fibrosis Trust. Login to comment
39 MATERIALS AND METHODS Cells and Cell Culture-For this study, we used mouse mammary epithelial cells (C127 cells) stably expressing either wild-type human CFTR or the CF mutant G1349D and Fischer rat thyroid (FRT) epithelial cells stably expressing the CF mutant G551D.3 C127 cells were generous gifts of Professor M. J. Welsh (University of Iowa, Iowa City, IA) and Dr C. R. O`Riordan (Genzyme, Framingham, MA), whereas FRT cells were a generous gift of Drs. L. J. V. Galietta and O. Zegarra-Moran (Istituto Giannina Gaslini, Genoa, Italy). Login to comment
49 To determine the relationship between phloxine B concentration and channel activity for G551Dand G1349D-CFTR, we used membrane patches containing multiple active channels. Login to comment
50 For all other studies, we used membrane patches containing small numbers of active channels (wild-type and G1349D-CFTR, Յ4; G551D-CFTR, Յ6). Login to comment
55 In contrast, the presence of the CFTR potentiators phloxine B or 2Ј-dATP were required to determine the number of G551D-CFTR Cl-channels in a membrane patch. Login to comment
56 Despite our precautions, we cannot exclude the possibility of unobserved G551Dand G1349D-CFTR Cl-channels in membrane patches. Login to comment
57 Therefore, values of Po for G551Dand G1349D-CFTR might possibly be overestimated. Login to comment
67 Po was calculated from either open- and closed-times, as described previously (26), or by using the equation: Po ϭ I/͑n ϫ i͒ (Eq. 2) where n represents the number of active channels in the membrane patch. For wild-type CFTR, only membrane patches that contained a single active channel were used for burst analysis, whereas for G551Dand G1349D-CFTRs, we used membrane patches containing no more than four active channels. Login to comment
73 4 Z. Cai, J.-H. Chen, and D. N. Sheppard, data not shown Rescue of G551D and G1349D-CFTR by CFTR Potentiators JANUARY 27, 2006•VOLUME 281•NUMBER 4 JOURNAL OF BIOLOGICAL CHEMISTRY 1971 and IBI í 2,090 Ϯ 481 ms (n ϭ 12) (p Ͼ 0.1 for both sets of data). Login to comment
88 RESULTS The Single-channel Activity of Wild-type, G551D-, and G1349D-CFTRs-Before investigating the rescue of the CF mutants G551D and G1349D by CFTR potentiators, we quantified their single-channel activity. Login to comment
89 Fig. 1A shows representative single-channel recordings of wild-type, G551D-, and G1349D-CFTR. Login to comment
90 Like other NBD mutants (3), G551Dand G1349D-CFTR were without effect on i but perturbed severely channel gating (Fig. 1, A and B). Login to comment
92 In contrast, G551Dand G1349D-CFTR both attenuated the duration of bursts and prolonged dramatically the interburst interval (Fig. 1A). Login to comment
93 As a result, the Po of G551Dand G1349D-CFTR were reduced markedly when compared with that of wild-type CFTR (Fig. 1C). Login to comment
95 Based on analyses of closed-time histograms (wild-type CFTR, ␶c ϭ 14.87 Ϯ 0.51 ms (n ϭ 10); G1349D-CFTR, ␶c ϭ 17.41 Ϯ 1.49 ms (n ϭ 5); p Ͼ 0.05), we used a burst delimiter (␶c) of 15 ms to discriminate inter-and intraburst closures.6 The MBD of G551Dand G1349D-CFTR were similar and both about one-third that of wild-type CFTR (Fig. 1D). Login to comment
96 In contrast, the IBI of G551Dand G1349D-CFTR were prolonged strikingly when compared with that of wild-type CFTR (Fig. 1E). Login to comment
97 Phloxine B Rescues the Gating Defect of G551D-CFTR but Not That of G1349D-CFTR-The fluorescein derivative phloxine B potentiates efficaciously wild-type and ⌬F508-CFTR Cl- currents (11). Login to comment
98 Like its effects on wild-type CFTR (11), phloxine B (0.1-5 ␮M) potentiated greatly G551D-CFTR Cl- currents, whereas phloxine B (10-40 ␮M) inhibited channel activity (Fig. 2, A and B). Login to comment
99 Interestingly, for both wild-type and G551D-CFTR, phloxine B (5 ␮M) potentiated the maximum Cl- cur- rent (Fig. 2B). Login to comment
100 However, because under control conditions the activity of G551D-CFTR is much lower than that of wild-type CFTR (Fig. 1), the potentiation of G551D-CFTR by phloxine B exceeded greatly that of wild-type CFTR (Fig. 2B). Login to comment
103 6 For G551D-CFTR, no membrane patches containing a single active CFTR Cl-channel were obtained. Login to comment
104 However, given the very large difference in the duration of intra-and interburst closures for G551D-CFTR, misclassification errors when defining bursts should be rare. Login to comment
106 The single-channel activity of wild-type (WT), G551Dand G1349D-CFTRs. Login to comment
107 A, representative single-channel recordings of wild-type, G551D-, and G1349D-CFTRs in excisedinside-outmembranepatchesfromC127cellsexpressingwild-typeandG1349D-CFTRandFRTcellsexpressingG551D-CFTR.Inthisandsubsequentfigures,unlessotherwise indicated, ATP (1 mM) and PKA (75 nM) were continuously present in the intracellular solution, voltage was -50 mV, and there was a large Cl-concentration gradient across the membrane patch (internal [Cl- ] ϭ 147 mM; external [Cl- ] ϭ 10 mM). Login to comment
110 For G551D-CFTR, four active channels were observed when phloxine B (5 ␮M) was added to the intracellular solution (not shown). Login to comment
111 B-E, i, Po, MBD, and IBI of wild-type, G551D-, and G1349D-CFTRs. Login to comment
112 Columns and error bars indicate means ϩ S.E. (wild-type, n ϭ 20 for Po and i, n ϭ 10 for MBD and IBI; G551D, n ϭ 35 for i, n ϭ 10 for Po, MBD, and IBI; G1349D, n ϭ 25 for i, Po, and MBD but n ϭ 5 for IBI). Login to comment
115 Rescue of G551D and G1349D-CFTR by CFTR Potentiators 1972 tiates G551D-CFTR Cl- currents, we measured i, Po, MBD, and IBI in the absence and presence of the drug. Login to comment
116 Phloxine B (0.1-40 ␮M) caused a concentration-dependent decrease in i equivalent to that observed with wild-type CFTR (Fig. 2C), suggesting that phloxine B does not potentiate G551D-CFTR by increasing current flow through the channel. Login to comment
117 Instead, phloxine B (1-5 ␮M) potentiated G551D-CFTR by enhancing dramatically channel gating, and thus, Po (Fig. 2, A and D, p Ͻ 0.01). Login to comment
119 Importantly, phloxine B (5 ␮M) prolonged the MBD of G551D-CFTR Cl-channels to a length equivalent to that of wild-type CFTR in the absence of drugs (Figs. 1D and 2E; p Ͼ 0.05). Login to comment
120 However, although phloxine B (5 ␮M) decreased the IBI of G551D-CFTR by 62%, it was still over 11-fold longer than that of wild-type CFTR in the absence of the drug (Figs. 1E and 2F; p Ͻ 0.001). Login to comment
121 Phloxine B (1-20 ␮M) also had biphasic effects on G1349D-CFTR Cl- currents with phloxine B (5 ␮M) potentiating the maximum current (Fig. 3, AandB)(11).Ofnote,themagnitudeofG1349D-CFTRCl- currentpoten- tiated by phloxine B (5 ␮M) was much smaller than that of G551D-CFTR but equivalent to that of wild-type CFTR (Figs. 2B and 3B). Login to comment
127 Phloxine B (PB) potentiates strongly the single-channel activity of G551D-CFTR. Login to comment
128 A, representative recordings show the effects of phloxine B (1 and 5 ␮M) on the activity of G551D-CFTR Cl-channels. Login to comment
129 B, effects of phloxine B concentration on G551D (filled circles and solid line) and wild-type CFTR (open circles and dotted line). Login to comment
130 Data are means Ϯ S.E. (G551D, n ϭ 5-8; wild-type CFTR, n ϭ 4-9) at each point. Login to comment
132 C, effect of phloxine B concentration on i of G551D- (filled circles and solid line) and wild-type CFTR (open circles and dotted line). Login to comment
134 D-F, effects of phloxine B (5 ␮M) on Po, MBD, and IBI of G551D-CFTR Cl-channels. Columns and error bars indicate means ϩ S.E. (n ϭ 7). Login to comment
148 Rescue of G551D and G1349D-CFTR by CFTR Potentiators JANUARY 27, 2006•VOLUME 281•NUMBER JOURNAL OF BIOLOGICAL CHEMISTRY 1973 of phloxine B (5 ␮M) was over 5-fold longer than that of wild-type CFTR in the absence of drug. Login to comment
149 Thus, our data demonstrate that phloxine B rescues the gating defect of G551D-CFTR but not that of G1349D-CFTR. Login to comment
150 Effects of PPi on the Single-channel Activity of G551Dand G1349D-CFTR-The non-hydrolyzable inorganic phosphate analogue, PPi, enhances robustly wild-type CFTR Cl- currents by (i) increasing the rate of channel opening and (ii) decreasing markedly the rate of channel closure (12, 13). Login to comment
151 Fig. 4A demonstrates that PPi (5 mM) augmented the single-channel activity of G551D-CFTR. Login to comment
154 PPi (5 mM) increased, but not significantly, the Po of G551D-CFTR by enhancing slightly MBD and curtailing weakly IBI (Fig. 4, C-E). Login to comment
158 2Ј-Deoxy-ATP Activates Both G551Dand G1349D-CFTR Cl-Channels-Because both phloxine B and PPi failed to potentiate G1349D-CFTR, we searched for other agents that might rescue this CF mutant. Login to comment
176 Fig. 7A suggests that 2Ј-dATP (1 mM) enhanced markedly G551D-CFTR channel gating with the result that the number of active channels increased. Login to comment
177 Quantification of the effects of 2Ј-dATP on G551D-CFTR revealed (i) no significant change in i (Fig. 7B), (ii) a 10-fold increase in Po (Fig. 7C), (iii) a 3.5-fold enhancement of MBD (Fig. 7D), and (iv) a 61% decrease in IBI (Fig. 7E). Login to comment
178 Of note, the MBD of G551D-CFTR in the presence of 2Ј-dATP (1 mM) was similar to that of wild-type CFTR in the presence of ATP (1 mM; Figs. 1D and 7D). Login to comment
179 However, the IBI of G551D-CFTR in the presence of 2Ј-dATP (1 mM) was over 17-fold longer than that of wild-type CFTR in the presence of ATP (1 mM; Figs. 1E and 7E). Login to comment
182 Effects of PPi on the single-channel activity of G551D-CFTR. Login to comment
183 A, representative recordings show the effects of PPi (5 mM) on the activity of G551D-CFTR Cl-channels. B-E, effects of PPi (5 mM) on i, Po, MBD, and IBI of G551D-CFTR. Login to comment
185 The asterisks indicate values that are significantly different from thecontrolvalue(pϽ0.05).IncreasingthePPi concentration to 10 mM failed to enhance further the single-channel activity of G551D-CFTR (nPo: control,0.07Ϯ0.03;PPi (10mM),0.23Ϯ0.10,nϭ6;pϭ 0.7 versus PPi (5 mM) data). Login to comment
187 Rescue of G551D and G1349D-CFTR by CFTR Potentiators 1974 out effect on i but augmented G1349D-CFTR channel gating, leading to an increase in the number of active channels (Fig. 8). Login to comment
190 We interpret our data to suggest that 2Ј-dATP potentiates wild-type, G551D-, and G1349D-CFTR channel gating by similar mechanisms but that 2Ј-dATP incompletely restores normal channel gating to either G551D-CFTR or G1349D-CFTR. Login to comment
191 DISCUSSION The CF mutants G551D and G1349D affect equivalent residues in the LSGGQ motifs of NBD1 and NBD2. Login to comment
193 The CFTR potentiators phloxine B and 2Ј-dATP, but not PPi, augment G551D-CFTR channel gating, whereas only 2Ј-dATP enhances that of G1349D-CFTR. Login to comment
194 Our results demonstrate that G551Dand G1349D-CFTR have distinct pharmacological profiles. Login to comment
195 Molecular Mechanisms of CFTR Dysfunction in CF Previous studies demonstrated that G551Dand G1349D-CFTR cause a loss of Cl-channel function by disrupting ATP binding, hydrolysis, and thus, channel gating (17, 19, 31). Login to comment
196 Building on these data, our quantitative analysis of channel gating reveals that these mutants have exceptionally slow opening rates and very fast closing rates when compared with those of wild-typeCFTR.Toexplaintheseverityofthesegatingdefects,weconsider how G551Dand G1349D-CFTR might perturb the ATP-driven NBD dimerization model of CFTR channel gating (5, 20). Login to comment
197 The exceptionally slow rates of G551Dand G1349D-CFTR channel opening suggest that these mutants impede ATP binding to sites 1 and 2 with the result that the rate of NBD dimerization is retarded. Login to comment
198 Moreover, once the NBD dimer forms in G551Dand G1349D-CFTR Cl-channels, it is inherently unstable. Login to comment
215 Rescue of G551D and G1349D-CFTR by CFTR Potentiators JANUARY 27, 2006•VOLUME 281•NUMBER 4 JOURNAL OF BIOLOGICAL CHEMISTRY 1975 reduced. Login to comment
216 Of note, using a molecular model of the NBD dimer, Moran et al. (15) demonstrated that G551Dand G1349D-CFTR each destabilize ATP binding to sites 1 and 2. Login to comment
220 Below, we use the model of Vergani et al. (5) to discuss the effects of CFTR potentiators on wild-type, G551D-, and G1349D-CFTR. Login to comment
223 Consistent with this idea, phloxine B prolonged the MBD of G551D-CFTR (present study). Login to comment
224 However, phloxine B also decreased the IBI of G551Dand G1349D-CFTR (present study), suggesting that the drug can promote NBD dimer formation for some, but not other, CF mutants (e.g. ⌬F508 (11)). Login to comment
229 2Ј-dATP potentiates strongly G551D-CFTR Cl-channels. Login to comment
230 A, single-channel recordings of G551D-CFTR in the presence of either ATP (1 mM) or 2Ј-dATP (1 mM). Login to comment
232 B-E, effects of 2Ј-dATP on i, Po, MBD, and IBI of G551D-CFTR, respectively. Login to comment
242 Rescue of G551D and G1349D-CFTR by CFTR Potentiators 1976 the stability of the NBD dimer. Login to comment
246 Consistent with this idea, G551D-CFTR markedly attenuated PPi potentiation of CFTR. Login to comment
254 First, among the agents that we tested, only 2Ј-dATP potentiated the gating behavior of both G551Dand G1349D-CFTR. Login to comment
256 Second, 2Ј-dATP prolonged the MBD of G551D-CFTR to a magnitude equivalent to that of wild-type CFTR in the presence of ATP. Login to comment
257 However, 2Ј-dATP only attenuated partially the extended IBI of G551D-CFTR. Login to comment
258 (Note that phloxine B had similar effects on G551D-CFTR). Login to comment
259 These data indicate that for G551D-CFTR, the defect in channel closing is easier to rescue than that of channel opening. Login to comment
260 An explanation for the effects of 2Ј-dATP on G551D-CFTR channel gating is provided by the C1 7 C2 7 O model of CFTR channel gating (30). Login to comment
262 Given that the IBI of G551D-CFTR is 45-fold longer than that of wild-type CFTR (Fig. 1E), the energy barrier opposing the opening of the G551D-CFTR Cl-channel must be considerably greater than that of wild-type CFTR. Login to comment
263 Thus, although 2Ј-dATP and phloxine B might provide sufficient energy to G551D-CFTR to normalize the rate of channel closing, this energy is insufficient to rescue the defect in channel opening. Login to comment

[hide] Nucleotide-binding domains of cystic fibrosis tran... J Biol Chem. 2006 Feb 17;281(7):4058-68. Epub 2005 Dec 16. Gross CH, Abdul-Manan N, Fulghum J, Lippke J, Liu X, Prabhakar P, Brennan D, Willis MS, Faerman C, Connelly P, Raybuck S, Moore J
Nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator, an ABC transporter, catalyze adenylate kinase activity but not ATP hydrolysis.
J Biol Chem. 2006 Feb 17;281(7):4058-68. Epub 2005 Dec 16., 2006-02-17 [PMID:16361259]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter family. CFTR consists of two transmembrane domains, two nucleotide-binding domains (NBD1 and NBD2), and a regulatory domain. Previous biochemical reports suggest NBD1 is a site of stable nucleotide interaction with low ATPase activity, whereas NBD2 is the site of active ATP hydrolysis. It has also been reported that NBD2 additionally possessed adenylate kinase (AK) activity. Knowledge about the intrinsic biochemical activities of the NBDs is essential to understanding the Cl(-) ion gating mechanism. We find that purified mouse NBD1, human NBD1, and human NBD2 function as adenylate kinases but not as ATPases. AK activity is strictly dependent on the addition of the adenosine monophosphate (AMP) substrate. No liberation of [(33)P]phosphate is observed from the gamma-(33)P-labeled ATP substrate in the presence or absence of AMP. AK activity is intrinsic to both human NBDs, as the Walker A box lysine mutations abolish this activity. At low protein concentration, the NBDs display an initial slower nonlinear phase in AK activity, suggesting that the activity results from homodimerization. Interestingly, the G551D gating mutation has an exaggerated nonlinear phase compared with the wild type and may indicate this mutation affects the ability of NBD1 to dimerize. hNBD1 and hNBD2 mixing experiments resulted in an 8-57-fold synergistic enhancement in AK activity suggesting heterodimer formation, which supports a common theme in ABC transporter models. A CFTR gating mechanism model based on adenylate kinase activity is proposed.

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10 Interestingly, the G551D gating mutation has an exaggerated nonlinear phase compared with the wild type and may indicate this mutation affects the ability of NBD1 to dimerize. Login to comment
32 In contrast, the second most common CF mutation G551D is only defective in channel function (6) and has a prevalence of 2-7% in the CF population (7). Login to comment
60 The G551D gating mutation has an exaggerated nonlinear phase as compared with wild type, and may indicate this mutation affects NBD1 ability to form dimers. Login to comment
103 The mNBD1 proteins (wild type and G551D) were purified to homogeneity as judged by a Coomassie-stained SDS-PAGE gel (Fig. 1A). Login to comment
104 A total of nine mNBD1 wild type and four mNBD1 G551D independent protein samples were purified for this study. Login to comment
132 A, Coomassie-stained SDS-PAGE of native mouse NBD1 (mNBD1) wild type and G551D mutant proteins (2 ␮g of protein). Login to comment
196 TABLE 1 Wild-type and mutant protein kinetic parameters for AK activity Enzymea Mutation ATP AMP ADP Km b,c Vmax b,c Km b,c Vmax b,c Km b,c Vmax b,c ␮M ␮M/min ␮M ␮M/min ␮M ␮M/min mNBD1 WT 30 Ϯ 2 1.2 Ϯ 0.01 30 Ϯ 4 0.9 Ϯ 0.02 450 Ϯ 60 1.4 Ϯ 0.06 mNBD1 G551D 80 Ϯ 7 0.4 Ϯ 0.02 70 Ϯ 7 0.4 Ϯ 0.02 360 Ϯ 50 1.0 Ϯ 0.05 hNBD1 WT 80 Ϯ 5 0.7 Ϯ 0.02 60 Ϯ 7 0.7 Ϯ 0.03 300 Ϯ 20 1.0 Ϯ 0.02 hNBD2 WT 70 Ϯ 3 0.6 Ϯ 0.01 40 Ϯ 5 0.7 Ϯ 0.03 280 Ϯ 20 0.7 Ϯ 0.02 a Enzyme concentrations are mNBD1 WT (900 nM), G551D (1800 nM), hNBD1 (4.7 nM), hNBD2 (30 nM). Login to comment
240 Next, we wanted to examine three key CFTR residues in mNBD1 (K464A, G551D, and ⌬F508) to determine what effect these mutations had on their ability to form homodimers or alter AK activity. Login to comment
245 In contrast, the mNBD1 G551D mutant protein purified with the same characteristics as the wild-type protein. Login to comment
246 The AK activity of G551D mutant was measured as a function of protein concentration (Fig. 6A). Login to comment
247 From this data, we find the G551D gating mutation lacks AK activity at low protein concentrations (Ͻ250 nM) when compared with the wild type protein, suggesting the mutation affects the ability of the NBD1 to dimerize. Login to comment
248 This was confirmed with two independently purified mNBD1 G551D samples. Login to comment
249 The kinetic parameters of mNBD1 G551D protein (1800 nM) were determined (Table 1) at saturating ATP (2 mM). Login to comment
250 The apparent AMP Km was 70 ␮M and Vmax was 0.4 ␮M/min. At high AMP (400 ␮M) the apparent ATP Km was 80 ␮M and Vmax was 0.4 ␮M/min. At high protein concentrations, the G551D mutation results in only a modest decrease in catalysis and 2-fold decrease in substrate affinity when compared with wild type protein. Login to comment
251 These data suggest the G551D mutation does not significantly disrupt catalytic function directly but rather the mutation may reduce the affinity between the two NBDs in the intact CFTR protein. Login to comment
253 We surmise that the G551D mutation renders NBD1 unable to associate or rearrange productively with NBD2 upon nucleotide substrate binding and that this subsequently leads to a defect in Cl-ion transport. Login to comment
255 However, we cannot formally rule out the possibility that the CFTR G551D folds less efficiently than the wild-type protein (i.e. that there are a lower percentage of active molecules in G551D preparation as compared with wild type preparation) as a good active site titrating compound is not available. Login to comment
266 A, mNBD1 wild type (square), mNBD1 G551D mutant (triangle) hNBD1 (B) and hNBD2 (C) proteins are shown. 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] 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
258 Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein. Login to comment

[hide] Flagellin of Pseudomonas aeruginosa inhibits Na+ t... FASEB J. 2006 Mar;20(3):545-6. Epub 2006 Jan 12. Kunzelmann K, Scheidt K, Scharf B, Ousingsawat J, Schreiber R, Wainwright B, McMorran B
Flagellin of Pseudomonas aeruginosa inhibits Na+ transport in airway epithelia.
FASEB J. 2006 Mar;20(3):545-6. Epub 2006 Jan 12., [PMID:16410345]

Abstract [show]
Pseudomonas aeruginosa causes severe life-threatening airway infections that are a frequent cause for hospitalization of cystic fibrosis (CF) patients. These Gram-negative pathogens possess flagella that contain the protein flagellin as a major structural component. Flagellin binds to the host cell glycolipid asialoGM1 (ASGM1), which appears enriched in luminal membranes of respiratory epithelial cells. We demonstrate that in mouse airways, luminal exposure to flagellin leads to inhibition of Na+ absorption by the epithelial Na+ channel ENaC, but does not directly induce a secretory response. Inhibition of ENaC was observed in tracheas of wild-type mice and was attenuated in mice homozygous for the frequent cystic fibrosis conductance regulator (CFTR) mutation G551D. Similar to flagellin, anti-ASGM1 antibody also inhibited ENaC. The inhibitory effects of flagellin on ENaC were attenuated by blockers of the purinergic signaling pathway, although an increase in the intracellular Ca2+ concentration by recombinant or purified flagellin or whole flagella was not observed. Because an inhibitor of the mitogen-activated protein kinase (MAPK) pathway also attenuated the effects of flagellin on Na+ absorption, we conclude that flagellin exclusively inhibits ENaC, probably due to release of ATP and activation of purinergic receptors of the P2Y subtype. Stimulation of these receptors activates the MAPK pathway, thereby leading to inhibition of ENaC. Thus, P. aeruginosa reduces Na+ absorption, which could enhance local mucociliary clearance, a mechanism that seem to be attenuated in CF.

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7 Inhibition of ENaC was observed in tracheas of wild-type mice and was attenuated in mice homozygous for the frequent cystic fibrosis conductance regulator (CFTR) mutation G551D. Login to comment
42 Mice, homozygous for the Cftr-G551D mutation (CF), or wild-type strain C57BL/6 (Charles River Laboratories, Wilmington, MA) were infected with P. aeruginosa using the agar bead protocol described in detail in McMorran et al. (20). Login to comment
82 Here we examined ASGM1 expression in airway epithelial cells of tracheas from both wild-type mice and mice homozygous for the common CFTR mutation G551D (CF mice). Login to comment
95 Effects of flagellin in CF mouse tracheas and tracheas from P. aeruginosa-infected mice Similar experiments were performed on tracheas from transgenic mice homozygous for the frequent CFTR mutation G551D (24). Login to comment
96 Tracheas from cftrG551D/G551D mice demonstrated a spontaneous transepithelial voltage of -16.2 ± 1.9 mV, a calculated Isc of -292.2 ± 26.1 µA/cm2 , and a transepithelial resistance of 55.2 ± 4.1 Ωcm2 (n=5), which is consistent with previous recordings made with this mouse strain. Login to comment
151 We found reduced inhibition of amiloride-sensitive Na+ absorption by flagellin of P. aeruginosa in the cftrG551D/G551D mice, lacking functional CFTR. Login to comment
152 This remains currently unexplained since expression of membrane ASGM1 receptors appeared not reduced in cftrG551D/G551D mice when compared with wild-type animals. Login to comment
154 We demonstrated previously that enhanced P. aeruginosa burden in the airways of cftrG551D/G551D mice was corrected by overexpression of wild-type CFTR, which enhanced fluid transport and normalized pathogen clearance (36). 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] Rescue of DeltaF508-CFTR trafficking and gating in... Am J Physiol Lung Cell Mol Physiol. 2006 Jun;290(6):L1117-30. Epub 2006 Jan 27. Van Goor F, Straley KS, Cao D, Gonzalez J, Hadida S, Hazlewood A, Joubran J, Knapp T, Makings LR, Miller M, Neuberger T, Olson E, Panchenko V, Rader J, Singh A, Stack JH, Tung R, Grootenhuis PD, Negulescu P
Rescue of DeltaF508-CFTR trafficking and gating in human cystic fibrosis airway primary cultures by small molecules.
Am J Physiol Lung Cell Mol Physiol. 2006 Jun;290(6):L1117-30. Epub 2006 Jan 27., [PMID:16443646]

Abstract [show]
Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in cftr, a gene encoding a PKA-regulated Cl(-) channel. The most common mutation results in a deletion of phenylalanine at position 508 (DeltaF508-CFTR) that impairs protein folding, trafficking, and channel gating in epithelial cells. In the airway, these defects alter salt and fluid transport, leading to chronic infection, inflammation, and loss of lung function. There are no drugs that specifically target mutant CFTR, and optimal treatment of CF may require repair of both the folding and gating defects. Here, we describe two classes of novel, potent small molecules identified from screening compound libraries that restore the function of DeltaF508-CFTR in both recombinant cells and cultures of human bronchial epithelia isolated from CF patients. The first class partially corrects the trafficking defect by facilitating exit from the endoplasmic reticulum and restores DeltaF508-CFTR-mediated Cl(-) transport to more than 10% of that observed in non-CF human bronchial epithelial cultures, a level expected to result in a clinical benefit in CF patients. The second class of compounds potentiates cAMP-mediated gating of DeltaF508-CFTR and achieves single-channel activity similar to wild-type CFTR. The CFTR-activating effects of the two mechanisms are additive and support the rationale of a drug discovery strategy based on rescue of the basic genetic defect responsible for CF.

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87 Fischer rat thyroid (FRT) epithelia expressing ⌬F508-CFTR or G551D-CFTR were grown on Costar Snapwell cell culture inserts for 4 days. Login to comment
263 This includes the missense mutation G551D, which results in defective gating but does not impair trafficking to the apical membrane (9). Login to comment
264 In FRT epithelial monolayers expressing G551D-CFTR, VRT-532 potentiated forskolin-stimulated Cl-secretion with an EC50 of 20 Ϯ 3 ␮M (n ϭ 3), which was about fivefold less potent than the EC50 observed for ⌬F508-CFTR in temperature-corrected FRT monolayers (EC50 ϭ 3.8 Ϯ 0.5 ␮M; n ϭ 46). Login to comment
358 Although several potent CFTR potentiators have been reported in the literature, CFTR potentiator VRT-532 appears to be distinguished by its ability to potentiate the gating of several forms of CFTR, including ⌬F508-, G551D-, and wt-CFTR. Login to comment
359 Like genistein (38), VRT-532 had an approximately fivefold lower affinity for G551D-CFTR than ⌬F508-CFTR. Login to comment
361 G551D (ϳ70 nM and ϳ1,100 nM against ⌬F508- and G551D-CFTR, respectively) or are inactive against G551D-CFTR (benzothiophenes and sulfonamides) (38, 62). 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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45 The glycine-to- aspartic acid missense mutations G551D and G1349D are class III mutations located within the signature sequence LSGGQ in NBD1 and LSHGH in NBD2, respectively [20]. Login to comment
46 G551D is one of the five most frequent CF mutations with a frequency of 2-5% depending of the population of origin. Login to comment
175 Importantly, NS004 is a modulator of several mutated forms of CFTR; P574H [60], K1250A [61], delF508 [31, 35, 61] and G551D [59]. Login to comment
178 In G551D-CFTR expressing CHO cells, in the presence of 10 µM forskolin, NS004 stimulated G551D-CFTR activity in a dose-dependent manner with an EC50 of 1.5 µM [59]. Login to comment
180 By contrast to genistein (see below), no inhibitory effect was observed at high concentrations of NS004 for both wtand G551D-CFTR suggesting that NS004 is not an allosteric activator [59]. Login to comment
207 By contrast, the response of the two mutants G1349D- and G551D-CFTR to genistein is dramatically altered [39]. Login to comment
208 Genistein is not able to stimulate G1349D- and CFTR bearing the double mutation G551D/G1349D whereas genistein stimulates G551D-CFTR [38, 39, 69] without any inhibition at high concentration [38, 39]. Login to comment
216 The best agent found was UCCF-0339 (Fig. 3) able to activate wt-CFTR with a Kd of 1.7 µM. UCCF-339 appears to be slightly more potent than apigenin (Kd of 4 µM) on wild-type but on the contrary to apigenin did not activate the mutant G551D [70]. Login to comment
220 Activation of CFTR by Benzoquinolizinium Derivatives In 1999 screening of a small library of heterocycle agents identified the benzoquinolizinium family (Fig. 5) of agents as activators of wt-CFTR [40] and later of the mutant G551D [71] and potent correctors of the abnormal trafficking of delF508-CFTR [50, 72]. Login to comment
221 A second SAR study was conducted with a series of benzo[c]quinolizinium and benzo[f]indolo[2, 3-a]quinolizinium salts and generated many derivatives that have been tested on both wtand G551D-CFTR chloride channel activity [46]. Login to comment
241 This compound is the most potent agent of the series, active with the same efficacy on both wtand G551D-CFTR. Login to comment
246 Additional experiments also showed that MPB-91 did not compete with the ATP binding, did not modulate the ATPase activity at NBD1 and NBD2 and had no significant effect on the reduced ATPase activity of G551D-NBD1 [71]. Login to comment
254 The therapeutic potential for CF of MPBs was demonstrated following studies conducted on delF508-CFTR and G551D-CFTR. Login to comment
255 For example, MPB-91 and MPB-104 but not MPB-07 activated G551D-CFTR [46, 71]. Login to comment
263 Further studies on the correlation between the structure of MPB and their effects will be needed but it is possible that part of the molecule that is common to MPB-07, MPB-91 and MPB-104 is required to interact with delF508-CFTR trafficking whereas an other part allows the activation of G551D-CFTR and Kir6.2 channels. Login to comment
283 Unfortunately, the compounds have no effect on G551D-CFTR [81]. Login to comment
288 A trifluoro- methylphenylbenzamine activated the CF-causing mutant G551D with a Kd > 10µM [47]. Login to comment

[hide] A large deletion in the CFTR gene in CBAVD. Genet Med. 2006 Feb;8(2):93-5. Hantash FM, Milunsky A, Wang Z, Anderson B, Sun W, Anguiano A, Strom CM
A large deletion in the CFTR gene in CBAVD.
Genet Med. 2006 Feb;8(2):93-5., [PMID:16481891]

Abstract [show]
PURPOSE: Most cystic fibrosis mutation screening methods do not detect large exon deletions or duplications in the cystic fibrosis transmembrane regulator gene. We looked for such mutations in congenital bilateral absence of the vas deferens patients in whom routine screening assays had identified only one or no cystic fibrosis transmembrane regulator gene mutations. METHODS: DNA samples from 48 men with congenital bilateral absence of the vas deferens were tested for exonic deletions and duplications in the cystic fibrosis transmembrane regulator gene using a laboratory-developed semiquantitative fluorescent PCR assay. RESULTS: Semi-quantitative fluorescent PCR identified a large deletion in one (2%) of the 48 patients. This patient, previously characterized as carrying only the IVS8-5T mutation, was found to have a deletion of exons 22-24 of the cystic fibrosis transmembrane regulator gene. In a second patient with the IVS8-5T mutation, we identified a one-base pair insertion in exon 17b that disrupted the reading frame. CONCLUSIONS: Analysis of the cystic fibrosis transmembrane regulator gene for exon deletions and duplications should be included for complete study of CBAVD patients, especially those considering assisted reproduction.

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14 Genotypes from Group 1 were as follows: ⌬F508/wt (N ϭ 13), 5T/wt (N ϭ 7), ⌬F508/5T (N ϭ 1), G551D/wt genotype (N ϭ 2), and N1303K/wt (N ϭ 1). 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] The glycine residues G551 and G1349 within the ATP... J Membr Biol. 2005 Dec;208(3):203-12. Epub 2006 Apr 7. Melin P, Norez C, Callebaut I, Becq F
The glycine residues G551 and G1349 within the ATP-binding cassette signature motifs play critical roles in the activation and inhibition of cystic fibrosis transmembrane conductance regulator channels by phloxine B.
J Membr Biol. 2005 Dec;208(3):203-12. Epub 2006 Apr 7., [PMID:16604470]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) protein contains a canonical ATP-binding cassette (ABC) signature motif, LSGGQ, in nucleotide binding domain 1 (NBD1) and a degenerate LSHGH in NBD2. Here, we studied the contribution of the conserved residues G551 and G1349 to the pharmacological modulation of CFTR chloride channels by phloxine B using iodide efflux and whole-cell patch clamp experiments performed on the following green fluorescent protein (GFP)-tagged CFTR: wild-type, delF508, G551D, G1349D, and G551D/G1349D double mutant. We found that phloxine B stimulates and inhibits channel activity of wild-type CFTR (Ks = 3.2 +/- 1.6 microM: , Ki = 38 +/- 1.4 microM: ) and delF508 CFTR (Ks = 3 +/- 1.8 microM: , Ki = 33 +/- 1 microM: ). However, CFTR channels with the LSGDQ mutated motif (mutation G551D) are activated (Ks = 2 +/- 1.13 microM: ) but not inhibited by phloxine B. Conversely, CFTR channels with the LSHDH mutated motif (mutation G1349D) are inhibited (Ki = 40 +/- 1.01 microM: ) but not activated by phloxine B. Finally, the double mutant G551D/G1349D CFTR failed to respond not only to phloxine B stimulation but also to phloxine B inhibition, confirming the importance of both amino acid locations. Similar results were obtained with genistein, and kinetic parameters were determined to compare the pharmacological effects of both agents. These data show that G551 and G1349 control the inhibition and activation of CFTR by these agents, suggesting functional nonequivalence of the signature motifs of NBD in the ABC transporter CFTR.

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2 Here, we studied the contribution of the conserved residues G551 and G1349 to the pharmacological modulation of CFTR chloride channels by phloxine B using iodide efflux and whole-cell patch clamp experiments performed on the following green fluorescent protein (GFP)-tagged CFTR: wild-type, delF508, G551D, G1349D, and G551D/G1349D double mutant. Login to comment
4 However, CFTR channels with the LSGDQ mutated motif (mutation G551D) are activated (Ks = 2 ± 1.13 lM) but not inhibited by phloxine B. Conversely, CFTR channels with the LSHDH mutated motif (mutation G1349D) are inhibited (Ki = 40 ± 1.01 lM) but not activated by phloxine B. Finally, the double mutant G551D/ G1349D CFTR failed to respond not only to phloxine B stimulation but also to phloxine B inhibition, confirming the importance of both amino acid locations. Login to comment
7 Key words: Cystic fibrosis - CFTR - ABC signature - G551D - G1349D - Phloxine B - Genistein - Non-Michaelis-Menten Introduction The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) chloride channel belongs to the ATP-binding cassette (ABC) transporters, a large evolutionarily conserved family of integral membrane proteins that catalyze the active transport of a variety of solutes across biological membranes coupled to hydrolysis of nucleotides as a source of energy (Riordan et al., 1989; Vankeerberghen, Cuppens & Cassima, 2002). Login to comment
22 Individual delF508, G551D and G1349D mutations were created by site-directed mutagenesis as previously described (Melin et al., 2004). Login to comment
23 Double mutant G551D/G1349D (named 2GD-CFTR) was derived from pEGFP-G551D-CFTR. Login to comment
61 Results PROPERTIES OF G1349D-CFTR CHLORIDE CURRENT Individual delF508, G551D and G1349D and double G551D/G1349D (named 2GD-CFTR) mutations were created by site-directed mutagenesis and inserted into the pEGFP-C1-CFTR mammalian expression vector, allowing fusion of GFP to the N terminus of CFTR as previously described (Melin et al., 2004). Login to comment
65 The class III mutation G1349D, like G551D, impaired activation of CFTR channels by the adenylate cyclase activator forskolin (Fsk). Login to comment
66 The corresponding mean current densities are 11 ± 4 pA/pF for G551D-CFTR and 9 ± 4 pA/pF for G1349D-CFTR at +40 mV in the presence of 10 lM Fsk (n = 3). Login to comment
71 Using iodide efflux experiments, we determined the concentration dependence of the effect of glibenclamide on G1349D-CFTR and G551D/ G1349D (noted 2GD) in cells activated by 10 lM Fsk + 100 lM IBMX. Login to comment
75 We explored the effect of PhlxB on the Cl) channel activity of the following CFTR mutants: delF508, G551D, G1349D and 2GD. Login to comment
87 EFFECTS OF PHLXB ON G551D, G1349D AND G551D/G1349D CHANNELS In the next series of experiments, we examined the consequence of the glycine-to-aspartic acid mutations in NBD1 and NBD2 on the response to PhlxB in the presence of Fsk. Login to comment
88 A classical sigmoidal concentration-response relationship describes the effect of PhlxB on G551D (Fig. 3B, black triangles). Login to comment
89 These results indicate a complete loss of inhibition at high PhlxB concentrations (>10 lM) of G551D-CFTR compared to wt-CFTR (see Fig. 3A). Login to comment
90 The calculated Ks = 2 ± 1.13 lM (n = 4) shows that PhlxB is a potent activator of G551D channels (Table 1). Login to comment
92 Contrary to the previous results with the NBD1 mutation G551D, we Fig. 1. Login to comment
99 The double mutant, carrying G551D and G1349D mutations, is also refractory to PhlxB stimulation (Fig. 3B, open squares). Login to comment
101 EFFECT OF PHLXB WHEN G1349D-, G551DAND 2GD-CFTR-EXPRESSING CELLS ARE STIMULATED BY FSK/IBMX Because the mutant G1349D can be activated by Fsk/IBMX but not by Fsk/PhlxB, we took advantage of this difference to investigate whether PhlxB could nevertheless inhibit the mutant channel after its activation by Fsk/IBMX, like the wt-CFTR channels. Login to comment
110 Then, similar experiments were performed with G551D-CFTR-expressing cells. Login to comment
111 As expected from iodide efflux experiments, a significant increase of Cl) current was recorded in G551D-expressing cells stimulated by 10 lM Fsk + 100 lM IBMX (Fig. 5A and B). Login to comment
112 However, contrary to G1349D-CFTR, PhlxB did not inhibit G551D-CFTR currents after stable activation of G551D (compare left and right whole-cell 0 100 200 300 400 -1000 0 1000 2000 3000 I(pA) Time (ms) 0 100 200 300 400 -1000 0 1000 2000 3000 I(pA) Time (ms) 0 100 200 300 400 -1000 0 1000 2000 3000I(pA) Time (ms) Fsk + IBMXbasal Fsk + IBMX + Glib -100-80 -60 -40 -20 20 40 60 80 100 -1000 1000 2000 basal Fsk+IBMX +Glib Vm (mV) I (pA) -6 -5 -4 0 25 50 75 100 2GD G1349D Log [glibenclamide] (M) %ofactivation ns ns ns ns ns A B D E C Fig. 2. Login to comment
123 PhlxB did not potentiate G551D-CFTR currents due to maximal stimulation of the current by Fsk/IBMX (Fig. 5A and C). Login to comment
124 Further application of 100 lM glibenclamide in the extracellular bath fully inhibited the currents (Fig. 5A and C), confirming that G551D-CFTR is refractory to inhibition only by PhlxB but not by glibenclamide. Login to comment
126 We also noted two differences between G551D and G1349D-CFTR channels stimulated by Fsk+IBMX. Login to comment
127 First, the time to maximal current stimulation with G551D-CFTR, 120 ± 9 s (n = 4), was significantly faster (P < 0.01) than that for G1349D (194 ± 8 s, n = 4). Login to comment
128 Second, the current density measured at +40 mV was 26 ± 5 pA/pF (n = 6) for G551D construct, a value approximately threefold smaller (P < 0.001) than the current density measured for G1349D, 88 ± 13 pA/pF (n = 4). Login to comment
142 In this configuration, each nucleotide is sandwiched between the two NBDs and each nucleotide-binding site is formed by the Walker A, Walker B, Q-loop and wt-CFTR delF508 (24h/27˚C) -7 -6 -5 -4 0.00 0.05 0.10 0.15 0.20 Log [PhlxB] (M) kpeak-kbasal(min-1 ) ** ns ns ** * -7 -6 -5 -4 0.00 0.05 0.10 0.15 0.20 G551D G1349D 2GD Log [PhlxB] (M) kpeak-kbasal(min-1 ) A B Fig. 3. Login to comment
147 (B) Concentration-response curves of PhlxB on COS-7 cells transiently transfected with GFP-CFTR with the following mutations: G551D, -G1349D and -2GD. Login to comment
151 Kinetic parameters for activation (Ks) and inhibition (Ki) of CFTR by PhlxB and genisteina PhlxB Genistein Construct Ks Ki Ks Ki wt-CFTR 3.2 ± 1.6 38 ± 1.4 10 ± 1b 106 ± 1b delF508 3 ± 1.8 33 ± 1 9.8 ± 1b 107 ± 1b G551D 2 ± 1.13 -d 13 ± 1.25b - G1349D - 40 ± 1.01c - 121 ± 1.14c 2GD - - - - a All data (in lM) are n = 4. b Data from Melin et al., 2004. c With activation by Fsk/IBMX. Login to comment
158 Interestingly our data show that G1349D-CFTR elicited a larger Cl) current than G551D-CFTR, these two glycine residues being located within the NBD1 and NBD2 active sites, respectively (Fig. 1B). Login to comment
171 According to these data, we show that the LSGDQ mutated sequence (with G551D) drastically impairs activation of CFTR Cl) channel activity by Fsk/IBMX compared to the LSHDH mutated sequence (with G1349D). Login to comment
174 Drugs like genistein and PhlxB are associated with direct binding at the NBDs (Randak et al., 1999; Cai & Sheppard, 2002), consistent with our observations that CFTR modulation is altered by NBD mutations G551D and G1349D. Login to comment
185 Effects of PhlxB on cAMP-activated G551D-CFTR channels. Login to comment
186 (A) Representative time course of G551D-CFTR amplitude current in the presence of various agonists. Login to comment
187 (B) Representative trace of current recorded on G551D channels with Fsk + IBMX (*) and with Fsk + IBMX + PhlxB (**). Login to comment
188 (C) I/V relationships for the G551D-CFTR channels (mean ± SEM) normalized by cell capacitance. Login to comment
189 (D) Summary of current amplitudes (mean ± SEM between +40 and )40 mV) recorded on cells transfected with GFP-G551D-CFTR in various conditions: basal (n = 10), Fsk + IBMX (n = 6), Fsk + IBMX + PhlxB (n = 5), Fsk + IBMX + PhlxB + glibenclamide (Glib, n = 3). 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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79 As hypothesized, the contribution of the enhancer polymorphism to the prediction of percent predicted FEV1 TABLE 2-Frequency of CFTR Alleles in Dublin and Belfast CF Populations Allele1 Frequency Dublin Belfast Full study population Disease category2 DF508 76.1% 54.3% 68.3% Nonmild G551D 6.1% 3.9% 5.3% Nonmild R117H 2.7% 4.8% 3.4% Mild 621 þ 1 G ! 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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114 In some mutations, e.g. G551D, there is minimal function and in some, e.g. S1255P, ATP is less potent at stimulating activity. Login to comment
125 Other mutations are often found in higher frequency in particular ethnic populations, such as the W1282X mutation in Ashkenazi Jewish populations and G551D in French Canadians.71 Such information is useful in designing mutation panels suitable for screening programs in different populations. 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] Acute Pseudomonas challenge in cystic fibrosis mic... J Allergy Clin Immunol. 2006 May;117(5):1163-9. Saadane A, Soltys J, Berger M
Acute Pseudomonas challenge in cystic fibrosis mice causes prolonged nuclear factor-kappa B activation, cytokine secretion, and persistent lung inflammation.
J Allergy Clin Immunol. 2006 May;117(5):1163-9., [PMID:16675347]

Abstract [show]
BACKGROUND: Cystic fibrosis (CF) is characterized by an excessive and prolonged inflammatory response to Pseudomonas aeruginosa in the lung. There are high levels of cytokines and chemokines and an exaggerated PMN influx causing significant morbidity and mortality. OBJECTIVE: To compare the kinetics of the inflammatory response with the kinetics of clearance of acute bacterial challenge in the lungs of CF and wild-type (WT) mice. METHODS: We challenged CF knockout (KO) and WT mice intratracheally with P aeruginosa in suspension and evaluated bacteria counts, nuclear factor-kappaB (NF-kappaB), and inhibitor of NF-kappaB alpha protein (I-kappaBalpha) in lung tissue, cytokines, and PMN in bronchoalveolar lavage (BAL). RESULTS: Both groups of mice cleared the infection with the same kinetics. CF-KO mice had more PMN in BAL than WT mice. CF-KO mice had high concentrations of proinflammatory cytokines in BAL on days 2 and 4, whereas cytokines in BAL from WT mice were only slightly elevated. CF-KO mice failed to regenerate I-kappaBalpha once it was degraded, and consequently had prolonged and excessive activation of NF-kappaB for the entire 6-day duration of the study. In contrast, WT mice showed only slight NF-kappaB activation, which plateaued at day 4. CONCLUSION: These data suggest that NF-kappaB is dysregulated in CF lung infection and could be a good target for therapy. Prolonged responses to initial acute infections may contribute to the eventual establishment of chronic persistent inflammation. CLINICAL IMPLICATIONS: Dysregulation of the I-kappaB/NF-kappaB pathway in cystic fibrosis leads to prolonged cytokine secretion and persistent inflammation in response to acute challenges and may be important in the development of chronic lung inflammation and infection.

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140 Two other studies, using mice with the G551D mutation in CFTR, also showed that those mice display an abnormal inflammatory response with increased cytokines and infiltrating cells.24,25 Because the genes for proinflammatory cytokines, such as TNF, IL-1b, and IL-6, are activated by NF-kB,22 we studied NF-kB activation in the lung tissue. 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] Molecular analysis of the IVS8-T splice variant 5T... Mol Hum Reprod. 2006 Jul;12(7):469-73. Epub 2006 May 19. Radpour R, Gilani MA, Gourabi H, Dizaj AV, Mollamohamadi S
Molecular analysis of the IVS8-T splice variant 5T and M470V exon 10 missense polymorphism in Iranian males with congenital bilateral absence of the vas deferens.
Mol Hum Reprod. 2006 Jul;12(7):469-73. Epub 2006 May 19., [PMID:16714368]

Abstract [show]
Congenital bilateral absence of the vas deferens (CBAVD) is responsible for 2-6% of male infertility in which mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been identified. To investigate CBAVD at the molecular level in Iran, we have characterized the mutations in the CFTR gene in 106 patients with this condition. None had clinical manifestations of cystic fibrosis (CF). We also analysed a DNA variant (the 5T allele) in a noncoding region of CFTR, which causes reduced levels of the normal CFTR protein and M470V exon 10 missense polymorphism. Five of the 106 patients with CBAVD had mutations in both copies of the CFTR gene, and none of them had the 5T allele. Eighty-five patients had a mutation in at least one copy of CFTR, and of these patients, 46 had one 5T allele (in 11 cases, two alleles and in 35 cases, just one allele of 5T was detected). In 21 patients, no CFTR and 5T mutations were found (19.81%). 5T/M470 genotype was found in 19 patients, 5T/V470 was found in 3 and 5T with heterozygote form of M470V was found in 24 CBAVD patients. In CBAVD patients, 28 F508del carriers were identified. Most of our patients with CBAVD have mutations in the CFTR gene. The combination of the 5T allele in one copy of the CFTR gene with a CF mutation in the other copy is the most common cause of CBAVD in Iran. The 5T allele mutation has a wide range of clinical presentations and revealed a high frequency, occurring in patients with CBAVD or moderate forms of CF and infertile men.

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90 Mutation geno types IVS8-PolyT M470V n (%) Two mutations detected F508del/R117H 9T/9T M/M 1 (0.94) F508del/621+1G>T 7T/7T V/V 1 (0.94) 1540A/G/1540A/G 7T/7T M/M 2 (1.89) R347H/R117H 9T/7T M/V 1 (0.94) G551D/IVS8-5T 7T/5T M/V 2 (1.89) F508del/IVS8-5T 7T/5T M/V 8 (7.55) 9T/5T M/M 6 (5.67) 1717-1G>A/IVS8-5T 7T/5T M/V 4 (3.77) R117H/IVS8-5T 7T/5T M/V 2 (1.89) 621+1G>T/IVS8-5T 7T/5T M/V 3 (2.83) 9T/5T M/M 2 (1.89) 1540A/G/IVS8-5T 7T/5T M/V 2 (1.89) R553X/IVS8-5T 7T/5T M/V 1 (0.94) IVS8-5T/IVS8-5T 5T/5T V/V 3 (2.83) 5T/5T M/M 8 (7.55) One mutation detected G85E/- 7T/7T V/V 2 (1.89) G551D/- 9T/7T V/V 1 (0.94) 621+1G>T/- 7T/7T M/M 2 (1.89) 9T/7T M/V 1 (0.94) R334W/- 7T/7T M/V 1 (0.94) F508del/- 7T/7T M/V 7 (6.60) 9T/7T M/M 3 (2.83) 9T/9T M/V 2 (1.89) IVS8-5T/- 5T/7T M/M 3 (2.83) 5T/9T M/V 2 (1.89) 1717-1G>A/- 7T/7T M/V 3 (2.83) 9T/7T M/V 2 (1.89) R117H/- 7T/7T M/M 2 (1.89) 9T/7T M/V 1 (0.94) 2789+5G>A/- 7T/7T M/M 1 (0.94) 3120+1G>A/- 9T/7T M/V 2 (1.89) R560T/- 9T/7T M/V 1 (0.94) N1303K/- 9T/7T V/V 1 (0.94) 1651A/G/- 7T/7T M/V 1 (0.94) R553X/- 9T/7T M/V 1 (0.94) No mutation detected -/- 7T/7T M/M 12 (11.32) -/- 9T/9T M/M 3 (2.83) -/- 9T/7T M/V 6 (5.66) Table IV. Login to comment

[hide] New insights into cystic fibrosis: molecular switc... Nat Rev Mol Cell Biol. 2006 Jun;7(6):426-36. Guggino WB, Stanton BA
New insights into cystic fibrosis: molecular switches that regulate CFTR.
Nat Rev Mol Cell Biol. 2006 Jun;7(6):426-36., [PMID:16723978]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-)-selective ion channel, is a prototypic member of the ATP-binding cassette transporter superfamily that is expressed in several organs. In these organs, CFTR assembles into large, dynamic macromolecular complexes that contain signalling molecules, kinases, transport proteins, PDZ-domain-containing proteins, myosin motors, Rab GTPases, and SNAREs. Understanding how these complexes regulate the intracellular trafficking and activity of CFTR provides a unique insight into the aetiology of cystic fibrosis and other diseases.

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111 Genistein, a flavinoid, activates G551D-CFTR channels - which are present in the plasma membrane, but are inactive - and ∆F508-CFTR channels. Login to comment
112 Therefore, genistein might be useful in individuals with the G551D mutation, and might enhance ∆F508-CFTR-mediated Cl-secretion in patients who also receive a drug that increases the membrane expression of ∆F508-CFTR. Login to comment

[hide] Chloroquine normalizes aberrant transforming growt... Pediatr Pulmonol. 2006 Aug;41(8):771-8. Perkett EA, Ornatowski W, Poschet JF, Deretic V
Chloroquine normalizes aberrant transforming growth factor beta activity in cystic fibrosis bronchial epithelial cells.
Pediatr Pulmonol. 2006 Aug;41(8):771-8., [PMID:16779853]

Abstract [show]
Cystic fibrosis (CF) remains a fatal progressive disease in spite of the discovery and characterization of the CFTR gene. Transforming growth factor beta (TGF-beta) has been implicated in pathophysiology of CF. Previous reports have shown the trans-Golgi network (TGN) is hyperacdified in CF epithelial cells in culture and that this hyperacidification can be corrected with the membrane permeant weak base, chloroquine. In this study bioactive TGF-beta produced by CF and normal cells was measured using a reporter cell line with a TGF-beta responsive promoter linked to luciferase. Increased levels of TGF-beta were detected in the conditioned media from CF epithelial cells compared to their matched controls-(IB3-1 vs. S9; pCEP-R vs. pCEP, CuFi-4 vs. NuLi-1). Levels of TGF-beta were normalized with chloroquine indicating that the hyperacidification of the TGN of CF cells is responsible for the altered TGF-beta levels.

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39 CuFi-4 and NuLi-1 cell lines were derived from primary airway epithelium and immortalized by infections with HPV-16 E6/E7; CuFi-4 cells are from a CF patient (d508/G551D) and NuFi-1 cells are from a normal individual.19 The cells were cultured on collagen-coated plastic dishes (type VI, human placental; catalog no. C-7521, Sigma St. Louis, MO) in serum-free bronchial epithelial cell growth medium with supplements (catalog no. CC3170, Clonetics/BioWhittaker, Walkersville, MD). Login to comment

[hide] CFTR chloride channel drug discovery--inhibitors a... Curr Pharm Des. 2006;12(18):2235-47. Verkman AS, Lukacs GL, Galietta LJ
CFTR chloride channel drug discovery--inhibitors as antidiarrheals and activators for therapy of cystic fibrosis.
Curr Pharm Des. 2006;12(18):2235-47., [PMID:16787252]

Abstract [show]
The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a cAMP-activated chloride channel expressed in epithelia in the lung, intestine, pancreas, testis and other tissues, where it facilitates transepithelial fluid transport. In the intestine CFTR provides the major route for chloride secretion in certain diarrheas. Mutations in CFTR cause the hereditary disease cystic fibrosis, where chronic lung infection and deterioration in lung function cause early death. CFTR is a well-validated targeted for development of inhibitors for therapy of secretory diarrheas and activators for therapy in cystic fibrosis. Our lab has identified and optimized small molecule inhibitors of CFTR, as well as activators of DeltaF508-CFTR, the most common mutant CFTR causing cystic fibrosis. High-throughput screening of small molecule collections utilizing a cell-based fluorescence assay of halide transport yielded thiazolidinone and glycine hydrazide CFTR inhibitors that block enterotoxin-mediated secretory diarrhea in rodent models, including a class of non-absorbable inhibitors that target the CFTR pore at its external entrance. Benzothiophene, phenylglycine and sulfonamide potentiators were identified that correct the defective gating of DeltaF508-CFTR chloride channels, and other small molecules that correct its defective cellular processing. Small molecule modulators of CFTR function may be useful in the treatment of cystic fibrosis, secretory diarrhea and polycystic kidney disease.

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221 Phenylglycines had another interesting property in being able to activate other CFTR mutants including G551D, G1349D, and D1152H (Fig. 7C). 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] Abnormal regulatory interactions of I148T-CFTR and... Am J Physiol Cell Physiol. 2007 Jan;292(1):C603-11. Epub 2006 Jul 5. Suaud L, Yan W, Rubenstein RC
Abnormal regulatory interactions of I148T-CFTR and the epithelial Na+ channel in Xenopus oocytes.
Am J Physiol Cell Physiol. 2007 Jan;292(1):C603-11. Epub 2006 Jul 5., [PMID:16822950]

Abstract [show]
The mechanisms underlying regulatory interactions of the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial Na(+) channel (ENaC) in Xenopus oocytes are controversial. CFTR's first nucleotide binding domain (NBD-1) may be important in these interactions, because mutations within NBD-1 impair these functional interactions. We hypothesized that an abnormal CFTR containing a non-NBD-1 mutation and able to transport chloride would retain regulatory interactions with murine ENaC (mENaC). We tested this hypothesis for I148T-CFTR, where the mutation is located in CFTR's first intracellular loop. I148T-CFTR has been associated with a severe CF phenotype, perhaps because of defects in its regulation of bicarbonate transport, but it transports chloride similarly to wild-type CFTR in model systems (Choi JY, Muallem D, Kiselyov K, Lee MG, Thomas PJ, Muallem S. Nature 410: 94-97, 2001). cRNAs encoding alphabetagamma-mENaC and I148T-CFTR were injected separately or together into Xenopus oocytes. mENaC and CFTR functional expression were assessed by two-electrode voltage clamp. mENaC whole oocyte expression was determined by immunoblotting, and surface expression was quantitated by surface biotinylation. Injection of I148T-CFTR cRNA alone yielded high levels of CFTR functional expression. In coinjected oocytes, mENaC functional and surface expression was not altered by activation of I148T-CFTR with forskolin/ IBMX. Furthermore, the CFTR potentiator genistein both enhanced functional expression of I148T-CFTR and restored regulation of mENaC surface expression by activated I148T-CFTR. These data suggest that the ability to transport chloride is not a critical determinant of regulation of mENaC by activated CFTR in Xenopus oocytes and provide further evidence that I148T-CFTR is dysfunctional despite maintaining the ability to transport chloride.

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31 These data are consistent with our observations that mutant CFTRs in which the mutation is within NBD-1, such as ⌬F508-CFTR and G551D-CFTR, lack characteristic regulatory interactions with ␣beta␥-mENaC when activated by forskolin/3-isobutyl-1-methylxanthine (IBMX) (33, 34). Login to comment
94 C604 REGULATORY INTERACTIONS OF I148T-CFTR AND ENaC AJP-Cell Physiol • VOL 292 • JANUARY 2007 • www.ajpcell.org onAugust,2011ajpcell.physiology.orgDownloadedfrom The isoflavone and CFTR potentiator genistein increases chloride transport by activated WT and mutant CFTRs, including ⌬F508-CFTR and G551D-CFTR (13, 33, 34). Login to comment
98 This enhancement of activated I148T-CFTR by genistein is similar in magnitude to the effect of genistein on WT-CFTR (ϳ2-fold; Ref. 34) but slightly smaller in magnitude compared with our previously observed enhancement of ⌬F508-CFTR-and G551D-CFTR-mediated currents by genistein (5.7and 4-fold, respectively) (33, 34). Login to comment
108 Our data regarding the regulatory interactions of ⌬F508- and G551D-CFTR also are consistent with the data of others suggesting that an intact NBD-1 is required for activated CFTR to inhibit mENaC-mediated conductance (3, 33, 34). Login to comment
116 These observations are similar to our group`s data regarding CFTRs with appropriate intracellular trafficking, such as WT-CFTR (34, 37) and G551D-CFTR (33). Login to comment
127 These data are similar to our previous data for the CFTR mutants ⌬F508 and G551D that have defective regulatory interactions with mENaC; activation of ⌬F508 and G551D with forskolin/IBMX also does not lead to acute decreases in mENaC functional expression (33, 34). Login to comment
159 Interregulation of I148T-CFTR and ␣beta␥ mENaC after IBMX/forskolin/genistein stimulation. Login to comment
160 We examined whether the CFTR potentiator genistein, which improves functional interactions between ␣beta␥-mENaC and the G551D-CFTR and ⌬F508-CFTR mutants (33, 34), is similarly able to restore I148T-CFTR`s regulatory interactions with mENaC. Login to comment
163 In contrast, I148T-CFTR/mENaC coinjected oocytes had ϳ5.5-fold greater current with addition of genistein (-5.29 Ϯ 0.81 ␮A, n ϭ 23) than in forskolin/IBMX-stimulated oocytes injected with I148T-CFTR alone (-0.97 Ϯ; 0.31 ␮A, n ϭ 19). Login to comment
164 These data suggest synergistic enhancement of I148T-CFTR functional expression by genistein and ␣beta␥-mENaC in the presence forskolin/IBMX, and are qualitatively similar to our previous observations of synergistic activation of G551D-CFTR by genistein and ␣beta␥-mENaC (33). Login to comment
171 C607REGULATORY INTERACTIONS OF I148T-CFTR AND ENaC AJP-Cell Physiol • VOL 292 • JANUARY 2007 • www.ajpcell.org -4.44 Ϯ 1.12 ␮A (ϩforskolin/IBMX/genistein) n ϭ 17, P ϭ 0.048] in oocytes injected with ␣beta␥-mENaC alone (Fig. 4B). Login to comment
173 These data are similar to our previous observations with WT-, ⌬F508-, and G551D-CFTR, where this forskolin/IBMX/genistein stimulation in mENaC-mediated current observed in oocytes injected with ␣beta␥- mENaC alone was not present in CFTR/␣beta␥-mENaC coinjected oocytes; such data are consistent with improved regulation of ␣beta␥-mENaC by the forskolin/IBMX-activated mutant CFTRs in the presence of genistein (33, 34). Login to comment
201 These findings are discussed in the context of our group`s previous observations regarding the interactions of mENaC with WT-, ⌬F508-, and G551D-CFTR, which are summarized in Table 1. Login to comment
225 Wild type N/A Same Decrease* Decrease* Increased 34, 37 32c;F508 NBD-1 Decreased No change No change No change 34 G551D NBD-1 Same Decrease No change Increased 33 I148T Intracellular loop 1 Same Decrease* No change* No change Present study WT, wild type; mENaC, murine epithelial sodium channel; NBD-1, nucleotide binding domain-1. Login to comment
228 That a similar NBD-1/R peptide fragment containing the G551D mutation (20) and the CFTR mutants G551D (33) and ⌬F508 (34) do not manifest forskolin/ IBMX-regulated inhibition of ENaC also suggests that a structurally and functionally intact NBD-1 is required for this regulatory interaction. Login to comment
235 Genistein potentiates CFTR (WT, ⌬F508, and G551D) chloride transport by increasing channel open probability (1, 36). Login to comment
239 These effects are similar to genistein`s actions to potentiate ⌬F508- and G551D-CFTR-mediated chloride transport and improve the regulatory interactions of these mutants with mENaC (33, 34). Login to comment
198 These findings are discussed in the context of our group`s previous observations regarding the interactions of mENaC with WT-, ⌬F508-, and G551D-CFTR, which are summarized in Table 1. Login to comment
222 Wild type N/A Same Decrease* Decrease* Increased 34, 37 ⌬F508 NBD-1 Decreased No change No change No change 34 G551D NBD-1 Same Decrease No change Increased 33 I148T Intracellular loop 1 Same Decrease* No change* No change Present study WT, wild type; mENaC, murine epithelial sodium channel; NBD-1, nucleotide binding domain-1. Login to comment
232 Genistein potentiates CFTR (WT, ⌬F508, and G551D) chloride transport by increasing channel open probability (1, 36). Login to comment
236 These effects are similar to genistein`s actions to potentiate ⌬F508- and G551D-CFTR-mediated chloride transport and improve the regulatory interactions of these mutants with mENaC (33, 34). Login to comment

[hide] Discovery of pyrrolo[2,3-b]pyrazines derivatives a... J Pharmacol Exp Ther. 2006 Oct;319(1):349-59. Epub 2006 Jul 7. Noel S, Faveau C, Norez C, Rogier C, Mettey Y, Becq F
Discovery of pyrrolo[2,3-b]pyrazines derivatives as submicromolar affinity activators of wild type, G551D, and F508del cystic fibrosis transmembrane conductance regulator chloride channels.
J Pharmacol Exp Ther. 2006 Oct;319(1):349-59. Epub 2006 Jul 7., [PMID:16829626]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) represents the main Cl(-) channel in the apical membrane of epithelial cells for cAMP-dependent Cl(-) secretion. Here we report on the synthesis and screening of a small library of 6-phenylpyrrolo[2,3-b]pyrazines (named RP derivatives) evaluated as activators of wild-type CFTR, G551D-CFTR, and F508del-CFTR Cl(-) channels. Iodide efflux and whole-cell patch-clamp recordings analysis identified RP107 [7-n-butyl-6-(4-hydroxyphenyl)[5H]-pyrrolo[2,3-b]pyrazine] as a submicromolar activator of wild-type (WT)-CFTR [human airway epithelial Calu-3 and WT-CFTR-Chinese hamster ovary (CHO) cells], G551D-CFTR (G551D-CFTR-CHO cells), and F508del-CFTR (in temperature-corrected human airway epithelial F508del/F508del CF15 cells). The structural analog RP108 [7-n-butyl-6-(4-chlorophenyl)[5H]pyrrolo[2,3-b]pyrazine], contrary to RP107, was a less potent activator only at micromolar concentrations. RP107 and RP108 did not have any effect on the cellular cAMP level. Activation was potentiated by low concentration of forskolin and inhibited by glibenclamide and CFTR(inh)-172 [3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl-)methylene]-2-thioxo-4- thiazolidinone]but not by calixarene or DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid). Finally, we found significant stimulation of short circuit current (I(sc)) by RP107 (EC(50) = 89 nM) and RP108 (EC(50) = 103 microM) on colon of Cftr(+)(/)(+) but not of Cftr(-/-) mice mounted in Ussing chamber. Stimulation of I(sc) was inhibited by glibenclamide but not affected by DIDS. These results show that RP107 stimulates wild-type CFTR and mutated CFTR, with submicromolar affinity by a cAMP-independent mechanism. Our preliminary structure-activity relationship study identified 4-hydroxyphenyl and 7-n-butyl as determinants required for activation of CFTR. The potency of these agents indicates that compounds in this class may be of therapeutic benefit in CFTR-related diseases, including cystic fibrosis.

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0 Discovery of Pyrrolo[2,3-b]pyrazines Derivatives as Submicromolar Affinity Activators of Wild Type, G551D, and F508del Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels Sabrina Noel, Christelle Faveau, Caroline Norez, Christian Rogier, Yvette Mettey, and Fre´ de´ ric Becq Institut de Physiologie et Biologie Cellulaires Centre National de la Recherche Scientifique (CNRS) Unite´ Mixte de Recherche 6187, Universite´ de Poitiers, Poitiers, France (S.N., C.F., C.N., C.R., Y.M., F.B.); and Faculte´ de Me´ decine et Pharmacie, Poitiers cedex, France (C.F., Y.M.) Received March 15, 2006; accepted July 6, 2006 ABSTRACT The cystic fibrosis transmembrane conductance regulator (CFTR) represents the main Cl-channel in the apical membrane of epithelial cells for cAMP-dependent Cl-secretion. Login to comment
1 Here we report on the synthesis and screening of a small library of 6-phenylpyr- rolo[2,3-b]pyrazines (named RP derivatives) evaluated as activators of wild-type CFTR, G551D-CFTR, and F508del-CFTR Cl-channels. Login to comment
2 Iodide efflux and whole-cell patch-clamp recordings analysis identified RP107 [7-n-butyl-6-(4-hydroxyphenyl)[5H]- pyrrolo[2,3-b]pyrazine] as a submicromolar activator of wild-type (WT)-CFTR [human airway epithelial Calu-3 and WT-CFTR-Chinese hamster ovary (CHO) cells], G551D-CFTR (G551D-CFTR-CHO cells), and F508del-CFTR (in temperature-corrected human airway epithelial F508del/F508del CF15 cells). Login to comment
36 Moreover, RP107 activates two CF-associated CFTR mutants (G551D-CFTR and F508del-CFTR mutants). Login to comment
61 CHO cells stably transfected with pNUT vector alone (mock-CHO) or containing wild-type CFTR (WT-CFTR-CHO) and the mutant G551D-CFTR were provided by J. R. Riordan and X. B. Chang (Mayo Clinic of Scottsdale, Scottsdale, AZ) (Tabcharani et al., 1991; Becq et al., 1994, 1999). Login to comment
66 maintained in ␣-minimal essential medium-GlutaMAX containing 7% FBS, 50 IU/ml penicillin and 50 ␮g/ml streptomycin, and methotrexate for cell selection (WT-CFTR-CHO, 100 ␮M; G551D-CHO, 20 ␮M; pNUT-CHO, 20 ␮M) (Tabcharani et al., 1991; Becq et al., 1994). Login to comment
83 Four different cell types, WT-CFTR-CHO, G551D-CFTR CHO, Calu-3, and CF-15, were incubated in Multiwell plates at 37°C in Krebs` solution containing 1 ␮M KI and 1 ␮Ci/ml Na125 I (NEN, Boston, MA) for 30 min (CHO cells) or 1 h (Calu-3 and CF15 cells) to permit the 125 I to reach equilibrium. Login to comment
196 To determine whether RP107 could activate CFTR mutants, we performed iodide efflux experiments with cells expressing two of the most common CF mutations, i.e., the class III mutation G551D-CFTR studied with CHO cells stably expressing G551D-CFTR and the class II deletion F508del-CFTR studied in human airway epithelial CF15 cells endogenously expressing F508del-CFTR protein. Login to comment
197 The mutation G551D-CFTR disrupts activation of CFTR, and phosphorylation alone is not able to achieve sufficient stimulation of G551D-dependent Cl-transport (Welsh and Smith, 1993; Becq et al., 1994; Illek et al., 1999; Marivingt-Mounir et al., 2004). Login to comment
198 Iodide efflux experiments on G551D-CFTR CHO cells have been performed in the presence of 10 ␮M fsk. Login to comment
200 However, RP107 stimulates an efflux in G551D-CFTR expressing cells with an EC50 of 1.5 Ϯ 1.1 nM in the presence of 10 ␮M fsk (n ϭ 4; Fig. 6A). Login to comment
202 This delay in response may be related to the gating defect of the G551D-CFTR channel (Welsh and Smith, 1993) and to its inability to response to high concentration of cAMP agonists (Becq et al., 1994; Illek et al., 1999; Marivingt-Mounir et al., 2004). Login to comment
216 A, effect of RP107 in G551D-CFTR-CHO cells. Login to comment
224 this case was not delayed as it was for G551D-CFTR but was similar to that of wild-type CFTR (t3 for WT-CFTR and F508del-CFTR). Login to comment
279 Discussion In the present study, we report on the discovery of 6-phe- nylpyrrolo[2,3-b]pyrazines, a novel family of wild-type CFTR, G551D, and F508del-CFTR activators. Login to comment
282 RP107 is the most potent with an affinity of 140 to 152 nM on wild-type CFTR (Calu-3 and WT-CFTR-CHO cells), 1.5 nM on G551D-CFTR, and 111 nM on temperature-corrected F508del-CFTR. Login to comment
324 It is noteworthy that trifluoromethylphe- nylbenzamine activated G551D-CFTR channels with a Kd Ͼ 10 ␮M (Ma et al., 2002). Login to comment
327 In conclusion, we have identified 6-phenylpyrrolo[2,3b]pyrazines as a new scaffold structure for the selective activation of CFTR, with submicromolar affinity on wild-type CFTR, G551D-, and F508del-CFTR, two of the most common CF mutations. 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

[hide] Cystic fibrosis mouse models. Am J Respir Cell Mol Biol. 2007 Jan;36(1):1-7. Epub 2006 Aug 3. Guilbault C, Saeed Z, Downey GP, Radzioch D
Cystic fibrosis mouse models.
Am J Respir Cell Mol Biol. 2007 Jan;36(1):1-7. Epub 2006 Aug 3., [PMID:16888286]

Abstract [show]
Animal models of cystic fibrosis (CF) are powerful tools that enable the study of the mechanisms and complexities of human disease. Murine models have several intrinsic advantages compared with other animal models, including lower cost, maintenance, and rapid reproduction rate. Mice can be easily genetically manipulated by making transgenic or knockout mice, or by backcrossing to well-defined inbred strains in a reasonably short period of time. However, anatomic and immunologic differences between mice and humans mean that murine models have inherent limitations that must be considered when interpreting the results obtained from experimental models and applying these to the pathogenesis of CF disease in humans. This review will focus on the different CF mouse models available that represent diverse phenotypes observed in humans with CF and that can help researchers elucidate the diverse functions of the CFTR protein.

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14 Several CF mouse models have been generated using a method of gene targeting in embryonic stem cells to disrupt the endogenous CFTR gene. These models have established animal models for the two most common human mutations, ⌬F508 and G551D, among others. Login to comment
34 CYSTIC FIBROSIS MOUSE MODELS Mutation Usefulness CFTRtm1UNC Exon 10 replacement Survival rates, using a liquid-nutrient diet and colyte No CFTR mRNA detectable Transgenic mice containing FABP-hCFTR gene to correct intestinal disease Susceptibility to S. aureus, B. cepacia, P. aeruginosa Resistance to V. cholerae Congenic strain B6 (lung disease) and BALB/C CFTRtm1HGU Exon 10 insertional Susceptibility to S. aureus, B. cepacia, P.aeruginosa 10% of WT CFTR mRNA CFTRtm1CAM Exon 10 replacement Transgenic mice containing hCFTR gene to correct No CFTR mRNA detectable intestinal pathology Resistance to V. cholerae CFTRtm1BAY Exon 3 insertional duplication Ͻ 2% WT CFTR mRNA CFTRtm3BAY Exon 2 replacement No CFTR mRNA detectable CFTRtm1HSC Exon 1 replacement Modifying genes for meconium ileus No CFTR mRNA detectable CFTRtm1EUR ⌬F508 exon 10 insertional "hit and run" Mutant CFTR mRNA normal levels CFTRtm2CAM ⌬F508 exon 10 replacement Resistance to S. typhi Mutant CFTR mRNA 30% of WT levels CFTRtm1KTH ⌬F508 exon 10 replacement Susceptibility to P. aeruginosa Mutant CFTR mRNA Low in intestine CFTRtm1G551D G551D exon 11 replacement Susceptibility to P. aeruginosa Mutant CFTR mRNA 53% of WT levels CFTRtm2HGU G480C exon 10 insertional "hit and run" Mutant CFTR mRNA normal levels Definition of abbreviations: CFTR, cystic fibrosis transmembrane conductance regulator; WT, wild type. Login to comment
48 The G551D mutation is a class III mutation, affecting the regulatory domain of the CFTR protein (18, 19). Login to comment
72 Furthermore, none of the ⌬F508 or the G551D models exhibit any obvious pancreatic pathology (6, 7, 13, 16-18). Login to comment
93 In the mice harboring the G551D mutation, about one-third of the animals exhibit inspissated eosinophilic material in the lumen of the pharyngeal submucosal glands. 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] 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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116 Patients With CFTR and PRSS1 Mutations CFTR Mutation 1 CFTR Mutation 2 PRSS1 Mutation 1 PRSS1 Mutation 2 No. of Patients 5T E79K 1 5T R122H 1 2789+17 C9T C139S 1 deltaF508 N29I 1 deltaF508 Q1352H G208A G208A 1 G551D R122H 1 T908N IVS4-8 C9T IVS4-11 C9T 1 Total patients 7 CFTR mutations in boldface would not have been detected by the ACOG/ACMG mutation panel. Login to comment

[hide] Immunoreactive trypsin/DNA newborn screening for c... Pediatrics. 2006 Nov;118(5):e1523-9. Epub 2006 Oct 2. Scotet V, Audrezet MP, Roussey M, Rault G, Dirou-Prigent A, Journel H, Moisan-Petit V, Storni V, Ferec C
Immunoreactive trypsin/DNA newborn screening for cystic fibrosis: should the R117H variant be included in CFTR mutation panels?
Pediatrics. 2006 Nov;118(5):e1523-9. Epub 2006 Oct 2., [PMID:17015492]

Abstract [show]
BACKGROUND: Cystic fibrosis newborn screening is now implemented universally in France, as well as in many states in the United States and in various areas of Europe and Australia. Because the screening protocol usually includes the analysis of the most common CFTR mutations, it is of the utmost importance that only mutations that result in classical cystic fibrosis are included in this test. The panels of mutations used in most cystic fibrosis newborn screening programs enable the detection of a relatively frequent CFTR variant (R117H) whose implication in cystic fibrosis remains unclear. Physicians, therefore, have difficulty managing detected compound heterozygotes with this variant, which raises the issue of the appropriateness of extended testing in families and of the legitimate use of prenatal diagnosis. OBJECTIVE: The aim of this study was to describe the clinical outcome of the children found to be compound heterozygous for R117H by screening in Brittany (western France), where cystic fibrosis newborn screening was set up in 1989, and to assess whether this CFTR variant should be included in the newborn screening mutation panels. METHODS: Data on clinical status were obtained by the referring pediatricians. RESULTS: Since our screening protocol has enabled detection of R117H (ie, in 1995), 360466 newborns have been screened for cystic fibrosis in Brittany, of whom 124 had elevated immunoreactive trypsin and 2 mutations in the CFTR gene. Nine of these children (7.3%) were compound heterozygous for R117H, which in all cases was linked to the 7T_11TG haplotype [IVS8-nT variant/m(TG) repeat]. Their genotypes were F508del/R117H (n = 7), I507del/R117H (n = 1), or G551D/R117H (n = 1). At the time of this writing, the mean age of these 9 children was 7.0 years (the oldest being >10 years of age), and none of them had yet developed any signs of cystic fibrosis; they have been pancreatic sufficient and have had good nutritional status and pulmonary function. Moreover, we observed that, in Brittany, all the patients carrying the R117H variant have been identified exclusively through cystic fibrosis newborn screening. CONCLUSIONS: In view of the high frequency of R117H-7T identified by cystic fibrosis newborn screening, the uncertain outcome of the asymptomatic children, and physicians' difficulty in managing these situations, we propose the withdrawal of the R117H variant from the panels of CFTR mutations used in cystic fibrosis newborn screening, given the expanding implementation of cystic fibrosis newborn screening.

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18 Their genotypes were F508del/R117H (n ϭ 7), I507del/R117H (n ϭ 1), or G551D/ R117H (n ϭ 1). Login to comment
67 Their genotypes were F508del/R117H (n ϭ 7), I507del/R117H (n ϭ 1), or G551D/R117H (n ϭ 1). Login to comment

[hide] CFTR (ABCC7) is a hydrolyzable-ligand-gated channe... Pflugers Arch. 2007 Feb;453(5):693-702. Epub 2006 Sep 26. Aleksandrov AA, Aleksandrov LA, Riordan JR
CFTR (ABCC7) is a hydrolyzable-ligand-gated channel.
Pflugers Arch. 2007 Feb;453(5):693-702. Epub 2006 Sep 26., [PMID:17021796]

Abstract [show]
As the product of the gene mutated in cystic fibrosis, the most common genetic disease of Caucasians, CFTR is an atypical ABC protein. From an evolutionary perspective, it is apparently a relatively young member of the ABC family, present only in metazoans where it plays a critical role in epithelial salt and fluid homeostasis. Functionally, the membrane translocation process it mediates, the passive bidirectional diffusion of small inorganic anions, is simpler than the vectorial transport of larger more complex substrates ("allocrites") by most ABC transporters. However, the control of the permeation pathway which cannot go unchecked is necessarily more stringent than in the case of the transporters. There is tight regulation by the phosphorylation/dephosphorylation of the unique CFTR R domain superimposed on the basic ABC regulation mode of ATP binding and hydrolysis at the dual nucleotide binding sites. As with other ABCC subfamily members, only the second of these sites is hydrolytic in CFTR. The phosphorylation and ATP binding/hydrolysis events do not strongly influence each other; rather, R domain phosphorylation appears to enable transduction of the nucleotide binding allosteric signal to the responding channel gate. ATP hydrolysis is not required for either the opening or closing gating transitions but efficiently clears the ligand-binding site enabling a new gating cycle to be initiated.

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244 Howell LD, Borchardt R, Cohn JA (2000) ATP hydrolysis by a CFTR domain: pharmacology and effects of G551D mutation. 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] Curcumin opens cystic fibrosis transmembrane condu... J Biol Chem. 2007 Feb 16;282(7):4533-44. Epub 2006 Dec 18. Wang W, Bernard K, Li G, Kirk KL
Curcumin opens cystic fibrosis transmembrane conductance regulator channels by a novel mechanism that requires neither ATP binding nor dimerization of the nucleotide-binding domains.
J Biol Chem. 2007 Feb 16;282(7):4533-44. Epub 2006 Dec 18., 2007-02-16 [PMID:17178710]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are essential mediators of salt transport across epithelia. Channel opening normally requires ATP binding to both nucleotide-binding domains (NBDs), probable dimerization of the two NBDs, and phosphorylation of the R domain. How phosphorylation controls channel gating is unknown. Loss-of-function mutations in the CFTR gene cause cystic fibrosis; thus, there is considerable interest in compounds that improve mutant CFTR function. Here we investigated the mechanism by which CFTR is activated by curcumin, a natural compound found in turmeric. Curcumin opened CFTR channels by a novel mechanism that required neither ATP nor the second nucleotide-binding domain (NBD2). Consequently, this compound potently activated CF mutant channels that are defective for the normal ATP-dependent mode of gating (e.g. G551D and W1282X), including channels that lack NBD2. The stimulation of NBD2 deletion mutants by curcumin was strongly inhibited by ATP binding to NBD1, which implicates NBD1 as a plausible activation site. Curcumin activation became irreversible during prolonged exposure to this compound following which persistently activated channels gated dynamically in the absence of any agonist. Although CFTR activation by curcumin required neither ATP binding nor heterodimerization of the two NBDs, it was strongly dependent on prior channel phosphorylation by protein kinase A. Curcumin is a useful functional probe of CFTR gating that opens mutant channels by circumventing the normal requirements for ATP binding and NBD heterodimerization. The phosphorylation dependence of curcumin activation indicates that the R domain can modulate channel opening without affecting ATP binding to the NBDs or their heterodimerization.

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6 Consequently, this compound potently activated CF mutant channels that are defective for the normal ATP-dependent mode of gating (e.g. G551D and W1282X), including channels that lack NBD2. Login to comment
44 CFTR constructs that are robustly activated by curcumin include two of the more common CF mutants, G551D (mutation in NBD1 (21)) and W1282X (nonsense mutation deleting most of NBD2 (22)). Login to comment
80 RESULTS ATP-independent Activation of G551D-CFTR and Wild Type CFTR Channels by Curcumin-In a previous study we observed that the G551D regulation mutant is resistant to activation by a class of CFTR agonist that strongly activates ⌬F508-CFTR and wild type channels (5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) analogs; Ref. 19). Login to comment
81 G551D-CFTR channels normally exhibit very low single channel open probabilities (Po) because this mutation disrupts the ABC signature sequence in NBD1, which lines the ATP-binding pocket at the NBD dimer interface in other ABC transporters (9-11). Login to comment
82 Fig. 1A shows that G551D-CFTR channels are strongly stimulated by curcumin. Login to comment
86 As noted previously, the neutral NPPB derivative (NPPB-AM) only weakly activates G551D-CFTR channels at a dose that maximally activates the wild type channel (10 ␮M; Ref. 19). Login to comment
87 Conversely, curcumin exhibited a strong and dose-dependent activation of the G551D-CFTR currents (see also single channel data in Fig. 3). Login to comment
91 Because the G551D-CFTR mutant is strongly disrupted for the normal ATP-dependent mode of channel gating, we tested whether curcumin also could activate this mutant in the absence of bath ATP. Login to comment
92 Fig. 1B shows that curcumin strongly stimulates G551D-CFTR currents after removing bath ATP and by adding hexokinase/glucose to enzymatically eliminate residual ATP. Login to comment
97 Thus, both wild type channels and G551D-CFTR channels can be activated by curcumin under these conditions. Login to comment
105 Curcumin Activates CFTR Channels That Lack NBD2-The finding that curcumin promotes the opening of G551D-CFTR channels and wild type channels in the absence of ATP raised the possibility that this compound acts at a step downstream of ATP binding and possibly NBD dimerization. Login to comment
107 To our initial surprise we found that CFTR channels that lack the entire NBD2 as well as the COOH-terminal tail (⌬1198-CFTR) express at high levels in HEK-293T cells where they localize to the cell surface as efficiently as wild type or G551D-CFTR channels (see schematic of constructs in Fig. 2A and surface biotinylation results in Fig. 2B). Login to comment
109 Like for G551D-CFTR channels, however, the currents mediated by ⌬1198-CFTR are markedly stimulated by curcumin (Fig. 2C). Login to comment
117 This feature was not unique to the activation of ⌬1198-CFTR currents, i.e. persistently activated currents were observed for every CFTR construct that was tested including G551D and wild type CFTR (e.g. Fig. 1A). Login to comment
124 A, curcumin strongly stimulates G551D-CFTR in a dose-dependent fashion. Login to comment
128 B, curcumin also activates G551D-CFTR channels in the absence of ATP. Login to comment
152 Gating Properties of Curcumin-activated Channels; Curcumin Increases the Opening Rates of G551D-CFTR Channels and the NBD2 Deletion Mutants-To explore how curcumin influences the gating properties of these mutant constructs, we tested its effects on G551D-CFTR, ⌬1198-CFTR, and W1282X-CFTR channels in excised micropatches containing small numbers of channels (less than eight). Login to comment
156 Individual records for G551D-CFTR, ⌬1198-CFTR, and W1282X-CFTR channels are shown in Fig. 3 (A, B, and D). Login to comment
157 Mean data for the stimulation of ⌬1198-CFTR channels by 5 ␮M curcumin are summarized in Fig. 3C. Not surprisingly, the most obvious effect of curcumin was to increase the opening rates of G551D-CFTR and the NBD2 deletion constructs, which otherwise open at extremely low rates. Login to comment
179 A, G551D-CFTR. Login to comment
187 It should be noted that ATP did not obviously blunt the curcumin activation of channels that possess NBD2 (e.g. G551D-CFTR and wild type channels; results not shown). Login to comment
208 A similar PKA dependence of the curcumin effect was observed for G551D-CFTR, W1282X-CFTR, and wild type channels (results not shown). Login to comment
214 DISCUSSION Our data indicate that curcumin strongly activates mutant CFTR channels that normally have very low activities because of defects in ATP binding and/or NBD heterodimerization (e.g. G551D-CFTR and W1282X-CFTR channels). Login to comment
222 Curcumin Activation Is Phosphorylation-dependent-Curcumin activates the NBD2 deletion constructs or G551D-CFTR channels in the absence of active PKA in the bath or following deletion of most of the R domain. Login to comment
253 Tight ATP binding to NBD1 is less likely for mutants that are disrupted for ATP binding and/or the dimerization of the two NBDs (e.g. G551D, W1282X, and ⌬1198-CFTR), given that dimerization would be expected to stabilize ATP binding to NBD1 (9-11). Login to comment
256 Interestingly, the activation of G551D-CFTR channels by curcumin is not blunted by ATP (results not shown). Login to comment
257 Presumably this difference is due to the presence of the second NBD in the G551D mutant that also modulates channel gating and that may inhibit the curcumin response in the absence of ATP (e.g. by reducing curcumin access to its binding site). Login to comment
266 If so, then curcumin (or more potent analogs) might have value for treating CF patients with mutations that primarily disrupt the normal ATP-dependent mode of channel regulation (e.g. G551D). Login to comment

[hide] Regulatory interactions of N1303K-CFTR and ENaC in... Am J Physiol Cell Physiol. 2007 Apr;292(4):C1553-61. Epub 2006 Dec 20. Suaud L, Yan W, Carattino MD, Robay A, Kleyman TR, Rubenstein RC
Regulatory interactions of N1303K-CFTR and ENaC in Xenopus oocytes: evidence that chloride transport is not necessary for inhibition of ENaC.
Am J Physiol Cell Physiol. 2007 Apr;292(4):C1553-61. Epub 2006 Dec 20., [PMID:17182731]

Abstract [show]
Regulatory interactions of the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial Na(+) channel (ENaC) are readily apparent in Xenopus oocytes. However, the mechanism underlying these interactions remains controversial. CFTR's first nucleotide binding fold (NBD-1) may be important in these interactions, as dysfunctional CFTRs containing mutations within NBD-1, such as DeltaF508 and G551D, lack such functional interactions with murine ENaC (mENaC). We hypothesized that a dysfunctional CFTR containing a non-NBD-1 mutation would retain regulatory interactions with mENaC and tested this hypothesis for N1303K-CFTR, where the mutation is located in CFTR's second nucleotide binding fold (NBD-2). cRNA for alphabetagamma-mENaC and N1303K-CFTR was injected separately or together into Xenopus oocytes. ENaC and CFTR functional expression was assessed by two-electrode voltage clamp. Injection of N1303K (class II trafficking mutation) yielded low levels of CFTR function on activation with forskolin and 3-isobutyl-1-methylxanthine (IBMX). In coinjected oocytes, N1303K did not alter mENaC functional expression or surface expression before activation of N1303K. This is similar to our prior observations with DeltaF508. However, unlike our observations with DeltaF508, activation of N1303K acutely decreased mENaC functional and surface expression, and N1303K currents were enhanced by coinjection of mENaC. Furthermore, genistein only mildly enhanced the functional expression of N1303K-CFTR and did not improve regulation of ENaC by N1303K-CFTR. These data suggest that a structurally and functionally intact CFTR NBD-1 in activated CFTR can regulate mENaC surface expression independent of Cl(-) transport in Xenopus oocytes.

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9 CFTR`s first nucleotide binding fold (NBD-1) may be important in these interactions, as dysfunctional CFTRs containing mutations within NBD-1, such as ⌬F508 and G551D, lack such functional interactions with murine ENaC (mENaC). Login to comment
29 A similar NBD-1/R peptide fragment containing the G551D mutation did not demonstrate forskolin/IBMX-regulated inhibition of ENaC (19). Login to comment
30 CFTRs containing NBD-1 mutations, such as ⌬F508-CFTR and G551D-CFTR, lack regulatory interactions with ␣beta␥-murine ENaC (mENaC) when activated by forskolin-IBMX (31, 32). Login to comment
31 These and other data (4, 29) suggest that form and/or function of NBD-1 is important in the inhibition of ENaC by activated CFTR. Interestingly, regulation of mENaC by ⌬F508 and G551D was restored by further activation or "repair" of these mutants with genistein (31, 32), which may interact with CFTR`s second nucleotide binding fold (NBD-2) (25). Login to comment
40 We also observed reduced activation of N1303K by genistein and a lack of improved N1303K regulation of mENaC with genistein, which differs significantly from our previous data with ⌬F508 and G551D (31, 32). Login to comment
102 The isoflavone genistein increases Cl-transport by activated WT and mutant CFTRs including ⌬F508-CFTR and G551D-CFTR (13, 31, 32). Login to comment
107 Interestingly, this stimulation of activated N1303K-CFTR by genistein is minimal compared with our previously observed stimulation of ⌬F508-CFTR and G551D-CFTR by genistein (5.7-fold and 4-fold stimulation by genistein, respectively) (31, 32). Login to comment
139 These observations directly contrast with our previous observations with G551D-CFTR, where a similar magnitude of stimulated G551D-mediated current did not result in a decrease in amiloride-sensitive whole oocyte current (31). Login to comment
189 We therefore examined whether the CFTR potentiator genistein, which activates G551D-CFTR, ⌬F508-CFTR, and WT CFTR Cl-conductance and restores functional interactions between ␣beta␥- mENaC and the G551D-CFTR and ⌬F508-CFTR mutants (31, 32), is able to activate N1303K-CFTR and improve its regulation with mENaC. Login to comment
192 In contrast, coinjection of ␣beta␥-mENaC increases forskolin/ IBMX/genistein-stimulated G551D-CFTR-mediated current in oocytes but does not alter G551D`s Po (31). Login to comment
206 These data contrast with our previous observations with WT, ⌬F508-, and G551D-CFTR, where such an increase in ␣beta␥-mENaC-mediated current after forskolin-IBMX-genistein in CFTR/mENaC-coinjected oocytes was not present (31, 32). Login to comment
207 These data are consistent with genistein not further improving regulation of ␣beta␥-mENaC by N1303K-CFTR and therefore being a less effective potentiator of N1303K-CFTR function than it is for ⌬F508- and G551D-CFTR. Login to comment
212 These data demonstrate synergistic enhancement of N1303K-CFTR functional expression by genistein and ␣beta␥-mENaC in the presence of forskolin-IBMX, and are similar to our previous data suggesting synergistic enhancement of G551D-CFTR (31) and I148T-CFTR (33) function by genistein and ␣beta␥-mENaC. Login to comment
256 A similar NBD-1/R peptide fragment containing the G551D mutation did not demonstrate forskolin/IBMX-regulated inhibition of ENaC (19). Login to comment
259 We previously showed (31, 32) that CFTRs with NBD-1 mutations (G551D-CFTR and ⌬F508-CFTR) lack the ability to regulate ␣beta␥-mENaC when activated by forskolin-IBMX. Login to comment
266 Genistein activates CFTR (WT, ⌬F508, and G551D) Cl-transport by increasing channel open time and decreasing channel closing, thereby increasing channel Po (1, 34). Login to comment
270 These data contrast with our previous observations that genistein enhanced Cl-transport by WT CFTR and the NBD-1 mutants ⌬F508- and G551D-CFTR by four- to sixfold (31, 32). Login to comment
271 Furthermore, while genistein improves the regulation of ␣beta␥-mENaC by activated ⌬F508- and G551D-CFTR (31, 32), it did not enhance regulation of ␣beta␥-mENaC by activated N1303K. Login to comment

[hide] Misassembled mutant DeltaF508 CFTR in the distal s... J Cell Sci. 2007 Feb 1;120(Pt 3):447-55. Epub 2007 Jan 9. Gentzsch M, Choudhury A, Chang XB, Pagano RE, Riordan JR
Misassembled mutant DeltaF508 CFTR in the distal secretory pathway alters cellular lipid trafficking.
J Cell Sci. 2007 Feb 1;120(Pt 3):447-55. Epub 2007 Jan 9., 2007-02-01 [PMID:17213331]

Abstract [show]
Most patients with cystic fibrosis (CF) have a single codon deletion (DeltaF508) in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) that impairs assembly of the multidomain glycoprotein. The mutant protein escapes endoplasmic reticulum (ER) quality control at low temperature, but is rapidly cleared from the distal secretory pathway and degraded in lysosomes. CF cells accumulate free cholesterol similar to Niemann-Pick disease type C cells. We show that this lipid alteration is caused by the presence of misassembled mutant CFTR proteins, including DeltaF508, in the distal secretory pathway rather than the absence of functional CFTR. By contrast, cholesterol distribution is not changed by either D572N CFTR, which does not mature even at low temperature, or G551D, which is processed normally but is inactive. On expression of the DeltaF508 mutant, cholesterol and glycosphingolipids accumulate in punctate endosomal structures and cholesterol esters are reduced, indicating a block in the translocation of cholesterol to the ER for esterification. This is overcome by Rab9 overexpression, resulting in clearance of accumulating intracellular cholesterol. Similar but less pronounced alterations in intracellular cholesterol distribution are observed on expression of a temperature-rescued mutant variant of the related ATP-binding cassette (ABC) protein multidrug resistance-associated protein 1 (MRP1). Thus, on escape from ER quality control, misassembled mutants of CFTR and MRP1 impair lipid homeostasis in endocytic compartments.

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18 By contrast, cholesterol distribution is not changed by either D572N CFTR, which does not mature even at low temperature, or G551D, which is processed normally but is inactive. Login to comment
51 Neither the severe disease-causing mutation G551D, which prevents CFTR channel activation although it is processed normally (Cutting et al., 1990; Gregory et al., 1991), nor the D572N mutation, which is retained at the ER at high or low temperature, changed cholesterol distribution from normal. Login to comment
76 Western blots showing maturation of CFTR and CFTR variants ⌬F508, 1410X, D572N and G551D grown at 37°C and 27°C. Login to comment
78 D572N CFTR is retained at the ER at high or low temperature and the severe-disease-causing mutation G551D, which prevents CFTR channel activation, is processed normally at 37°C and 27°C. Login to comment
86 Microscopy showing filipin staining of CFTR and CFTR variants ⌬F508, 1410X, D572N and G551D grown at 27°C. Login to comment
162 Stable BHK-21 cell lines expressing G551D and D572N variants of CFTR or the C-terminal truncation 1410X CFTR were established as described previously (Chang et al., 1993; Gentzsch and Riordan, 2001; Loo et al., 1998). 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] Functional analysis of mutations in the putative b... J Biol Chem. 2007 Mar 23;282(12):9098-104. Epub 2007 Jan 23. Zegarra-Moran O, Monteverde M, Galietta LJ, Moran O
Functional analysis of mutations in the putative binding site for cystic fibrosis transmembrane conductance regulator potentiators. Interaction between activation and inhibition.
J Biol Chem. 2007 Mar 23;282(12):9098-104. Epub 2007 Jan 23., 2007-03-23 [PMID:17244607]

Abstract [show]
An increasing number of compounds able to potentiate the activity of mutants of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have been identified by high throughput screening or by individual search of derivatives of known active compounds. Several lines of evidence suggest that most CFTR potentiators act through the same mechanism, probably by binding to the nucleotide binding domains to promote the activity of the protein and then, with lower affinity, to an inhibitory site. With the aim of identifying the activating binding site, we recently modeled the nucleotide binding domain dimer and predicted a common binding site for potentiators in its interface. To validate this model experimentally, we mutated some of the residues involved in the putative binding site, i.e. Arg(553), Ala(554), and Val(1293). The activity of CFTR potentiators was measured as apical membrane currents on polarized cells stably expressing wild type or mutated proteins. CFTR activity was elicited by application of a membrane-permeable cAMP analogue followed by increasing concentrations of potentiators. We found that all three mutants responded to cAMP, although the affinity of R553Q was higher than that of wild type CFTR. In R553Q and V1293G mutants, the dissociation constant of potentiators for the activating site was increased, whereas the dissociation constant for the inhibitory site was reduced. Our results show that the mutated residues are part of the activating binding site for potentiators, as suggested by the molecular model. In addition, these results suggest that the activating and inhibitory sites are not independent of each other.

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37 Printed in the U.S.A. 9098 tion that mutations in conserved residues of the NBDs such as G551D and G1349D exhibit a shift in the affinity for potentiators (5, 15-18). Login to comment
41 After in silico docking of several compounds, we compared the theoretical binding free energy measured on the model, with the experimental binding free energy obtained from dissociation constants from wild type, G551D, and G1349D proteins. We found a good correlation between these two parameters for a putative binding site located in the interface of the NBD1-NBD2 dimer, embedded in a cavity on NBD1, and interacting also with the NBD2 surface. Login to comment
99 TABLE 1 Parameters obtained from the fit to Equation 1 of CPTcAMP dose-response relationship on wild type and mutant CFTRs For comparison, the parameters of G551D, a severe CF-causing mutation, are included. Login to comment
100 Wild type A554E V1293G R553Q G551D n ϭ 5 n ϭ 4 n ϭ 4 n ϭ 6 n ϭ 7 Imax (␮A/cm2 ) 282.2 Ϯ 13.5 66.9 Ϯ 16.4a 70.2 Ϯ 14a 93.8 Ϯ 19a 10.1 Ϯ 2.8a Kd (␮M) 54.2 Ϯ 11.5 40.5 Ϯ 5.9 48.4 Ϯ 13.5 21.5 Ϯ 4.5a 74.2 Ϯ 12.1 I(20)/I(max) 0.3 Ϯ 0.04 0.34 Ϯ 0.03 0.32 Ϯ 0.05 0.51 Ϯ 0.05a 0.23 Ϯ 0.03 a p Ͻ 0.05. Login to comment
150 Mutations of NBDs, like the CF mutation G551D (see Table 1 and Ref. 15) or the other mutations studied here, A554E and V1293G, do not seem to change significantly the sensitivity of the protein to CPTcAMP. Login to comment
155 An even more marked reduction had been found previously for G551D (see Table 1 and Refs. Login to comment
156 15 and 18), but in that case we measured the expression of both G551D and wild type proteins. We concluded that the small amount of G551D current was due to a severe gating defect of the mutant and not to reduced expression levels, as confirmed by the reduced open channel probability estimated from single channel recording. Login to comment
186 Probably, the NBD with higher affinity for potentiators is NBD1, because CF mutation G551D, situated on NBD1 near the potentiator binding site, causes a more pronounced effect on the equilibrium constant for the activation site than the symmetrical CF mutation on NBD2, G1349D (16). Login to comment

[hide] Molecular study of (TG)m(T)n polymorphisms in Iran... J Androl. 2007 Jul-Aug;28(4):541-7. Epub 2007 Feb 21. Radpour R, Gourabi H, Gilani MA, Dizaj AV
Molecular study of (TG)m(T)n polymorphisms in Iranian males with congenital bilateral absence of the vas deferens.
J Androl. 2007 Jul-Aug;28(4):541-7. Epub 2007 Feb 21., [PMID:17314234]

Abstract [show]
Congenital bilateral absence of the vas deferens (CBAVD) is a frequent cause of obstructive azoospermia. Nearly 75% of men with CBAVD have at least 1 detectable common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The different alleles at the (TG)(m)(T)(n) polymorphic locus at the 3' end of human CFTR intron 8 determine the efficiency of exon 9 splicing. To study the CFTR gene mutations and (TG)(m)(T)(n) polymorphisms in Iranian CBAVD patients with presumed low CF frequency and to better understand the complex regulation of exon 9 splicing among our study population, we analyzed CFTR mutations and (TG)(m)(T)(n) polymorphisms in 112 Iranian CBAVD, 7 congenital unilateral absence of the vas deferens males from Iran, and 84 fertile males as controls. Moreover, we compared the rate of CFTR transcripts with exon 9 (9+) with reduction of the (T)(n) repeat in our study population. Our study showed that the 5T mutation was present with high frequency in our patients. Longer (TG)(m) polymorphic tracts increase the proportion of exon 9 deletion transcripts but only when activated by the 5T allele. The combination of the 5T allele in 1 copy of the CFTR gene with a CF mutation in the other copy is the most common cause of CBAVD in the Iranian population. We also observed the highest level of exon 9+ splicing efficiency among the tested samples with the (TG)(12)(T)(7) allele, which represents the most common intron 8 splice variant allele in the general population. Our results support the idea that a putative role of the (T)(n) repeat is to distance the (TG)(m) repeat from the 3' splice site and that the different alleles at the (T)(n) locus affect the efficiency by which the splice acceptor consensus sequence is recognized.

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77 CFTR gene mutations in 112 CBAVD patients and 7 CBAVD patients* Samples Mutation genotype3 (TG)m(T)n n (%) CBAVD Two mutations detected (5 /112 5 4.46%) F508del / R117H (TG)10 9T / (TG)10 9T 1 (0.89) F508del / 621+1G.T (TG)11 7T / (TG)11 7T 1 (0.89) 1540A/G / 1540A/G (TG)11 7T / (TG)11 7T 2 (1.79) R347H / R117H (TG)10 9T / (TG)11 7T 1 (0.89) One mutation detected with one 5T allele (32 / 112 5 28.57%) G551D / - (TG)10 7T/ (TG)13 5T 2 (1.79) F508del / - (TG)12 7T/ (TG)13 5T 8 (7.14) (TG)11 9T/ (TG)13 5T 6 (5.36) 1717-1G.A / - (TG)11 7T/ (TG)12 5T 4 (3.57) R117H / - (TG)12 7T/ (TG)13 5T 2 (1.79) 621+1G.T / - (TG)11 7T/ (TG)13 5T 3 (2.68) 2 (1.79) 1540A/G / - (TG)11 7T/ (TG)13 5T 2 (1.79) R553X / - (TG)12 7T/ (TG)13 5T 1 (0.89) Y122H / -4 (TG)11 7T / (TG)13 5T 1 (0.89) T338A / -4 (TG)10 7T / (TG)13 5T 1 (0.89) No mutation detected with two 5T alleles (11 / 112 5 9.82%) - / - (TG)12 5T / (TG)13 5T 3 (2.68) - / - (TG)13 5T / (TG)13 5T 8 (7.14) One mutation detected without 5T allele (35 / 112 5 31.25%) G85E / - (TG)11 7T / (TG)11 7T 2 (1.79) G551D / - (TG)10 9T / (TG)12 7T1 1 (0.89) 621+1G.T / - (TG)11 7T / (TG)11 7T 2 (1.79) (TG)10 9T / (TG)11 7T 1 (0.89) R334W / - (TG)12 7T / (TG)10 7T 1 (0.89) F508del / - (TG)11 7T / (TG)11 7T 7 (6.25) (TG)11 9T / (TG)12 7T 3 (2.68) (TG)10 9T / (TG)10 9T 2 (1.79) 1717-1G.A / - (TG)11 7T / (TG)12 7T 3 (2.68) (TG)10 9T / (TG)11 7T 2 (1.79) R117H/- (TG)12 7T / (TG)12 7T 2 (1.79) (TG)10 9T / (TG)11 7T 1 (0.89) 2789+5G.A / - (TG)10 7T / (TG)11 7T 1 (0.89) 3120+1G.A / - (TG)10 9T / (TG)11 7T 2 (1.79) R560T / - (TG)10 9T / (TG)11 7T 1 (0.89) N1303K / - (TG)10 9T / (TG)11 7T 1 (0.89) 1651A/G / - (TG)11 7T / (TG)12 7T 1 (0.89) R553X / - (TG)10 9T / (TG)10 7T 1 (0.89) K536X / -4 (TG)10 9T / (TG)10 9T 1 (0.89) No mutation detected with one 5T alleles (7 / 112 5 6.25%) - / - (TG)13 5T / (TG)12 7T 3 (2.68) - / - (TG)13 5T / (TG)10 9T 4 (3.57) No mutation detected (22 / 112 5 19.64%) - / - (TG)11 7T / (TG)11 7T 12 (10.71) - / - (TG)11 7T / (TG)12 7T 1 (1.79) - / - (TG)10 9T / (TG)10 9T 3 (2.68) - / - (TG)10 9T / (TG)11 7T 6 (5.36) CUAVD One mutation detected without 5T allele (2 / 7 5 28.57%) R334W / - (TG)10 9T / (TG)11 7T 1 (14.29) R117H / - (TG)11 7T / (TG)11 7T 1 (14.29) No mutation detected with one 5T alleles (3 / 7 5 42.86%) - / - (TG)11 9T / (TG)13 5T 2 (28.57) - / - (TG)10 7T / (TG)13 5T 1 (14.29) No mutation detected (2 / 7 5 28.57%) - / - (TG)10 9T / (TG)12 7T 2 (28.57) * CBAVD indicates congenital bilateral absence of the vas deferens; CUAVD, congenital unilateral absence of the vas deferens. 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

[hide] G551D and G1349D, two CF-associated mutations in t... J Gen Physiol. 2007 Apr;129(4):285-98. Epub 2007 Mar 12. Bompadre SG, Sohma Y, Li M, Hwang TC
G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects.
J Gen Physiol. 2007 Apr;129(4):285-98. Epub 2007 Mar 12., [PMID:17353351]

Abstract [show]
Mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR) result in cystic fibrosis (CF). CFTR is a chloride channel that is regulated by phosphorylation and gated by ATP binding and hydrolysis at its nucleotide binding domains (NBDs). G551D-CFTR, the third most common CF-associated mutation, has been characterized as having a lower open probability (Po) than wild-type (WT) channels. Patients carrying the G551D mutation present a severe clinical phenotype. On the other hand, G1349D, also a mutant with gating dysfunction, is associated with a milder clinical phenotype. Residues G551 and G1349 are located at equivalent positions in the highly conserved signature sequence of each NBD. The physiological importance of these residues lies in the fact that the signature sequence of one NBD and the Walker A and B motifs from the other NBD form the ATP-binding pocket (ABP1 and ABP2, named after the location of the Walker A motif) once the two NBDs dimerize. Our studies show distinct gating characteristics for these mutants. The G551D mutation completely eliminates the ability of ATP to increase the channel activity, and the observed activity is approximately 100-fold smaller than WT-CFTR. G551D-CFTR does not respond to ADP, AMP-PNP, or changes in [Mg(2+)]. The low activity of G551D-CFTR likely represents the rare ATP-independent gating events seen with WT channels long after the removal of ATP. G1349D-CFTR maintains ATP dependence, albeit with a Po approximately 10-fold lower than WT. Interestingly, compared to WT results, the ATP dose-response relationship of G1349D-CFTR is less steep and shows a higher apparent affinity for ATP. G1349D data could be well described by a gating model that predicts that binding of ATP at ABP1 hinders channel opening. Thus, our data provide a quantitative explanation at the single-channel level for different phenotypes presented by patients carrying these two mutations. In addition, these results support the idea that CFTR's two ABPs play distinct functional roles in gating.

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1 (c) The Rockefeller University Press $15.00 Volume 129 Number 4 April 2007 285-298 http://www.jgp.org/cgi/doi/10.1085/jgp.200609667 285 A RT I C L E G551D and G1349D, Two CF-associated Mutations in the Signature Sequences of CFTR, Exhibit Distinct Gating Defects Silvia G. Bompadre,1,2 Yoshiro Sohma,2,3 Min Li,1,2 and Tzyh-Chang Hwang1,2 1Department of Medical Pharmacology and Physiology, 2Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO 65211 3Department of Physiology, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan Mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR) result in cystic fibrosis (CF). Login to comment
3 G551D-CFTR, the third most common CF-associated mutation, has been characterized as having a lower open probability (Po) than wild-type (WT) channels. Login to comment
4 Patients carrying the G551D mutation present a severe clinical phenotype. Login to comment
9 The G551D mutation completely eliminates the ability of ATP to increase the channel activity, and the observed activity is ‫-001ف‬fold smaller than WT-CFTR. Login to comment
10 G551D-CFTR does not respond to ADP, AMP-PNP, or changes in [Mg2+]. Login to comment
11 The low activity of G551D-CFTR likely represents the rare ATP-independent gating events seen with WT channels long after the removal of ATP. Login to comment
25 These mutations can be divided into four classes based on the mechanisms that disrupt CFTR function (Welsh and Smith, 1993): defective protein production (I); defective protein processing (II); defective activation and regulation (III), including G551D and G1349D; and defective conductance (IV). Login to comment
26 G551D is the third overall most common CF mutation with a worldwide frequency of ‫%3ف‬ (www.genet .sickkids.on.ca/cftr). Login to comment
28 The result of this glycine-to-aspartate mutation at position 551 is a significantly decreased Correspondence to Tzyh-Chang Hwang: hwangt@health.missouri.edu Abbreviations used in this paper: ABC, ATP-binding cassette; ABP, ATP binding pocket; CFTR, cystic fibrosis transmembrane conductance regulator; NBD, nucleotide binding domain. Login to comment
33 Although G551D-CFTR and G1349D-CFTR have been used in pharmacological studies for years (e.g., Illek et al., 1999; Galietta et al., 2001; Moran et al., 2005; Pedemonte et al., 2005), little is known about the mechanism responsible for their dysfunction due to limited functional studies of these two mutants at a single-channel level (e.g., Cai et al., 2006). Login to comment
36 Using a fluorescence-based assay they observed that G551D-CFTR channels lack functional activity, even though the channels can be normally phosphorylated in the R domain (Chang et al., 1993). Login to comment
37 Logan et al. (1994) reported that as a consequence of the G551D, or G1349D mutation, nucleotide binding to isolated recombinant NBD1 and NBD2 is decreased. Login to comment
39 Wilkinson et al. (1996) measured the rates of activation and deactivation of macroscopic CFTR currents in Xenopus oocytes and found that the apparent on rate of channel activation by cAMP for G551D-CFTR or G1349D-CFTR was drastically reduced. Login to comment
42 We have studied the response of G551D-CFTR and G1349D-CFTR to ATP, ADP, and AMP-PNP in excised inside-out membrane patches. Login to comment
44 The G551D mutation completely abolishes the response of the channel to ATP and ADP, whereas the G1349D mutation remains responsive to ATP and the ATP-induced activity can be inhibited by ADP. Login to comment
50 Point mutations (G551D and G1349D) were introduced into WT-CFTR by QuikChange XL method (Stratagene). Login to comment
60 Thus the G551D mutation is located in ABP2 and the G1349D mutation is located in ABP1. Login to comment
102 AMP-PNP was purchased from Roche and stored as 250 mM stock in H2O at -20°C. R E S U LT S Expression of G551D-CFTR and G1349D-CFTR It has been previously reported that neither G551D-CFTR nor G1349D-CFTR exhibit trafficking defects in COS cells (Gregory et al., 1991). Login to comment
105 G551D-CFTR and G1349D-CFTR expression in CHO cells. Login to comment
106 (A) Western blot analysis for WT-, G551D-, and G1349D-CFTR. Login to comment
110 (B) Mean current densities obtained from whole-cell experiments for G551D (n = 30), G1349D (n = 29), and WT (n = 20). Login to comment
111 Columns and error bars indicate means ± SEM, * indicates P < 0.01 between G551D and G1349D. Login to comment
115 We transfected CHO cells with WT-CFTR, G551D-CFTR, and G1349-CFTR, in parallel, using the same amount of DNA. Login to comment
119 Fig. 2 B shows that the current densities of G551D (n = 30) and G1349D (n = 29) mutants are lower than that of WT-CFTR (n = 20). Login to comment
120 However, the current density of G1349D-CFTR is significantly larger than that of G551D-CFTR (P < 0.01), suggesting that the Po of G1349D-CFTR is higher than that of G551D-CFTR. Login to comment
121 ATP-dependent Gating of G551D-CFTR and G1349D-CFTR To investigate the mechanism responsible for the different gating behavior of G551D and G1349D mutants, we studied both mutants in excised inside-out membrane patches. Login to comment
125 Gating of G551D-CFTR and G1349D-CFTR in excised inside-out membrane patches. Login to comment
126 (A) A recording of G551D-CFTR channel currents in an excised inside-out membrane patch. Login to comment
128 Expanded current traces show the G551D-CFTR channel activity in the presence of ATP + PKA and during washout. Login to comment
129 (B) Repeated addition and removal of ATP in the same patch did not result in significant changes of the channel activity of G551D-CFTR. Login to comment
135 (E) Comparisons of mean macroscopic current amplitude for G551D- (n = 16), G1349D- (n = 18), and WT-CFTR (n = 13). Login to comment
137 In the case of G551D-CFTR, the elicited currents were usually small compared with the macroscopic current seen with WT-CFTR (Fig. 3, compare A and D). Login to comment
138 Surprisingly, when ATP and PKA were removed, the G551D-CFTR current remained unchanged for tens of minutes (n = 46). Login to comment
139 Fig. 3 A shows a 25-min continuous recording of G551D-CFTR. Login to comment
140 We are confident that the observed current arises from G551D-CFTR channels because no activity was observed in nontransfected cells and the observed currents were PKA dependent. Login to comment
141 The single-channel amplitude of G551D-CFTR is similar to that of WT channels (see below), and furthermore, glibenclamide, a CFTR blocker (Zhou et al., 2002), blocks G551D-CFTR currents to the same extent as it does to WT-CFTR (unpublished data). Login to comment
142 Repeated addition and removal of ATP in the same patch did not result in significant changes of the channel activity (Fig. 3 B), suggesting that G551D-CFTR channel activity is ATP independent. Login to comment
144 When the same experiment was performed with G1349D-CFTR, we observed generally larger currents than that of G551D-CFTR (e.g., Fig. 3 C). Login to comment
145 In addition, unlike G551D-CFTR, G1349D-CFTR channel current decreases immediately upon ATP washout, a property shared with the WT channels (Fig. 3 D). Login to comment
148 The steady-state mean current amplitude (a product of the number of functional channels, Po and single-channel amplitude) shows a similar pattern as that of whole-cell recordings (i.e., WT > G1349D > G551D). Login to comment
153 Since the Po for WT channels in the presence of 1 mM ATP is 0.45 ± 0.04 (see Fig. 9 D), the estimated Po are ≅ 0.0037 for G551D (120 times smaller than the WT Po), and ≅ 0.045 (10 times smaller than the WT Po) for G1349D. Login to comment
155 Comparison of the mean open times of G551D-, G1349D-, and WT-CFTR. Login to comment
156 (A) Expanded single-channel current traces in the presence of 1 mM ATP for G551D-, G1349D-, and WT-CFTR. Login to comment
159 Figure 5. Effect of [Mg2+] in the closing rate of WT and G551D-CFTR. Login to comment
163 (B) G551D-CFTR channel openings remain unaltered when ATP, PKA, and Mg2+ are removed from the perfusion solution (n = 6). Login to comment
168 It is interesting to note that Cai et al. (2006) found that the G551D and G1349D mutations shorten the open time of the channels. Login to comment
171 Fig. 5 shows representative traces for WT and G551D-CFTR. Login to comment
173 That is not the case for G551D-CFTR, where the current remains unaltered when we switch from Mg2+-containing solution to Mg2+- free solution without ATP (n = 6). Login to comment
174 The mean open time for G551D-CFTR in the Mg2+-free solution is 311 ± 85 ms (n = 3), similar to the open time observed in the Mg2+- containing solution. Login to comment
176 Effect of ADP and AMP-PNP on G551D-CFTR and G1349D-CFTR Channels The results shown above indicate that G551D and G1349D, mutations at the equivalent position in the signature sequence of NBD1 and NBD2, respectively, exhibit different gating defects. Login to comment
181 However, ADP had little effect on G551D-CFTR currents (n = 10, Fig. 6 B), further confirming that G551D channel currents are ATP independent. Login to comment
185 In excised patches, after activation with 1 mM ATP + PKA, we applied 2 mM AMP-PNP + 1 mM ATP to WT (Fig. 7 A), G551D (Fig. 7 B), Figure 6. Effect of ADP on G551D, G1349D, and WT-CFTR. Login to comment
186 Representative current traces for WT-CFTR (A), G551D-CFTR (B), and G1349D-CFTR (C). Login to comment
187 Currents from both WTand G1349D-CFTR are reduced by ADP, but ADP fails to inhibit G551D-CFTR channel currents. Login to comment
188 (D) Summary of percent inhibition by 500 μM ADP for G551D- (n =10), G1349D- (n = 8), and WT-CFTR (n = 8). Login to comment
196 In contrast, AMP-PNP failed to increase either the G551D- or G1349D-CFTR currents (Fig. 7, B and C). Login to comment
199 [ATP] Dependence of G551Dand G1349D-CFTR Our data suggest that the G551D mutation abolished nucleotide-dependent gating. Login to comment
201 Since the signature sequence itself is not an ATP binding site before the two NBDs dimerize (Locher et al., 2002; Smith et al., 2002; Lewis et al., 2004), it seems unlikely that the G551D mutation will affect ATP dose-response relationship, which reflects ATP affinity of the closed state when the NBDs are in monomeric configurations. Login to comment
202 G551D-CFTR activity was examined at different [ATP]. Login to comment
203 Fig. 8 A shows that the channel activity of G551D-CFTR is not altered even in the presence of 10 mM ATP. Login to comment
218 Qualitatively speaking, as the [ATP] is increased, the activity of the G1349D mutant does not increase as much as that of WT channels, as if the increasing activation of G1349D-CFTR upon Figure 7. Effect of AMP-PNP on G551D, G1349D, and WT-CFTR. Login to comment
219 Representative current traces for WT-CFTR (A), G551D-CFTR (B), and G1349D-CFTR (C). Login to comment
221 Neither G551D- nor G1349D-CFTR responds to AMP-PNP (n = 4 each). Login to comment
238 ATP dose-response relationship for G551D-CFTR. Login to comment
239 (A) Representative trace for G551D-CFTR in the presence of 10 mM ATP. Login to comment
241 (B) Normalized ATP dose-response relationship for G551D-CFTR. Login to comment
257 The parameters for the spontaneous openings were obtained from Bompadre et al. (2005b) (measured for ∆R-CFTR), and we assumed that ATP binding to ABP1 has little effect on the ATP-independent opening (i.e., kO(SPT0) = kO(SPT1) = kO(SPT)), an assumption supported by Zhou et al. (2006) and the current results with the G551D mutation (see Discussion). Login to comment
258 We also assumed that the closing rate of both types of spontaneous openings is the same (kc(SPT0) = kc(SPT1) = kc(SPT)) since the mean open time of G551D-CFTR is approximately the same as the mean open time of WT channels in the absence of ATP. Login to comment
288 D I S C U S S I O N The data presented in the current studies strongly suggest that the G551D mutation of NBD1 completely eliminates ATP-dependent gating. Login to comment
289 The residual low activity of G551D-CFTR represents ATP-independent gating events. Login to comment
290 On the other hand, G1349D-CFTR exhibits a fairly different behavior than G551D-CFTR; it maintains some ATP dependence, but with a lower Po than WT channels due to a lower opening rate. Login to comment
311 We showed that G551D channels, though still need to be phosphorylated to be functional, do not respond to ATP at all. Login to comment
312 This lack of response to ATP also explains why the G551D mutant is insensitive to ADP since the main effect of ADP is to competitively inhibit channel opening by ATP (Bompadre et al., 2005a). Login to comment
314 If we propose that all the activity observed with the G551D channels represents spontaneous ATP-independent openings, the Po of G551D should be compatible with that of the ATP-independent activity of WT channels. Login to comment
318 Although it is very difficult, if not impossible, to estimate precisely the Po for G551D from single-channel kinetic analysis, we made a rough estimation by comparing macroscopic current amplitudes between WT and G551D channels if we assume the number of channels in the membrane patches is about the same. Login to comment
319 Fig. 3 E shows that the average mean current of G551D-CFTR in excised patches is ‫021ف‬ times smaller than that of WT channels. Login to comment
320 This result is consistent quantitatively with our hypothesis that G551D-CFTR only exhibits ATP-independent openings. Login to comment
325 Thus, the G551D mutation more likely hampers the conformational changes at ABP2 that facilitate NBD dimerization (i.e., channel opening by ATP). Login to comment
326 On the other hand, if we assume, here, that the G551D mutation does not affect ATP binding at ABP1 (since the mutation is located at ABP2), the failure of ATP to increase the activity of this mutant supports the notion that ATP binding at ABP1 alone indeed does not catalyze channel opening (Zhou et al., 2006; Scheme B, Fig. 11). Login to comment
327 Then, G551D-CFTR openings represent spontaneous openings either with no ATP bound at the ABPs or with ATP bound at ABP1. Login to comment
330 It is worth to note that Cai et al. (2006) reported that 2`-deoxy ATP can potentiate G551D-CFTR channel activity. Login to comment
332 In contrast to G551D-CFTR, G1349D-CFTR still maintains some ATP dependence, but has a maximal Po ‫-01ف‬fold lower than WT-CFTR. Login to comment
348 It is interesting to note that AMP-PNP does not lock open either G551D or G1349D channels. Login to comment
351 In the case of G551D, it is not surprising that we do not see any effects with AMP-PNP because this mutation completely eliminates ATP-dependent gating. Login to comment
356 In addition to the mechanistic insights into how CFTR`s two NBDs work concertedly to gate the channel, our observations also provide a quantitative explanation for the different phenotypes exhibited in CF patients carrying the G551D or G1349D mutation. Login to comment
364 Since genistein increases the activity of G551D-CFTR (e.g., Illek et al., 1999) our results suggest that genistein`s action may involve more than a simple effect on ATP-dependent gating as previously proposed (Weinreich, et al., 1997; Wang et al., 1998). Login to comment
367 On the other hand, 20 μM genistein increases G551D current density by 6.5 ± 2.4-fold (n = 19). Login to comment
413 2006. Differential sensitivity of the cystic fibrosis (CF)-associated mutants G551D and G1349D to potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl-channel. Login to comment
473 Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein. Login to comment
507 Phenylglycine and sulfonamide correctors of defective ∆F508 and G551D cystic fibrosis transmembrane conductance regulator chloride-channel gating. 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] Structure-activity relationship of 1,4-dihydropyri... Mol Pharmacol. 2007 Jul;72(1):197-207. Epub 2007 Apr 23. Pedemonte N, Boido D, Moran O, Giampieri M, Mazzei M, Ravazzolo R, Galietta LJ
Structure-activity relationship of 1,4-dihydropyridines as potentiators of the cystic fibrosis transmembrane conductance regulator chloride channel.
Mol Pharmacol. 2007 Jul;72(1):197-207. Epub 2007 Apr 23., [PMID:17452495]

Abstract [show]
Mutations occurring in the CFTR gene, encoding for the cystic fibrosis transmembrane conductance regulator chloride channel, cause cystic fibrosis (CF). Mutations belonging to class II, such as DeltaPhe508, give rise to a protein with both a defective maturation and altered channel gating. Mutations belonging to class III, such as G551D and G1349D, cause only a gating defect. We have previously identified antihypertensive 1,4-dihydropyridines (DHPs), a class of drugs that block voltage-dependent Ca(2+) channels, as effective potentiators of CFTR gating, able to correct the defective activity of CFTR mutants (Mol Pharmacol 68:1736-1746, 2005). However, optimization of potency for CFTR versus Ca(2+) channels is required to design selective compounds for CFTR pharmacotherapy. In the present study, we have established DHP structure-activity relationship for both CFTR potentiation and Ca(2+) channel inhibition using cell-based assays for both types of channels. A panel of 333 felodipine analogs was studied to understand the effect of various substitutions and modifications in the DHP scaffold. Our results show that alkyl substitutions at the para position of the 4-phenyl ring lead to compounds with very low activity on Ca(2+) channels and strong effect as potentiators on the DeltaPhe508, G551D, and G1349D CFTR mutants.

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2 Mutations belonging to class III, such as G551D and G1349D, cause only a gating defect. Login to comment
7 Our results show that alkyl substitutions at the para position of the 4-phenyl ring lead to compounds with very low activity on Ca2ϩ channels and strong effect as potentiators on the ⌬Phe508, G551D, and G1349D CFTR mutants. Login to comment
27 The most common class III mutation is G551D (glycine-to-aspartic acid change at position 551), with a worldwide frequency of 3.1% of CF alleles (Hamosh et al., 1992). Login to comment
31 For example, high concentrations of flavonoids such as genistein can improve ⌬Phe508- and G551D-CFTR channel gating (Hwang et al., 1997; Illek et al., 1999; Zegarra-Moran et al., 2002). Login to comment
34 Conversely, phenylglycines, another class of compounds identified by high-throughput screening, are effective also on G551D and G1349D channels. Login to comment
36 Among them, felodipine was the most potent compound, having activity on ⌬Phe508- and G551D-CFTR. Login to comment
45 To this purpose, we screened a set of 333 felodipine analogs using cell-based assays to determine their activity on three CFTR mutants (⌬Phe508, G551D, and G1349D) and on DHP-sensitive Ca2ϩ channels. Login to comment
48 Fischer rat thyroid (FRT) cells were stably transfected with ⌬Phe508, G551D, or G1349D-CFTR, and the halide-sensitive yellow fluorescent protein mutant YFP-H148Q/I152L (Galietta et al., 2001a). Login to comment
66 Measurements of CFTR activity were carried out on FRT cells expressing mutant CFTR and the halide-sensitive YFP 24 h (G551D and G1349D) or 48 h (⌬Phe508) after plating on microplates. Login to comment
124 The panel of compounds was tested on FRT cells coexpressing the halide-sensitive mutant H148Q/I152L of the fluorescent protein YFP and the CFTR mutants ⌬Phe508, G551D, and G1349D. Login to comment
186 We found consistently that G551D was the most refractory to potentiation; the concentrations needed to stimulate this mutant were always 10 to 20 times higher than those active on ⌬Phe508 (Fig. 8A, see also Supplemental Data). Login to comment
187 Conversely, the G1349D mutant displayed a sensitivity between that of ⌬Phe508 and G551D (Fig. 8, B and C). Login to comment
195 In agreement with all previous observations, activity on G551D required higher concentrations, with Ka values of 1 to 6 ␮M (Fig. 9D). Login to comment
199 DHP-194 was the most potent compound, with apparent Ka values of approximately 0.11 and 1.2 ␮M for ⌬Phe508-CFTR and G551D-CFTR, respectively, in agreement with the values derived from the fluorescence assay (0.11 and 2.0 ␮M). Login to comment
201 For example, DHP-229 displayed Ka values of 0.32 and 2.6 ␮M for ⌬Phe508 and G551D channels, respectively, in fluorescence experiments and of 0.31 and 4.0 ␮M in short-circuit current recordings. Login to comment
202 In contrast, genistein had a reduced potency for ⌬Phe508 (Ka of 18 ␮M in fluorescence assays and 14 ␮M in short-circuit current experiments) and for G551D (Ka of 94 ␮M in fluorescence assays and 124 ␮M in short-circuit current experiments). Login to comment
203 The maximal response to the compounds in ⌬Phe508-CFTR cells was comparable with that to genistein, whereas in G551D-CFTR cells, the activity elicited by DHPs was at least 2-fold larger than that evoked by genistein, as already described for antihypertensive DHP drugs (Pedemonte et al., 2005b). Login to comment
206 In particular, CFTR potentiators are needed to restore activity in those CFTR mutants affected by impaired channel gating (class III mutants), like G551D and G1349D. Login to comment
209 In a previous study, we described the effect of antihypertensive 1,4-dihydropyridines as potentiators of ⌬Phe508 and G551D-CFTR (Pedemonte et al., 2005b). Login to comment
214 Note that the concentrations needed to stimulate the G551D mutant are always 10 to 20 times higher than those effective on ⌬Phe508 or G1349D. Login to comment
223 In parallel, we have used the functional assay based on the halide-sensitive YFPs to measure activity of compounds on ⌬Phe508, G551D, and G1349D CFTR mutants. Login to comment
225 Our first result is that DHP-based structures activate also G1349D-CFTR, besides ⌬Phe508 and G551D mutants. Login to comment
229 In contrast, G551D required consistently higher concentrations, as already found for other CFTR activators (Zegarra-Moran et al., 2002, Pedemonte et al., 2005a; Zegarra-Moran et al., 2007). Login to comment
237 B-E, dose-response relationships obtained for indicated compounds on VDCC (B), ⌬Phe508-CFTR (C), G551D-CFTR (D), and G1349D-CFTR (E). 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] 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] 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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54 Eighteen of the heterozygous group were ⌬F508 heterozygotes with another described mutation of the CFTR gene; 11 patients were heterozygous with one allele (10 ⌬F508; one G551D) identified at the time of genotype testing, with the other allele remained unidentified. Login to comment
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] Coincidental CFTR mutation and Sjogren syndrome re... Pancreas. 2007 Jul;35(1):94-5. Frossard JL, Dumonceau JM, Spahr L, Giostra E, Hadengue A
Coincidental CFTR mutation and Sjogren syndrome revealing a pancreatic disease.
Pancreas. 2007 Jul;35(1):94-5., [PMID:17575550]

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26 Genetic testing revealed a double-allele CFTR mutations (G551D/IVS8-5T). Login to comment

[hide] Discovery of alpha-aminoazaheterocycle-methylglyox... J Pharmacol Exp Ther. 2007 Sep;322(3):1023-35. Epub 2007 Jun 19. Routaboul C, Norez C, Melin P, Molina MC, Boucherle B, Bossard F, Noel S, Robert R, Gauthier C, Becq F, Decout JL
Discovery of alpha-aminoazaheterocycle-methylglyoxal adducts as a new class of high-affinity inhibitors of cystic fibrosis transmembrane conductance regulator chloride channels.
J Pharmacol Exp Ther. 2007 Sep;322(3):1023-35. Epub 2007 Jun 19., [PMID:17578899]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) represents the main Cl(-) channel in the apical membrane of epithelial cells for cAMP-dependent Cl(-) secretion. Here we report on the synthesis and screening of a small library of nontoxic alpha-aminoazaheterocycle-methylglyoxal adducts, inhibitors of wild-type (WT) CFTR and G551D-, G1349D-, and F508del-CFTR Cl(-) channels. In whole-cell patch-clamp experiments of Chinese hamster ovary (CHO) cells expressing WT-CFTR, we recorded rapid and reversible inhibition of forskolin-activated CFTR currents in the presence of the adducts 5a and 8a,b at 10 pM concentrations. Using iodide efflux experiments, we compared concentration-dependent inhibition of CFTR with glibenclamide (IC(50) = 14.7 microM), 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl-)methylene]-2-thioxo-4-t hiazolidinone (CFTR(inh)-172) (IC(50) = 1.2 microM), and alpha-aminoazaheterocycle-methylglyoxal adducts and identified compounds 5a (IC(50) = 71 pM), 8a,b (IC(50) = 2.5 nM), and 7a,b (IC(50) = 3.4 nM) as the most potent inhibitors of WT-CFTR channels. Similar ranges of inhibition were also found when these compounds were evaluated on CFTR channels with the cystic fibrosis mutations F508del (in temperature-corrected human airway epithelial F508del/F508del CF15 cells)-, G551D-, and G1349D-CFTR (expressed in CHO and COS-7 cells). No effect of compound 5a was detected on the volume-regulated or calcium-regulated iodide efflux. Picomolar inhibition of WT-CFTR with adduct 5a was also found using a 6-methoxy-N-(3-sulfopropyl)-quinolinium fluorescent probe applied to the human tracheobronchial epithelial cell line 16HBE14o-. Finally, we found comparable inhibition by 5a or by CFTR(inh)-172 of forskolin-dependent short-circuit currents in mouse colon. To the best of our knowledge, these new nontoxic alpha-aminoazaheterocycle-methylglyoxal adducts represent the most potent compounds reported to inhibit CFTR chloride channels.

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1 Here we report on the synthesis and screening of a small library of nontoxic ␣-aminoazaheterocycle-methylglyoxal adducts, inhibitors of wild-type (WT) CFTR and G551D-, G1349D-, and F508del-CFTR Cl-channels. Login to comment
4 Similar ranges of inhibition were also found when these compounds were evaluated on CFTR channels with the cystic fibrosis mutations F508del (in temperature-corrected human airway epithelial F508del/ F508del CF15 cells)-, G551D-, and G1349D-CFTR (expressed in CHO and COS-7 cells). Login to comment
25 Here we present the synthesis and identification of ␣-aminoazaheterocycle-methylglyoxal adducts as highly potent inhibitors of wild-type CFTR and CFTR affected by the following CF mutations: F508del, G551D, and G1349D. Login to comment
73 All cell lines were grown under standard culture conditions, as follows. CHO cells stably transfected with pNUT vector alone (mock-CHO) or containing wild-type CFTR (WT-CFTR-CHO) or the mutant G551D-CFTR were provided by J. R. Riordan and X. B. Chang (Scottsdale, AZ) (Tabcharani et al., 1991; Becq et al., 1994, 1999). Login to comment
74 They were maintained at 37°C in 5% CO2 in ␣-minimal essential medium-GlutaMAX containing 7% fetal bovine serum (FBS), 50 IU/ml penicillin and 50 ␮g/ml streptomycin, and methotrexate for cell selection (WT-CFTR-CHO: 100 ␮M, G551D-CHO: 20 ␮M) (Tabcharani et al., 1991; Becq et al., 1994). Login to comment
116 CFTR-dependent iodide efflux was stimulated either by forskolin (WT-CFTR) or by a cocktail containing forskolin with genistein (F508del- and G551D-CFTR) or with the benzo[c]quinolizinium derivative MPB-91 (G1349D-CFTR). Login to comment
190 The present study was undertaken on the following cells: CHO cells overexpressing WT-CFTR or mutated G551D-CFTR and COS-7 cells expressing G1349D-CFTR (both G551D and G1349D are class III CF mutations); two human airway epithelial cells, Calu-3 and 16HBE14o-, expressing endogenous WT-CFTR; and the human airway epithelial cells JME/CF15, endogenously expressing F508del-CFTR (class II CF mutation). Login to comment
209 Experiments were performed in the presence of 1 ␮M Fsk (WT-CFTR), 10 ␮M Fsk ϩ 30 ␮M genistein (F508del- and G551D-) or 10 ␮M ϩ 250 ␮M MPB-91 (G1349D-CFTR). Login to comment
210 Compounds (Ratio a,b Determined by NMR Spectrometry) Endogenous CFTR Heterologous Expression of CFTR WT-CFTR F508del-CFTR WT-CFTR G551D-CFTR G1349D-CFTR Glibenclamide 11.7 Ϯ 1.1 ␮M 11.8 Ϯ 1.1 ␮M 14.7 Ϯ 1.3 ␮M 7.9 Ϯ 1.6 ␮M 9.0 Ϯ 1.4 ␮M CFTRinh-172 N.D. N.D. 1.2 Ϯ 0.9 ␮M N.D. N.D. 5a 93.3 Ϯ 1.3 pM 67.3 Ϯ 1.3 pM 71.0 Ϯ 1.2 pM 43.1 Ϯ 1.5 nM 72.3 Ϯ 1.1 pM 8a,b (60:40) 15.7 Ϯ 1.1 nM 8.7 Ϯ 1.2 nM 2.5 Ϯ 1.7 nM 190 Ϯ 2 nM 79.4 Ϯ 2 nM 7a,b (60:40) 2.3 Ϯ 1.5 nM 6.3 Ϯ 2.1 nM 3.4 Ϯ 1.2 nM 154 Ϯ 2 nM 7.0 Ϯ 1.1 nM 3a,b (75:25) 11.2 Ϯ 1.6 ␮M 7.0 Ϯ 1.4 ␮M 4.4 Ϯ 1.3 M 35.5 Ϯ 1.6 ␮M 9.0 Ϯ 1.8 ␮M 4a,b (60:40) 11.9 Ϯ 1.7 ␮M 6.0 Ϯ 3.3 ␮M 5.7 Ϯ 1.3 ␮M 110 Ϯ 2 ␮M 2.9 Ϯ 1.4 ␮M N.D., complete dose-response curve was not determined, but the inhibition for each cell type with 10 ␮M concentrations of inhibitors was confirmed. Login to comment
258 G551D-CFTR activity, expressed in CHO cells, was analyzed in response to ␣-aminoazaheterocycle-methylglyoxal adducts. Login to comment
260 Glibenclamide inhibited G551D-CFTR with an IC50 of 7.9 Ϯ 1.6 ␮M, similar to that for WT-CFTR (Table 1). Login to comment
261 It is interesting to note that to inhibit G551D-CFTR (whereas glibenclamide is equally potent com- Fig. 7. Login to comment
270 In contrast with G551D, adducts 3a,b, 4a,b, 5a (IC50 of 72.3 Ϯ 1.1 pM), 7a,b, and 8a,b inhibited G1349D-CFTR with affinities similar to that of WT-CFTR (Table 1). Login to comment
293 B, with CHO cells overexpressing G551D-CFTR and COS-7 cells expressing G1349D-CFTR. Login to comment
296 molar affinity in G551D-CFTR cells. 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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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

[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 transmembrane conductance regulato... Sheng Li Xue Bao. 2007 Aug 25;59(4):431-42. Bompadre SG, Hwang TC
Cystic fibrosis transmembrane conductance regulator: a chloride channel gated by ATP binding and hydrolysis.
Sheng Li Xue Bao. 2007 Aug 25;59(4):431-42., 2007-08-25 [PMID:17700963]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that belongs to the ATP-binding cassette (ABC) transporter superfamily. Defective function of CFTR is responsible for cystic fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasian populations. The disease is manifested in defective chloride transport across the epithelial cells in various tissues. To date, more than 1400 different mutations have been identified as CF-associated. CFTR is regulated by phosphorylation in its regulatory (R) domain, and gated by ATP binding and hydrolysis at its two nucleotide-binding domains (NBD1 and NBD2). Recent studies reveal that the NBDs of CFTR may dimerize as observed in other ABC proteins. Upon dimerization of CFTR's two NBDs, in a head-to-tail configuration, the two ATP-binding pockets (ABP1 and ABP2) are formed by the canonical Walker A and B motifs from one NBD and the signature sequence from the partner NBD. Mutations of the amino acids that interact with ATP reveal that the two ABPs play distinct roles in controlling ATP-dependent gating of CFTR. It was proposed that binding of ATP to the ABP2, which is formed by the Walker A and B in NBD2 and the signature sequence in NBD1, is critical for catalyzing channel opening. While binding of ATP to the ABP1 alone may not increase the opening rate, it does contribute to the stabilization of the open channel conformation. Several disease-associated mutations of the CFTR channel are characterized by gating defects. Understanding how CFTR's two NBDs work together to gate the channel could provide considerable mechanistic information for future pharmacological studies, which could pave the way for tailored drug design for therapeutical interventions in CF.

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47 In the S.S., there are two important disease-associated mutations, G551D in NBD1 and G1349D in NBD. Login to comment
208 G551D, located in the signature sequence of NBD1, is the third most common CF-associated mutation (www.genet. Login to comment
210 Patients carrying the G551D mutation present a severe phenotype[63,64] . Login to comment
213 [55] found that the G551D mutation (located at the ABP2) completely eliminates the ability of ATP to increase the opening rate of the channel (Fig.5). Login to comment
214 ADP does not inhibit G551D-CFTR currents and AMP-PNP does not lock the channel open. Login to comment
215 The observed low activity of G551D-CFTR channels likely represents the ATP-independent openings also seen with the wild-type channels[38,55] . Login to comment
217 Interestingly, the high affinity ATP analog P-ATP increases G551D-CFTR currents mainly by increasing the open time of the channel[66] . Login to comment
218 Introducing the mutationY1219G (located at ABP2) in the G551D background, which reduces the ATP-binding affinity by more than 50-fold in wild-type channels[56] , does not alter significantly the effect of P-ATP. Login to comment
219 However, the mutation W401G/G551D (W401G is located at ABP1) decreases remarkably the effect of P-ATP, suggesting that it is at this binding site (ABP1) where P-ATP binds to increase the G551D-CFTR activity[66] . Login to comment
224 Gating of G551D-CFTR and G1349D-CFTR. Login to comment
225 A: Recording of G551D-CFTR current in an excised inside-out membrane patch. Login to comment
263 Lately, our observation that P-ATP enhances G551D by binding to ABP1 implicates that ABP1 can potentially be a target for drugs to bind and increase the channel activity. 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
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

[hide] The cystic fibrosis transmembrane recruiter the al... Pflugers Arch. 2007 Nov;455(2):215-21. Epub 2007 Sep 6. Mehta A
The cystic fibrosis transmembrane recruiter the alter ego of CFTR as a multi-kinase anchor.
Pflugers Arch. 2007 Nov;455(2):215-21. Epub 2007 Sep 6., [PMID:17805562]

Abstract [show]
This review focuses on a newly discovered interaction between protein kinases involved in cellular energetics, a process that may be disturbed in cystic fibrosis for unknown reasons. I propose a new model where kinase-mediated cellular transmission of energy provides mechanistic insight to a latent role of the cystic fibrosis transmembrane conductance regulator (CFTR). I suggest that CFTR acts as a multi-kinase recruiter to the apical epithelial membrane. My group finds that, in the cytosol, two protein kinases involved in cell energy homeostasis, nucleoside diphosphate kinase (NDPK) and AMP-activated kinase (AMPK), bind one another. Preliminary data suggest that both can also bind CFTR (function unclear). The disrupted role of this CFTR-kinase complex as 'membrane transmitter to the cell' is proposed as an alternative paradigm to the conventional ion transport mediated and CFTR/chloride-centric view of cystic fibrosis pathogenesis. Chloride remains important, but instead, chloride-induced control of the phosphohistidine content of one kinase component (NDPK, via a multi-kinase complex that also includes a third kinase, CK2; formerly casein kinase 2). I suggest that this complex provides the necessary near-equilibrium conditions needed for efficient transmission of phosphate energy to proteins controlling cellular energetics. Crucially, a new role for CFTR as a kinase controller is proposed with ionic concentration acting as a signal. The model posits a regulatory control relay for energy sensing involving a cascade of protein kinases bound to CFTR.

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113 For example, the G551D mutation is an example that Fig. 1 CFTR modelled as a pair of scissors with each ATP trapped in a finger hole and sandwiched between the two ATP binding domains. Login to comment
123 In G551D, for example, the G551 residue lies within a molecular lid that traps one gamma phosphate of ATP between the first and second nucleotide binding domains. Login to comment
124 The extra negative charge in G551D means that this lid is malformed, perhaps being more repulsive to phosphate. 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
8 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. Login to comment
141 Each genotype traces a different path, with the exception of p.G551D and p.R553X. Login to comment
162 The exceptions included 3-bp deletions, p.I507del/p.F508del, and 2 single-base variant pairs (p.G551D/p.R553X and p.W1282X/ c.4002AϾG). Login to comment
165 Fig. 4 in the online Data Supplement details an unlabeled probe assay to differentiate p.G551D from p.R553X. Login to comment
205 The substitutions were 5 bp (p.G551D and p.R553X) and 24 bp (p.W1282X and c.4002AϾG) apart, and the variants within each pair were of the same type (16). 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
173 All of the mutations in the clone mix appear homozygous (mutant band present, wild-type band not present) with the exception at the G551D mutation site, which appears heterozygous (mutant and wild-type bands present). Login to comment
174 This is expected, since, as discussed above, clone 1 contains the G551D mutation sequence and clone 3 contains the G551 wild-type sequence. Login to comment
203 The G551/R553 gene region is represented by four bead populations, one for each normal allele, G551 and R553, and one for each mutant allele, G551D and R553X. Login to comment
228 See results for a further description of the G551D and R553X heterozygous sites on clones 1 and 3. Login to comment

[hide] Chronic inflammation in the cystic fibrosis lung: ... Clin Rev Allergy Immunol. 2008 Apr;34(2):146-62. Nichols D, Chmiel J, Berger M
Chronic inflammation in the cystic fibrosis lung: alterations in inter- and intracellular signaling.
Clin Rev Allergy Immunol. 2008 Apr;34(2):146-62., [PMID:17960347]

Abstract [show]
A vicious cycle of airway obstruction, infection, and inflammation continues to cause most of the morbidity and mortality in cystic fibrosis (CF). Mutations that result in decreased expression or function of the membrane Cl(-) channel, cystic fibrosis transmembrane regulator (CFTR), result in a decrease in the volume (and hence the depth) of liquid on the airway surface, impaired ciliary function, and dehydrated glandular secretions. In turn, these abnormalities contribute to a milieu, which promotes chronic infection with a limited but unique spectrum of microorganisms. Defects in CFTR also perturb regulation of several intracellular signaling pathways including signal transducers and activator of transcription, I-kappaB and nuclear factor-kappa B, and low molecular weight GTPases. Together, these abnormalities result in excessive production of NF-kappaB dependent cytokines such as interleukin (IL)-1, tumor necrosis factor (TNF), IL-6, and IL-8. There are decreased responses to interferon gamma and transforming growth factor beta leading to decreased production of iNOS and NO. Abnormalities of lipid mediators and decreased secretion of counter/regulatory cytokines have also been reported. Together, these effects combine to create a chronic inflammatory process, which damages and obstructs the airways, and eventually claims the life of the patient.

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300 Studies in (G551D) cftr-/- mice in which normal human CFTR was selectively expressed in particular cell types 154 using transgenes with tissue-specific promoters suggested that defective CFTR function in the epithelium was necessary and sufficient to cause characteristic abnormalities in the response to LPS in the lung [136]. Login to comment

[hide] Novel pharmaceutical approaches for treating patie... Curr Pharm Des. 2007;13(31):3252-63. Saeed Z, Wojewodka G, Marion D, Guilbault C, Radzioch D
Novel pharmaceutical approaches for treating patients with cystic fibrosis.
Curr Pharm Des. 2007;13(31):3252-63., [PMID:18045175]

Abstract [show]
Before the cloning of the CFTR gene in 1989, there were relatively few treatment options for the many phenotypes associated with cystic fibrosis (CF). The advancement of research in areas such as immunology, molecular biology and pharmacology have provided new insights into the mechanism and evolution of CF. More than 40 systematic clinical trials evaluating new therapies for CF are presently registered with the NIH. A great deal of effort is focused on the main cause of mortality: chronic and persistent lung infections. Intestinal malabsorption, pancreatic insufficiency, reduced bone mineral density and reproductive abnormalities are other manifestations of this disease that have been targeted by innovated treatments which are giving renewed hope to CF patients and their families. The following review is a summary of the novel pharmaceutical approaches for the treatment of cystic fibrosis aimed at improving both the quality and the longevity of the lives of patients afflicted with this devastating disease.

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14 These mutations have been grouped into 5 different classes of protein dysfunctions as shown in Fig. (1) [3]: • Class I- defective protein maturation and premature degradation • Class II- deregulation of the CFTR protein processing, for example diminished ATP binding and hydrolysis ( F508 mutation found in 70-80% of CF patients) • Class III- block in regulation which causes a disruption of activation and regulation of the CFTR protein at the plasma membrane, for example the protein may be defective in ATP binding and hydrolysis, or phosphorylation (G551D mutation). Login to comment
52 Class I is described when there is defective protein maturation and premature degradation; Class II is a deregulation of the CFTR protein processing, for example diminished ATP binding and hydrolysis ( F508 mutation found in 70-80% of CF patients); Class III occurs when regulation is blocked causing a disruption in activation and regulation of CFTR protein at the plasma membrane, for example the protein may be defective in respect to ATP binding and hydrolysis, or phosphorylation (G551D mutation). Login to comment

[hide] The role of the UPS in cystic fibrosis. BMC Biochem. 2007 Nov 22;8 Suppl 1:S11. Turnbull EL, Rosser MF, Cyr DM
The role of the UPS in cystic fibrosis.
BMC Biochem. 2007 Nov 22;8 Suppl 1:S11., [PMID:18047735]

Abstract [show]
CF is an inherited autosomal recessive disease whose lethality arises from malfunction of CFTR, a single chloride (Cl-) ion channel protein. CF patients harbor mutations in the CFTR gene that lead to misfolding of the resulting CFTR protein, rendering it inactive and mislocalized. Hundreds of CF-related mutations have been identified, many of which abrogate CFTR folding in the endoplasmic reticulum (ER). More than 70% of patients harbor the DeltaF508 CFTR mutation that causes misfolding of the CFTR proteins. Consequently, mutant CFTR is unable to reach the apical plasma membrane of epithelial cells that line the lungs and gut, and is instead targeted for degradation by the UPS. Proteins located in both the cytoplasm and ER membrane are believed to identify misfolded CFTR for UPS-mediated degradation. The aberrantly folded CFTR protein then undergoes polyubiquitylation, carried out by an E1-E2-E3 ubiquitin ligase system, leading to degradation by the 26S proteasome. This ubiquitin-dependent loss of misfolded CFTR protein can be inhibited by the application of 'corrector' drugs that aid CFTR folding, shielding it from the UPS machinery. Corrector molecules elevate cellular CFTR protein levels by protecting the protein from degradation and aiding folding, promoting its maturation and localization to the apical plasma membrane. Combinatory application of corrector drugs with activator molecules that enhance CFTR Cl- ion channel activity offers significant potential for treatment of CF patients. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).

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145 For example, the potentiator VRT-532 was able to potentiate ion channel activity in the CFTR mutants ΔF508 and G551D, inferring its potential as a drug treatment for CF patients in combination with VRT-325 [81]. Login to comment

[hide] Mechanism of G551D-CFTR (cystic fibrosis transmemb... J Biol Chem. 2008 Feb 29;283(9):5364-9. Epub 2007 Dec 30. Bompadre SG, Li M, Hwang TC
Mechanism of G551D-CFTR (cystic fibrosis transmembrane conductance regulator) potentiation by a high affinity ATP analog.
J Biol Chem. 2008 Feb 29;283(9):5364-9. Epub 2007 Dec 30., 2008-02-29 [PMID:18167357]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel gated by ATP binding and hydrolysis at its nucleotide binding domains (NBD). The NBDs dimerize in a head-to-tail configuration, forming two ATP binding pockets (ABP) with the ATP molecules buried at the dimer interface. Previous studies have indicated that ABP2, formed by the Walker A and B motifs of NBD2 and the signature sequence of NBD1, is the site critical for the ATP-dependent opening of CFTR. The G551D mutation in ABP2, the third most common cystic fibrosis-associated mutation, abolishes ATP-dependent gating, resulting in an open probability that is approximately 100-fold lower than that of wild-type channels. Interestingly, we found that the ATP analog N6-(2-phenylethyl)-ATP (P-ATP) increases G551D currents mainly by increasing the open time of the channel. This effect is reduced when P-ATP is applied together with ATP, suggesting a competition between ATP and P-ATP for a common binding site. Introducing mutations that lower the nucleotide binding affinity at ABP2 did not alter significantly the effects of P-ATP on G551D-CFTR, whereas an equivalent mutation at ABP1 (consisting of the Walker A and B motifs of NBD1 and the signature sequence of NBD2) dramatically decreased the potency of P-ATP, indicating that ABP1 is the site where P-ATP binds to increase the activity of G551D-CFTR. These results substantiate the idea that nucleotide binding at ABP1 stabilizes the open channel conformation. Our observation that P-ATP enhances the G551D activity by binding at ABP1 implicates that ABP1 can potentially be a target for drugs to bind and increase the channel activity.

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0 Mechanism of G551D-CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) Potentiation by a High Affinity ATP Analog*□S Received for publication,November 16, 2007 Published, JBC Papers in Press,December 30, 2007, DOI 10.1074/jbc.M709417200 Silvia G. Bompadre‡1 , Min Li‡ , and Tzyh-Chang Hwang‡§ From the ‡ Dalton Cardiovascular Research Center and the § Department of Medical Pharmacology and Physiology, University of Missouri-Columbia, Columbia, Missouri 65211 Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel gated by ATP binding and hydrolysis at its nucleotide binding domains (NBD). Login to comment
3 The G551D mutation in ABP2, the third most common cystic fibrosis-associated mutation, abolishes ATP-dependent gating, resultinginanopenprobabilitythatisϳ100-foldlowerthanthatof wild-type channels. Login to comment
4 Interestingly, we found that the ATP analog N6 -(2-phenylethyl)-ATP (P-ATP) increases G551D currents mainly by increasing the open time of the channel. Login to comment
6 Introducingmutationsthatlowerthenucleotidebindingaffinityat ABP2 did not alter significantly the effects of P-ATP on G551D-CFTR, whereas an equivalent mutation at ABP1 (consisting of the Walker A and B motifs of NBD1 and the signature sequence of NBD2) dramatically decreased the potency of P-ATP, indicating that ABP1 is the site where P-ATP binds to increase the activity of G551D-CFTR. Login to comment
16 The importance of the signature sequence in CFTR gating is attested by the fact that mutations such as G551D and G1349D in thisregionoftheproteinareassociatedwithCF.G551D,locatedin the signature sequence of NBD1, is one of the most common CF-associated mutations (the Cystic Fibrosis Mutation Database). Login to comment
17 G551D-CFTR channels exhibit much lower open probability than wild-type(WT)channels(7-9),andconsequently,thepatientscar- rying this mutation present a severe phenotype (10, 11). Login to comment
18 G551D has been used as a model in pharmacological characterizations of CFTR (9,12-15),butthefundamentalmechanismforthefunctionaldefect has been unclear until recently. Login to comment
19 We found that the G551D mutation completely eliminates the ability of ATP to increase the opening rate of the channel (7), consistent with the idea that ATP binding to ABP2 is critical for the ATP-dependent opening of CFTR channels(6).TheobservedlowactivityofG551D-CFTRlikelyrep- resents the rare ATP-independent openings seen with the WT-CFTR channels in the absence of ATP (7, 16). Login to comment
20 Although G551D-CFTR does not respond to ATP, interestingly Cai et al. (9) showed that the ATP analog 2Ј-deoxy-ATP increases the Po of G551D-CFTR. Login to comment
21 It remains unclear which nucleotide binding sites this ATP analog acts on to potentiate G551D-CFTR. Login to comment
23 Since the G551D mutation, located in ABP2, may not affect the function of ABP1, we tested the hypothesis that P-ATP potentiates G551D-CFTR by binding to ABP1. Login to comment
24 We found that P-ATP increases G551D cur- * This work was supported by National Institutes of Health Grants NIHR01DK55835 and NIHR01HL53455 (to T. C. H.) and NIHK01DK075408 (to S. G. Login to comment
38 By introducing mutations that lower the nucleotide binding affinity at each NBD, we were able to conclude that this effect of P-ATP on G551D-CFTR is through binding of the nucleotide to ABP1. Login to comment
40 EXPERIMENTAL PROCEDURES Site-directed Mutagenesis-The constructs containing single mutations (G551D, W401G, and Y1219G) have been described previously (6, 7). Login to comment
41 Additional point mutations were introduced into G551D-CFTR by using a QuikChange XL kit (Stratagene, La Jolla, CA) according to the manufacturer`s protocol. Login to comment
55 Since the activity of G551D is extremely low, the number of channels in the membrane patch cannot be ascertained. Login to comment
57 To better estimate the mean closed timeweemployedastrategybasedonourpreviousresults(7).Wefirst obtained␶o inthecontrolconditionasdescribedabove.SincethePo of G551D-CFTRisϳ0.004(i.e.1/120 of the Po for WT-CFTR), ␶c can be calculated based on the equation, ␶c ϭ (␶o/Po) - ␶o. Login to comment
62 RESULTS Effect of P-ATP on G551D-CFTR-Fig. 1A shows a continuous current trace from an excised inside-out patch of a Chinese hamster ovary cell expressing G551D-CFTR. Login to comment
64 Reversible activation of G551D-CFTR by P-ATP. Login to comment
65 A, P-ATP, a high affinity ATP analog, increases G551D-CFTR activity. Login to comment
68 C, P-ATP dose-response relationship for G551D-CFTR channels. Login to comment
71 ATP Analog Potentiates G551D-CFTR FEBRUARY 29, 2008•VOLUME 283•NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 5365 atUniversityofNorthCarolinaatChapelHill,onAugust,2011www.jbc.orgDownloadedfrom protein kinase (not shown). Login to comment
74 Similar results were obtained for G551D-CFTR expressed in human epithelial cells CFPAC-1 (see supplemental materials). Login to comment
75 To further quantify the effect of P-ATP on G551D currents, we measured the channel activity at different [P-ATP] and calculated the ratio between the mean current in the presence of P-ATP and the mean control current (washout). Login to comment
76 The P-ATP dose-response relationship (Fig. 1C) shows that G551D-CFTR currents can be enhanced as much as 7-fold at 50 ␮M P-ATP with a K1/2 of ϳ6 ␮M. In patches with sporadic openings, we could observe in detail the effect of P-ATP on the channels. Login to comment
83 The results indicate that the increase of the G551D channel activity by P-ATP is achieved mainly by an increase in the open time of the channel. Login to comment
84 G551D channels have extremely long closed times, ␶o ϽϽ ␶c; consequently, the channel activity is directly proportional to the open time (i.e. Po ϭ ␶o/(␶o ϩ ␶c) Х ␶o/␶c). Login to comment
86 Based on these results, we can safely conclude that the increase in G551D-CFTR activity in the presence of P-ATP is mainly due to an increase in ␶o. ATP and P-ATP Compete for the Same Biding Sites-We next tested whether the effect of P-ATP on G551D-CFTR is through binding to the same site(s) ATP may bind. Login to comment
87 Since ATP has little effect on G551D, one expects that the effect of P-ATP will be diminished by the presence of ATP if they share a common binding site. Login to comment
89 In the presence of ATP, the effect of P-ATP is dramatically reduced, but removal of ATP rapidly restores the potentiation of P-ATP on G551D-CFTR, suggesting that these two nucleotides can readily exchange at a binding site. Login to comment
92 Where Does P-ATP Bind to Increase the G551D-CFTR Current?-To determine to which ABP P-ATP binds to exert its effect on G551D-CFTR channels, we introduced mutations that lower the nucleotide biding affinity at each ABP. Login to comment
96 Although the Y1219G mutation decreases the apparent affinity of ATP and P-ATP in WT background, introducing this mutation into the G551D background has little influence on the FIGURE 2. Login to comment
97 P-ATP dependent gating of G551D-CFTR in excised patches. Login to comment
98 A, single-channel current traces for G551D-CFTR in the presence and absence of 10 ␮M P-ATP. Login to comment
99 B, summary of the mean open time and the mean closed time for G551D-CFTR in the presence and absence of P-ATP (n ϭ 11). Login to comment
104 The presence of ATP reduces the potentiation effect of P-ATP, but removal of ATP rapidly restores the effect of P-ATP on G551D-CFTR, indicating that these two nucleotides can readily exchange at a binding site (n ϭ 6). Login to comment
105 ATP Analog Potentiates G551D-CFTR 5366 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283•NUMBER •FEBRUARY effect of P-ATP. Login to comment
106 Fig. 4A shows a representative trace of G551D/ Y1219G-CFTR channels in the absence and presence of 10 ␮M P-ATP. Login to comment
107 10 ␮M P-ATP increased the activity of this double mutant by 5.0 Ϯ 0.6-fold (n ϭ 7), and as in the case of G551D channels, this increase in activity is mainly due to an increase in the open time of the channel (Fig. 4B). Login to comment
109 The G551D/Y1219G-CFTR P-ATP dose response is very similar to the G551D-CFTR dose response, suggesting that lowering the binding affinity at the ABP2 site does not alter the effect of P-ATP. Login to comment
110 In contrast, introducing W401G in the G551D background significantly reduced the effect of P-ATP. Login to comment
111 The activity of W401G/G551D-CFTR channels in the presence of 10 ␮M P-ATP is smaller than the activity of G551D channels under the same conditions. Login to comment
112 The mean current -fold increase is 1.9 Ϯ 0.2 for W401G/G551D-CFTR when compared with 6.2 Ϯ 0.7 for G551D-CFTR. Login to comment
116 We thus conclude that P-ATP binds to ABP1 to increase the open time of G551D channels and consequently enhance the activity of this mutant. Login to comment
117 DISCUSSION The current results show that P-ATP, a high affinity ATP analog, binds to ABP1 to enhance G551D-CFTR, and it does so mainly by increasing the open time of the channel. Login to comment
119 Mechanistically, it establishes that nucleotide binding at ABP1 can stabilize the open state even for G551D-CFTR, a mutant irresponsive to ATP; pharmacologically, it suggests that ABP1 may be a potential molecular target for drugs that could potentiate G551D-CFTR. Login to comment
125 Previously, we showed that ATP fails to increase the opening rate of G551D-CFTR (7), and this observation led us to conclude that G551D-CFTR presents only ATP independent openings. Login to comment
127 These results are consistent with the ideas that ABP2 is the site that couples ATP binding to the channel opening and that the G551D mutation specifically abolishes the function of ABP2. Login to comment
128 Thus, other nucleotides such ADP or AMP-PNP, which presumably act on ABP2 (7, 16), were not effective on G551D-CFTR (7). Login to comment
130 P-ATP effect on G551D/Y1219G-CFTR. Login to comment
131 A, single-channel traces of Y1219G/G551D-CFTR in the presence or absence of P-ATP. Login to comment
132 The mutation Y1219G (located in ABP2) decreases the apparent P-ATP binding affinity under the WT background (see supplemental materials), but when introduced into the G551D background, the P-ATP potentiation is not affected. Login to comment
133 B, comparison of the mean currents and mean open times for G551D and G551D/Y1219G in the presence of 10 ␮M P-ATP (n ϭ 5-11). Login to comment
135 P-ATP dose-response relationships for G551D/Y1219G-(Œ) and W401G/G551D-CFTR (f). Login to comment
138 The K1/2 values are 13 Ϯ 5 and 79 Ϯ 30 ␮M for G551D/ Y1219G-CFTR and W401G/G551D-CFTR, respectively. Login to comment
139 The dashed line represents the P-ATP dose-response relationship for G551D-CFTR shown in Fig. 1. Login to comment
140 ATP Analog Potentiates G551D-CFTR FEBRUARY 29, 2008•VOLUME 283•NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 5367 that for G551D-CFTR, the function of ABP1 may not be jeopardized since P-ATP can bind to this site to stabilize the open channel conformation. Login to comment
141 The significance of this conclusion is that the G551D construct presents a great opportunity to investigate specifically the interaction of nucleotides with ABP1, a yet challenging issue for several reasons. Login to comment
145 It is interesting to note that the effects of P-ATP echo what has been reported by Cai et al. (9), that 2Ј-deoxy-ATP increased the activity of G551D channels. Login to comment
148 However, it should be pointed out that it is extremely difficult to accurately estimate ␶c because of the low Po of G551D. Login to comment
150 Given the extremely low Po of G551D, this practice inevitably results in an underestimation of the closed time. Login to comment
152 If it turns out that 2Ј-deoxy-ATP also binds to ABP1 to potentiate G551D-CFTR, it may be possible to design even more effective nucleotides for this mutant. Login to comment
153 If we assume that the ATP-independent openings of G551D involve the dimerization of the NBDs, an outstanding question remains to be answered. Login to comment
154 When G551D channels open, is ABP2 occupied or empty? Login to comment
156 Since the G551D mutation is located in the signature sequence of NBD1 (ABP2), one could assume that the nucleotide binding at ABP2 is not affected by this mutation, but instead, the post-binding events may be impaired. Login to comment
160 Alternatively, if an NBD dimer can form in the presence of ATP (or P-ATP) bound at ABP2, we need to explain why ATP (or P-ATP) fails to increase the opening rate of G551D channels. Login to comment
162 Since we do not know whether the G551D open state involves the dimerization of the NBDs, we need to consider the possibility that the openings of G551D may be unrelated to the dimerization of the NBDs. Login to comment
164 Thus, we cannot rule out the possibility that the openings of G551D are decoupled from the NBD dimerization. Login to comment
165 However, since binding of P-ATP to ABP1 decreases the closing rate of the G551D channels, we have to conclude that even under this scenario, the transmembrane domains are not totally decoupled from the NBDs in G551D-CFTR. Login to comment
168 G551D is a disease-associated mutation that traffics normally to the membrane (7, 25) but with defective gating. Login to comment
170 Many compounds have been shown to increase G551D-CFTR currents (9, 12, 13, 15, 26), but none of them seems to be potent enough to completely restore the Po of the channel to WT levels. Login to comment
172 P-ATP effect on W401G/G551D-CFTR. Login to comment
173 A, single-channel traces of W401G/G551D-CFTR in the presence or absence of P-ATP. Login to comment
175 ATP Analog Potentiates G551D-CFTR 5368 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283•NUMBER 9•FEBRUARY understanding of how these compounds work or where they bind, it is very difficult to design a drug that may accomplish the task of rescuing the defective channel. Login to comment
178 Our observation that P-ATP potentiates G551D-CFTR by binding to ABP1 suggests that ABP1 may be a target for drugs to bind and enhance the mutant channel activity. Login to comment
181 It should be interesting to examine biochemically whether the G551D mutation abolishes nucleotide occlusion. Login to comment
182 Nevertheless, we believe that these new findings will likely serve as a structural framework for the future development of potentiators for CFTR mutants such as G551D. Login to comment

[hide] Diversity of the basic defect of homozygous CFTR m... J Med Genet. 2008 Jan;45(1):47-54. Stanke F, Ballmann M, Bronsveld I, Dork T, Gallati S, Laabs U, Derichs N, Ritzka M, Posselt HG, Harms HK, Griese M, Blau H, Mastella G, Bijman J, Veeze H, Tummler B
Diversity of the basic defect of homozygous CFTR mutation genotypes in humans.
J Med Genet. 2008 Jan;45(1):47-54., [PMID:18178635]

Abstract [show]
BACKGROUND: Knowledge of how CFTR mutations other than F508del translate into the basic defect in cystic fibrosis (CF) is scarce due to the low incidence of homozygous index cases. METHODS: 17 individuals who are homozygous for deletions, missense, stop or splice site mutations in the CFTR gene were investigated for clinical symptoms of CF and assessed in CFTR function by sweat test, nasal potential difference and intestinal current measurement. RESULTS: CFTR activity in sweat gland, upper airways and distal intestine was normal for homozygous carriers of G314E or L997F and in the range of F508del homozygotes for homozygous carriers of E92K, W1098L, R553X, R1162X, CFTRdele2(ins186) or CFTRdele2,3(21 kb). Homozygotes for M1101K, 1898+3 A-G or 3849+10 kb C-T were not consistent CF or non-CF in the three bioassays. 14 individuals exhibited some chloride conductance in the airways and/or in the intestine which was identified by the differential response to cAMP and DIDS as being caused by CFTR or at least two other chloride conductances. DISCUSSION: CFTR mutations may lead to unusual electrophysiological or clinical manifestations. In vivo and ex vivo functional assessment of CFTR function and in-depth clinical examination of the index cases are indicated to classify yet uncharacterised CFTR mutations as either disease-causing lesions, risk factors, modifiers or neutral variants.

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89 Several well characterised severe mutations occur in the evolutionarily conserved Walker (G1244E, G1249E) or dodecapeptide motifs (G551D, G1349D) of the ABC transporter CFTR.1 The missense mutants G622D23 in the regulatory domain and G314E in the fifth transmembrane region led to no clinical symptoms of CF. Login to comment

[hide] Misfolding of the cystic fibrosis transmembrane co... Biochemistry. 2008 Feb 12;47(6):1465-73. Epub 2008 Jan 15. Cheung JC, Deber CM
Misfolding of the cystic fibrosis transmembrane conductance regulator and disease.
Biochemistry. 2008 Feb 12;47(6):1465-73. Epub 2008 Jan 15., 2008-02-12 [PMID:18193900]

Abstract [show]
Understanding the structural basis for defects in protein function that underlie protein-based genetic diseases is the fundamental requirement for development of therapies. This situation is epitomized by the cystic fibrosis transmembrane conductance regulator (CFTR)-the gene product known to be defective in CF patients-that appears particularly susceptible to misfolding when its biogenesis is hampered by mutations at critical loci. While the primary CF-related defect in CFTR has been localized to deletion of nucleotide binding fold (NBD1) residue Phe508, an increasing number of mutations (now ca. 1,500) are being associated with CF disease of varying severity. Hundreds of these mutations occur in the CFTR transmembrane domain, the site of the protein's chloride channel. This report summarizes our current knowledge on how mutation-dependent misfolding of the CFTR protein is recognized on the cellular level; how specific types of mutations can contribute to the misfolding process; and describes experimental approaches to detecting and elucidating the structural consequences of CF-phenotypic mutations.

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89 In contrast, the mutant G551D (also located in NBD1) affects mainly the function of CFTR but not its trafficking (71), while the mutant G1349D, at an equivalent position in NBD2 as G551 in NBD1, similarly affects the function of CFTR (72). Login to comment

[hide] Proteasome-dependent pharmacological rescue of cys... J Pharmacol Exp Ther. 2008 Apr;325(1):89-99. Epub 2008 Jan 29. Norez C, Bilan F, Kitzis A, Mettey Y, Becq F
Proteasome-dependent pharmacological rescue of cystic fibrosis transmembrane conductance regulator revealed by mutation of glycine 622.
J Pharmacol Exp Ther. 2008 Apr;325(1):89-99. Epub 2008 Jan 29., [PMID:18230692]

Abstract [show]
The most common mutation (F508del) causing cystic fibrosis (CF) results in misfolding of the CF transmembrane conductance regulator (CFTR), leading to its degradation via the proteasome pathway. To study the mechanism of action of several pharmacological chaperones benzo[c]quinolizinium (MPB), we analyzed their effects on two CF mutations; F508del-CFTR and G622D-CFTR. The replacement of Gly622 by an aspartic acid (G622D) alters the trafficking and activity of the protein. G622D, similar to F508del, was functionally rescued by the glucosidase inhibitor miglustat but, unlike F508del, could not be rescued by MPB. A structure-activity relationship for F508del functional correction revealed the following profile: MPB-104-91-07-80 > 05 > 89 >> 9-hydroxyphenanthrene = phenanthrene. Coimmunoprecipitation experiments on human airway epithelial F508del/F508del CF15 cells showed that MPB did not prevent the interaction of F508del-CFTR with heat shock protein (HSP)70, HSP90, or calnexin. Functional rescue of F508del-CFTR by MPB and miglustat was abolished by brefeldin A (BFA) but potentiated by thapsigargin (TG) and geldanamycin. The proteasome inhibitor MG132 potentiated the effect of miglustat but only modestly affected that of MPB. It is noteworthy that MPB inhibited proteasome activity in F508del-CFTR-expressing cells but did not directly affect the activity of purified 20S proteasome. With the mutant G622D-CFTR, MPB did not inhibit proteasome activity, as in mock-transfected cells. Inhibition of cellular degradation machinery by MPB is not only CFTR-dependent, but it also follows similar structure-activity relationship as demonstrated by functional correction. We conclude that G622D is a partial trafficking-deficient mutant with dysfunctional chloride channel activity, and that Gly622 is part of the putative site for interaction of MPB with CFTR, protecting the channel from proteasome-mediated degradation.

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25 Several other less frequent CF mutations such as G551D or G1349D are classified as class 3 because the corresponding proteins, although correctly located at the plasma membrane, have a dysfunctional regulation (see for a recent review, MacDonald et al., 2007). Login to comment
34 In previous studies, we identified benzo[c]quinolizinium (MPB) derivatives (several chemical structures are shown in Fig. 1B together with two phenanthrene derivatives) as activators of wild-type CFTR, and G551D, G1349D, and F508del mutants (Becq et al., 1999; Dormer et al., 2001a; Marivingt-Mounir et al., 2004; Melin et al., 2004). Login to comment
234 In a previous study analyzing the effect of CFTR channel activators, we reported a structure-activity relationship of MPB compounds (Marivingt-Mounir et al., 2004), and we demonstrated that some of these derivatives were able to stimulate the channel activity of wt-, G551D-, G1349D-, G551D/ G1349D-, and F508del-CFTR (Becq et al., 1999; Dormer et al., 2001a; Marivingt-Mounir et al., 2004; Melin et al., 2004). Login to comment

[hide] Nasal abnormalities in cystic fibrosis mice indepe... Am J Respir Cell Mol Biol. 2008 Jul;39(1):19-25. Epub 2008 Jan 31. Hilliard TN, Zhu J, Farley R, Escudero-Garcia S, Wainwright BJ, Jeffery PK, Griesenbach U, Bush A, Davies JC, Alton EW
Nasal abnormalities in cystic fibrosis mice independent of infection and inflammation.
Am J Respir Cell Mol Biol. 2008 Jul;39(1):19-25. Epub 2008 Jan 31., [PMID:18239192]

Abstract [show]
It is not known whether the progressive airway changes in cystic fibrosis (CF) are all secondary to infection and inflammation. The CF mouse nose shares electrophysiologic and cellular properties with human CF airway epithelium. In the present work, we tested the hypothesis that structural abnormalities in the nasal mucosa of CF mice develop independent of infection and inflammation. We performed nasal lavage and subsequent serial coronal section through the nasal tissue of adult CF (mutations Cftr(TgHm1G551D) and Cftr(tm1Unc)-TgN((FABPCFTR))) and wild-type mice raised under normal housing conditions. Nasal tissue was also obtained from Day 17 embryos and newborn pups. Detailed histologic examination of the respiratory and olfactory epithelium within the nasal cavity was performed. Bacterial culture, cell count, and macrophage inflammatory protein-2 (MIP-2) concentration were assessed in nasal lavage fluid. Significantly thickened respiratory epithelium and increased mucous cell density was found in adult CF mice of both mutations compared with wild-type animals. In contrast, the olfactory epithelium was thinner, with a decreased cell density. Areas of lymphoid aggregates were found in CF mice but not in non-CF mice. There were no differences in bacterial growth, cell count, or MIP-2 concentrations. No genotype differences were observed in the embryonic or newborn periods. There are significant histologic changes in the nasal mucosa of adult CF mice, not associated with increased lumenal inflammation or bacterial content, and which are not present perinatally. These may be novel therapeutic targets.

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33 Adult Animals Two genotypes of adult CF mice were studied: (1) CftrTgHm1G551D (mixed background [5], termed G551D, n 5 12); (2) Cftrtm1Unc-TgN(FABPCFTR) (mixed background [23], termed FABP, n 5 6). Login to comment
34 Wild-type littermates (Cftr1/1, n 5 12) of G551D mice were used as controls. Login to comment
49 Nasal Lavage Nasal lavage was performed on adult G551D and wild-type mice. Login to comment
54 Pup and Embryo Investigation Timed mating was performed between G551D heterozygotes (C57BL/6 background). Login to comment
56 After genotyping of tail tips, two 5-mm sections were cut at 50-mm intervals from six G551D and seven wild-type pup heads, and the same number of embryo heads. Login to comment
69 RE (arrowed) at depth of 4,600 mm from (A) wild-type nose and (B) G551D CF nose. Login to comment
75 Heads from CF mice tended to be smaller then wild-type animals; the lateral sinus in G551D mice closed at a median depth of 5,000 mm compared with wild type at 5,800 mm (P , 0.001); and 5,000 mm in FABP (P 5 0.053 versus wild type). Login to comment
77 Respiratory Epithelium RE was significantly thicker in both types of CF mice (G551D 14.8 mm, FABP 14.9 mm) compared with wild-type mice (10.0 mm; P , 0.01 for both), as shown in Figure 2. Login to comment
78 Mucous cell density was significantly greater in both types of CF mice (G551D 89.6 cells/mm, FABP 84.7 cells/mm) compared with wild type (54.0 cells/mm; P , 0.001 and P 5 0.001, respectively), as shown in Figure 3. Login to comment
79 Collections of inflammatory cells under the RE, all with the appearance of lymphoid aggregates, were present in at least one section in 11 out of the 12 G551D mice and in 2 out of the 6 FABP mice, compared with none of the 12 wild-type mice (x2520.8, P , 0.001). Login to comment
81 Olfactory Epithelium OE was significantly thinner in both types of CF mice (G551D 32.6 mm, FABP 39.0 mm) compared with wild-type (50.8 mm; P , 0.001 for both), as shown in Figure 5. Login to comment
82 OE cell density was also decreased in both types of CF mice (G551D 27.4 cells/ 1,000 mm2, FABP 28.6 cells/1,000 mm2) compared with wild-type (36.4 cells/1,000 mm2; P , 0.001 and P 5 0.001, respectively), as shown in Figure 6. Login to comment
84 Nasal Lavage in Adult Mice The median cell count in nasal lavage was not significantly different between G551D (5,000 cells/ml, range 0-18,000) and wild-type mice (2,000 cells/ml, 0-20000, P 5 0.11). Login to comment
87 MIP-2 concentrations were not significantly different between G551D (9.8 pg/ml, 7.2-82.0) and wild-type (10.1 pg/ml, 7.3-15.5). Login to comment
89 Embryo and Pup Investigation RE and OE starting points were similar between G551D and wild-type pups and embryos. Login to comment
91 Mucous cells (arrowed) in RE at depth of 3,400 mm from (A) wild-type nose and (B) G551D CF nose. Login to comment
94 Area of lymphoid aggregates (arrowed) on the lateral wall of the nose in a G551D CF head at a depth of 4,200 mm. H&E, scale bar 5 100 mm. Login to comment
95 different between G551D and wild-type pups or embryos (combined thickness: 11.7 mm versus 14.1 mm). Login to comment
97 OE was well developed in both Day 17 embryos and pups and the thickness was similar between G551D and wild-type heads (combined thickness 50.9 mm versus 52.0 mm). Login to comment
100 OE (arrowed) in the dorsum of the nose of (A) a wild-type head and (B) a G551D CF head at a depth of 4,600 mm. H&E, scale bar 5 100 mm. Login to comment
102 OE (arrowed) at high power in the dorsum of the nose in (A) a wild-type head and (B) a G551D CF head at a depth of 4,600 mm. H&E, scale bar 5 100 mm. Login to comment
167 Acknowledgments: The authors thank Sara Escudio Garcia and Luci Somerton for managing the G551D breeding colony and for performing the tail tip genotype analysis; John Williams for the processing of the adult tissue; Yusheng Qiu for the processing of the embryo tissue; the staff of the Central Biosciences and NHLI animal facilities; Neil Madden for help with the microbiological analysis; and Abraham Jacob, Brian Faddis, and Richard Chole, Department of Otolaryngology, Washington University School of Medicine, St. Login to comment

[hide] The intact CFTR protein mediates ATPase rather tha... Biochem J. 2008 Jun 1;412(2):315-21. Ramjeesingh M, Ugwu F, Stratford FL, Huan LJ, Li C, Bear CE
The intact CFTR protein mediates ATPase rather than adenylate kinase activity.
Biochem J. 2008 Jun 1;412(2):315-21., 2008-06-01 [PMID:18241200]

Abstract [show]
The two NBDs (nucleotide-binding domains) of ABC (ATP-binding-cassette) proteins function in a complex to mediate ATPase activity and this activity has been linked to their regulated transport activity. A similar model has been proposed for CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel defective in cystic fibrosis, wherein ATP binding and hydrolysis regulate the channel gate. Recently, it was shown that the individual NBDs isolated from CFTR primarily mediate adenylate kinase activity, raising the possibility that this activity may also contribute to gating of the CFTR channel. However, this present study shows that whereas the isolated NBDs exhibit adenylate kinase activity, the full-length purified and reconstituted CFTR protein functions as an ATPase, arguing that the enzymatic activity of the NBDs is dependent on their molecular context and appropriate domain-domain assembly. As expected, the disease-causing mutant bearing a mutation in the ABC signature motif, CFTR-G551D, exhibited a markedly reduced ATPase activity. Furthermore, mutation of the putative catalytic base in CFTR caused a reduction in ATPase activity, with the CFTR-E1371Q mutant supporting a low level of residual activity. Neither of these mutants exhibited detectable adenylate kinase activity. Together, these findings support the concept that the molecular mechanism of action of CFTR is dependent on ATP binding and hydrolysis, and that the structure of prokaryotic ABC ATPases provide a useful template for understanding their mechanism of action.

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4 As expected, the disease-causing mutant bearing a mutation in the ABC signature motif, CFTR-G551D, exhibited a markedly reduced ATPase activity. Login to comment
26 Furthermore, the consequences of the disease-causing mutation G551D and the catalytic base mutation E1371Q on the enzymatic activity of the full-length protein were also evaluated. Login to comment
36 Purification and reconstitution of wt (wild-type), CFTR-G551D and CFTR-E1371Q Detailed protocols regarding the generation of wt and mutant CFTR-His10 proteins are described elsewhere [29,30]. Login to comment
118 Interestingly, the addition of AMP (400 μM) to PKA-treated CFTR did not exert any significant effect on its ATPase activity (Figures 3A and 5B), a finding which is consistent with previous Figure 4 The disease-causing mutant CFTR-G551D and the putative catalytic base mutant CFTR-E1371Q exhibit reduced ATPase activity (A) Silver-stained gel showing purification of CFTR-E1371Q. Login to comment
120 (C) ATPase activity (Results are means + - S.E.M.) of CFTR-G551D (n = 6) or CFTR-E1371Q (n = 6). Login to comment
125 The enzymatic activity of full-length mutant CFTR proteins: CFTRG551D and CFTR-E1371Q Gly551 is located in the signature motif of NBD1 [1] and is thought to exist at the interface through which NBD1 and NBD2 interact to form the catalytic site for ATP hydrolysis [4,7]. Login to comment
126 The mutation, CFTR-G551D, is associated with a severe form of CF. Login to comment
127 We reported previously that the enzymatic activity of purified PKA phosphorylated CFTR-G551D, measured as the generation of [α-32 P]ADP from [α-32 P]ATP, was severely abrogated [4]. Login to comment
136 As for the wt and CFTR-G551D proteins, there was no measurable adenylate kinase activity stimulated in CFTR-E1371Q by the addition of 400 μM AMP (Figure 4B). Login to comment
207 The CF disease-causing mutant, CFTR-G551D, exhibits defective ATPase activity, a result which is consistent with the putative location of the signature motif (L548 SGGQ552 ) relative to ATP in the NBD dimer interface [7,21]. Login to comment

[hide] Drugs and their molecular targets: an updated over... Fundam Clin Pharmacol. 2008 Feb;22(1):1-18. Landry Y, Gies JP
Drugs and their molecular targets: an updated overview.
Fundam Clin Pharmacol. 2008 Feb;22(1):1-18., [PMID:18251718]

Abstract [show]
About 330 targets bind approved drugs, 270 encoded by the human genome and 60 belonging to pathogenic organisms. A large number of druggable targets have been recently proposed from preclinical and first clinical data, but a huge reservoir of putative drug targets, possibly several thousands, remains to be explored. This overview considers the different types of ligands and their selectivity in the main superfamilies of drug targets, enzymes, membrane transporters and ion channels, and the various classes of membrane and nuclear receptors with their signalling pathway. Recently approved drugs such as monoclonal antibodies, tyrosine kinase and proteasome inhibitors, and major drugs under clinical studies are reviewed with their molecular target and therapeutic interest. The druggability of emerging targets is discussed, such as multidrug resistance transporters and cystic fibrosis transmembrane conductance regulator (CFTR), hyperpolarization-activated cyclic nucleotides-gated (HCN), cyclic nucleotide-gated (CNG) and transient receptor potential (TRP) ion channels, tumour necrosis factor (TNF) and receptor activator of NFkappaB (RANK) receptors, integrins, and orphan or recently deorphanized G-protein-coupled and nuclear receptors. Large advances have been made in the therapeutical use of recombinant cytokines and growth factors (i.e. tasonermin, TNFalpha-1a; becaplermin, platelet-derived growth factor (PDGF); dibotermin-alpha, bone morphogenetic proteins (BMP)2; anakinra, interleukin-1 receptor antagonist protein (IRAP), and in enzyme replacement therapy, i.e. algasidase (alpha-galactosidase) and laronidase (alpha-l-iduronidase). New receptor classes are emerging, e.g. membrane aminopeptidases, and novel concepts are stimulating drug research, e.g. epigenetic therapy, but the molecular target of some approved drugs, such as paracetamol and imidazolines, still need to be identified.

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92 Other mutations of CFTR, like G551D and G1349D (glycine to aspartic acid change at position 551 or 1349), cause only a gating defect. Login to comment

[hide] Alpha-aminoazaheterocyclic-methylglyoxal adducts d... J Pharmacol Exp Ther. 2008 May;325(2):529-35. Epub 2008 Feb 13. Sonawane ND, Zegarra-Moran O, Namkung W, Galietta LJ, Verkman AS
Alpha-aminoazaheterocyclic-methylglyoxal adducts do not inhibit cystic fibrosis transmembrane conductance regulator chloride channel activity.
J Pharmacol Exp Ther. 2008 May;325(2):529-35. Epub 2008 Feb 13., [PMID:18272811]

Abstract [show]
Inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have potential applications in the therapy of secretory diarrheas and polycystic kidney disease. In a recent study, several highly polar alpha-aminoazaheterocyclic-methylglyoxal adducts were reported to reversibly inhibit CFTR chloride channel activity with IC50 values in the low picomolar range (J Pharmacol Exp Ther 322:1023-1035, 2007), more than 10,000-fold better than that of thiazolidinone and glycine hydrazide CFTR inhibitors previously identified by high-throughput screening. In this study, we resynthesized and evaluated the alpha-aminoazaheterocyclic-methylglyoxal adducts reported to have high CFTR inhibition potency (compounds 5, 7, and 8). We verified that the reported synthesis procedures produced the target compounds in high yield. However, we found that these compounds did not inhibit CFTR chloride channel function in multiple cell lines at up to 100 microM concentration, using three independent assays of CFTR function including short-circuit current analysis, whole-cell patch-clamp experiments, and yellow fluorescence protein-fluorescence quenching. As positive controls, approximately 100% of CFTR inhibition was found by thiazolidinone and glycine hydrazide CFTR inhibitors. Our data provide direct evidence against CFTR inhibition by alpha-aminoazaheterocyclic-methylglyoxal adducts.

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179 An interior binding site is mandated by the data in Fig. 8 of Routaboul et al. (2007), showing remarkably weaker CFTR inhibition after mutation (G551D) at a residue located in a cytosolic domain of CFTR. 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] Evidence for direct CFTR inhibition by CFTR(inh)-1... Biochem J. 2008 Jul 1;413(1):135-42. Caci E, Caputo A, Hinzpeter A, Arous N, Fanen P, Sonawane N, Verkman AS, Ravazzolo R, Zegarra-Moran O, Galietta LJ
Evidence for direct CFTR inhibition by CFTR(inh)-172 based on Arg347 mutagenesis.
Biochem J. 2008 Jul 1;413(1):135-42., 2008-07-01 [PMID:18366345]

Abstract [show]
CFTR (cystic fibrosis transmembrane conductance regulator) is an epithelial Cl- channel inhibited with high affinity and selectivity by the thiazolidinone compound CFTR(inh)-172. In the present study, we provide evidence that CFTR(inh)-172 acts directly on the CFTR. We introduced mutations in amino acid residues of the sixth transmembrane helix of the CFTR protein, a domain that has an important role in the formation of the channel pore. Basic and hydrophilic amino acids at positions 334-352 were replaced with alanine residues and the sensitivity to CFTR(inh)-172 was assessed using functional assays. We found that an arginine-to-alanine change at position 347 reduced the inhibitory potency of CFTR(inh)-172 by 20-30-fold. Mutagenesis of Arg347 to other amino acids also decreased the inhibitory potency, with aspartate producing near total loss of CFTR(inh)-172 activity. The results of the present study provide evidence that CFTR(inh)-172 interacts directly with CFTR, and that Arg347 is important for the interaction.

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24 However, NBD mutants such as G551D, G1349D or P574H, do not show an altered sensitivity to CFTRinh-172 [9,12]. Login to comment
25 In contrast, the potency of CFTR activators, whose effect involves direct interaction with the NBDs, is greatly altered by mutations such as G551D which resides in NBD1 [13-15]. 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] CLC-0 and CFTR: chloride channels evolved from tra... Physiol Rev. 2008 Apr;88(2):351-87. Chen TY, Hwang TC
CLC-0 and CFTR: chloride channels evolved from transporters.
Physiol Rev. 2008 Apr;88(2):351-87., [PMID:18391167]

Abstract [show]
CLC-0 and cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels play important roles in Cl(-) transport across cell membranes. These two proteins belong to, respectively, the CLC and ABC transport protein families whose members encompass both ion channels and transporters. Defective function of members in these two protein families causes various hereditary human diseases. Ion channels and transporters were traditionally viewed as distinct entities in membrane transport physiology, but recent discoveries have blurred the line between these two classes of membrane transport proteins. CLC-0 and CFTR can be considered operationally as ligand-gated channels, though binding of the activating ligands appears to be coupled to an irreversible gating cycle driven by an input of free energy. High-resolution crystallographic structures of bacterial CLC proteins and ABC transporters have led us to a better understanding of the gating properties for CLC and CFTR Cl(-) channels. Furthermore, the joined force between structural and functional studies of these two protein families has offered a unique opportunity to peek into the evolutionary link between ion channels and transporters. A promising byproduct of this exercise is a deeper mechanistic insight into how different transport proteins work at a fundamental level.

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846 Most notable are G551D and G1349D, two mutations located in the signature sequence of NBD1 (ABP2) and NBD2 (ABP1), respectively. Login to comment
847 G551D is the third overall most common CF mutation, with a worldwide frequency of ϳ3% (www.genet.sickkids.on.ca/ cftr). Login to comment
849 The G551D mutation results in a significantly decreased chloride current due to a drastic reduction of the channel activity (41, 73, 334, 337). Login to comment
853 (35) indicate that the G551D mutation completely abolishes ATP-dependent gating of CFTR (also see Ref. 329), whereas G1349D-CFTR retains some ATP dependence albeit with a Po ϳ10-fold lower than that of WT-CFTR. Login to comment
866 It is interesting to note that, although the activity of G551D-CFTR is ATP independent, 2Ј-deoxy-ATP increases the Po of this mutant mainly by prolonging the open time (41). Login to comment

[hide] Validation of nasal potential difference measureme... Am J Respir Cell Mol Biol. 2008 Oct;39(4):490-6. Epub 2008 May 5. Griesenbach U, Smith SN, Farley R, Singh C, Alton EW
Validation of nasal potential difference measurements in gut-corrected CF knockout mice.
Am J Respir Cell Mol Biol. 2008 Oct;39(4):490-6. Epub 2008 May 5., [PMID:18458238]

Abstract [show]
Attempts at correcting the nasal potential difference (PD) in cystic fibrosis (CF) mice have long been used in preclinical gene and small molecule therapy development. However, in general, CF mice suffer from intestinal disease, are runted, and have high mortality rates; they are therefore difficult to work with, especially if large numbers are required. Because of this, large-scale PD studies in CF mice have not been performed. Working with CF mice has become substantially easier after the generation of the gut-corrected CF-knockout mouse. Fatty acid-binding promoter (FABp)-mediated expression of CFTR in the gut, but not the airways, prevents the intestinal disease of the CF knockout mouse. This model has given us the unique opportunity to systematically study PDs in large numbers of CF mice. The nose, but not the lungs, of these animals mimic the bioelectric defect seen in humans. We have therefore assessed the bioelectrics of the respiratory epithelium comparing FABp-CF and wild-type mice. The large body of data gathered in CF and wild-type mice allowed us, for the first time, to establish power calculations that should inform sample sizes required in gene and small molecule therapy development. In addition, we address the important issues of intra-animal variability as well as intra- and inter-operator variability for scoring the traces, and the effect of age and sex on nasal PD in CF mice. These data should allow a more informed use of CF animals in future studies.

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15 In these mice the cystic fibrosis transmembrane conductance regulator (Cftr) gene, which encodes an epithelial chloride channel, is either completely knocked out (nulls) or naturally occurring mutations such as DF508 or G551D have been introduced (4), leading to altered chloride and sodium transport across epithelial cells. Login to comment
21 To assess the effects of gene and small molecule therapy on the respiratory tract, it is useful to use a strain that has very low, or absent, residual CFTR function such as the UNC-null (7), G551D (8), or DF508 mice (9). Login to comment
42 E-mail: u.griesenbach@imperial.ac.uk Am J Respir Cell Mol Biol Vol 39. pp 490-496, 2008 Originally Published in Press as DOI: 10.1165/rcmb.2007-0385OC on May 5, 2008 Internet address: www.atsjournals.org strain dependent, we therefore, first compared PD in C57Bl/6 congenic G551D CF mice and C57Bl/6 wild-type mice, and subsequently compared PD in C57Bl/6 congenic G551D and FABp-CF mice. Login to comment
50 MATERIALS AND METHODS Mouse Models Male and female wild-type C57Bl/6 mice and G551D CF knockout mice (8) were purchased from Charles River UK (Margate, Kent, UK). Login to comment
98 RESULTS Comparison of Nasal PD in Wild-Type, GD-B6, and FABp-CF-Knockout Mice Transepithelial nasal potential difference was measured in C57Bl/6 wild-type (WT-B6), C57Bl/6 congenic G551D (GD-B6), and FABp-CF mice. Login to comment
101 There was no difference in nasal PD between G551D and FABp CF mice (Figure 1). Login to comment
112 Nasal PD was measured in wild-type C57Bl/6 mice (WT-B6), C56Bl/6 congenic G551D CF knockout mice (GD-B6), and gut-corrected CF knockout mice (FABp). Login to comment
178 We were aware that the absence of genetically matched wild-type littermates might affect the interpretation of our results. To address this point, we compared nasal PDs of FABp-CF mice, C57Bl/6 congenic G551D mice, and C57Bl/6 WT mice. Login to comment
179 Importantly, both strains of CF mice were significantly different from WT for all PD-related measurements, and there was no difference between FABp and G551D CF mice. Login to comment
181 Animal numbers in the G551D cohort were lower than in WT and FABp cohorts due to the restricted availability and poor health of these mice. Login to comment

[hide] Cystic fibrosis: ironing out the problem of infect... Am J Physiol Lung Cell Mol Physiol. 2008 Jul;295(1):L23-4. Epub 2008 May 16. Reid DW, Anderson GJ, Lamont IL
Cystic fibrosis: ironing out the problem of infection?
Am J Physiol Lung Cell Mol Physiol. 2008 Jul;295(1):L23-4. Epub 2008 May 16., [PMID:18487353]

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8 Further evidence to support a problem with iron trafficking comes from the CF G551D mouse model. Login to comment
16 Further evidence to support a problem with iron trafficking comes from the CF G551D mouse model. 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 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] 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] 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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35 CFTR mutations may Table 1 Methods for CFTR gene mutation detection most frequently used in Europe Methods for the detection of known mutations Mutations detected Advantages Limits and pitfalls Heteroduplex analysis (strictly speaking a scanning method) Mainly F508del and I507del Other microinsertions/deletions (2 bp minimum): 394delTT (Northern Europe), 1677delTA (Black Sea countries), 1609delCA (Spain) Simple and rapid Migration pattern not specific for a given mutation Restriction enzyme analysis (restriction sites can be natural or created by the use of modified primers) Mainly specific individual mutations Possibly a small number of mutations can be combined in one assay Simple and rapid Useful for cascade carrier testing in case of rare mutations Not specific, especially if site abolition (eg, G551D and R553X abolish the same Hinc II site, and W1282X and R1283M the same Mnl I site) Reverse dot blot hybridization Up to 20 mutations per multiplex Appropriate for large series Innogenetics (Inno LiPA)a 36 mutations Good specificity ARMS (amplification refractory mutation system) Up to 20 mutations Appropriate for large series Design of primers is difficult Results are based on the absence of PCR product Tepnel (Elucigene)a 28-30 mutations Good specificity OLA (oligonucleotide ligation assay) Appropriate for large series Abbott Molecular (Cystic Fibrosis Genotyping Assay)a 32 mutations Good specificity Methods for the detection of unknown mutations DGGE (denaturing gradient gel electrophoresis) DGGE, DHPLC, SSCP and Sequencing: High sensitivity (495%) Difficult to set up; difficult automation Can miss isostable mutations in the homozygous state DHPLC (denaturing high performance liquid chromatography) Aiming to detect all mutations of small bp in the coding regions and intronic boundaries High sensitivity (495%) Generally miss homozygous mutations Need sequencing of polymorphism-rich regions SSCP (single strand conformation polymorphism) Simple and rapid to set up Sensitivity 80-85% Sequencing (as a first-line method or confirmation after a scanning technique) Close to 100% sensitivity Quantitative fluorescent multiplex PCR MLPA (multiple ligation-dependent probe amplification) Aiming to detect deletions, insertions, and duplications All coding regions Simple and rapid Sensitive to extraction methods Duplications may be difficult to evidence a Commercially available methods are indicated in italics be missed by scanning techniques, especially when homozygous, and even direct sequencing cannot identify 100% of mutations. Login to comment
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
64 R117H, R553X, N1303K & G551D were identified by ARMS on a total of five chromosomes. Login to comment
96 Table 2 Genotypes of CF subjects (n=50) Genotype Number of subjects Delta F508/Delta F508 5 Delta F508/3849+10kb C-T 1 Delta F508/S549N 2 Delta F508/S158N 1 Delta F508/Y1381H 1 Delta F508/1525-1 G-A 2 V520F/R117H 1 I530L/I530L 1 876-6del4/876-6del4 1 1792ins A/1792insA 1 3986-3987delC/3986-3987delC 1 Delta F508/U 10 1161 delC/U 2 L69H/U 1 R117H/U 1 Q493L/U 1 Y517C/U 1 S549N/U 3 G551D/U 1 E1329Q/U 1 N1303K/U 1 Y1381H/U 1 L218X/U 1 R553X/U 1 1525-1G-A/U 3 3120+1G-A/U 2 3849+10kb C-T/U 2 U/U 1 U-unidentified Table 3 Outcome prediction scores of novel substitution mutations identified in Indian CF patients Wild type Mutant Position Output Reliablity Prediction L H 69 0.5210 0 Pathological S N 158 0.3304 3 Neutral Q L 493 0.7784 5 Pathological I L 530 0.0591 8 Neutral E Q 1329 0.1018 7 Neutral Molecular Modelling and Bioinformatics (MMB) program (http://mmb.pcb.ub.es/PMut/) was used for pathological predictions of novel sequence variants. Login to comment
113 We first identified five of the mutations by ARMS (Delta F508, R117H, R553X, N1303K & G551D) and one by restriction digestion (3849+10kbC-T) and later identified by SSCP eight known (Y517C, V520F, S549N, Y1381H, 1525-1G-A, 3120+1G-A, 1161delC and L218X) and eight previously unreported mutations (L69H, S158N, Q493L, I530L, E1329Q, 876-6del4, 1792insA and 3986-3987delC). 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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142 Several CF mouse models have been generated using gene targeting to disrupt the murine CFTR locus by homologous recombination (Dorin et al. 1992, Snouwaert et al. 1992, O`Neal et al. 1993, Ratcliff et al. 1993, Hasty et al. 1995, Rozmahel et al. 1996) and by introducing specific human mutations into the equivalent mouse loci, including DF508 (Colledge et al. 1995, van Doorninck et al. 1995, Zeiher et al. 1995, French et al. 1996) and the G551D mutation (Delaney et al. 1996, Dickinson et al. 2000). Login to comment
163 To address this question, a collection of recombinant CF mouse models was generated containing clinically relevant mutations in CFTR by introducing specific human mutations into the equivalent mouse loci including DF508 (Colledge et al. 1995, van Doorninck et al. 1995, Zeiher et al. 1995, French et al. 1996), G551D (substitution of a glycine with an aspartic acid) (Delaney et al. 1996) and G480C (missense mutation) (Dickinson et al. 2000) (Table 2). 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] cAMP-mediated regulation of cholesterol accumulati... Am J Physiol Lung Cell Mol Physiol. 2008 Nov;295(5):L809-19. Epub 2008 Sep 12. Manson ME, Corey DA, White NM, Kelley TJ
cAMP-mediated regulation of cholesterol accumulation in cystic fibrosis and Niemann-Pick type C cells.
Am J Physiol Lung Cell Mol Physiol. 2008 Nov;295(5):L809-19. Epub 2008 Sep 12., [PMID:18790990]

Abstract [show]
The goal of this study was to identify a mechanism regulating cholesterol accumulation in cystic fibrosis (CF) cells. Both CFTR activation and expression are regulated by the cAMP pathway, and it is hypothesized that a feedback response involving this pathway may be involved in the phenotype of cholesterol accumulation. To examine the role of the cAMP pathway in cholesterol accumulation, we treated two CF model cell lines with the Rp diastereomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS) and visualized by filipin staining. Rp-cAMPS treatment eliminated cholesterol accumulation in CF cells, whereas 8-bromo-cAMP treatment led to cholesterol accumulation in wild-type cells. To confirm these findings in an independent model system, we also examined the role of cAMP in modulating cholesterol accumulation in Niemann-Pick type C (NPC) fibroblasts. Expression of the protein related to NPC, NPC1, is also directly regulated by cAMP; therefore, it is postulated that NPC cells exhibit the same cAMP-mediated control of cholesterol accumulation. Cholesterol accumulation in NPC cells also was reduced by the presence of Rp-cAMPS. Expression of beta-arrestin-2 (betaarr2), a marker of cellular response to cAMP signaling, was significantly elevated in CF model cells, Cftr(-/-) MNE, primary tissue obtained by nasal scrapes from CF subjects, and in NPC fibroblasts compared with respective controls.

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210 The expression of the inactive but properly trafficked G551D CFTR mutant has no effect on cholesterol transport. Login to comment
219 We have shown that Cftr-/- mice exhibit cholesterol processing defects (41), and we have demonstrated that cholesterol processing in IB3 (W1282X/⌬F508) is restored in CFTR-corrected IB3 cells (S9 cells) (40). Login to comment
222 Expression of G551D CFTR in CHO cells is unlikely to Fig. 6. Correction of cholesterol transport in CF model IB3 cells with Rp-cAMPS. Login to comment
207 The expression of the inactive but properly trafficked G551D CFTR mutant has no effect on cholesterol transport. Login to comment

[hide] Generation of a conditional null allele for Cftr i... Genesis. 2008 Oct;46(10):546-52. Hodges CA, Cotton CU, Palmert MR, Drumm ML
Generation of a conditional null allele for Cftr in mice.
Genesis. 2008 Oct;46(10):546-52., [PMID:18802965]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes a cAMP-regulated chloride channel that is important in controlling the exchange of fluid and electrolytes across epithelial cells. Mutation of CFTR can lead to cystic fibrosis (CF), the most common lethal genetic disease in Caucasians. CF is a systemic illness with multiple organ systems affected including pulmonary, gastrointestinal, pancreatic, immune, endocrine, and reproductive systems. To understand the role of CFTR in the various tissues in which it is expressed, we generated a murine conditional null allele of Cftr (Cftr(fl10)) in which loxP sites were inserted around exon 10 of the Cftr gene. The Cftr(fl10) allele was validated by generating constitutive Cftr null (Cftr(Delta10)) mice using the protamine-cre system. The Cftr(Delta10/Delta10) mice displayed almost identical phenotypes to previously published CF mouse models, including poor growth, decreased survival, intestinal obstruction, and loss of Cftr function as assessed by electrophysiology measurements on gut and nasal epithelium. Mice containing the conditional null Cftr allele will be useful in future studies to understand the role of Cftr in specific tissues and developmental time points and lead to a better understanding of CF disease.

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178 Cystic fibrosis mice carrying the missense mutation G551D replicate human genotype-phenotype correlations. 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] Update on gene modifiers in cystic fibrosis. Curr Opin Pulm Med. 2008 Nov;14(6):559-66. Collaco JM, Cutting GR
Update on gene modifiers in cystic fibrosis.
Curr Opin Pulm Med. 2008 Nov;14(6):559-66., [PMID:18812833]

Abstract [show]
PURPOSE OF REVIEW: Cystic fibrosis (CF) is a common, life-limiting monogenic disease, which typically manifests as progressive bronchiectasis, exocrine pancreatic dysfunction, and recurrent sinopulmonary infections. Although the gene responsible for CF (CFTR) was described in 1989, it has become increasingly evident that modifier genes and environmental factors play substantial roles in determining the severity of disease, particularly lung disease. Identifying these factors is crucial in devising therapies and other interventions to decrease the morbidity and mortality associated with this disorder. RECENT FINDINGS: Although many genes have been proposed as potential modifiers of CF, only a handful have withstood the test of replication. Several of the replicated findings reveal that genes affecting inflammation and infection response play a key role in modifying CF lung disease severity. Interactions between CFTR genotype, modifier genes, and environmental factors have been documented to influence lung function measures and infection status in CF patients. SUMMARY: Several genes have been demonstrated to affect disease severity in CF. Furthermore, it is likely that gene-gene and gene-environment interactions can explain a substantial portion of the variation of lung disease. Ongoing genome-wide studies are likely to identify novel genetic modifiers. Continued exploration of the role of genetic and nongenetic modifiers of CF is likely to yield new options for combating this debilitating disease.

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118 As a similar example in CF, VX-770, which is in phase II trials as of early 2008, is being tested on individuals with the G551D mutation in CFTR. Login to comment

[hide] Direct interaction of a small-molecule modulator w... Biochem J. 2009 Feb 15;418(1):185-90. Pasyk S, Li C, Ramjeesingh M, Bear CE
Direct interaction of a small-molecule modulator with G551D-CFTR, a cystic fibrosis-causing mutation associated with severe disease.
Biochem J. 2009 Feb 15;418(1):185-90., 2009-02-15 [PMID:18945216]

Abstract [show]
CF (cystic fibrosis) is caused by mutations in CFTR (CF transmembrane conductance regulator), which cause its mistrafficking and/or dysfunction as a regulated chloride channel on the apical surface of epithelia. CFTR is a member of the ABC (ATP-binding-cassette) superfamily of membrane proteins and a disease-causing missense mutation within the ABC signature sequence; G551D-CFTR exhibits defective phosphorylation and ATP-dependent channel gating. Studies of the purified and reconstituted G551D-CFTR protein revealed that faulty gating is associated with defective ATP binding and ATPase activity, reflecting the key role of G551 in these functions. Recently, high-throughput screens of chemical libraries led to identification of modulators that enhance channel activity of G551D-CFTR. However, the molecular target(s) for these modulators and their mechanism of action remain unclear. In the present study, we evaluated the mechanism of action of one small-molecule modulator, VRT-532, identified as a specific modulator of CF-causing mutants. First, we confirmed that VRT-532 causes a significant increase in channel activity of G551D-CFTR using a novel assay of CFTR function in inside-out membrane vesicles. Biochemical studies of purified and reconstituted G551D-CFTR revealed that potentiation of the ATPase activity of VRT-532 is mediated by enhancing the affinity of the mutant for ATP. Interestingly, VRT-532 did not affect the ATPase activity of the Wt (wild-type) CFTR, supporting the idea that this compound corrects the specific molecular defect in this mutant. To summarize, these studies provide direct evidence that this compound binds to G551D-CFTR to rescue its specific defect in ATP binding and hydrolysis.

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1 CFTR is a member of the ABC (ATP-binding-cassette) superfamily of membrane proteins and a disease-causing missense mutation within the ABC signature sequence; G551D-CFTR exhibits defective phosphorylation and ATP-dependent channel gating. Login to comment
2 Studies of the purified and reconstituted G551D-CFTR protein revealed that faulty gating is associated with defective ATP binding and ATPase activity, reflecting the key role of G551 in these functions. Login to comment
3 Recently, high-throughput screens of chemical libraries led to identification of modulators that enhance channel activity of G551D-CFTR. Login to comment
6 First, we confirmed that VRT-532 causes a significant increase in channel activity of G551D-CFTR using a novel assay of CFTR function in inside-out membrane vesicles. Login to comment
7 Biochemical studies of purified and reconstituted G551D-CFTR revealed that potentiation of the ATPase activity of VRT-532 is mediated by enhancing the affinity of the mutant for ATP. Login to comment
9 To summarize, these studies provide direct evidence that this compound binds to G551D-CFTR to rescue its specific defect in ATP binding and hydrolysis. Login to comment
15 The CF-causing missense mutation G551D-CFTR is associated with severe disease [2]. Login to comment
16 Unlike the more common F508-CFTR, G551D-CFTR is not mistrafficked and exhibits normal expression on the cell surface [1]. Login to comment
19 Therefore it has been suggested that the disease-causing mutation G551D will interfere with ATP binding, NBD1 and NBD2 heterodimerization and the ATPase activity that results from this domain-domain interaction [9]. Login to comment
20 In fact, we showed in studies of purified and reconstituted G551D-CFTR that it is severely defective as an ATPase [10,11]. Login to comment
23 A subgroup of these molecules is effective in modulating the functional expression of two disease-causing mutants: F508-CFTR and G551D-CFTR [13]. Login to comment
24 As we have optimized the purification and functional reconstitution of G551D-CFTR, we have an important reagent with which to evaluate whether certain of these pharmacological compounds act directly to modify the mutant CFTR protein. Login to comment
27 EXPERIMENTAL Vesicle-based flux assay of Wt (wild-type) CFTR and G551D-CFTR function The measurement of iodide efflux from membrane vesicles was adapted from a previously described cellular iodide efflux method [14]. Login to comment
38 Purification and reconstitution of Wt CFTR and G551D-CFTR Detailed protocols regarding the generation of Wt and mutant CFTR-His proteins are described elsewhere [15]. Login to comment
44 ATPase assay of purified Wt CFTR and G551D-CFTR protein ATPase activity was measured as the production of [γ -32 P]Pi from [γ -32 P]ATP as described by Gross et al. [17a]. Login to comment
51 RESULTS A novel iodide efflux assay reports potentiating activity of VRT-532 on G551D-CFTR VRT-532 has been shown to potentiate cAMP-activated chloride ion flux through G551D-CFTR in Fischer rat thyroid epithelial cells grown in monolayers and studied in voltage-clamp experiments conducted in the Ussing chamber apparatus [13]. Login to comment
52 The increase in chloride flux described above could report changes in the function of other membrane proteins that have an impact on the driving force for chloride ion flux through G551D-CFTR rather than changes in the intrinsic activity of the mutant protein. Login to comment
54 Therefore, as the first step towards understanding the mechanism of action of this small molecule in G551D-CFTR function, we were prompted to re-evaluate the effect of this modulator on the channel activity of G551D-CFTR in a more simplified system. Login to comment
55 Typically, activation of the channel function of CFTR and CF-causing mutants (including G551D-CFTR) by membrane-permeable agonists of cAMP-dependent PKA has been studied in intact cells using a convenient method known as the iodide efflux assay [18-20]. Login to comment
58 At the other end of the spectrum, patch-clamp studies of single channels in isolated membrane patches provide exquisite resolution of the kinetics of channel gating; however, as the channel activity of certain mutants, including G551D-CFTR, is severely impaired, acquisition of sufficient data to draw conclusions regarding the pharmacological effects of interesting compounds is time-consuming and often limited to a small number of channel proteins. Login to comment
60 Vesicles were prepared from Sf9 cells infected with baculovirus containing cDNA coding for Wt CFTR or G551D-CFTR using methods that have been described previously [21]. Login to comment
78 Figure 2 shows an example of the regulated efflux from membrane vesicles expressing G551D-CFTR. Login to comment
81 VRT-532 directly binds reconstituted G551D-CFTR and modifies its ATPase activity It has been shown that ATP-dependent heterodimerization of the two NBDs of CFTR is required for their activity as an ATPase and is permissive for subsequent conformational changes leading to opening of the chloride channel gate [22-24]. Login to comment
82 Further, it has been suggested that the failure of G551D-CFTR to bind ATP accounts for its defective ATPase activity and channel gating [5]. Login to comment
83 These findings prompted us to test the hypothesis that VRT-532 potentiates channel opening of G551D-CFTR by directly binding to enhance ATP affinity and hydrolysis. Login to comment
84 As previously determined, purified and reconstituted G551D-CFTR exhibits a very low level of ATPase activity relative to the Wt protein (0.1 +- 0.1 versus 1.4 +- 0.1 nmol·μg·h-1 respectively) in the presence of 1 mM MgATP [15]. Login to comment
88 Although the ATPase activity of G551D-CFTR was significantly enhanced by the addition of VRT-532, the activity of the treated mutant protein failed to achieve the level of activity exhibited by the Wt CFTR protein (measured at 1 mM MgATP) as shown in Figure 4(A). Login to comment
90 In Figure 5 and Table 1, the apparent ATP dependence of the ATPase activity exhibited by G551D-CFTR after treatment with VRT-532 (5 μM) is shown. Login to comment
91 In contrast with the dose response obtained for untreated G551D-CFTR, which failed to exhibit saturable ATP dependence, the data corresponding to the VRT-532-treated protein could be fitted using a Michaelis-Menten function (r2 = 0.9). Login to comment
92 These findings suggest that VRT-532 acts to enhance the ATPase activity of G551D-CFTR by increasing the apparent affinity for ATP. Login to comment
93 Figure 2 VRT-532 enhances channel activity in G551D-CFTR (A)Asexpected,iodideeffluxmediatedbyvesiclesexpressingG551D-CFTRwasnotsignificantly enhanced by PKA and MgATP plus valinomycin. Login to comment
96 (B) The histogram compares iodide efflux rates of membrane vesicles containing G551D-CFTR treated with MgATP (1 mM) and PKA (200 nM) before and after VRT-532 treatment (n = 5). Login to comment
101 In these 'proof of principle` studies, we revealed the direct action of one such compound, VRT-532, to enhance the ATP affinity of the CF-causing mutant, G551D-CFTR. Login to comment
102 As previously mentioned, VRT-532 is a particularly interesting small-molecule modulator as it enhances the channel activity of at least two different CFTR genotypes, including F508 and G551D-CFTR, suggesting that it may have a general role in repairing the molecular defects in multiple disease variants [13]. Login to comment
106 Therefore the findings of the present Figure 3 VRT-532 enhances intrinsic ATPase activity of G551D-CFTR (A) ATPase activity of purified, reconstituted and phosphorylated G551D-CFTR measured as the production of radioactive Pi from radioactive ATP. Login to comment
109 (B) The activation of G551D-CFTR is dependent on the dose of VRT-532, and this relationship can be fitted with a sigmoidal function (r2 = 0.94), yielding an EC50 of 2.7 μM (mean of duplicate measurements, from two different protein preparations; bars indicate range). Login to comment
110 Figure 4 VRT-532 significantly enhances the ATPase activity of G551D-CFTR but not Wt CFTR (A) VRT-532 (5 μM) significantly enhances ATPase activity of G551D-CFTR in the presence of 500 μM MgATP (n = 3, P < 0.05) but this activity stimulated by G551D-CFTR is less than the activity of the Wt protein measured at the same MgATP concentration, as indicated by the broken line [15]. Login to comment
112 study suggest that VRT-532 directly binds to purified reconstituted G551D-CFTR to modulate its activity, providing the impetus for future studies to determine whether it binds directly to F508-CFTR and to define the site for binding on these two mutants. Login to comment
113 Both the chloride channel gating and ATPase functions of CFTR likely report multiple dynamic conformational changes occurring throughout the whole molecule and, hence, the specific site at 5 VRT-532 enhances the ATPase activity of G551D-CFTR by increasing the apparent affinity for ATP The graph shows the effect of VRT-532 (5 μM) on ATP dose dependence of the ATPase activity of G551D-CFTR. Login to comment
124 Since the VRT-532 compound exerts a profound effect on ATPase activity by the G551D-CFTR protein rather than on the Wt protein, we speculate that it may be binding to or indirectly stabilizing an intramolecular interaction which is important for ATPase activity and is altered in the mutant protein. Login to comment
128 We are currently optimizing the purification and reconstitution of Table 1 Kinetic parameters obtained from analyses of ATP dependence of the ATPase activity of Wt CFTR and VRT-532-treated G551D-CFTR The ATPase activity of G551D-CFTR cannot be fitted using the Michaelis-Menten equation (see Figure 5 and [10,15]). Login to comment
131 Protein Vmax (nmol · μg · h-1 ) Km ATP (mM) CFTR [10,15] 4.6 0.7 G551D-CFTR N.A. NA G551D-CFTR + VRT-532 1.6 3.6 the major CF mutant for the purpose of testing this hypothesis directly. Login to comment
132 While G551D-CFTR is not a common mutation in the CF patient population, it does account for disease in approx. Login to comment
134 Hence, the insights regarding the molecular mechanisms for functional repair of the G551D-CFTR mutant by the small molecule, VRT-532, generated in the present study will provide the template for future drug development and potentially improvement in the clinical care of these severely affected patients. Login to comment
135 Currently, a clinical trial is in progress that is investigating the efficacy of a different molecule (VX-770) in patients bearing the G551D-CFTR mutation (http://www.cff.org/research/ClinicalResearch/ FAQs/VX-770). 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] Potentiation of cystic fibrosis transmembrane cond... Mol Membr Biol. 2008 Sep;25(6-7):528-38. Hughes LK, Ju M, Sheppard DN
Potentiation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- currents by the chemical solvent tetrahydrofuran.
Mol Membr Biol. 2008 Sep;25(6-7):528-38., [PMID:18989824]

Abstract [show]
The chemical solvent tetrahydrofuran (THF) increases short-circuit current (I(sc)) in renal epithelia endogenously expressing the cystic fibrosis transmembrane conductance regulator (CFTR). To understand how THF increases I(sc), we employed the Ussing chamber and patch-clamp techniques to study cells expressing recombinant human CFTR. THF increased I(sc) in Fischer rat thyroid (FRT) epithelia expressing wild-type CFTR with half-maximal effective concentration (K(D)) of 134 mM. This THF-induced increase in I(sc) was enhanced by forskolin (10 microM), inhibited by the PKA inhibitor H-89 (10 microM) and the thiazolidinone CFTR(inh)-172 (10 microM) and attenuated greatly in FRT epithelia expressing the cystic fibrosis mutants F508del- and G551D-CFTR. By contrast, THF (100 mM) was without effect on untransfected FRT epithelia, while other solvents failed to increase I(sc) in FRT epithelia expressing wild-type CFTR. In excised inside-out membrane patches, THF (100 mM) potentiated CFTR Cl(-) channels open in the presence of ATP (1 mM) alone by increasing the frequency of channel openings without altering their duration. However, following the phosphorylation of CFTR by PKA (75 nM), THF (100 mM) did not potentiate channel activity. Similar results were obtained with the triangle upR-S660A-CFTR Cl(-) channel that is not regulated by PKA-dependent phosphorylation and using 2'deoxy-ATP, which gates wild-type CFTR more effectively than ATP. Our data suggest that THF acts directly on CFTR to potentiate channel gating, but that its efficacy is weak and dependent on the phosphorylation status of CFTR.

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3 This THF-induced increase in Isc was enhanced by forskolin (10 mM), inhibited by the PKA inhibitor H-89 (10 mM) and the thiazolidinone CFTRinh-172 (10 mM) and attenuated greatly in FRT epithelia expressing the cystic fibrosis mutants F508del- and G551D-CFTR. Login to comment
28 Materials and methods Cells and cell culture For this study, we used Fischer rat thyroid (FRT) epithelial cells expressing wild-type, F508del- and G551D- human CFTR [8] and mouse mammary epithelial (C127) cells stably expressing either wild-type human CFTR or the variant DR-S660A-CFTR Figure 1. Login to comment
102 To provide further evidence that THF increases Isc by acting on CFTR, we tested its effects on the cystic fibrosis (CF) mutants F508del and G551D, both of which are located in NBD1 of CFTR and associated with a severe disease phenotype. Login to comment
104 However, like G551D [11], F508del also perturbs severely CFTR channel gating [14]. Login to comment
106 Figure 2H demonstrates that THF (100 mM), itself, had little or no effect on Isc in FRT epithelia expressing F508del- and G551D-CFTR, whereas forskolin (10 mM) elicited a modest increase in Isc in F508del- and G551D-expressing FRT epithelia. Login to comment
107 Moreover, THF (100 mM) failed to potentiate Isc following treatment of F508del- and G551D-expressing FRT epithelia with forskolin (10 mM; Figure 2H). Login to comment
108 By contrast, genistein (50 mM) enhanced robustly cAMP-stimulated Isc in FRT epithelia expressing F508del- and G551D-CFTR (Figure 2H). Login to comment
115 (G, H) The change in Isc evoked in wild-type, F508del- and G551D-CFTR FRT epithelia by different agents. 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