ABCMdb

ABCC7 p.Gly85Glu

Admin's notes:	Class II (maturation defect) Veit et al.
ClinVar:	c.254G>A , p.Gly85Glu D , Pathogenic c.254G>T , p.Gly85Val ? , not provided
CF databases:	c.254G>A , p.Gly85Glu D , CF-causing ; CFTR1: This mutation was detected in family #26, a French Canadian family classified as PI. This Gly to Glu change is associated with a group IIb haplotype. The mutation destroys a Hinfl site. The PCR product derived from the 3i-5 and 3i-3 primers is cleaved by this enzyme into 3 fragments, 172, 105, and 32 bp, respectively, for the normal sequence; a fragment of 277 bp would be present for the mutant sequence. They analyzed 54 CF chromosomes, 8 from group II and 50 normal chromosomes, 44 from group II and did not find another eample of G85E. c.254G>T , p.Gly85Val (CFTR1) ? , The patient with G85V carried G542X on the other chromosome, and presented mild phenotype with PI.
Predicted by SNAP2:	A: D (95%), C: D (95%), D: D (95%), E: D (59%), 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: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: N, 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: N, T: D, V: D, W: D, Y: D,

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II (maturation 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] 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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359 G85E and G91R affected folding by insertion of a charged residue within the plane of the bilayer [169]. 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] 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
92 The technique developed demonstrates excellent single-strand separation and non-radioactive visualisation on polyacrylamide gels, and is time-saving and directly Table 2 Known mutations identified in 198 CF patients analysed investigatively Exon (E) Number of CFTR mutations intron (I) chromosomes Patient`s nationality Highest prevalence ∆F508 E10 212 miscellaneous 3905insT E20 025 Swiss Swiss, Amish, Arcadian R553X E11 020 Swiss, German German 1717-1G->A I10 017 Swiss, Italian Italian N1303K E21 011 Swiss, French, Italian Italian W1282X E20 014 Swiss, Italian, Israelit Jewish-Askhenazi G542X E11 009 Swiss, Spanish, Italian Spanish 2347delG E13 008 Swiss R1162X E19 006 Swiss, Italian, Russian Italian 3849+10kbC->T I19 005 German, French R347P E07 004 Swiss T5 I08 004 Swiss R334W E07 003 Swiss Q525X E10 003 Swiss 3732delA E19 003 Swiss S1235R E19 003 Italian, Turkish G85E E03 002 Italian, Greek I148T E04 002 Austrian, Turkish French-Canadian 621+1G->T I04 002 French French-Canadian 1078delT E07 002 Swiss E585X E12 002 Italian 2176insC E13 002 Swiss, Italian 2789+5G->A I14b 002 Italian Spanish D1152H E18 002 Swiss, French 4016insT E21 002 Turkish Q39X E02 001 Swiss 394delTT E03 001 Swiss Nordic, Finnish R117H E04 001 Swiss A120T E04 001 Swiss G126D E04 001 Swiss 711+5G->A I05 001 Russian M348K E07 001 Italian L568F E12 001 Italian 2183AA->G E13 001 Italian Italian K710X E13 001 Swiss S945L E15 001 French 3272-26A.->G I17a 001 Swiss M1101K E17b 001 Swiss Huttite 3601-17C->T I18 001 Swiss R1158X E19 001 Swiss 4005+1G-A I20 001 Italian applicable to early diagnostic testing, carrier detection and prenatal diagnosis. Login to comment

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

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

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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] Defects in processing and trafficking of the cysti... Kidney Int. 2000 Mar;57(3):825-31. Skach WR
Defects in processing and trafficking of the cystic fibrosis transmembrane conductance regulator.
Kidney Int. 2000 Mar;57(3):825-31., [PMID:10720935]

Abstract [show]
Cystic fibrosis (CF) is caused by inherited mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel expressed in epithelial tissues. Most mutations in CF patients result in rapid intracellular degradation of the CFTR protein. While this defect is thought to result from abnormal protein folding, it is unclear how mutant and wild-type (WT) proteins differ in structure, how the cell is able to distinguish these differences, and how the fate of the mutant protein is determined. By examining the initial steps of CFTR assembly into the endoplasmic reticulum (ER) membrane, it has recently been shown that CFTR utilizes two redundant translocation pathways to direct N-terminus folding events. Mutations that block one pathway therefore do not alter transmembrane topology, but rather appear to disrupt intracellular trafficking through perturbations in higher order tertiary structure. These studies suggest that cellular quality control machinery acts at least in part, by monitoring proper interactions between CFTR subdomains. The end result of this process is the conversion of misfolded CFTR into a membrane-bound, polyubiquitinated complex. This complex recruits cytosolic degradation machinery to the endoplasmic reticulum membrane where CFTR is degraded as it is extracted from the lipid bilayer. Understanding how cellular machinery mediates this process will be an important step in designing strategies to modify protein folding and degradation in CF and related ion channelopathies.

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52 The post-translational pathway is utilized by most (Ͼ60%) of WT chains and essentially all G85E and G91R mutant chains. Login to comment
76 While it is often tempting Two CF mutations, G85E and G91R, each introduce to view the acquisition of protein function as a criteria an additional charged residue within the hydrophobic for "normal" folding, in the case of CFTR this is not core of TM1. Login to comment
79 This suggested that G85E and G91R CFTR mutants [35]. Login to comment
93 In addition, they indicated that G85E and Golgi and lysosome-independent compartment. Login to comment

[hide] A new approach for identifying non-pathogenic muta... Hum Genet. 2000 Feb;106(2):172-8. Bombieri C, Giorgi S, Carles S, de Cid R, Belpinati F, Tandoi C, Pallares-Ruiz N, Lazaro C, Ciminelli BM, Romey MC, Casals T, Pompei F, Gandini G, Claustres M, Estivill X, Pignatti PF, Modiano G
A new approach for identifying non-pathogenic mutations. An analysis of the cystic fibrosis transmembrane regulator gene in normal individuals.
Hum Genet. 2000 Feb;106(2):172-8., [PMID:10746558]

Abstract [show]
Given q as the global frequency of the alleles causing a disease, any allele with a frequency higher than q minus the cumulative frequency of the previously known disease-causing mutations (threshold) cannot be the cause of that disease. This principle was applied to the analysis of cystic fibrosis transmembrane conductance regulator (CFTR) mutations in order to decide whether they are the cause of cystic fibrosis. A total of 191 DNA samples from random individuals from Italy, France, and Spain were investigated by DGGE (denaturing gradient gel electrophoresis) analysis of all the coding and proximal non-coding regions of the gene. The mutations detected by DGGE were identified by sequencing. The sample size was sufficient to select essentially all mutations with a frequency of at least 0.01. A total of 46 mutations was detected, 20 of which were missense mutations. Four new mutations were identified: 1341+28 C/T, 2082 C/T, L1096R, and I11131V. Thirteen mutations (125 G/C, 875+40 A/G, TTGAn, IVS8-6 5T, IVS8-6 9T, 1525-61 A/G, M470V, 2694 T/G, 3061-65 C/A, 4002 A/G, 4521 G/A, IVS8 TG10, IVS8 TG12) were classified as non-CF-causing alleles on the basis of their frequency. The remaining mutations have a cumulative frequency far exceeding q; therefore, most of them cannot be CF-causing mutations. This is the first random survey capable of detecting all the polymorphisms of the coding sequence of a gene.

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79 Out of the 20 missense mutations, three (G85E, ∆F508, and N1303K) are certainly CF-causing, and several (R31C, K68E, R75Q, I148T, V562L, G576A-R668C, L997F, F1052V, S1235R) have been described in congenital bilateral absence of the vas deferens, in disseminated bronchiectasis, in pancreatitis, or in atypical CF cases mutations as reported in the CFGAC website (). Login to comment
80 Many (13 out of 20) of the missense mutations change highly conserved (5/5 species analyzed) amino acid residues (R75Q, G85E, I148T, I506V, R668C, G622D, L997F, I1027T, F1052V, L1096R, I1131V, R1162L, N1303K); others affect amino acid residues conserved in 4/5 species (K68 E, R170H, M470V, V562L, S1235R), or in 3/5 species (R31C and G576A; Tucker et al. 1992). Login to comment
91 The remaining 30 (after having excluded the certainly CF-causing mutations G85E, ∆F508 and N1303K) have been found a few times only: once (20 mutations), twice (five mutations), three times (three mutations), four times (one mutation), and eight times (one mutation). 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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119 Certain missense mutations such as G85E [32] may confer a variable pancreatic phenotype. Login to comment

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

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570 SYLVAIN R RIVARD* CHRISTIAN ALLARD† JEAN-PIERRE LEBLANC† MARCEL MILOT† GERVAIS AUBIN† FERNAND SIMARD† CLAUDE FÉREC‡ MARC DE BRAEKELEER†§¶ *Département des Sciences Fondamentales, Université du Québec à Chicoutimi, Canada Table 1 Distribution of cystic fibrosis patients diagnosed before the age of 5 by age groups in Saguenay-Lac-Saint-Jean, (A) by genotype, (B) by mutation 0-10 years 10.1-20 years Over 20 years All ages No % No % No % No % (A) Genotype F508/ F508 15 (1) 40.5 21 (2) 36.2 18 (3) 42.9 54 (6) 39.4 F508/621+1G→T 12 (1) 32.4 16 (1) 27.6 10 (1*) 23.8 38 (3*) 27.7 F508/A455E 1 2.7 6 10.3 5 11.9 12 8.8 F508/I148T 1 2.7 1 1.7 2 1.5 F508/Y1092X 3 (1) 5.2 1 2.4 4 (1) 2.9 F508/Q890X 1 2.4 1 0.7 F508/R1158X 1 2.4 1 0.7 621+1G→T/621+1G→T 2 (1) 5.4 4 6.9 1 2.4 7 (1) 5.1 621+1G→T/A455E 1 2.7 4 6.9 3 7.1 8 5.8 621+1G→T/711+1G→T 2 (1) 5.4 2 (1) 3.4 4 (2) 2.9 621+1G→T/Y1092X 1 2.7 1 0.7 621+1G→T/S489X 1 2.7 1 0.7 621+1G→T/G85E 1 (1) 1.7 1 (1) 2.4 2 (2) 1.5 A455E/R117C 1 2.7 1 0.7 N1303K/I148T 1 2.4 1 0.7 Total 37 58 42 137 Death (4) 10.8 (6) 10.3 (5*) 11.9 (15*) 10.9 (B) Mutation F508 16 (1) 43.2 25 (3) 43.1 21 (3) 51.2 62 (7) 45.6 621+1G→T 18 (3) 48.6 23 (3) 39.7 12 (2*) 29.3 53 (8*) 39.0 A455E 3 8.1 10 17.2 8 19.5 21 15.4 Total 37 58 41 136 Death (4) 10.8 (6) 10.3 (5*) (12.2) (15*) (11.0) ( ): Number of deaths. 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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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

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

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

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

[hide] Inhibition of cystic fibrosis transmembrane conduc... J Clin Invest. 2000 Jun;105(12):1711-21. Hallows KR, Raghuram V, Kemp BE, Witters LA, Foskett JK
Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase.
J Clin Invest. 2000 Jun;105(12):1711-21., [PMID:10862786]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated Cl(-) channel that regulates other epithelial transport proteins by uncharacterized mechanisms. We employed a yeast two-hybrid screen using the COOH-terminal 70 residues of CFTR to identify proteins that might be involved in such interactions. The alpha1 (catalytic) subunit of AMP-activated protein kinase (AMPK) was identified as a dominant and novel interacting protein. The interaction is mediated by residues 1420-1457 in CFTR and by the COOH-terminal regulatory domain of alpha1-AMPK. Mutations of two protein trafficking motifs within the 38-amino acid region in CFTR each disrupted the interaction. GST-fusion protein pull-down assays in vitro and in transfected cells confirmed the CFTR-alpha1-AMPK interaction and also identified alpha2-AMPK as an interactor with CFTR. AMPK is coexpressed in CFTR-expressing cell lines and shares an apical distribution with CFTR in rat nasal epithelium. AMPK phosphorylated full-length CFTR in vitro, and AMPK coexpression with CFTR in Xenopus oocytes inhibited cAMP-activated CFTR whole-cell Cl(-) conductance by approximately 35-50%. Because AMPK is a metabolic sensor in cells and responds to changes in cellular ATP, regulation of CFTR by AMPK may be important in inhibiting CFTR under conditions of metabolic stress, thereby linking transepithelial transport to cell metabolic state.

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388 Structural cues involved in endoplasmic reticulum degradation The Journal of Clinical Investigation | June 2000 | Volume 105 | Number 12 of G85E and G91R mutant cystic fibrosis transmembrane conductance regulator. J. Clin. Invest. 100:1079-1088. 34. Login to comment

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

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

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104 In a small group of G85E/- 7T/7T 1 patients, alpha-glucosidase activity was 18.5 Ϯ 2.7 mU/ml in 2752-15C→G/- 7T/7T 1 CUAVD (n ϭ 4), and 26.7 Ϯ 5.5 mU/ml in CBAVD (n ϭ 7).L997F/-a 7T/7T 1 1677delTA/- 7T/7T 1 After reclassification of the patients according to the presence Y1014C/- 7T/9T 1 of zero, one or two mutations, none of the variables showed N1303K/- 7T/9T 1 significant differences either in CUAVD or CBAVD (notNegative CFTR mutation 16 (15) -/- 7T/7T 12 (11) shown). 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] 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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65 N. Engl. J. Med., 332, 1475-1480.∆F 508 G85E 296ϩ12T→C Daigneault, J., Aubin, G., Simard, F. and DeBraekeleer, M. (1992) TheN1303K E6OX 711ϩ1G→T incidence of cystic fibrosis in Saguenay-Lac-St-Jean (Quebec, Canada).G542 L88s 711ϩ3A→G Hum. Biol., 64, 115-119.R117H P67L A455E 621ϩ16→T R75X 1461ins4 DeBraekeleer, M. and Daigneault, J. (1992) Spatial distribution of the ∆F508 mutation of cystic fibrosis. 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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9 One rare mutation, G85E, in exon 3 was detected in 2 members of the same family. Login to comment
10 One was a patient (complex heterozygous, DF508/ G85E) and the other was a carrier (G85E/wt) (Table 1). Login to comment
20 Results of CF mutation identification Location of the mutation in the CFTR geneMutation %CF chromosomes with mutation (n)* DF508 65.039exon 10 exon 11G542X 5.03 N1303K exon 21 2 3.3 1717-1GA intron 10 2 3.3 2exon 4R117H 3.3 G85E 1.71exon 3 R1162X exon 19 1 1.7 Total identified - 50 83.3 *Sum of CF chromosomes from 30 patients is 60 (30×2). Login to comment
21 333 Letter to the Editor Mutation G85E has not previously been found in Slovenia and Hungary, but in Italy, Spain and southern Greece, it has a frequency of between 1.33 and 0.8% (9, 13, 14, 16, 19). 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

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

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

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33 These were: delF508 (Kerem et al., 1989), W1282X (Vidaud et al., 1990), G542X, 1717-1G R A, S549R (Kerem et al., 1990), N1303K (Osborn et al., 1991), 3849 1 10Kb C R T (Highsmith et al., 1994), T359K/Q360K (Shoshani et al., 1992), G85E (Zielenski et al., 1991), 405 1 1G R A (Dork et al., 1993), W1089X (Shosani et al., 1994), and D1152H (Highsmith et al., 1993). Login to comment
40 THE CONSENSUS POLICY OF SCREENING OF CF MUTATIONS IN ETHNIC GROUPS OF ISRAELI JEWSa Buchara and Iran Georgia Libya Morocco Tunis Turkey Egypt Sephardi W1089X 1 1 1 G85E 1 1 405-1 G® A 1 1 1 S549R 1 1 D1152H 1 1 1 1 T360K 1 Individuals of all ethnic groups were screened for the mutations W1282X, delF508, G5429X, N1303K, 3849110Kb C® T and 1717-1G® A. Login to comment
45 There were 173 (46.3%) carriers of the W1282X mutation; 110 (29.4%) carriers of delF508; 23 (6.1%) carriers of G542X; 10 (2.7%) carriers of N1303K; and 22 (5.9%) carriers of 3849 1 10KbC R T. Twenty (5.3%) were found to carry D1152H; 11 (2.9%) carried 405 1 1G R A; 4 (1.1%) carried W1089X; and 1 (0.3%) carried S549R. No carriers were detected for the mutations 1717-1G R A, G85E, and T360K, which were tested for in 7,383, 1,436, and 41 individuals, respectively. Login to comment
86 On the other hand, some mutations that were found in CF patients were not detected in the healthy cohort we studied, namely, G85E, 1717-1G R A, and T360K. Login to comment
91 One might reconsider the cost effectiveness of testing for infrequent mutations such as 1717-1G R A, W1089X, S549R, and G85E when screening the general Jewish population. Login to comment

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

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

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56 5T, G542X, R334W, N1303K, L206W, 3659-C, and G85E were identified in the remaining nine patients. Login to comment
64 Other genotypes present in our series ⌬F508/711+1G>T 2A 5T/5T 1B ⌬F508/5T 2B ⌬1507/- 1A ⌬F508/R117H 2B R1162X/1898+1G>A 1A ⌬F508/R1162X 1A 2183A/- 1A ⌬F508/N1303K 1A 1609-CA/1811+1.6kbA>G 1A ⌬F508/3272-26A>G 1B 1609-CA/R347P 1A ⌬F508/D836Y 1B Q890X/- 1A ⌬F508/1717-1G>A 1A R334W/- 1B G542X/W1282X 1A N1303K/2789+5G>A 1B G542X/2789+5G>A 1B 3659-C/- 1B G542X/P205S 1B G85E/- 1B G542X/D1270N 1B Negative 1A, 20B L206W/- 1B Unknown 2A creatic insufficiency was highly prevalent, affecting 33 patients (84.6%). Login to comment

[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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72 Two positive samples, with mutations localised in the two different domains, were selected (E60X and G85E). Login to comment
74 At 57°C, only the low melting domain represented by the mutation G85E in this example is analysed. Login to comment
87 At 57°C, the 3` part is studied (low melting) represented by mutation G85E (green), whereas at 58°C, the 5` part with mutation E60X (pink) is analysed gene. Login to comment

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

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

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

[hide] Nasal potential difference measurements in patient... Eur Respir J. 2001 Jun;17(6):1208-15. Wilschanski M, Famini H, Strauss-Liviatan N, Rivlin J, Blau H, Bibi H, Bentur L, Yahav Y, Springer H, Kramer MR, Klar A, Ilani A, Kerem B, Kerem E
Nasal potential difference measurements in patients with atypical cystic fibrosis.
Eur Respir J. 2001 Jun;17(6):1208-15., [PMID:11491166]

Abstract [show]
The diagnosis of cystic fibrosis (CF) is based on characteristic clinical and laboratory findings. However, a subgroup of patients present with an atypical phenotype that comprises partial CF phenotype, borderline sweat tests and one or even no common cystic fibrosis transmembrane conductance regulator (CFTR) mutations. The aim of this study was to evaluate the role of nasal potential difference (PD) measurements in the diagnosis of CF patients with an atypical presentation and in a population of patients suspected to have CF. Nasal PD was measured in 162 patients from four different groups: patients with classical CF (n = 31), atypical phenotype (n = 11), controls (n = 50), and patients with questionable CF (n = 70). The parameter, or combination of nasal PD parameters was calculated in order to best discriminate all CF patients (including atypical CF) from the non-CF group. The patients with atypical CF disease had intermediate values of PD measurements between the CF and non-CF groups. The best discriminate model that assigned all atypical CF patients as CF used: e(response to chloride-free and isoproterenol/response to amiloride) with a cut-off >0.70 to predict a CF diagnosis. When this model was applied to the group of 70 patients with questionable CF, 24 patients had abnormal PD similar to the atypical CF group. These patients had higher levels of sweat chloride concentration and increased rate of CFTR mutations. Nasal potential difference is useful in diagnosis of patients with atypical cystic fibrosis. Taking into account both the sodium and chloride transport elements of the potential difference allows for better differentiation between atypical cystic fibrosis and noncystic fibrosis patients. This calculation may assist in the diagnostic work-up of patients whose diagnosis is questionable.

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55 - Clinical data of patients with atypical cystic fibrosis disease Age yrs Sex Genotype Sweat chloride mmol?L-1 PS/PI FEV1 % pred 22 M 3849z10kbCRT/405z1GRA 112 PS 60 21 M 3849z10kbCRT/405z1GRA 70 PS 43 19 M G85E/5T 57 PS 72 15 M 5T/? 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] 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] 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] ATB(0)/SLC1A5 gene. Fine localisation and exclusio... Eur J Hum Genet. 2001 Nov;9(11):860-6. Larriba S, Sumoy L, Ramos MD, Gimenez J, Estivill X, Casals T, Nunes V
ATB(0)/SLC1A5 gene. Fine localisation and exclusion of association with the intestinal phenotype of cystic fibrosis.
Eur J Hum Genet. 2001 Nov;9(11):860-6., [PMID:11781704]

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

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

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

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

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

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

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

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136 Another patient presented a G85E mutation, which has been associated with both ps and pi phenotypes.35 In our study, this mutation was considered to be a mild mutation, although this patient had severe disease with a sweat chloride concentration of 157 mEq/L, pi, and severe respiratory disease (FEV1 at 26% of predictive value and Pseudomonas colonization). 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
40 Mutation Frequency (%) DelF508 54 N1303K 8 G542X 6.25 1717-1G ® A 2.50 R334W 1.75 2183AA ® G 1.50 R117H, L1077P, W1282X 1.25 D110E, R347P, E585X, 2789 ‡ 5G ® A 0.75 R352Q, R553X, R1066H, D1152H, R1158X, 1782delA, 1898 ‡ 1G ® A, 3659delC 0.50 G85E, R117L, G178R, D579G, H609R, Y1032C, V1153E, R1162X, 621 ‡ 1G ® T, 711 ‡ 1G ® T, 1845delAG o 1846delGA, 2143delT 0.25 Table2.Differencesinthethreestrategiesofneonatalscreening(audit1990-1999). Login to comment

[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] 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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56 Frequency of cystic fibrosis transmembrane regulator mutations in the Argentine population: 440 chromosomes analysed Mutation Localization Chromosome Number Percentage DF508 Exon 10 258 58.64 G542X Exon 11 18 4.10 W1282X Exon 20 12 2.73 N1303K Exon 21 12 2.73 R334W Exon 7 5 1.14 1717-1G»A Intron 10 5 1.14 3849π10KbC»T Intron 19 4 0.91 1811π1.6KbA»G Intron 11 4 0.91 IVS8-5T Intron 8 4 0.91 G85E Exon 3 3 0.68 621π1G»T Intron 4 3 0.68 2789π5G»A Intron 14b 3 0.68 DI507 Exon 10 3 0.68 2184delA Exon 13 2 0.45 2566insT Exon 13 2 0.45 2686insT Exon 14a 2 0.45 3659delC Exon 19 2 0.45 R1162X Exon 19 2 0.45 4016insT Exon 21 2 0.45 2789π2insA Intron 14b 2 0.45 L6V Exon 1 1 0.23 297π2A»G Intron 2 1 0.23 W57X Exon 3 1 0.23 R75Q Exon 3 1 0.23 Q220X Exon 6a 1 0.23 Y362X Exon 7 1 0.23 D426C Exon 9 1 0.23 1460delAT Exon 9 1 0.23 1353insT Exon 9 1 0.23 1782delA Exon 11 1 0.23 R553X Exon 11 1 0.23 S549R Exon 11 1 0.23 1898π3A»G Intron 12 1 0.23 2594delGT Exon 13 1 0.23 2183AA»G Exon 13 1 0.23 I1027T Exon 17a 1 0.23 R1066C Exon 17b 1 0.23 G1061R Exon 17b 1 0.23 4005-1G»A Intron 20 1 0.23 Total 367 83.45 209 nificant differences were observed among the compared populations (Table2). Login to comment

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

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

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

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

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50 G, R1162X, 3659delC, W1282X, 3905insT, N1303K, G85E, 621 þ 1G ! Login to comment

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

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

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46 A series of mutations usually associated with pancreatic sufficiency have been identified and defined as ''mild`` with reference to pancreatic status [Kerem et al., 1989c]: G85E, G91R, R117H, E193K, P205S, R334W, T338I, R347H, R347L, R347P, R352Q, A455E, S492F, S549N, P574H, D579G, 711 þ 5 G > A, C866Y, F1052V, H1054D, R1066H, R1068H, H1085R, D1152H, S1159P, S1251N, F1286S, G1349D, 2789 þ 5 G > A, and 3849 þ 10kb C > T [Dean et al., 1990; Cutting et al., 1990a; Cremonesi et al., 1992; Highsmith et al., 1994]. 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]

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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
301 We think that the difference in prevalence over the two periods has been because of the greater chance of recognising subjects with only one identified CFTR mutation, who can then be forwarded for a sweat test, owing to the wider DNA strategy (one case with a recall IRT under the cut off value had a genotype G85E/unknown), and because of the new definition of the upper normal value for the chloride sweat test in our laboratory (30 mmol/l). Login to comment
310 Since 1998, in our CF centre, an expanded DNA CFTR gene analysis and repeat sweat test after 6-12 months of life have been performed in hypertrypsinaemic Table 1 Genotypes of 33 patients with CF identified in the 15 month period Two CFTR mutations identified (18 patients) by PCR/OLA: ∆F508/∆F508 6 N1303K/N1303K 2 ∆F508/N1303K 3 R334W/R334W 1 ∆F508/G542X 2 G542X/G542X 1 ∆F508/3659delC 1 2183AA→G/ 2183AA→G 1 ∆F508/R1162X 1 One CFTR mutation identified (13 patients) by PCR/OLA: ∆F508/D1152H* 1 ∆F508/Y1032C* 1 ∆F508/R1066H* 1 ∆F508/UN 6 ∆F508/R1066C* 1 W1282X/L1077P* 1 ∆F508/D579G* 1 G85E/UN 1 No CFTR mutation identified (two patients) by PCR/OLA: 711+3A→G*/UN 1 D110E*/D110E* 1 *CFTR alleles identified by analysis by denaturing gradient gel electrophoresis and sequencing. Login to comment

[hide] 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]

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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
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

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

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

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

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

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

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

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

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

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

[hide] Comparison of two different protocols of neonatal ... Clin Genet. 2002 Sep;62(3):245-9. Narzi L, Lucarelli M, Lelli A, Grandoni F, Lo Cicero S, Ferraro A, Matarazzo P, Delaroche I, Quattrucci S, Strom R, Antonelli M
Comparison of two different protocols of neonatal screening for cystic fibrosis.
Clin Genet. 2002 Sep;62(3):245-9., [PMID:12220442]

Abstract [show]
The results of two different protocols of neonatal cystic fibrosis (CF) screening in the Lazio region of Italy are reported. The first study, conducted from 1992 to 2000 on about 200,000 newborns, consisted of an immunoreactive trypsin (IRT) protocol without mutation analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, referred to as the IRT/IRT protocol. Approximately 5% of the newborns with a positive first IRT test were also positive at the second test; approximately 57% of the newborns with a high IRT level at the second test were subsequently found to be affected by CF. In September 1998, a second protocol that included mutation analysis (IRT/DNA/IRT protocol) was started. Comparison of the two different screening protocols in terms of sensitivity in detecting CF patients demonstrated that the IRT/DNA/IRT protocol is more effective because it is able to detect a higher number of CF patients than the IRT/IRT protocol. In the same period, in addition to the overall diagnosis performed on a screening basis, 64 other subjects were identified as being affected by CF on the basis of symptomatic findings. The overall incidence of CF (screening + symptoms) was 1 : 2982, while that for carriers was 1 : 27. The sensitivity of the screening program increased over the period from 1992 to 2000, with the enhanced sensitivity in the past 2 years being due to the introduction of the IRT/DNA/IRT protocol.

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39 Mutations known to produce variable or mild clinical manifestations (e.g. G85E, R347P, 2789π 5GtoA) were observed in newborns with pancreatic sufficiency (PS). Login to comment
40 Affected subjects with initial PS (32%) were further evaluated for a period of time (from 6months to 1year): PS persisted in all except one (with DF508/G85E genotype) who, after the first year, showed PI with a decrement to borderline value of stool chymotrypsin (6.4U/g) but to pathological value of stool elastase1 (77mg/ g, with pathological values Ͻ 200mg/g). 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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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

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

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

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

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

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

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

[hide] 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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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

[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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67 Frequency of rarer CF mutations and polymorphisms Mutation Numberof chromosomes Frequency (%) Polymorphism Frequency (%) E60X 1 0.24 IVS6a-8 25.0 P67L 1 0.24 (TG)m 37.5 G85E 1 0.24 IVS8-Tn 23.8 6211G >T 1 0.24 M470V 41.3 IVS8^5T 5 1.21 V520F 2 0.48 18981G >A 2 0.48 R117H 1 ^ DF508 17 ^ Total 80 15 Frequencypercentages areadjustedtorepresent 85%. Login to comment
78 There are no reports of the E60X, P67L, G85E, IVS8-5T, V520F or 1898 1G> A mutations in previously published CF mutation frequency reports for the Republic of Ireland (11, 12). Login to comment
81 In UK reports (15), the incidence of the E60X mutation is reported at 0.16%, G85E at 0.21%, 621 1G> T and V520F at 0.17% and 1898 1G> A at 0.46%. Login to comment

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

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

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

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

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

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47 Analysis of mutations in the CFTR gene as tested by the multiplex polymerase chain reaction (PCR), followed by the reverse dot-blot technique, which searches for 29 of the most frequent mutations (DF508, N1303K, G542X, W1282X, 1717±1 G-A, R553X, 2183 AA-G, DI507, G551D, R560T, 3849 10kbC > T, R1162X, 3659delC, 3905insT, G85E, 621 1GT, R117H, R347P, R334W, A455E, 2789 5GA, Q552X, S1251N, 3905insT, 394delTT, E60X, 2143delT, 2184delA, 711 5G > A), and by ASO dot-blot for the following mutations: I148T, R1158X, 4016 1T, G1244E G >A.26 Statistical analysis was performed using multivariate analysis, by forward stepwise comparison; it was done to ®nd out which of the examined characteristics could be associated (P < 0.01) to nasal polyposis. Login to comment

[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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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
98 Number Frequency Number Frequency 1652_1655del 3 bp F508del 966 76.5% 226 81.3% 1192 77.4% 1784G>A G551D 82 6.5% 27 9.7% 109 7.1% 482G>A R117H 38 3.0% 4 1.4% 42 2.7% 1811G>C R560T 30 2.4% 2 0.7% 32 2.1% 621+1G>T 21 1.7% 21 1.4% 1648_1653delATC I507del 10 0.8% 1 0.4% 11 0.7% 1717-1G>A 9 0.7% 9 0.6% 1756G>T G542X 8 0.6% 8 0.5% 1187G>A R352Q 3 0.2% 2 0.7% 5 0.3% 1461ins4 5 0.4% 5 0.3% 4041C>G N1303K 5 0.4% 5 0.3% 310G>T E60X 4 0.3% 4 0.3% 1690G>T V520F 4 0.3% 4 0.3% 3007delG 4 0.3% 4 0.3% 3272-26A>G 2 0.2% 2 0.7% 4 0.3% 386G>A G85E 3 0.2% 3 0.2% 3849+10kbC>T 3 0.2% 3 0.2% Unidentified Unidentified 41 3.2% 11 4.0% 52 3.4% Total Total 1262 100.0% 278 100.0% 1540 100.0% Dublin cohort Cork cohort Ireland Amino acid change Nucleotide change We noted similar high frequencies of the F508del and G551D mutations in the three cohorts studied. Login to comment

[hide] 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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294 Less common mutations included G85E and 5T (n=5 chromosomes), A455E and R1162X (n=4 chromosomes), R347, Y1092X, R334W, and V520F (n=3 chromosomes). Login to comment
307 This includes one patient who carries the missense mutation G85E which appears to have an indeterminate effect on pancreatic phenotype as it has been observed in patients with PS and PI phenotypes.23 All but one of 49 individuals carrying at least one mutation belonging to classes IV or V have remained PS. Login to comment
364 A small number of mutations (for example, G85E) are considered to be indeterminate due to their inconsistent effect on pancreatic phenotype. 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
249 (Top), exon 3 wild-type and G85E mutant analytes; (bottom), exon 13.2A wild-type and 2184⌬A mutant analytes. 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
69 Table 3 Sequence variations identified in the PRSS1, CFTR, and SPINK1 genes in 34 ALD patients CFTR Patients n PRSS1 Mutant Poly T SPINK1 1 1 Fa F NDb P55S 2 1 F F 7/7 P55S 3 1 F F 7/7 MIT 4 1 F G85E 5/7 F 5 1 F F 5/5 F 6 1 F F 5/9 F 7-8 2 F F 5/7 F 9-27 19 F F 7/7 F 28 1 F F 7/7 ND 29-34 6 F F 7/9 F See Table 2. heterozygote). Login to comment

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

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

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63 The 3849+10kbC˚T mutation can lead to the inclusion of a cryptic 84-bp exon, which contains a stop codon.24 Another eight patients were heterozygous for stop mutations: six were ∆F508/W1282X, one was G85E/ W1282X, and one was W1282X/unknown. 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] 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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61 In this study, we also found that only four among 70 CFTR mutations included in the screening panel were present in CF patients born in the three different states of Brazil and in the two population subgroups (Euro- and Afro-Brazilians) studied; that is, DF508, G542X, R1162X, and G85E. Login to comment
63 FREQUENCIES OF 70 CFTR MUTATIONS IN DIFFERENT STATES OF BRAZIL, BY CONTINENTA L GROUP CFTR mutations SC PR MG detected n n n n % n % N % DF508 53 39 54 146 47.1 8 10.5 154 39.9 G542X 6 9 8 23 7.4 1 1.3 24 6.2 R1162X 9 2 4 15 4.8 2 2.6 17 4.4 N1303K 5 5 0 10 3.2 0 0 10 2.6 R334W 5 1 4 10 3.2 0 0 10 2.6 G85E 2 2 4 8 2.6 1 1.3 9 2.3 1717-1G®A 1 3 2 6 1.9 0 0 6 1.6 W1282X 4 1 1 6 1.9 0 0 6 1.6 3849110kbC®T 1 3 1 5 1.6 0 0 5 1.3 R553X 0 2 0 2 0.7 0 0 2 0.5 1812-1G®A 0 1 3 4 1.3 1 1.3 5 1.3 2183AA®G 2 1 0 3 1.0 0 0 3 0.8 312011G®A 0 0 2 2 0.7 2 2.6 4 1.0 Y1092X 0 1 1 2 0.7 1 1.3 3 0.8 G551D 0 0 0 0 0 0 0 0 0 W1089X 0 0 1 1 0.3 0 0 1 0.3 6211G®T 0 1 0 1 0.3 0 0 1 0.3 Q1238X 0 1 0 1 0.3 0 0 1 0.3 711-1G®T 0 1 0 1 0.3 0 0 1 0.3 R347P 1 0 0 1 0.3 0 0 1 0.3 189811G®A 1 0 0 1 0.3 0 0 1 0.3 I507 0 0 1 1 0.3 0 0 1 0.3 Subtotal 91 73 86 250 80.7 16 21.1 266 68.9 Alleles with CFTR 5 27 28 60 19.4 60 79.0 120 31.1 mutations not detected Total 96 100 114 310 100.0 76 100.0 386 100.0 Detection rate (%) 94.8 73.0 75.4 250 80.7 16 21.1 266 68.9 The following 70 CFTR mutations were selected and tested on the basis of frequency in various populations, known association with CF, or predicted deleterious effect on the CFTR protein product; DF508, G542X, N1303K, G551D, R553X, DI507, A455E, A559T, C524X, D1270N, E60X, G178R, G330X, G85E, 2307insA, I148T, K710X, P574H, Q1238X, Q493X, Q890X, R1158X, R1162X, R117H, R334W, R347H, R347P 2307insA, I148T, K710X, P574H, Q1238X, Q493X, Q890X, R1158X, R1162X, R117H, R334W, R347H, R347P 2307insA, 1148T, K710X, P574H, Q1238X, Q493X, Q890X, R1158X, R1162X, R117H, R334W, R347H, R347P, R352Q, R560T, S1196X, S1255X, S364P, S549N, S549R, V520F, W1089X, W1282X, W1310X, W1316X, Y1092X, Y122X, Y563D, 1078delT,1677delTA,1717-1G-A,1812-1G-A,1898 1 1G-A, 2043delG,2183delAA-G, 2184delA, 2789 1 5G-A, 2869insG, 2909delT, 3120 1 1G-A, 3120G-A, 3358delAC, 3659delC, 3662delA, 3750delAG, 3791delC, 3821delT, 3849 1 10KbC-T, 3849 1 4A-G, 3905insT, 405 1 1G-A, 444delA, 556delA, 574delA, 621 1 1G-T, and 711 1 1G-T. aSC, Santa Catarina State; PR, Parana State; MG, Minas Gerais State; n, number of chromosomes. Login to comment
78 G85E With an overall frequency of 2.3%, G85E is the sixth most common mutation identified to date in the Euro-Brazilian and Afro-Brazilian CF patients, (2.6% and 1.3%, respectively). Login to comment
79 Although G85E is not included in the 10 most frequent CF mutations worldwide (CFGAC, 1994) and neither is it among the five most frequent mutations in our sample, it is interesting to note that this mutation was found in all states and both population subgroups analyzed in this study, probably as the result of admixture with Euro-Brazilians. Login to comment
101 The presence in Afro-Brazilians of at least four CF alleles that are common in Caucasians (i.e., DF508, G542X, G85E, R1162X) indicates that the incidence of CF in Afro-Brazilians is due, at least in part, to genetic admixture. Login to comment

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

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

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

[hide] 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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115 It will soon be upgraded to include a further 10 mutations (2789 þ 5G>A, 394delT, G85E, 1811 þ 1,6kbA>G, Y122X, 711 þ 1G>T, W846X2, Y1092X C>A, 3272 À 26A>G, 3120 þ 1G>A 320pb). Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Hum Reprod. 2004 Feb;19(2):250-3. Wu CC, Hsieh-Li HM, Lin YM, Chiang HS
Cystic fibrosis transmembrane conductance regulator gene screening and clinical correlation in Taiwanese males with congenital bilateral absence of the vas deferens.
Hum Reprod. 2004 Feb;19(2):250-3., [PMID:14747162]

Abstract [show]
BACKGROUND: In Taiwan, an area with a very low incidence of cystic fibrosis (CF), we first screened for the most common mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene and looked for clinical correlations in 27 patients with clinically diagnosed congenital bilateral absence of the vas deferens (CBAVD). METHODS AND RESULTS: The clinical results showed that none of the 27 patients had CF symptoms. We did not detect any definite renal anomaly ultrasonographically. Mutation analysis was carried out on these 27 cases and 46 normal fertile males as controls. No mutations of Delta F508 or R117H were identified in any of the samples analysed. In the screening of IVS8-poly T, five of the 27 CBAVD patients showed the homozygous genotype for 5T/5T, 14 showed the heterozygous genotype for 5T/7T and eight showed the homozygous genotype for 7T/7T. The frequency of 5T alleles was 44.4%, which was significantly higher than in the 46 normal fertile males, for which there was a 5T frequency of 5.4%. CONCLUSIONS: The absence of major mutations of CFTR genes could be related to the much lower CF incidence in Taiwan. Further investigations into differences in the mutation spectrum of other CFTR genes are needed for a better understanding of the development of Taiwanese-Oriental CBAVD.

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42 Screening by an INNO-LiPA CFTR17+Tn kit For further con®rmation, we used a commercial kit (INNO-LiPA CFTR17+Tn; Innogenetics, Ghent, Belgium) that allowed the detection of 17 mutations (including 394delTT, G85E, E60X, 621+1G®T, R117H, 711+5G®A, 1078delT, R347P, R334W, A455E, 2143delT, 2183AA®G, 2184delA, 2789+5G®A, R1162X, 3659delC and 3849+10kbC®T) associated with IVS8-Tn polymorphisms (5T/7T/9T) in the CFTR gene to analyse our 27 patients and 46 normal, fertile control males. 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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37 b) The G85E, R334W and 3120+1G→Amutations, common in the Spanish population, were analyzed by restriction enzyme analysis (Gasparini et al., 1991; Zielenski et al., 1991). Login to comment
42 RESULTS Genetic analysis led to the detection of 15 different mutations: ∆F508, G542X, R1162X, G85E, N1303K, R334W, R75Q, R74W, D1270N, W1282X, ∆I507, 2789+5G→A, R1066C, R553X and -816C/T. Login to comment
47 Mutation Cumulative (%)%N ∆F508 G542X R1162X G85E N1303K R334W R75Q Other mutations* Unknown 42 6 3 3 3 2 2 13 30 40.4 5.7 2.9 2.9 2.9 1.9 1.9 12.5 28.9 40.4 46.1 49.0 51.9 54.9 56.7 58.6 71.1 99.9 *R74W, D1270N, W1282X, ∆I507, 2789+5G→A, R1066C, -816C/T, R553X, 5T (3 cases associated to other mutations, 2 cases without known second mutation). Login to comment
59 Genotypes N Percent ∆F508/∆F508 ∆F508/R1162X ∆F508/G85E ∆F508/G542X ∆F508/5T ∆F508/R334W ∆F508/1303X ∆F508/R1066C ∆F508/Unknown ∆I507/2789+G-A R74W/D1270N N1303K/G542X N1303K/R553K -816C-T/5T 5T/Unknown G542X/Unknown R75Q/Unknown W1282X/Unknown Unknown/Unknown 8 3 3 3 2 2 1 1 11 1 1 1 1 1 2 2 2 1 6 15.4 5.8 5.8 5.8 3.9 3.9 1.9 1.9 21.2 1.9 1.9 1.9 1.9 1.9 3.9 3.9 3.9 1.9 11.5 All individuals had pulmonary symptoms.All those carrying the ∆F508/∆F508 genotype had pancreatic insufficiency. Login to comment

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

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

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

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

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

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178 The optimal spotting conditions for each probe are indicated by the boxes around spots in C. wild-type controls and heterozygotes for each ACMG mutation and polymorphism, DNA from 12 compound heterozygotes (⌬F508/1898 ϩ 1GϾA, 711 ϩ 1GϾT/⌬F508, G85E/621 ϩ 1GϾT, 3659delC/⌬F508, 3120 ϩ 1GϾA/ 621 ϩ 1GϾT, R347P/G551D, A455E/⌬F508, R560T/ dF508, R553X/⌬F508, 621 ϩ 1GϾT/⌬F508, 621 ϩ 1GϾT/ 711 ϩ 1GϾT, R117H/⌬F508, and I506V/⌬F508) and DNA from 4 homozygous patients (⌬F508 and 2789 ϩ 5GϾA, 3849 ϩ 10kbCϾT, and G542X) was used in validation experiments. Login to comment
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] Mutations of the CFTR gene in Turkish patients wit... Hum Reprod. 2004 May;19(5):1094-100. Epub 2004 Apr 7. Dayangac D, Erdem H, Yilmaz E, Sahin A, Sohn C, Ozguc M, Dork T
Mutations of the CFTR gene in Turkish patients with congenital bilateral absence of the vas deferens.
Hum Reprod. 2004 May;19(5):1094-100. Epub 2004 Apr 7., [PMID:15070876]

Abstract [show]
BACKGROUND: Mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) can cause congenital bilateral absence of the vas deferens (CBAVD) as a primarily genital form of cystic fibrosis. The spectrum and frequency of CFTR mutations in Turkish males with CBAVD is largely unknown. METHODS: We investigated 51 Turkish males who had been diagnosed with CBAVD at the Hacettepe University, Ankara, for the presence of CFTR gene mutations by direct sequencing of the coding region and exon/intron boundaries. RESULTS: We identified 27 different mutations on 72.5% of the investigated alleles. Two-thirds of the patients harboured CFTR gene mutations on both chromosomes. Two predominant mutations, IVS8-5T and D1152H, accounted for more than one-third of the alleles. Five mutations are described for the first time. With one exception, all identified patients harboured at least one mutation of the missense or splicing type. Presently available mutation panels would have uncovered only 7-12% of CFTR alleles in this population cohort. CONCLUSIONS: Although cystic fibrosis is relatively rare in Turkey, CFTR mutations are responsible for the majority of CBAVD in Turkish males. Because of a specific mutation profile, a population-specific panel should be recommended for targeted populations such as CBAVD in Turkey or elsewhere.

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35 We next screened for six further CFTR gene mutations of the coding region and ¯anking intron sequences by previously described restriction-enzyme based methods: G85E, D110H, R347H, 2789+5G®A, D1152H, N1303K (DoÈrk et al., 1994a, 1997). 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] 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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80 The 27-mutation panel included all of the 16 except S549N and the following additional mutations: 3120 ϩ 1GϾA, 3659delC, A559T, R1162X, S1255X, 405 ϩ 3AϾC, 711 ϩ 1GϾT, 2789 ϩ 5GϾA, G480C, 2307insA, G85E, and 1078delT. Login to comment
141 One of these carries a mutation (G85E) that was not included on the 16-mutation panel (used at the time of testing) but is on the 27-mutation panel. 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
173 # One infant`s genotype: G85E/R117C screening done with 16-mutation panel before inclusion of G85E in the 27-mutation panel; other 2 infant`s genotypes: "not present in additional mutation analysis and yet to be identified" for the 2 alleles. Login to comment

[hide] Genotype/phenotype correlation of the G85E mutatio... Eur Respir J. 2004 May;23(5):679-84. Decaestecker K, Decaestecker E, Castellani C, Jaspers M, Cuppens H, De Boeck K
Genotype/phenotype correlation of the G85E mutation in a large cohort of cystic fibrosis patients.
Eur Respir J. 2004 May;23(5):679-84., [PMID:15176679]

Abstract [show]
In this European study, the phenotype in 68 patients, homozygous or compound heterozygous for the G85E mutation, was investigated. Each index case was compared with two cystic fibrosis (CF) patients from the same clinic, matched for age and sex: one with pancreatic sufficiency (PS) and one with pancreatic insufficiency (PI). When comparing 31 G85E/F508del and F508del/F508del patients, there were no differences in median age at diagnosis, mean sweat chloride value, most recent weight for height, most recent forced expiratory volume in one second % predicted, prevalence of chronic Pseudomonas aeruginosa colonisation and typical CF complications. However, PI was less frequent in the G85E/F508del group. Comparison of 55 G85E patients (with second mutation known and not classified as mild) with PS controls (n=44) showed that the G85E patients had a significantly higher sweat chloride, more often failure to thrive at diagnosis, higher prevalence of PI, worse current weight for height, higher prevalence of chronic P. aeruginosa colonisation and liver cirrhosis. Pulse-chase experiments revealed that G85E cystic fibrosis transmembrane conductance regulator failed to mature on a M470 as well as on a V470 background. Therefore, G85E is a class II mutation. Although there is variability in its clinical presentation, G85E mutation results in a severe phenotype.

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25 Worldwide, of all mutations reported, the G85E mutation has a frequency y0.2%. Login to comment
27 The G85E mutation is a missense mutation: in exon 3, at nucleotide position 386, guanine is replaced by adenosine, resulting in the substitution of glycine by negatively charged glutamic acid in the first membrane-spanning domain [9]. Login to comment
28 Earlier reports about G85E concern a small number of patients. Login to comment
30 The purpose of the present study was to determine the clinical outcome of CF patients, homozygous or compound heterozygous for the G85E mutation, in a large study group. Login to comment
31 In addition, the effect of G85E mutation constructs on CFTR protein expression was evaluated in in vitro cell lines. Login to comment
33 They were asked to report clinical data on their patients homozygous or compound heterozygous for the G85E mutation and, per index case, on two control CF patients. Login to comment
47 In order to have a clear-cut evaluation of the influence of the G85E mutation, a primary analysis for G85E/F508del patients versus F508del/F508del PI patients was performed. The G85E patients were also compared with the groups with PI and PS. Login to comment
49 From the group of G85E patients, thirteen with unknown second mutation or with a second mutation known to be associated with PS were eliminated. Login to comment
54 Apart from studying the phenotype in patients carrying the G85E mutation, the G85E mutation was expressed in cell lines, and the influence of the mutation on CFTR protein biosynthesis and expression in vitro was studied. Login to comment
55 This technique helps to determine which mutation class G85E belongs to, at least in the chosen expression system [18]. Login to comment
56 G85E-CFTR cDNA/pcDNA3 expression vectors, either on a M470-CFTR or V470-CFTR background, were made by means of the TransformerTM Site-Directed Mutagenesis Kit (Clontech, Palo Alto, CA, USA), using the mutagenesis primer 59-GGA GAT TTA TGT TCT ATG AAA TCT TTT-39, according to previously described protocols [19]. Login to comment
61 Data were collected on the phenotype of 68 patients homozygous or compound heterozygous for the G85E mutation. Login to comment
64 Out of 68 patients in the G85E group, 34 carried the G85E/ F508del mutations. Login to comment
68 One index case was homozygous for the G85E mutation and was PS. Login to comment
69 All but two patients in the G85E group were Caucasian: one was Caucasian/African (Antilles) and one was African. Login to comment
78 G85E/F508del compared to F508del/F508del This analysis concerns 31 patients and is presented in table 2: three of the 34 patients with G85E/F508del genotype in whom the PI control patient did not have a F508del/ F508del genotype were excluded from analysis. Login to comment
80 However, PI was less frequent in the G85E/F508del group. Login to comment
81 In addition, at time of diagnosis, steatorrhea and failure to thrive were less frequent in G85E/ F508del patients. Login to comment
83 G85E patients (with second mutation known and not classified as mild) compared to PI and PS matched controls Comparison of this larger G85E patient group with their PI controls (n=55; table 3) showed the same results as the comparison between the G85E/F508del subgroup versus the F508del/ F508del group. Login to comment
86 Comparison of the G85E group with PS controls (n=44; table 3) showed that the G85E patients have a significantly higher sweat chloride (pv0.0018), more often present symptoms of steatorrhea and failure to thrive at diagnosis (pv0.0001), higher prevalence of pancreatic insufficiency (pv0.0001), worse current weight for height (pv0.02), higher prevalence of chronic P. aeruginosa colonisation (pv0.0083) and liver cirrhosis (pv0.05). Login to comment
87 The most recent FEV1 % pred was 69¡4.8% in the G85E group and 85¡6.6% in the PS group (p=0.08). Login to comment
88 Median age at diagnosis was 22 months in the G85E group and 44 months in the PS group (p=0.13). Login to comment
90 If all G85E patients were considered (n=68) in the analysis, these last differences again reached statistical significance (data not shown). Login to comment
91 Three patients of the G85E group died: two patients died at the age of 10 yrs, of whom one after Burkholderia cepacia colonisation; and one patient died at Table 1. Login to comment
92 - Cystic fibrosis transmembrane conductance regulator genotypes of patients in the three study groups G85E PI PS Genotype Subjects n Genotype Subjects n Genotype Subjects n G85E/F508del# 34 F508del# /F508del# 58 F508del# /unknown 15 G85E/unknown 8 F508del# /unknown 2 F508del# /3849z10 kbCRT} 5 G85E/G542X# 5 F508del# /1717-1GR A 1 F508del# /R117H} 3 G85E/W1282X# 4 F508del# /N1303K# 1 T338I/L1065P 2 G85E/I507del# 3 F508del*/H139R 1 E585X/3272-26ARG} 2 G85E/R1162X# 3 F508del# /R1066C # 1 2183AARG/2789z5GRA 2 G85E/2183AARG 2 F508del# /G542X# 1 F508del# /711z5GRA 1 G85E/G85E 1 F508del# /712-1GRT 1 F508del# /D1152H} 1 G85E/E585X# 1 F508del# /621z1GRT 1 F508del# /1898z3ARG 1 G85E/711z1GRT# 1 F508del# /1898z1 1 F508del# /R347H} 1 G85E/712-1GRT# 1 Total 68 F508del# /2789z5GRA 1 G85E/621z1GRT# 1 2789z5GRA/? Login to comment
93 1 G85E/W496X 1 F508del# /N1303K# 1 G85E/N1303K# 1 T388I/R1158X 1 G85E/711z5GRA} 1 3272-26AwG} /E822X 1 G85E/R334W} 1 F508del# /R334W} 1 Total 68 574delA/2789z5GRA 1 F508del# /3272-26ARG} 1 F508del# /R352Q 1 F508del# /3272-26AwG} 1 R334W} /444delA 1 L206W/3272-26ARG} 1 F508del# /F508del# 1 L206W/? Login to comment
98 - Current age and clinical variables in a group of European cystic fibrosis (CF) patients heterozygous for G85E and F508del compared to CF patients homozygous for F508del G85E/F508del F508del/F508del G85E/F508del versus F508del/F508del Subjects n 31 31 Current age yrs 19.5¡7.2 (2-40) 19.5¡6.9 (3-39) 0.05# Age at time of diagnosis months 5 (2-40) 9 (2.5-23) 0.2} Sweat chloride mEq?L-1 100 (90-110) 100 (88-110) 0.95} Presenting symptoms Steatorrhea 38 86 0.0003z Failure to thrive 38 70 0.017z Meconium ileus 4 7 0.38z Respiratory 82 62 0.88z Pancreatic insufficiency 60 100 v0.001z Most recent weight for height percentile 60¡5 58¡6 0.68# Most recent FEV1 % pred 69¡5 65¡6 0.78# Chronic P. aeruginosa colonisation 41 45 0.33z Complications Liver cirrhosis 13 7 0.119z CF-DM 9 6 0.298z DIOS 16 6 0.16z Pancreatitis n 1 0 0.5z Data are presented as mean¡SD (range), median (interquartile range), mean¡SEM or % unless otherwise stated. Subjects were matched for centre, sex and age. FEV1: forced expiratory volume in one second; pred: predicted; P. aeruginosa: Pseudomonas aeruginosa; CF-RD: cystic fibrosis-related diabetes; DIOS: distal intestinal obstruction syndrome. Statistical analysis by paired t-test# , Wilcoxon signed-ranks test} and Fisher exact testz . Login to comment
100 This further documents the severe lung disease in the G85E patients. Login to comment
101 The frequency of pancreatic insufficiency was 57% in the total G85E group. Login to comment
102 Within the total G85E group, the age of the patients with PI was 23.0¡1.7 yrs and of the patients with PS 16.1¡1.7 yrs. Login to comment
104 Sibling pairs There were six sibling pairs in the G85E group. Login to comment
105 Two sibpairs, all compound heterozygous for the G85E and F508del mutations, were discordant for pancreatic disease manifestation. Login to comment
113 In vitro functional properties of G85E-CFTR The degree of G85E-CFTR maturation was investigated. Login to comment
115 However, the haplotype background of G85E-CFTR genes is mostly unknown. Login to comment
116 G85E was, therefore, studied either on a M470 or V470 background. Login to comment
117 G85E-V470-, G85E-M470- and wildtype-V470-CFTR were transiently expressed in COS cells. Login to comment
119 In contrast, G85E-V470- and G85E-M470-CFTR failed to mature to the complex-glycosylated C-form at all time periods (fig. 1). Login to comment
120 Discussion CF patients with the G85E mutation have a severe phenotype. Login to comment
121 When comparing G85E/F508del and F508del/ F508del patients, there are no differences in age at diagnosis, sweat chloride value, parameters evaluating lung disease, most recent weight for height, nor CF complications. Login to comment
122 Conversely, comparing G85E patients with PS patients it Table 3. Login to comment
123 - Current age and clinical variables in a group of European cystic fibrosis (CF) patients, carrying the G85E mutation and a known second mutation not associated with pancreatic sufficiency (PS), compared to two groups of CF patients not carrying the G85E mutation and having either pancreatic insufficiency (PI) or PS G85E PS PI G85E versus PS Subjects n 55 44 55 Current age yrs 18.2¡9 (2-51) 18.2¡9 (2-52) 18.9¡9 (3-46) 0.06# Age at time of diagnosis months 22 (3-124) 44 (6-169) 6 (2-28) 0.13} Sweat chloride mEq?L-1 106 (95-115) 100 (78-109) 102 (94-110) 0.0018} Presenting symptoms Steatorrhea 40 5 84 0.0001z Failure to thrive 40 14 71 0.0001z Meconium ileus 2 0 12 0.49z Respiratory 78 74 63 0.17z Pancreatic insufficiency 54 0 100 v0.0001z Most recent weight for height percentile 57¡4 72¡5 58¡5 0.02# Most recent FEV1 % pred 69¡5 85¡7 66¡5 0.08# Chronic P. aeruginosa colonisation 42 23 46 0.0083z Complications Liver cirrhosis 9 0 11 v0.05z CF-RD 11 2 13 0.1z DIOS 15 7 9 0.11z Pancreatitis n 2 2 0 0.55z Data are presented as mean¡SD (range), median (interquartile range), mean¡SEM or % unless otherwise stated. Subjects were matched for centre, sex and age. FEV1: forced expiratory volume in one second; pred: predicted; P. aeruginosa: Pseudomonas aeruginosa; CF-RD: cystic fibrosis-related diabetes; DIOS: distal intestinal obstruction syndrome. Statistical analysis by paired t-test# , Wilcoxon signed-ranks test} and Fisher exact testz . Login to comment
124 A+B 0.5 1.5 3.5 0.5 1.5 3.5 0.5 1.5 3.5 Time h G85E V470 G85E M470 WT-CFTR V470 C Fig. 1. Login to comment
125 - Analysis of biogenesis and degradation of G85E-cystic fibrosis transmembrane conductance regulator (CFTR). Login to comment
126 COS cells transfected with either G85E-V470-, G85E-M470- or wild type-V470-CFTR were metabolically labelled and chased for the times indicated. Login to comment
129 has been shown here that the G85E group has more severe disease when considering the same parameters. Login to comment
130 Conversely, there are some differences with classical PI patients: 43% of the G85E patients are PS; and steatorrhea and failure to thrive are less frequent presenting symptoms. Login to comment
136 In the total G85E group studied, the frequency of PI was 57%. Login to comment
143 In accordance with this report, the G85E PS patients in the present study are significantly younger than the G85E PI patients. Login to comment
146 Modifying genes can, at present, not yet be studied, but they could explain the rather infrequent occurrence of meconium ileus in the larger group of G85E patients. Login to comment
147 In a previous study, it was found that G85E-CFTR fails to mature and, therefore, is a class II mutation [31]. Login to comment
149 The heterogeneity in disease severity in the group of G85E-CF patients is thus remarkable for a class II mutation, unless if it is conferred by the mutation on the other chromosome. Login to comment
151 However, the haplotype background of G85E-CFTR genes is mostly unknown. Login to comment
152 G85E-CFTR was, therefore, studied both on a M470 and a V470 background, in order to investigate if G85E-CFTR properties are affected by the amino acid found at the M470V position and in this way could explain the observed disease variability. Login to comment
153 Both G85E- M470- and G85E-V470-CFTR, however, completely failed to mature. Login to comment
154 On the basis of the present in vitro findings, as well as on the basis of the clinical findings in the patient group, G85E can be classified as a mutation associated with severe disease. Login to comment
156 Even so, the age at diagnosis was lower in G85E patients, supporting the idea of a more severe phenotype. Login to comment
158 In the present study G85E patients have more severe lung disease as assessed both by more frequent chronic P. aeruginosa infection and worse most recent FEV1 % pred. Login to comment
159 In conclusion, G85E is associated with a severe phenotype. Login to comment
164 European G85E survey contributors: T. Bienvenu (Hoˆpital Cochin, Paris, France); R. Cabanas (Hospital Clinico, Santiago, Spain); T. Casals (Hospital Duran I Reynals, Barcelona, Spain); G. Castaldo (Universita Federico II - Ceinge, Napoli, Italy); C. Castellani (Ospedale Civile Maggiore, Verona, Italy); J. Dapena (Hospital U. Login to comment

[hide] A low prevalence of cystic fibrosis in Uruguayans ... Genet Mol Res. 2004 Jun 30;3(2):258-63. Cardoso H, Crispino B, Mimbacas A, Cardoso ME
A low prevalence of cystic fibrosis in Uruguayans of mainly European descent.
Genet Mol Res. 2004 Jun 30;3(2):258-63., [PMID:15266396]

Abstract [show]
Cystic fibrosis is the most common hereditary disease in populations of European descent, with its prevalence depending on the populations and ethnic groups studied. In contrast to Europe and North America, there is little information about this disease in Latin America. Uruguay currently has a human population of 3,000,000, with a low rate of miscegenation and no remaining isolated Amerindian groups. In the present study, we estimated the prevalence of cystic fibrosis in this country based on the detection of DeltaF508 mutation carriers in 500 unrelated individuals and on the frequency of individuals homozygous for this mutation within the affected population. The latter was calculated from the frequency of the different mutations and genotypes observed in a sample of 52 previously described patients with confirmed cystic fibrosis. A theoretical estimate of the prevalence of cystic fibrosis based on anthropological data suggested a frequency of 25 affected individuals/100,000 inhabitants. However, our data indicated that the true prevalence in the population was considerably lower (6.9 cases/100,000 inhabitants).

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38 *G542X, R1162X, G85E, N1303K, R334W, R75Q, R74W, D1270N, W1282X, ∆I507, 2789+5G->A, R1066C, -816C/T, and R553X. Table 1. Login to comment
56 *G542X, R1162X, G85E, N1303K, R334W, R75Q, R74W, D1270N, W1282X, ∆I507, 2789+5G->A, R1066C, -816C/T, R553X. Table 2. Login to comment

[hide] Relation of sweat chloride concentration to severi... Pediatr Pulmonol. 2004 Sep;38(3):204-9. Davis PB, Schluchter MD, Konstan MW
Relation of sweat chloride concentration to severity of lung disease in cystic fibrosis.
Pediatr Pulmonol. 2004 Sep;38(3):204-9., [PMID:15274098]

Abstract [show]
In cystic fibrosis (CF), sweat chloride concentration has been proposed as an index of CFTR function for testing systemic drugs designed to activate mutant CFTR. This suggestion arises from the assumption that greater residual CFTR function should lead to a lower sweat chloride concentration, as well as protection against severe lung disease. This logic gives rise to the hypothesis that the lower the sweat chloride concentration, the less severe the lung disease. In order to test this hypothesis, we studied 230 patients homozygous for the DeltaF508 allele, and 34 patients with at least one allele associated with pancreatic sufficiency, born since January 1, 1955, who have pulmonary function data and sweat chloride concentrations recorded in our CF center database, and no culture positive for B. cepacia. We calculated a severity index for pulmonary disease, using an approach which takes into account all available pulmonary function data as well as the patient's current age and survival status. Patients with alleles associated with pancreatic sufficiency had significantly better survival (P = 0.0083), lower sweat chloride concentration (81.4 +/- 23.8 vs. 103.2 +/- 14.2 mEq/l, P < 0.0001), slower rate of decline of FEV(1) % predicted (-0.75 +/- 0.34 vs. -2.34 +/- 0.17% predicted per year), and a better severity index than patients homozygous for the DeltaF508 allele (median 73rd percentile vs. median 55th percentile, P = 0.0004). However, the sweat chloride concentration did not correlate with the severity index, either in the population as a whole, or in the population of patients with alleles associated with pancreatic sufficiency, who are thought to have some residual CFTR function. These data suggest that, by itself, sweat chloride concentration does not necessarily predict a milder pulmonary course in patients with cystic fibrosis.

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26 T (12); A455E (1); D1270N; G85E (3); R117H (4); R334W (1); R347H (1); T347P (6); 2859 þ 5 G ! Login to comment

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

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

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33 There are mutations associated mainly with pancreatic insufficiency (PI), while others are associated mainly with pancreatic sufficiency (PS).3 However, some mutations such as G85E or 3849þ10 kb C ! Login to comment
69 T (1), G542X/G542X (1), G85E/G85E (1), 3849þ10 kb C ! Login to comment
71 A (1), S549R/S549R (1), Q359K/Q360K (1), DF508/Unknown (4), W1282X/Unknown (3), G542X/Unknown (2), G85E/Unknown (1), and Q359K/ Unknown (1). Login to comment

[hide] Use of fecal elastase-1 to classify pancreatic sta... J Pediatr. 2004 Sep;145(3):322-6. Borowitz D, Baker SS, Duffy L, Baker RD, Fitzpatrick L, Gyamfi J, Jarembek K
Use of fecal elastase-1 to classify pancreatic status in patients with cystic fibrosis.
J Pediatr. 2004 Sep;145(3):322-6., [PMID:15343184]

Abstract [show]
OBJECTIVE: To test the hypothesis that some patients with cystic fibrosis (CF) are misclassified as pancreatic insufficient, using fecal elastase-1 (FE-1) to define pancreatic status. STUDY DESIGN: Subjects with CF at 33 CF centers filled out questionnaires and submitted a stool specimen that was analyzed for FE-1. Subjects taking pancreatic enzyme supplements (PES) were asked to discontinue them and perform a 3-day fecal fat balance study if their FE-1 was >200 microg/g stool and they had never had pancreatitis. RESULTS: The median value for FE-1 in 1215 subjects was 0 microg/g stool (range, 0-867). There was a significant difference between patients who had been prescribed PES (n=1131) and those who had FE-1 <200 microg/g stool (n=1074; P<.0001). Sixty-seven subjects met criteria for discontinuation of PES. The mean coefficient of fat absorption for these subjects was 96.1%. CONCLUSIONS: FE-1 is an accurate, easily obtained screening test to classify pancreatic status in patients with CF. This information is important for prognostication, treatment, and to avoid misclassification in clinical research. Measurement of FE-1 should become a standard of care for patients with CF.

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116 FE-1 values in subjects with CFTR mutations associated with pancreatic sufficiency11 N Mean (mg/g stool) Median (mg/g stool) Range (mg/g stool) Subjects with at least one PS allele* FE-1 >200 mg/g stool 16 584 582.9 349-773 FE-1 <200 mg/g stool 5 64.4 74.8 0-125 Subjects with at least one PS variable alleley FE-1>200 mg/g stool 29 496.2 493.6 224-798 FE-1 <200 mg/g stool 13 76.1 65.9 0-187 *Pancreatic sufficient dominant CF alleles G551S R117H R347H P574H R334W R352Q T3381 yVariable pancreatic sufficient CF mutations G85E 3849 + 10 kb C fi T R347P 2789 + 5G fi A A455E In summary, FE-1 is an accurate, easily obtained screening test to classify patients with CF as PI or PS. 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

[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] 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

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

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

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240 Tracings are shown for two CF patients with pancreatic insufficiency (top panels) and FEV1 ,50% pred (left panel) or FEV1 .50% pred (right panel), both of whom were homozygous for the F508D mutation and belonged to the ''severe`` genotype group; and two CF patients with pancreatic sufficiency (bottom panels) and FEV1 ,50% pred (left panel) or FEV1 .50% pred (right panel), both of whom were compound heterozygous for the F508D mutation and the R117H and G85E mutations, respectively, and belonged to the ''mild`` genotype group. 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] A foldable CFTR{Delta}F508 biogenic intermediate a... J Cell Biol. 2004 Dec 20;167(6):1075-85. Younger JM, Ren HY, Chen L, Fan CY, Fields A, Patterson C, Cyr DM
A foldable CFTR{Delta}F508 biogenic intermediate accumulates upon inhibition of the Hsc70-CHIP E3 ubiquitin ligase.
J Cell Biol. 2004 Dec 20;167(6):1075-85., 2004-12-20 [PMID:15611333]

Abstract [show]
CFTRDeltaF508 exhibits a correctable protein-folding defect that leads to its misfolding and premature degradation, which is the cause of cystic fibrosis (CF). Herein we report on the characterization of the CFTRDeltaF508 biogenic intermediate that is selected for proteasomal degradation and identification of cellular components that polyubiquitinate CFTRDeltaF508. Nonubiquitinated CFTRDeltaF508 accumulates in a kinetically trapped, but folding competent conformation, that is maintained in a soluble state by cytosolic Hsc70. Ubiquitination of Hsc70-bound CFTRDeltaF508 requires CHIP, a U box containing cytosolic cochaperone. CHIP is demonstrated to function as a scaffold that nucleates the formation of a multisubunit E3 ubiquitin ligase whose reconstituted activity toward CFTR is dependent upon Hdj2, Hsc70, and the E2 UbcH5a. Inactivation of the Hsc70-CHIP E3 leads CFTRDeltaF508 to accumulate in a nonaggregated state, which upon lowering of cell growth temperatures, can fold and reach the cell surface. Inhibition of CFTRDeltaF508 ubiquitination can increase its cell surface expression and may provide an approach to treat CF.

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451 Structural cues involved in endoplasmic reticulum degradation of G85E and G91R mutant cystic fibrosis transmembrane conductance regulator. J. Clin. Login to comment

[hide] Side chain and backbone contributions of Phe508 to... Nat Struct Mol Biol. 2005 Jan;12(1):10-6. Epub 2004 Dec 26. Thibodeau PH, Brautigam CA, Machius M, Thomas PJ
Side chain and backbone contributions of Phe508 to CFTR folding.
Nat Struct Mol Biol. 2005 Jan;12(1):10-6. Epub 2004 Dec 26., [PMID:15619636]

Abstract [show]
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane protein, cause cystic fibrosis (CF). The most common CF-causing mutant, deletion of Phe508, fails to properly fold. To elucidate the role Phe508 plays in the folding of CFTR, missense mutations at this position were generated. Only one missense mutation had a pronounced effect on the stability and folding of the isolated domain in vitro. In contrast, many substitutions, including those of charged and bulky residues, disrupted folding of full-length CFTR in cells. Structures of two mutant nucleotide-binding domains (NBDs) reveal only local alterations of the surface near position 508. These results suggest that the peptide backbone plays a role in the proper folding of the domain, whereas the side chain plays a role in defining a surface of NBD1 that potentially interacts with other domains during the maturation of intact CFTR.

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337 Xiong, X., Bragin, A., Widdicombe, J.H., Cohn, J. & Skach, W.R. Structural cues involved in endoplasmic reticulum degradation of G85E and G91R mutant cystic fibrosis transmembrane conductance regulator. J. Clin. Login to comment
336 Xiong, X., Bragin, A., Widdicombe, J.H., Cohn, J. & Skach, W.R. Structural cues involved in endoplasmic reticulum degradation of G85E and G91R mutant cystic fibrosis transmembrane conductance regulator. J. Clin. 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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62 A procedure for the large-scale analysis of several mutations peculiar to southern Italy is also indicated Mutation Analytical CF alleles Campania Basilicata Puglia Total procedure n = 340 n = 52 n = 350 n = 742 DF508 55.6 55.8 46.8 51.5 N1303K 7.3 3.8 7.7 7.3 G542X 5.0 3.8 7.1 5.9 W1282X 3.5 3.8 0.6 2.2 2183 AA>G 2.3 5.8 0.8 1.9 852del22 0 5.8 3.2 1.9 3% agarose 1717-1G>A 2.3 1.9 1.1 1.8 4382delA 0 0 3.7 1.8 RE (Ear I -) 1259insA 0 0 3.1 1.5 4016insT 2.1 0 1.1 1.5 ASO R553X 1.5 0 1.7 1.5 R1158X 1.5 0 1.3 1.2 ASO or RE (Sfa N 1 -) L1077P 0.6 0 1.9 1.2 I502T 0.3 0 2.0 1.1 RE (Mse I -) 3849+10kbC>T 0 1.9 1.6 0.9 D579G 0 0 1.6 0.8 RE (Avr II +) G1244E 0.9 3.8 0.3 0.8 ASO or RE (Mbo II +) G1349D 0 0 1.7 0.8 RE (Sty I -) 2789+5 G>A 0.6 0 0.8 0.7 711+1 G>T 1.5 0 0 0.7 ASO L1065P 1.2 0 0 0.5 ASO or RE (Mnl I +) R347P 0.3 0 0.9 0.5 2522insC 0.9 0 0 0.4 E585X 0.6 0 0 0.3 G85E 0.6 0 0 0.3 G178R 0.6 0 0 0.3 D1152H 0.3 0 0.3 0.3 I148T-3195del6 0.6 0 0 0.3 I148T (alone) 0 0 0.3 0.1 R334W 0 0 0.3 0.1 DI507 0 0 0.3 0.1 I1005R 0 0 0.3 0.1 3272-26A>G 0.3 0 0 0.1 2711delT 0.3 0 0 0.1 L558S 0 1.9 0 0.1 W1063X 0 0 0.3 0.1 D110H 0.3 0 0 0.1 S549R (A>C) 0 1.9 0 0.1 2184insA 0.3 0 0 0.1 3131del22 0.3 0 0 0.1 R709N 0 0 0.3 0.1 A349V 0 0 0.3 0.1 4015insA 0 0 0.3 0.1 Y849X 0 1.9 0 0.1 Cumulative 91.6 92.1 91.7 91.5 Unknown 8.4 7.9 8.3 8.5 Total 100,0 100,0 100,0 100,0 RE: restriction enzyme (-/+: abolition or introduction of a RE site); ASO: allele specific oligonucleotide Figure 2 Multiplex denaturing gradient gel electrophoretic analysis of exons 8, 5 and 18 of the cystic fibrosis transmembrane regulator gene in a cystic fibrosis patient (case n. Login to comment
97 Due to the presence of 'local` mutations, the detection rate with commercial kits for CF chromosomes in Table 3 Mutations linked to different haplotypes possibly due to slippage events, characteryzed at the level of three CFTR intragenic loci (IVS8CA, IVS17bTA, IVS17bCA) by the indication of the repeats number Present study Other studies Cases Haplotype cases (n) (n. of repeats) (n) Haplotype references* (n. of repeats) R347P 4/4 16-32-13 3 16-32-13 1,2,3 1 16-31-13 3 2 17-28-13 1 1 16-45-13 1 L1077P 3/3 17-7-17 1 17-7-17 1 1 17-7-16 1 G85E 2/2 16-24-13 9 16-24-13 2,3 1 16-25-13 2 2183AA>G 14/14 16-31-13 1 16-31-13 3 4 16-30-13 1 R553X 6/11 17-55-13 3 17-58-13 3 3/11 18-55-13 1 17-57-11 1 1/11 16-55-13 2 17-55-13 1,3 1/11 16-55-11 6 17-55-11 1 1 17-52-11 1 1 17-54-11 1 1 17-56-13 3 G1244E 5/6 16-32-13 1 17-34-13 1 1/6 16-34-13 711 +1 G>T 5/5 16-25-13 7 16-25-13 1,2,3 1 16-26-13 1 G1349D 5/6 16-30-13 1/6 16-32-13 G178R 1/2 16-32-13 1 16-30-13 3 1/2 16-32-13 2 16-32-13 1 * References 1: Morral et al. 1996. Login to comment
115 Other mutations (see Table 2, group b) also seem to derive from a founding event, and haplotypes differing by a single microsatellite could depend on slippage phenomena, as already proposed for mutations G85E (Claustres et al. 1996) and L1077P (Morral et al. 1996). Login to comment

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

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

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193 However, certain missense mutations (such as G85E) may confer a variable panereatic phenotype. Login to comment

[hide] Cystic fibrosis: an overview. J Clin Gastroenterol. 2005 Apr;39(4):307-17. Turcios NL
Cystic fibrosis: an overview.
J Clin Gastroenterol. 2005 Apr;39(4):307-17., [PMID:15758625]

Abstract [show]
Cystic fibrosis (CF) is one of the most common inherited disorders of white populations. The isolation and cloning of the gene in CF that encodes the production of a transport protein that acts as an apical membrane chloride channel, termed cystic fibrosis transmembrane conductance regulator (CFTR), have improved our understanding of the disorder's pathophysiology and has aided diagnosis, but has also revealed the disease's complexity. Gene replacement therapy is still far from being used in patients with CF, mostly because of difficulties in targeting the appropriate cells. Life expectancy of patients with this disorder has greatly improved over past decades because of better symptomatic treatment strategies. This article summarizes advances in understanding and treatment of CF.

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55 Pancreatic Sufficient CF Mutations Dominant Pancreatic-Sufficient Variable Pancreatic-Sufficient G551S G85E P574H R347P R117H 3849 + 10kb C ! Login to comment

[hide] Pancreatitis among patients with cystic fibrosis: ... Pediatrics. 2005 Apr;115(4):e463-9. Epub 2005 Mar 16. De Boeck K, Weren M, Proesmans M, Kerem E
Pancreatitis among patients with cystic fibrosis: correlation with pancreatic status and genotype.
Pediatrics. 2005 Apr;115(4):e463-9. Epub 2005 Mar 16., [PMID:15772171]

Abstract [show]
OBJECTIVE: Pancreatitis is an infrequent complication among patients with cystic fibrosis (CF). It has mainly been reported for patients with pancreatic sufficiency (PS). Previous studies involved only a small number of patients because they contained data from single centers. The aim of this study was to evaluate the incidence of pancreatitis in a large heterogeneous CF population, to determine the relationship with pancreatic function, and to assess whether pancreatitis is associated with specific CFTR mutations. METHODS: Physicians caring for patients with CF were approached through the CF Thematic Network or through the European Cystic Fibrosis Foundation newsletter. They were asked to provide data on their current patient cohort through a standardized questionnaire and to report how many patients they had ever diagnosed as having pancreatitis. A detailed questionnaire was then sent, to be filled out for all of their patients for whom pancreatitis had ever occurred. We defined pancreatitis as an episode of acute abdominal pain associated with serum amylase levels elevated above the ranges established by each participating center's laboratory. General clinical data included age, genotype, age at diagnosis of CF, sweat chloride concentrations, pancreatic status, biometric findings, and respiratory status. CFTR mutations were also reported according to the functional classification of classes I to V. Patients were categorized as having PS, pancreatic insufficiency (PI), or PI after an initial period of PS. PI was defined as a 72-hour stool fat loss of >7 g/day, fat absorption of <93%, or fecal elastase levels of <200 microg/g feces. Clinical data on pancreatitis included age at the first episode, amylase and lipase levels, possible triggers, and occurrence of relapses or complications. RESULTS: A total of 10071 patients with CF, from 29 different countries, who were undergoing follow-up monitoring in 2002 were surveyed. Among this group, pancreatitis had ever been diagnosed for 125 patients (1.24%; 95% confidence interval [CI]: 1.02-1.46%). There was variability in the reported rates of pancreatitis for different countries. Twenty-six centers in 15 different countries sent detailed clinical data on their patients with pancreatitis and on their whole CF clinic. This involved 3306 patients with CF and 61 cases of pancreatitis, leading to a prevalence of 1.84% (95% CI: 1.39-2.30%). The mean age of the patients with pancreatitis ever was 24.4 years (SD: 10.8 years). The first episode of pancreatitis occurred at a mean age of 19.9 years (SD: 9.6 years). The median serum amylase level at the time of pancreatitis was 746 IU/L (interquartile range: 319-1630 IU/L), and the median lipase level was 577 IU/L (interquartile range: 229-1650 IU/L). The majority of patients had PS (34 of 61 patients, 56%; 95% CI: 43-68%). Pancreatitis occurred for 15 patients with PI (25%; 95% CI: 14-35%). Eight patients developed PI after initial PS. The occurrence of pancreatitis among patients with PS was 34 cases per 331 patients, ie, 10.27% (95% CI: 7.00-13.55%); the occurrence of pancreatitis among patients with PI was 15 cases per 2971 patients, ie, 0.5% (95% CI: 0.25-0.76%). The mean age (in 2002) of the CF cohort with pancreatitis did not differ between the PS and PI subgroups. The forced expiratory volume in 1 second was significantly lower among the patients with PI than among the patients with PS, ie, 65% (SEM: 7%) vs 79% (SEM: 4%). The mean age at the occurrence of pancreatitis and the amylase and lipase levels during pancreatitis were not different for patients with pancreatitis and PI versus PS. In the group with PS, 31 of 34 patients carried at least 1 class IV or V CFTR mutation. In the groups with PI and PI after PS, 5 of 15 patients and 3 of 8 patients, respectively, carried 2 class I, II, or III CFTR mutations. Relapses and/or evolution to chronic pancreatitis occurred for 42 patients. Pancreatitis preceded the diagnosis of CF in 18 of 61 cases. These patients were significantly older than the rest of the cohort, ie, age of 28.4 years (SEM: 3.4 years) vs 22.7 years (SEM: 1.3 years). Their median age at the diagnosis of CF was also significantly greater, ie, 21.5 years (interquartile range: 11.9-31 years) vs 7.6 years (interquartile range: 0.4-17.0 years). However, the ages at the occurrence of pancreatitis were similar, ie, 21.0 years (SEM: 3.0 years) vs 19.5 years (SEM: 1.2 years). CONCLUSIONS: This study of 10071 patients with CF from 29 different countries revealed an estimated overall occurrence of pancreatitis among patients with CF of 1.24% (95% CI: 1.02-1.46%). The incidence of pancreatitis was much higher among patients with PS. However, pancreatitis was also reported for 15 patients with PI from 11 centers in 9 different countries. A correct diagnosis of pancreatitis for the reported patients with PI was supported by amylase and lipase levels increased above 500 IU/L, similar to those for patients with PS and pancreatitis. A correct diagnosis of PI for these patients with pancreatitis was supported by the adequacy of the methods used. We chose the cutoff values used to distinguish between patients with PI and control subjects without gastrointestinal disease. For one half of the patients, the diagnosis of PI was established on the basis of low levels of stool elastase (mean: 97 mug/g stool). With a cutoff value of 200 microg/g stool, this noninvasive test has high sensitivity (>95%) and high specificity (>90%) to differentiate patients with PI from control subjects with normal pancreatic function. For the other one half of the patients with PI in the cohort, the pancreatic status was determined on the basis of the 3-day fecal fat balance, with the widely used cutoff value of >7 g of fat loss per day. The most likely reason for pancreatitis occurring among patients with PI is that some residual pancreatic tissue is present among these patients. Pancreatitis is a rare complication among patients with CF. It occurred for 1.24% (95% CI: 1.02-1.46%) of a large CF cohort. Pancreatitis occurs mainly during adolescence and young adulthood. It is much more common among patients with CF and PS (10.3%), but it can occur among patients with PI (0.5%). Pancreatitis can be the first manifestation of CF. Pancreatitis was reported for patients carrying a wide range of mutations.

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188 A455E, the Dutch mutation, is considered a class IV mutation but 50% to 75% of patients were reported to have PI.26 G85E, the Mediterranean mutation, was reported by Durno et al13 with PS, but it is actually a class II mutation with some variability in pancreatic function.28 In the present study, well-accepted methods of stool analysis (fat loss of Ͼ7 g/day or elastase levels of Ͻ200 ␮g/g stool) were chosen to determine pancreatic status as PS or PI. Login to comment

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

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

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275 Unexpectedly, the mechanism of dysfunction of p.L206W resembles that of p.G85E and p.P205S, which are misprocessed without altering ion conductance [Sheppard et al., 1996; Xiong et al., 1997]. Login to comment

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

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

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

[hide] 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

[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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119 Like ∆F508, several other clinically important mutations - such as N1303K, G85E, and G91R - lead to misfolded CFTR protein that is prematurely degraded. 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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62 G, R1162X, 3659delC, W1282X, 3905insT, N1303K, 1078delT, R347P, R347H and R334W labelled with TET (green) and A455E, 1898þ1G.A, 2183AA.G, 2789þ5G.A, G85E, 621þ1G.T, R117H, Y122X and 711þ1G.T labelled with HEX (yellow). Login to comment

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

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

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

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

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

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

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

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

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47 CFTR gene alterations were first scored by PCR and reverse dot blot (Chehab and Wall, 1992), targeted to the detection of the following mutations: ∆F508, G85E, 541∆C, D110H, R117H, 621+1G→T, 711+5G→A, R334W, R334Q, T338I, 1078∆T, R347H, R352Q, ∆I507, 1609∆CA, E527G, 1717-1G→A, 1717-8G→A, G542X, R347P, S549N, S549R A→C, Q552X, R553X, A559T, D579G, Y577F, E585X, 1898+3A→G, 2183AA→G, R709X, 2789+5G→A, 3132∆TG, 3272-26A→G, L1077P, L1065P, R1070Q, R1066H, M1101K, D1152H, R1158X, R1162X, 3849+10KbC→T, G1244E, W1282R, W1282X, N1303K and 4016∇T. Login to comment
79 Concerning instead the mutations found in the male group, besides ∆F508 the following have been found: 2789+5 g/a, 711+5 g/a, D1152H, G85E, N1303K, Q552X, R1158X, R117H, R334Q, R334W and R553X. 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
66 yThis mutation was not initially detected because G85E was not included in the original MA CF NBS program multimutation panel. Login to comment

[hide] Two-tiered immunoreactive trypsinogen-based newbor... J Pediatr. 2005 Sep;147(3 Suppl):S83-8. Sontag MK, Hammond KB, Zielenski J, Wagener JS, Accurso FJ
Two-tiered immunoreactive trypsinogen-based newborn screening for cystic fibrosis in Colorado: screening efficacy and diagnostic outcomes.
J Pediatr. 2005 Sep;147(3 Suppl):S83-8., [PMID:16202790]

Abstract [show]
OBJECTIVE: To examine immunoreactive trypsinogen (IRT)-based screening for cystic fibrosis (CF) for recall rate, genotype distribution, and "borderline" sweat test results. STUDY DESIGN: CF newborn screening in Colorado began in 1982, and >1,153,000 infants were screened through 2002 with an IRT-based screen (IRT/IRT). RESULTS: We have identified 313 infants with CF, giving an overall incidence of 1 in 3684 and a Hispanic incidence of 1 in 6495. Fifty-five infants with meconium ileus (17.6%) were excluded from analysis. Fourteen infants with false-negative results were identified (5.4%). The average recall rate was 0.6%, with a positive predictive value of 4.7%. Ninety-three percent of the infants had at least 1 DeltaF508 mutation, and 98% of the infants had at least 1 mutation from the American College of Medical Genetics recommended panel. Six infants had hypertrypsinogenemia and borderline results on sweat tests (30-60 mmol/L). Increased variability in sweat chloride levels were seen in these infants compared with infants with homozygous DeltaF508. Three children with initial borderline results on sweat tests had CF diagnosed. CONCLUSIONS: The recall and false-negative rates of our IRT/IRT CF screening program are reported. Additionally, genotypes of the patients identified mirror the CF population genotypes, reflecting similar disease severity in the screened population. Finally, infants with persistent hypertrypsinogenemia and borderline sweat test results need long-term follow-up.

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86 The pancreatic sufficient mutations identified were 18981 5G>T, 278915G>A, A455E, G551S, G85E, I336K, P67L, R117C, R117H, R334W, R347P. Login to comment

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

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

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

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

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

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51 The 29 Mutations and Tn Polymorphism That Can Be Detected by INNO-LiPA Assays Mutation Exon/Intron (i) E60X, G85E, 394delTT 3 621 + 1 G ., R117H (i) 4, 4 711 + 5 G . 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] Indirect CFTR mutation identification by PCR/OLA a... Genet Test. 2005 Winter;9(4):285-91. Stanziale P, Savino M, De Bonis P, Granatiero M, Zelante L, Bisceglia L
Indirect CFTR mutation identification by PCR/OLA anomalous electropherograms.
Genet Test. 2005 Winter;9(4):285-91., [PMID:16379540]

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

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

[hide] 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

[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] 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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63 Mild CFTR mutations (Class IV and V) ϭ R117H, R334W, G85E, R347P, 3849ϩ10KbC→T, 2789ϩ5G→A, A455E. 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] 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] Derlin-1 promotes the efficient degradation of the... J Biol Chem. 2006 Dec 1;281(48):36856-63. Epub 2006 Sep 5. Sun F, Zhang R, Gong X, Geng X, Drain PF, Frizzell RA
Derlin-1 promotes the efficient degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR folding mutants.
J Biol Chem. 2006 Dec 1;281(48):36856-63. Epub 2006 Sep 5., 2006-12-01 [PMID:16954204]

Abstract [show]
A complex involving Derlin-1 and p97 mediates the retrotranslocation and endoplasmic reticulum (ER)-associated degradation of misfolded proteins in yeast and is used by certain viruses to promote host cell protein degradation (Romisch, K. (2005) Annu. Rev. Cell Dev. Biol. 21, 435-456; Lilley, B. N., and Ploegh, H. L. (2004) Nature 429, 834-840; Ye, Y., Shibata, Y., Yun, C., Ron, D., and Rapoport, T. A. (2004) Nature 429, 841-847). We asked whether the components of this pathway are involved in the endoplasmic reticulum-associated degradation of the mammalian integral membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), a substrate for the ubiquitin-proteasome system. We report that Derlin-1 and p97 formed complexes with CFTR in human airway epithelial cells. Derlin-1 interacted with nonubiquitylated CFTR, whereas p97 associated with ubiquitylated CFTR. Exogenous expression of Derlin-1 led to its co-localization with CFTR in the ER where it reduced wild type (WT) CFTR expression and efficiently degraded the disease-associated CFTR folding mutants, DeltaF508 and G85E (>90%). Consistent with this, Derlin-1 also reduced the amount of WT or DeltaF508 CFTR appearing in detergent-in-soluble aggregates. An approximately 70% knockdown of endogenous Derlin-1 by RNA interference increased the steady-state levels of WT and DeltaF508 CFTR by 10-15-fold, reflecting its significant role in CFTR degradation. Derlin-1 mediated the degradation of N-terminal CFTR fragments corresponding to the first transmembrane domain of CFTR, but CFTR fragments that incorporated additional domains were degraded less efficiently. These findings suggest that Derlin-1 recognizes misfolded, nonubiquitylated CFTR to initiate its dislocation and degradation early in the course of CFTR biogenesis, perhaps by detecting structural instability within the first transmembrane domain.

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5 Exogenous expression of Derlin-1 led to its co-localization with CFTR in the ER where it reduced wild type (WT) CFTR expression and efficiently degraded the disease-associated CFTR folding mutants, ⌬F508 and G85E (>90%). Login to comment
49 CFTR G85E was generated using the site-directed mutagenesis kit (Stratagene) and the forward primer 5Ј-GAGGTTTATGTTCTATGAAATC- TTTTTATATTTAGGG (where the underlined GAA represents the base change that produces the mutation from glycine to glutamine at position 85). Login to comment
171 To further evaluate this idea, we expressed Derlin-1 with a disease-associated CFTR folding mutant that introduces a charged residue into the first membrane-spanning segment of TM1, G85E CFTR (32). Login to comment
172 As shown in Fig. 5D, only the immature form of G85E was observed at steady state, and co-expression of this mutant with Derlin-1 reduced its expression level by 90%. Login to comment
175 Derlin-1 and its degradation complex partner, p97, interacted physically with WT CFTR and reduced its maturation, and Derlin-1 initiated the efficient ER degradation of two CFTR folding mutants, ⌬F508 and G85E. Login to comment
209 D, CFTR folding mutant G85E was co-expressed with GFP or Derlin-1 in HEK 293 cells, as in Fig. 2A. Login to comment
214 Rather, the avid degradation of G85E and TM1 fragments by Derlin-1 suggests that it may sense the organization of the transmembrane ␣-helices. Login to comment
215 G85E CFTR introduces a charged residue in the first membrane-spanning ␣-helix, and although not altering its transmembrane topology, this mutation appears to disrupt the helical packing or in other ways destabilize TM1 (30). 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] 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] Patterns of GI disease in adulthood associated wit... Gut. 2007 Aug;56(8):1153-63. Epub 2007 Apr 19. Wilschanski M, Durie PR
Patterns of GI disease in adulthood associated with mutations in the CFTR gene.
Gut. 2007 Aug;56(8):1153-63. Epub 2007 Apr 19., [PMID:17446304]

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90 A few missense mutations (eg, G85E) confer a variable pancreatic phenotype. 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] Correlation of chest radiograph pattern with genot... Chest. 2007 Aug;132(2):569-74. Epub 2007 Jun 15. Kaza V, Katz MF, Cumming S, Frost AE, Safdar Z
Correlation of chest radiograph pattern with genotype, age, and gender in adult cystic fibrosis: a single-center study.
Chest. 2007 Aug;132(2):569-74. Epub 2007 Jun 15., [PMID:17573513]

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

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

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

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

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

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

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

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2 Among 96 tested alleles, we found nine different mutations: ⌬F508, 58.33%; G542X, 3.12%; N1303K, 2.08%; R1162X; 621 ؉ 1G→T; G85E; Y569C; E585X; and S466X, 1.04%. Login to comment
39 INNOGENETICS INNO-LIPA CFTR 12 and INNO-LIPA CFTR 7 ϩ Tn diagnostic kits were used to assess the presence of the 29 mutations in CF patients; ⌬F508, ⌬I507, G542X, N1303K, 1717-1G Ǟ A, W1282X, G551D, R553X, S1251N, R560T, 3905insT, Q552X, 394delTT, G85E, E60X, 621 ϩ 1G Ǟ T, R117H, 1078delT, R347P, R334W, 2143delT, 2183AA Ǟ G, 2184delA, 711 ϩ 5G Ǟ A, 2789 ϩ 5G Ǟ A, R1162X, 3659delC, 3849 ϩ 10kbC Ǟ T, and A455E. Login to comment
50 Nine different mutations were found: ⌬F508 (58.33%), G542X (3.12%), N1303K (2.08%), R1162X, 621 ϩ 1G Ǟ T, G85E, Y569C, E585X, and S466X (1.04%). Login to comment
77 G85E was associated with haplotype 22-16-13 and 621 ϩ 1G Ǟ T was found on haplotype 21-2113 (Table 4). Login to comment
81 MUTATIONS AND CORRESPONDING GENOTYPES OBSERVED IN A CROATION COHORT OF CF PATIENTS Number of affected Number of detected Mutation alleles (%) Genotype genotypes (%) ⌬F508 56 (58.33) ⌬F508/⌬F508 19 (39.58) G542X 3 (3.12)0 ⌬F508/Na 7 (14.58) N1303K 2 (2.08)0 ⌬F508/G542X 3 (6.25)0 R1162X 1 (1.04)0 ⌬F508/N1303K 2 (4.17)0 621ϩ1G→T 1 (1.04)0 ⌬F508/R1162X 1 (2.08)0 G85E 1 (1.04)0 ⌬F508/621ϩ1G→T 1 (2.08)0 Y569C 1 (1.04)0 ⌬F508/G85E 1 (2.08)0 E585X 1 (1.04)0 ⌬F508/Y569C 1 (2.08)0 S466X 1 (1.04)0 ⌬F508/E585X 1 (2.08)0 Na 29 (30.21) ⌬F508/S466X 1 (2.08) Na/Na 11 (22.92) Total 96b Total 48c aAlleles without mutation. Login to comment
112 ASSOCIATION BETWEEN MICROSATELLITE HAPLOTYPES AND CFTR GENE MUTATIONS IN A CROATIAN COHORT OF CF PATIENTS Haplotype Number of haplotypesa (%) Mutationsb No mutationc 21-23-13 20 (40) ⌬F508 (15) 3 G542X (2) 21-17-13 12 (24) ⌬F508 (10) 2 21-25-13 3 (6)0 ⌬F508 (3) / 21-21-13 2 (4)0 ⌬F508 (1) / 621ϩ1G→T (1) / 20-23-13 2 (4)0 ⌬F508 (2) / 22-17-13 1 (2)0 ⌬F508 (1) / 22-16-13 1 (2)0 G85E (1) / 25-17-13 1 (2)0 1 25-16-14 1 (2)0 1 26-16-13 1 (2)0 1 21-16-13 2 (4)0 2 22-23-13 1 (2)0 1 23-23-13 1 (2)0 1 19-23-13 1 (2)0 1 22-16-17 1 (2)0 1 aNumber of alleles with the corresponding haplotype. Login to comment

[hide] 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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55 Nine mutations showed a frequency higher than 1%, F508del (45.5%), G542X (6.3%), N1303K (4.5%), G85E, R334W and R1162X (3.6%), 2183AA.G and W1282X Table1.FrequenciesoftheCFTRmutations,theirmicrosatellitehaplotypesandIVS8-6(T)nallelesintheBrazilianCFpatientsa MutationExon/intron ChromosomesParana State/SantaCatarina State(total)%HaplotypesIVS8CA,IVS17bTA,IVS17bCA(n)(T)nlocus(n) DF508Exon1027/24(51)45.5416-7-17(1)/16-29-14(1)/16-31-13(1)/17-30-13 (1)/17-31-13(20)/17-32-13(7)23-31-13(15)/23-32-14 (1)/23-46-13(1)/25-30-13(1)/26-31-13(1)/unknown(1) 9T(44)/7T(3)unknown(4) G542XExon115/2(7)6.2523-32-13(1)/23-33-13(5)/23-34-13(1)9T(7) N1303KExon212/3(5)4.4616-30-13(1)/23-30-13(1)/23-31-13(3)9T(4)/7T(1) G85EExon32/2(4)3.5716-24-13(4)7T(4) R334WExon71/3(4)3.5716-34-13(1)/(16-48-13)(1)/17-33-13(1)/17-41-13(1)7T(3)/unknown(1) R1162XExon191/3(4)3.5717-31-13(4)7T(4) 2183AA.GExon131/2(3)2.6816-31-13(2)/16-31-14(1)7T(2)/unknown(1) W1282XExon201/2(3)2.6817-7-17(3)7T(2)/9T(1) R553XExon112/0(2)1.7817-44-11(1)/17-47-11(1)7T(1)/unknown(1) S4XExon11/0(1)0.89(16-__-13)(1)Unknown(1) 232del18Exon20/1(1)0.8921-36-13(1)Unknown(1) 62111G.TIntron41/0(1)0.89__-34-13(1)Unknown(1) 71111G.TIntron51/0(1)0.8916-25-13(1)7T(1) 71115G.AIntron51/0(1)0.89__-7-17(1)Unknown(1) R347PExon70/1(1)0.8916-32-13(1)7T(1) 1717-1G.AIntron101/0(1)0.8916-7-17(1)7T(1) 1717-8G.AIntron101/0(1)0.8916-33-13(1)9T(1) 1812-1G.AIntron111/0(1)0.8916-31-14(1)9T(1) A561EExon121/0(1)0.8916-44-13(1)7T(1) E585XExon121/0(1)0.89Unknown(1)7T(1) 189811G.AIntron120/1(1)0.8916-45-13(1)7T(1) G1069RExon17b1/0(1)0.8917-30-13(1)Unknown(1) Y1092XExon17b1/0(1)0.8916-30-137T(1) 3849110kbC.TIntron191/0(1)0.8916-7-17(1)7T(1) W1282GExon201/0(1)0.8916-32-14(1)7T(1) Unknown13/0(13)11.6016-7-17(1)/16-29-13(2)/16-30-13(1)/16-31-13 (1)/16-32-13(3)/16-33-13(1)16-34-13(1)/16-38-16 (1)/18-35-13(2) Unknown(13) Total112100 Ôn`,thetotalnumberofchromosomesbearingeachhaplotypeor(T)nlocus;Ôunknown`,usedwhenthehaplotype/(T)nlocuscannotbecharacterized;Ô_`,usedwhenaspecific alleleofthehaplotypecannotbecharacterized. Login to comment
80 Mutations G85E, R334W, R553X, 62111G.A, 1717-8G.A, G1069R and W1282G were associated with haplotypes not observed in the normal CFTR genes. 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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35 6 k b A .G , 3 2 7 2 - 2 6 A .G , 2 7 8 9 15 G .A , 312011G.A, 71111G.T, G85E, Y122X and W846X). 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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127 G85E is in a low-temperature domain compared with R75X, resulting in deviation of the melting curves at different temperatures. The PCR product was 169 bp in length. Login to comment
145 2 223CϾT R31C 3 355CϾT R75X 386GϾA G85E 4 482GϾA R117H 575TϾC I148T 621 ؉ 1GϾTb 5 711 ؉ 1GϾT 7 1078delT 1132CϾT R334W 1150delA 1172GϾC R347P 8 1341 ϩ 18AϾCc 9 1496CϾA A455E 10 1651-1653del I507del 1653-1655del F508deld 11 1717 - 1GϾA 1756GϾT G542Xe 1784GϾA G551Db 1789CϾT R553Xf 1811GϾC R560T 12 1898 ؉ 1GϾA 13 2184delA 14b 2789 ؉ 5GϾAe 16 3120 ؉ 1GϾA 18 3500 - 2AϾTg 19 3616CϾT R1162X 3659delC Intron 19 3849 ؉ 10kbCϾTe 20 3978GϾA W1282X 21 4041CϾG N1303K 22 4178GϾA G1349Dc a Disease-causing variants recommended for genotyping by the ACMG (4) are in bold. Login to comment
107 p.G85E is in a region of lower stability and the change in melting curve shape is most distinctive at lower temperatures. 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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34 Other identified class II disease-causing mutations in CFTR include N1303K, G85E and G91R [18]. Login to comment
36 The exact mechanism by which these mutations disrupt folding is not completely clear [21], but both the G85E and G91R mutations have been shown to affect folding due to the insertion of a charged residue in the plane of the lipid bilayer [9]. Login to comment

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

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

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23 The mutations DF508, W1282X, G542X, S549R, Q359K/T360K, 405 + 1G, 1717, and N1303K were defined as severe and the mutations 3849 + 10 kb, D1152H, G85E, I1234V, R334W, and 5T were defined as mild/variable. Login to comment
46 of patients W1282X/3849 + 10 kb 15 W1282X/5T 15 DF508/5T 7 DF508/D1152H 6 DF508/3849 + 10 kb 5 W1282X/D1152H 5 W1282X/I1234V 2 3849 + 10 kb/405 + 1G- > A 2 R334W/R334W 2 5T/5T 2 D1152H/D1152H 1 D1152H/5T 1 D1152H/3849 + 10 kb 1 DF508/UKN 13 W1282X/UKN 11 5T/UKN 7 D1152H/UKN 3 1717/UNK 1 G85E/UKN 1 Q359K/T360K/UKN 1 S549R/UKN 1 3849 + 10 kb/UKN 1 UKN/UKN 36 CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane regulator. Login to comment
69 Table 3 The clinical characteristics of the PS patients who became PI Age at CF diagnosis Presenting symptom Age at first pancreatitis event Age at transition PS-PI Genetic profile Sweat Clmmol/l 19 years Pulmonary 19 yearsa 22 years W1282X/G85E 66 6 months Affected sibling 3 yearsa 14 years W1282X/I1234V 82 1 month Affected sibling (None) 12 years DF508/G85E 32 28 years Pancreatitis 28 yearsa 34 years D1152H/5T 30 CF, cystic fibrosis; PI, pancreatic insufficient; PS, pancreatic sufficient. Login to comment
72 The other mutations carried by the PS group (D1152H, G85E, I1234V, and R334W) were also found to cause variable clinical expressions [7-10]. Login to comment

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

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

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116 Moreover, some nascent/immature CFTR protein present in the ER and A B 501 E92K Y89C G85E/V P5L S50P E60K R75Q NBD1 NBD2 R FIGURE 1. Login to comment
156 Confocal images from representative xy sections taken from1of 3 independent experiments show the subcellular distribution of wild-type CFTR (WT), p.F508del mutant (F508del), and variants p.S50P (S50P), p.E60K (E60K), p.G85E (G85E), p.G85V (G85V), p.E92K (E92K), p.P5L (P5L), p.R75Q (R75Q), and p.Y89C (Y89C). Login to comment
180 B: Recordings fromvariants p.S50P (S50P), p.E60K (E60K), p.G85E (G85E), p.G85V (G85V), and p.E92K (E92K) (superimposed recordings). Login to comment
3 By site-directed mutagenesis, we generated four CFTR variants in the N-terminal cytoplasmic tail (p.P5L, p.S50P, p.E60K, and p.R75Q) and four in the first transmembrane segment of membrane-spanning domain 1 (p.G85E/V, p.Y89C, and p.E92K). Login to comment
4 Immunoblot analysis revealed that p.S50P, p.E60K, p.G85E/V, and p.E92K produced only core-glycosylated proteins. Login to comment
40 The eight CFTR variants included in this study: p.P5L, p.S50P, p.E60 K, p.R75Q, p.G85E, p.G85V, p.Y89C, and p.E92K (Fig. 1A) were generated by oligonucleotide-directed mutagenesis in pCMVCFTRNot6.2wt using the QuickChangeTM XL-Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the manufacturer`s instructions (see Supplementary Table S1 for a detailed description of the mutagenesis primers employed; available online at http://www.interscience.wiley.com/jpages/1059-7794/supp mat). Login to comment
102 RESULTS Description and Cross-Species Analysis of Natural N-Terminus CFTR Variants We chose eight naturally occurring sequence variants, four located across the N-terminal CFTR tail (p.P5L, p.S50P, p.E60K, and p.R75Q), and four within the first segment of MSD1 (p.G85E, p.G85V, p.Y89C, and p.E92 K) (Fig. 1A; Table 1). Login to comment
110 In contrast, variants p.S50P, p.E60K, p.G85E, p.G85V, and p.E92K, produced only higher mobility band B proteins suggesting that, like the p.F508del mutant, the resulting misfolded channels are retained and degraded in the cytoplasm (Fig. 2A). Login to comment
118 B: Alignment of the N-terminus (amino acids 1 to 100) of the CFTR protein derived from di¡erent species.The sequences derived from human (Homo sapiens, Gen- BankNM_000492), mouse (Mus musculus,GenBankNM_021050), Norway rat (Rattusnovergicus,GenBankNM_031506), European rabbit (Oryctolagus cuniculus, GenBank NM_001082716), cow (Bos taurus, GenBank NM_174018), sheep (Ovis aries, GenBank NM_001009781), African clawed frog (Xenopus laevis, GenBank X65256), and spiny dog'sh (Squalus acanthias, GenBank M83785) were aligned using the ClustalW multiple sequence alignment program.The amino acid residue a¡ected by each of the variants analyzed (p.P5L, p.S50P, p.E60K, p.R75Q, p.G85E, p.G85V, p.Y89C, and p.E92K) is indicated in bold. Login to comment
122 Similarly, variants p.S50P, p.E60K, p.G85V, p.G85E, and p.E92K displayed a yellow colocalization pattern clearly compatible with retention of the anomalous CFTR proteins within the intracellular compartments and no detection of PM staining (Fig. 3). Login to comment
130 Likewise, none of the five variants (p.S50P, p.E60K, p.G85E, p.G85V, and p.E92K) in which severe misprocessing was previously demonstrated (Figs. 2 and 3), was able to generate cAMP-activated currents (Fig. 4B). Login to comment
133 Genotype^Phenotype Correlation in the N-Terminal CFTR MissenseVariants Under StudyÃ Missense varianta Phenotype Second allele (number of patients)b p.P5L CF p.F508del (1), p.P205S (1) p.S50P CBAVD p.F508del (1), p.E115del (1) p.E60K CF p.G542X (1), p.I507del (1) p.R75Q HT p.F508del (3), p.E725K (1) B p.R347H (1), p.R75Q (1), n.i. (4) Br c.1584G4A (2), c.1210-7_1210-6delTT (1), n.i.(3) NT p.F508del (1) CP c.1584G4A (1), n.i. (3) MI n.i. (1) CUAVD n.i. (2) OZ n.i. (2) Normal p.R75Q (1), c.2052_2053insA (1), n.i. (1) p.G85E CF p.F508del (8), p.G542X (2), p.I507del (1), c.580-1G4T (1), p.G85E (1), c.1477_ 1478delCA (1) CBAVD p.G576A (1) HT p.L997F (1),WT (1) p.G85V CF p.F508del (2), p.G542X (2), p.Y1092X (1), c.265715G4A (1), p.A1006E, c.1210-7_1210- 6delTT (1), n.i. (1) p.Y89C CF n.i. (1)c p.E92K CF p.F508del (2), p.Q890X (1), p.L206W (1) CBAVD c.1210-7_1210-6delTT (1) ÃThe recommendations for mutation nomenclature (www.hgvs.org/mutnomen/) were used to name CFTR gene sequence variations at both the nucleotide level and the protein level. Login to comment
136 Besides being present in CF patients, we have found both p.G85E and p.E92K variants in CBAVD patients [Casals et al., 2000]. Login to comment
137 Moreover, the p.G85E variant was also detected in one newborn with hypertrypsinemia [Gartner et al., 2003]. Login to comment
213 Four of the variants (p.P5L, p.S50P, p.E60 K, and p.R75Q) are localized within the cytosolic N-terminal tail, and the remaining four (p.G85E, p.G85V, p.Y89C, and p.E92K) are embedded in three positions within the first transmembrane segment (TM1) of MSD1. Login to comment
215 Accordingly, using three different approaches (immunoblotting, immunocytochemistry, and electrophysiology) we found that 5 (p.S50P, p.E60K, p.G85E, p.G85V, and p.E92K) out of the 8 variants failed to mature, showing an analogous behavior than the most common F508del mutation. Login to comment
219 MSD1-A¡ectingVariants Concerning the p.G85E mutation, our results are in agreement with those reported by Decaestecker et al. [2004] through pulse-chase experiments. Login to comment
220 Moreover, although both p.G85E and p.G85V, similarly to p.E92K, do not appear to affect TM1 topology [Xiong et al., 1997], they are also localized within or adjacent to the bilayer. Login to comment
222 Indeed, it has been recently shown that Derlin-1, an ERAD component, efficiently degrades p.G85E and p.F508del folding mutants [Sun et al., 2006]. Login to comment
228 Regarding the p.Y89C variant, based on the structural features of the amino acids involved (the bulky hydrophobic tyrosine residue is substituted by the smaller hydrophilic cysteine residue) and its position within TM1 (between the severe folding mutations p.G85E/V and p.E92K) it would also be expected a major structural rearrangement and folding defect of MSD1 conferred by this variant. 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] 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] Genetic determinants and epidemiology of cystic fi... Diabetes Care. 2008 Sep;31(9):1789-94. Epub 2008 Jun 5. Adler AI, Shine BS, Chamnan P, Haworth CS, Bilton D
Genetic determinants and epidemiology of cystic fibrosis-related diabetes: results from a British cohort of children and adults.
Diabetes Care. 2008 Sep;31(9):1789-94. Epub 2008 Jun 5., [PMID:18535191]

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

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54 Genotypes associated with cystic fibrosis were coded into five established classes reflecting CFTR function of defective production, processing, regulation, conductance, and quantity of CFTR protein (12) as follows: I: G542X, R553X, W1282X, R1162X, 621-1G3T, 1717- 1G3 A, 1078⌬T, and 3659⌬C; II: ⌬F508, ⌬I507, N1303K, and S549N; III: G551Dand R560T; IV: R117H, R334W, G85E, and R347P; V: 3849ϩ5G3A, and A455E; and unknown: 711ϩIG3 T, 2184DA, and 1898ϩIG3 A. Login to comment

[hide] Patterns of gastrointestinal disease associated wi... Curr Gastroenterol Rep. 2008 Jun;10(3):316-23. Wilschanski M
Patterns of gastrointestinal disease associated with mutations of CFTR.
Curr Gastroenterol Rep. 2008 Jun;10(3):316-23., [PMID:18625144]

Abstract [show]
This review focuses on the pathobiology of the gastrointestinal and hepatic manifestations of cystic fibrosis (CF) disease in relation to their genetic basis in mutations of the CF transmembrane conductance regulator (CFTR) gene. It reviews the nature of the CFTR protein, a categorization of the types of gene mutations underlying CF's various manifestations, and the ways in which absent or reduced CFTR produces various functional abnormalities in the different organs affected by CF. Subsequently, the particular organ-related clinical manifestations of CF directly associated with loss of CFTR function are addressed. Thereafter, the review discusses some of the complexities of the genotype-phenotype relationships related to milder mutations or complex genetic disorders in which CFTR abnormalities interact with other genetic and environmental factors, and the potential diagnostic roles of sweat testing or other electrophysiologic testing. This discussion examines secondary gastrointestinal manifestations of CF and the particular cases of diseases that may be related to abnormalities of CFTR.

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78 A few missense mutations (eg, G85E) confer a variable pancreatic phenotype. 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] 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] 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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56 Liver disease is second to pulmonary disease (plus organ transplantation complications) as a cause of mortality in CF (1.7% of deaths).26 Table 3 Core mutation panel carrier recommended by the ACMG for routine CF diagnostic testing and carrier screening of the general population7 Intronic mutations Exonic mutations Missense Nonsense In-Frame Deletion 621ϩ1GϾT G85E G542X ⌬I507 711ϩ1GϾT R117H R553X ⌬F508 1717-1GϾA R334W R1162X 1898ϩ1GϾA R347P W1282X 2184delA A455E 2789ϩ5GϾA G551D 3120ϩ1GϾA R560T 3659delC N1303K 3849ϩ10kbCϾT Endocrine manifestations of CF CF-related diabetes mellitus (CFRDM) may present in adolescence. Login to comment

[hide] IL1B polymorphisms modulate cystic fibrosis lung d... Pediatr Pulmonol. 2009 Jun;44(6):580-93. Levy H, Murphy A, Zou F, Gerard C, Klanderman B, Schuemann B, Lazarus R, Garcia KC, Celedon JC, Drumm M, Dahmer M, Quasney M, Schneck K, Reske M, Knowles MR, Pier GB, Lange C, Weiss ST
IL1B polymorphisms modulate cystic fibrosis lung disease.
Pediatr Pulmonol. 2009 Jun;44(6):580-93., [PMID:19431193]

Abstract [show]
RATIONALE: Variability in pulmonary disease severity is found in patients with cystic fibrosis (CF) who have identical mutations in the CF transmembrane conductance regulator (CFTR) gene. We hypothesized that one factor accounting for heterogeneity in pulmonary disease severity is variation in the family of genes affecting the biology of interleukin-1 (IL-1), which impacts acquisition and maintenance of Pseudomonas aeruginosa infection in animal models of chronic infection. METHODS: We genotyped 58 single nucleotide polymorphisms (SNPs) in the IL-1 gene cluster in 808 CF subjects from the University of North Carolina and Case Western Reserve University (UNC/CWRU) joint cohort. All were homozygous for DeltaF508, and categories of "severe" (cases) or "mild" (control subjects) lung disease were defined by the lowest or highest quartile of forced expired volume (FEV(1)) for age in the CF population. After adjustment for age and gender, genotypic data were tested for association with lung disease severity. Odds ratios (ORs) comparing severe versus mild CF were also calculated for each genotype (with the homozygote major allele as the reference group) for all 58 SNPs. From these analyses, nine SNPs with a moderate effect size, OR < or =0.5 or >1.5, were selected for further testing. To replicate the case-control study results, we genotyped the same nine SNPs in a second population of CF parent-offspring trios (recruited from Children's Hospital Boston), in which the offspring had similar pulmonary phenotypes. For the trio analysis, both family-based and population-based associations were performed. RESULTS: SNPs rs1143634 and rs1143639 in the IL1B gene demonstrated a consistent association with lung disease severity categories (P < 0.10) and longitudinal analysis of lung disease severity (P < 0.10) in CF in both the case-control and family-based studies. In females, there was a consistent association (false discovery rate adjusted joint P-value <0.06 for both SNPs) in both the analysis of lung disease severity in the UNC/CWRU cohort and the family-based analysis of affection status. CONCLUSION: Our findings suggest that IL1beta is a clinically relevant modulator of CF lung disease.

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111 The other 12% of the CFTR mutations included G551D, G542X, G85E, W1282X, and N1303K. Login to comment

[hide] Mechanisms for rescue of correctable folding defec... Mol Biol Cell. 2009 Sep;20(18):4059-69. Epub 2009 Jul 22. Grove DE, Rosser MF, Ren HY, Naren AP, Cyr DM
Mechanisms for rescue of correctable folding defects in CFTRDelta F508.
Mol Biol Cell. 2009 Sep;20(18):4059-69. Epub 2009 Jul 22., [PMID:19625452]

Abstract [show]
Premature degradation of CFTRDeltaF508 causes cystic fibrosis (CF). CFTRDeltaF508 folding defects are conditional and folding correctors are being developed as CF therapeutics. How the cellular environment impacts CFTRDeltaF508 folding efficiency and the identity of CFTRDeltaF508's correctable folding defects is unclear. We report that inactivation of the RMA1 or CHIP ubiquitin ligase permits a pool of CFTRDeltaF508 to escape the endoplasmic reticulum. Combined RMA1 or CHIP inactivation and Corr-4a treatment enhanced CFTRDeltaF508 folding to 3-7-fold greater levels than those elicited by Corr-4a. Some, but not all, folding defects in CFTRDeltaF508 are correctable. CHIP and RMA1 recognize different regions of CFTR and a large pool of nascent CFTRDeltaF508 is ubiquitinated by RMA1 before Corr-4a action. RMA1 recognizes defects in CFTRDeltaF508 related to misassembly of a complex that contains MSD1, NBD1, and the R-domain. Corr-4a acts on CFTRDeltaF508 after MSD2 synthesis and was ineffective at rescue of DeltaF508 dependent folding defects in amino-terminal regions. In contrast, misfolding caused by the rare CF-causing mutation V232D in MSD1 was highly correctable by Corr-4a. Overall, correction of folding defects recognized by RMA1 and/or global modulation of ER quality control has the potential to increase CFTRDeltaF508 folding and provide a therapeutic approach for CF.

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183 Thus, we asked to what extent can disease related folding defects caused by mutations in MSD1 (G85E, G91R, and V232D), MSD2 (M1137R), and NBD2 (N1303K) be corrected relative to those caused by deletion of F508 in NBD1 (Figure 4A). Login to comment
184 The G85E, G91R, M1137R, and N1303K mutations all hinder folding of the nascent B-form of CFTR via a mechanism that involves insertion of a charged amino acid into an inappropriate region (Gregory et al., 1991; Xiong et al., 1997; Vankeerberghen et al., 1998). Login to comment
189 The CFTR G85E and G91R point mutations are contained within TM1, whereas the V232D mutation lies within TM4 of CFTR`s MSD1 domain. Login to comment
191 The addition of Corr-4a slightly increased the steady-state levels of CFTR G85E B-form, but Corr-4a-dependent folding of this mutant was not detected. Login to comment
317 Furthermore, the folding defect of the MSD1 mutant G85E appeared to be largely noncorrectable by Corr-4a. Login to comment

[hide] Non-classic cystic fibrosis associated with D1152H... Clin Genet. 2010 Apr;77(4):355-64. Epub 2009 Oct 15. Burgel PR, Fajac I, Hubert D, Grenet D, Stremler N, Roussey M, Siret D, Languepin J, Mely L, Fanton A, Labbe A, Domblides P, Vic P, Dagorne M, Reynaud-Gaubert M, Counil F, Varaigne F, Bienvenu T, Bellis G, Dusser D
Non-classic cystic fibrosis associated with D1152H CFTR mutation.
Clin Genet. 2010 Apr;77(4):355-64. Epub 2009 Oct 15., [PMID:19843100]

Abstract [show]
BACKGROUND: Limited knowledge exists on phenotypes associated with the D1152H cystic fibrosis transmembrane conductance regulator (CFTR) mutation. METHODS: Subjects with a D1152H allele in trans with another CFTR mutation were identified using the French Cystic Fibrosis Registry. Phenotypic characteristics were compared with those of pancreatic insufficient (PI) and pancreatic sufficient (PS) cystic fibrosis (CF) subjects in the Registry (CF cohort). RESULTS: Forty-two subjects with D1152H alleles were identified. Features leading to diagnosis included chronic sinopulmonary disease (n = 25), congenital absence of the vas deferens (n = 11), systematic neonatal screening (n = 4), and genetic counseling (n = 2). Median age at diagnosis was 33 [interquartile range (IQR, 24-41)] years in D1152H subjects. Median sweat chloride concentrations were 43.5 (39-63) mmol/l in D1152H subjects and were markedly lower than in PI and PS CF subjects (p < 0.05). Bronchiectasis was present in 67% of D1152H subjects, but Pseudomonas aeruginosa colonization and pancreatic insufficiency were present in <30% of subjects. Estimated rates of decline in forced expiratory volume in 1 s (FEV(1)) were lower in D1152H subjects vs PI CF subjects (p < 0.05). None of the D1152H subjects identified since 1999 had died or required lung transplantation. CONCLUSIONS: When present in trans with a CF-causing mutation, D1152H causes significant pulmonary disease, but all subjects had prolonged survival.

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42 The CF genetic analysis panel used in France seeks for 32 mutations: G85E, 394delTT, 621+1G>T, 711+1G>T, R334W, R347P, R347H, 1078delT, 5T/7T/9T, A455E, F508del, I507del, V520F, 1717-1G>A, G542X, G551D, R553X, R560T, S549R (T>G), S549N, 1898+1G>A, 2183AA>G, 2184delA, 2789+5G>A, 3120+1G>A, R1162X, 3659delC, 3849+10kbC>T, W1282X, 3905insT, 3876delA, N1303K. Login to comment

[hide] A 10-year large-scale cystic fibrosis carrier scre... J Cyst Fibros. 2010 Jan;9(1):29-35. Epub 2009 Nov 7. Picci L, Cameran M, Marangon O, Marzenta D, Ferrari S, Frigo AC, Scarpa M
A 10-year large-scale cystic fibrosis carrier screening in the Italian population.
J Cyst Fibros. 2010 Jan;9(1):29-35. Epub 2009 Nov 7., [PMID:19897426]

Abstract [show]
BACKGROUND: Cystic Fibrosis (CF) is one of the most common autosomal recessive genetic disorders, with the majority of patients born to couples unaware of their carrier status. Carrier screenings might help reducing the incidence of CF. METHODS: We used a semi-automated reverse-dot blot assay identifying the 47 most common CFTR gene mutations followed by DGGE/dHPLC analysis. RESULTS: Results of a 10-year (1996-2006) CF carrier screening on 57,999 individuals with no prior family history of CF are reported. Of these, 25,104 were couples and 7791 singles, with 77.9% from the Italian Veneto region. CFTR mutations were found in 1879 carriers (frequency 1/31), with DeltaF508 being the most common (42.6%). Subjects undergoing medically assisted reproduction (MAR) had significantly (p<0.0001) higher CF carrier frequency (1/22 vs 1/32) compared to non-MAR subjects. CONCLUSIONS: If coupled to counselling programmes, CF carrier screening tests might help reducing the CF incidence.

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48 Forty-seven different CFTR mutations/gene alterations were chosen and analysed: ΔF508, G85E, 541delC, D110H, R117H, 621+1G→T, 711+5G→A, R334W, R334Q, T338I, R347H, R347P, R352Q, S466X, ΔI507, E527G, 1717-1G→A, 1717-8G→A, G542X, S549N, S549R A→C, G551D, Q552X, R553X, D579G, 1874insT, E585X, 1898+3A→G, 2183AA→G, 2184delA, R709X, 2789+5G→A, 3132delTG, 3199del6, 3272-26A→G, L1077P, L1065P, R1066H, M1101K, D1152H, R1158X, R1162X, 3849+10KbC→T, G1244E, W1282X, N1303K and 4016insT. Login to comment
97 CF mutation General adult population MAR population n=1879 n=236 ΔF508 42.6 45.7 2183AA→G 5.9 5.9 R1162X 5.7 8.2 N1303K 5.4 5.9 G542X 4.2 3.7 D1152H 3.9 5.0 R553X 3.7 3.1 R117H 3.3 1.8 711+5G→A 2.8 4.1 Q552X 2.8 0.4 2789+5G→A 2.2 3.1 1717-1G→A 2.6 2.8 E527G 2.4 - G85E 2.4 0.9 R334Q 0.9 0.4 W1282X 0.7 0.9 R334W 0.6 - 1898+3A→G 0.5 0.4 R1158X 0.4 - R1066H 0.4 0.4 T338I 0.4 1.8 3849+10Kb C→T 0.4 1.3 3272-26 A→G - 0.9 3132delTG - 0.9 3659 del C - 0.4 4016 ins T - 0.4 1717-8G→A - 0.4 R347H - 0.4 ΔI507 - 0.4 R1070Q - 0.4 Other (16) 5.4 - Table 2a List of CFTR compound heterozygotes in the adult general population. Mutation Health status Disorder Gender Age (years) Notes and refs ΔF508/A238V Infertile CBAVD M 36 (A) ΔF508/R352W Infertile CBAVD M 45 (A) R553X/R334Q M 38 ΔF508/R347H M 53 [17] S42F/D372E (1251T→G) M 39 (A) (B) ΔF508/D110H Infertile M 38 ΔF508/L1414S (4373T→C) Infertile CBAVD M 44 (A) (B) ΔF508/V201M, D1270N & R74W Infertile CBAVD M 44 (A) [18,19] 2183AA→G/L206W Infertile CBAVD M 40 (A) 711+5G→A/ L206W Infertile CBAVD M 40 (A) Table 2b List of CFTR compound heterozygotes in the population enrolled for medically assisted reproduction. Login to comment

[hide] Deletion of CFTR translation start site reveals fu... Cell Physiol Biochem. 2009;24(5-6):335-46. Epub 2009 Nov 4. Ramalho AS, Lewandowska MA, Farinha CM, Mendes F, Goncalves J, Barreto C, Harris A, Amaral MD
Deletion of CFTR translation start site reveals functional isoforms of the protein in CF patients.
Cell Physiol Biochem. 2009;24(5-6):335-46. Epub 2009 Nov 4., [PMID:19910674]

Abstract [show]
BACKGROUND/AIMS: Mutations in the CFTR gene cause Cystic Fibrosis (CF) the most common life-threatening autosomal recessive disease affecting Caucasians. We identified a CFTR mutation (c.120del23) abolishing the normal translation initiation codon, which occurs in two Portuguese CF patients. This study aims at functionally characterizing the effect of this novel mutation. METHODS: RNA and protein techniques were applied to both native tissues from CF patients and recombinant cells expressing CFTR constructs to determine whether c.120del23 allows CFTR protein production through usage of alternative internal codons, and to characterize the putative truncated CFTR form(s). RESULTS: Our data show that two shorter forms of CFTR protein are produced when the initiation translation codon is deleted indicating usage of internal initiation codons. The N-truncated CFTR generated by this mutation has decreased stability, very low processing efficiency, and drastically reduced function. Analysis of mutants of four methionine codons downstream to M1 (M82, M150, M152, M156) revealed that each of the codons M150/M152/M156 (exon 4) can mediate CFTR alternative translation. CONCLUSIONS: The CFTR N-terminus has an important role in avoiding CFTR turnover and in rendering effective its plasma membrane traffic. These data correlate well with the severe clinical phenotype of CF patients bearing the c.120del23 mutation.

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172 Altogether, these results for c.120del23-CFTR suggest an important role of the N-terminus in CFTR folding, stability and processing, which was also evidenced by other studies demonstrating that point mutations in this region - S50P; E60K; G85E/V; E92K - prevent CFTR maturation [35, 36]. Login to comment

[hide] CFTR allelic heterogeneity in Brazil: historical a... J Hum Genet. 2010 Feb;55(2):71-6. Epub 2009 Nov 27. Faucz FR, Souza DA, Olandoski M, Raskin S
CFTR allelic heterogeneity in Brazil: historical and geographical perspectives and implications for screening and counseling for cystic fibrosis in this country.
J Hum Genet. 2010 Feb;55(2):71-6. Epub 2009 Nov 27., [PMID:19942933]

Abstract [show]
The goal of the present study was to provide a complete and updated spectrum of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene mutations in the Brazilian population combining all available in silico data for patients with CF in Brazil, including founder background and migration flow that consisted of the actual genetic pool of the Brazilian population. Information sources in international databases (PUBMED and SCIELO) were searched. The Brazilian population shows a wide variation in the frequency of CFTR mutations in states Rio Grande do Sul (RS), Santa Catarina (SC), Parana (PR), Sao Paulo (SP), Rio de Janeiro (RJ), Minas Gerais (MG), Para (PA) and Bahia (BA); this variation includes the most common mutation p.F508del. Apparently, this frequency variation is because of the different ethnic compositions. States such as SC and PR have a greater European admixture with almost 90% of CF alleles identified. In other states, such as BA, higher frequency of alleles that are common among African populations is seen. Overall, the CFTR mutational spectrum indicates the presence of European, African and Amerindian ethnic groups in the contemporary Brazilian CF patients. Here, we present an analysis of the CFTR allelic heterogeneity and discuss the origin of its genetic composition, in an attempt to provide improved perspective for the CF population screening in Brazil and genetic counseling.

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46 cHomogeneity test between the two previous PR and SC results and RS35: mutations p.N1303 K, p. R1162X, p.W1282X and p.R553X and the mutations p.G85E, c.2183AA4G and 'other` were grouped for the test. Login to comment
42 Table 1 Frequencies of some mutations in different regions from Brazil South Southeast North Northeast Mutation PR and SC19 PR and SC11 RS35 SP34 RJ36 MG11 PA37 BA38 p.F508del 45.54% (51/112) 46.94% (92/196) 48.7% (75/154) 50.00% (96/192) 28.42% (54/190) 47.37% (54/114) 22.73% (15/66) 8.68% (25/288) p.G542X 6.25% (7/112) 7.65% (15/196) 3.25% (5/154) 4.17% (8/192) 2.10% (4/190) 7.02% (8/114) 0.00% (0/66) nt p.N1303K 4.46% (5/112) 5.10% (10/196) 0.00% (0/154) 2.08% (4/192) nt 0.00% (0/114) nt nt p.G85E 3.57% (4/112) 2.04% (4/196) nt nt 4.73% (9/190) 3.51% (4/114) nt nt p.R334W 3.57% (4/112) 3.06% (6/196) 1.30% (2/154) nt 2.63% (5/190) 3.51% (4/114) nt nt p.R1162X 3.57% (4/112) 5.61% (11/196) 0.00% (0/154) nt 0.53% (1/190) 3.51% (4/114) nt nt c.2183AA4G 2.68% (3/112) 1.53% (3/196) nt nt 0.00% (0/190) 0.00% (0/114) nt nt p.W1282X 2.68% (3/112) 2.55% (5/196) 0.65% (1/154) 0.52% (1/192) 0.00% (0/190) 0.88% (1/114) nt nt p.R553X 1.78% (2/112) 1.02% (2/196) 0.65% (1/154) 0.52% (1/192) 0.00% (0/190) 0.00% (0/114) 0.00% (0/66) nt p.G551D 0.00% (0/112) 0.00% (0/196) 0.00% (0/154) 1.04% (2/192) 0.53% (1/190) 0.00% (0/114) 4.55% (3/66) nt Othera 25.89% (29/112) 24.49% (48/196) 45.45% (70/154) 56.25% (108/192) 61.05% (116/190) 65.79% (54/114) 72.73% (48/66) 91.32% (263/288) P¼0.9226b P¼0.0007c Abbreviations: BA, Bahia state; MG, Minas Gerais state; nt, not tested; PA, Para´ state; PR, Parana´ state; RJ, Rio de Janeiro state; RS, Rio Grande do Sul state; SC, Santa Catarina state; SP, Sa˜o Paulo state. Login to comment
45 bHomogeneity test between the PR and SC19 and PR and SC11: mutations p.G85E and p.R334W, and the mutations c.2183AA4G, p.W1282X, p.R553X and p.G551D were grouped for the test. Login to comment
53 This can be showed when we compare the occurrence of the eight most frequent mutations in Italy (which consists of B70% of all mutations in this country) with those of other populations (Table 3).14,53,54 Faucz et al.19 found nine mutations with a frequency higher than 1% (p.F508del: 45.5%; p.G542X: 6.3%; p.N1303K: 4.5%; p.G85E, p.R334W and p.R1162X: total of 3.6%; c.2183AA4G and p.W1282X: 2.7%; and p.R553X: 1.8%) in CF patients from PR and SC (south of Brazil). Login to comment

[hide] Cystic fibrosis genotype and assessing rates of de... Radiology. 2009 Dec;253(3):813-21. Cleveland RH, Zurakowski D, Slattery D, Colin AA
Cystic fibrosis genotype and assessing rates of decline in pulmonary status.
Radiology. 2009 Dec;253(3):813-21., [PMID:19952026]

Abstract [show]
PURPOSE: To evaluate the hierarchical phenotypic expression of cystic fibrosis transmembrane conductance regulator (CFTR) genotypes in the respiratory system as has been documented in the pancreas. MATERIALS AND METHODS: This study was institutional review board approved; informed consent was not required. HIPAA guidelines were followed. Genotype effects were assessed by using chest radiographic and pulmonary function test (PFT) results in 93 patients. Serial chest radiographic and PFT (percentage of predicted forced expiratory volume in 1 second [FEV(1)], percentage of predicted forced vital capacity [FVC]) results were compared by using analysis of variance with repeated measures. By using CFTR class of mutations, two groups were created: group S (severe disease) and group M (mild disease). Within group S, three subgroups were created: A consisted of patients with two class I alleles; B, class I allele and class II or III allele; C, class II allele and class II or III allele. Group M consisted of patients with at least one allele from class IV-VI. RESULTS: Within group S, subgroup A had a faster deterioration than B or C according to radiographic data (A vs B, P = .014; A vs C, P = .009), with only a borderline difference in FEV(1) for subgroups A versus C (P = .031). Otherwise, PFTs were not sensitive for distinguishing subgroups. Only radiographic results identified that subgroup B had faster progression than C (P = .003); all parameters had trends of decline in the same direction. Group S had a faster decline than group M (radiography, P = .005; FVC, P = .011; FEV(1), P = .529). CONCLUSION: Disease progressed more rapidly with gene class hierarchical correlations seen in pancreatic disease. Radiography was more sensitive for identifying differences.

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56 Measurement Tools All chest radiographic, FEV1, and FVC studies were performed at the study institution during the observed life spans Table 2 Patients according to CF Genotype Group Parameter Genotype Class Pancreatic Exocrine Status* No. of Patients Group S (severe pancreatic and pulmonary phenotypes) Subgroup A (class I and class I) 5 G542X/W1282X I/I PI 2 W1282X/W1282X I/I PI 1 621ϩ1G-T/Y1092X I/I PI 1 3120ϩ1G-A/3120ϩ1G-A I/I PI 1 Subgroup B (class I and class II or III) 16 G542X/⌬F508 I/II PI 6 W1282X/⌬F508 I/II PI 3 Q493X/⌬F508 I/II PI 2 R553X/⌬F508 I/II PI 2 1717-1G/⌬F508 I/II PI 1 621ϩ1G-T/⌬F508 I/II PI 1 2184delA/G551D I/III PI 1 Subgroup C (class II and class II or III) 68 D1507/⌬F508 II/II PI 3 N1303K/⌬F508 II/II PI 2 ⌬F508/⌬F508 II/II PI 57 G551D/⌬F508 II/III PI 6 Group M (mild pancreatic and pulmonary phenotypes) Miscellaneous severe and miscellaneous mild 4 ⌬F508/G85E II/IV PS 2 ⌬F508/R117H II/IV PS 1 D1507/R352Q II/IV PS 1 Miscellaneous mild and miscellaneous mild . Login to comment

[hide] Do common in silico tools predict the clinical con... Clin Genet. 2010 May;77(5):464-73. Epub 2009 Jan 6. Dorfman R, Nalpathamkalam T, Taylor C, Gonska T, Keenan K, Yuan XW, Corey M, Tsui LC, Zielenski J, Durie P
Do common in silico tools predict the clinical consequences of amino-acid substitutions in the CFTR gene?
Clin Genet. 2010 May;77(5):464-73. Epub 2009 Jan 6., [PMID:20059485]

Abstract [show]
Computational methods are used to predict the molecular consequences of amino-acid substitutions on the basis of evolutionary conservation or protein structure, but their utility in clinical diagnosis or prediction of disease outcome has not been well validated. We evaluated three popular computer programs, namely, PANTHER, SIFT and PolyPhen, by comparing the predicted clinical outcomes for a group of known CFTR missense mutations against the diagnosis of cystic fibrosis (CF) and clinical manifestations in cohorts of subjects with CF-disease and CFTR-related disorders carrying these mutations. Owing to poor specificity, none of tools reliably distinguished between individual mutations that confer CF disease from mutations found in subjects with a CFTR-related disorder or no disease. Prediction scores for CFTR mutations derived from PANTHER showed a significant overall statistical correlation with the spectrum of disease severity associated with mutations in the CFTR gene. In contrast, PolyPhen- and SIFT-derived scores only showed significant differences between CF-causing and non-CF variants. Current computational methods are not recommended for establishing or excluding a CF diagnosis, notably as a newborn screening strategy or in patients with equivocal test results.

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64 Mutations in the CFTR gene grouped by clinical category Cystic fibrosis CFTR-related disease No disease T338I D614G L320V V920L L90S M470V H199R S1251N I203M G550R P111A I148T Q1291H R560K L1388Q L183I R170H I1027T S549R D443Y P499A L1414S T908N R668C S549N A455E E1401K Q151K G27E I1234L Y563N R347P C866R S1118C P1290S R75Q A559T V520F P841R M469V E1401G P67L G85E S50Y E1409K R933G G458V G178R Y1032C R248T I980K G85V V392G L973P L137H T351S R334W I444S V938G R792G R560T R555G L1339F D1305E P574H V1240G T1053I D58G G551D L1335P I918M F994C S945L L558S F1337V R810G D1152H G1247R P574S R766M D579G W1098R H949R F200I R352Q L1077P K1351E M244K L206W M1101K D1154G L375F N1303K R1066C E528D D110Y R347H R1070Q A800G P1021S S549K A1364V V392A damaging` (is supposed to affect protein function or structure) and 'probably damaging` (high confidence of affecting protein function or structure). Login to comment
57 PI prevalence and in silico prediction scores for 13 most frequent missense mutations identified in Canadian CF patients Mutation Total PI Total (PI + PS) PI prevalence Class PANTHER scorea POLYPHENa SIFTa p.R334W 1 9 0.11 CF-PS -7.4419 Possibly damaging 0.01 p.P67L 2 14 0.14 CF-PS -4.1736 Probably damaging 0 p.R347P 2 12 0.17 CF-PS -7.5259 Possibly damaging 0.01 p.R347H 1 5 0.20 CF-PS -6.8327 Possibly damaging 0 p.A455E 8 39 0.21 CF-PS -8.8641 Probably damaging 0 p.L206W 4 19 0.21 CF-PS -8.5817 Possibly damaging 0 p.P574H 4 7 0.57 CF-PI/PSb -8.1252 Probably damaging 0 p.G85E 15 24 0.63 CF-PI/PSb -7.3194 Possibly damaging 0 p.M1101K 22 33 0.67 CF-PI/PSb -5.8849 Probably damaging 0.01 p.R1066C 7 8 0.88 CF-PI -7.7424 Probably damaging 0 p.G551D 56 59 0.95 CF-PI -9.5654 Probably damaging 0 p.N1303K 47 49 0.96 CF-PI -9.7687 Probably damaging 0 p.V520F 7 7 1.00 CF-PI -7.1652 Benign 0 aPANTHER scores range from zero to negative values (maximum -12). Login to comment

[hide] Acidification-dependent activation of CD1d-restric... Immunology. 2010 Jun;130(2):288-95. Epub 2010 Jan 19. Rzemieniak SE, Hirschfeld AF, Victor RE, Chilvers MA, Zheng D, van den Elzen P, Turvey SE
Acidification-dependent activation of CD1d-restricted natural killer T cells is intact in cystic fibrosis.
Immunology. 2010 Jun;130(2):288-95. Epub 2010 Jan 19., [PMID:20102408]

Abstract [show]
CD1d-restricted natural killer T (NKT) cells are emerging as critical regulators of the immune response to infectious agents, including Pseudomonas aeruginosa; and therapies to augment NKT-cell activation may represent a novel approach to treat chronic, antibiotic-resistant bacterial infections. We examined the capacity of dendritic cells (DCs) from people with cystic fibrosis (CF) to activate NKT cells. Our study was motivated by three lines of evidence: (i) NKT cells play a critical role in clearing P. aeruginosa infection; (ii) activation of NKT cells requires acidification-dependent processing of glycolipid antigens within the endolysosomal compartment; and (iii) endolysosomal acidification may be reduced in CF. We demonstrated that NKT-cell activation was dependent upon intact organelle acidification as inhibitors of the vacuolar (H(+))-ATPases prevented DCs from activating NKT cells with two glycolipid antigens, alpha-galactosylceramide and galactose-galactosylceramide. In contrast, cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel dysfunction had no significant biological impact on the capacity of DCs to activate NKT cells. Dendritic cells from subjects with CF and DCs treated with the thiazolidinone CFTR(inh)-172 inhibitor showed no reduction in their ability to activate NKT cells. Based on these data, we find no evidence for an inherent defect in glycolipid antigen presentation to NKT cells in CF subjects.

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21 The CF subjects enrolled in this study had the following CFTR mutations: DF508/DF508 = 4, DF508/621 + 1G> T = 1, DF508/ G85E = 1, DF508/unknown = 1, unknown/unknown = 1. Login to comment

[hide] Incidence, prevalence, etiology, and prognosis of ... Dig Dis Sci. 2010 Oct;55(10):2988-98. Epub 2010 Jan 28. Joergensen M, Brusgaard K, Cruger DG, Gerdes AM, de Muckadell OB
Incidence, prevalence, etiology, and prognosis of first-time chronic pancreatitis in young patients: a nationwide cohort study.
Dig Dis Sci. 2010 Oct;55(10):2988-98. Epub 2010 Jan 28., [PMID:20108119]

Abstract [show]
BACKGROUND/AIMS: Publications on etiology of chronic pancreatitis (CP) are infrequent. Etiologies today encompass genetic disorders. We wanted to describe etiologies of today and identify patients with genetic disorders like hereditary pancreatitis (HP), mutations in Serine Protease Inhibitor Kazal type1 (SPINK1), and the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) among patients formerly considered to have idiopathic CP. METHODS: Data on patients diagnosed with first-time CP < 30 years of age in Denmark identified in the Danish National Registry of Patients were retrieved. Patients previously considered to have idiopathic pancreatitis were offered genetic counseling and evaluation for HP, SPINK1, and CFTR mutations. RESULTS: In the period 1980-2004, 580 patients < 30 years of age presented with CP, the standardized prevalence ratio of CP increased from 11.7 per 100,000 person years in 1980-1984 to 17.0 per 100,000 in 2000-2004 (p < 0.001). The odds ratio (OR) having gallstone-related CP increased in the latter time period, especially in women, that of alcohol-induced CP decreased over time. OR having idiopathic CP increased in the latter period; 50% of patients with idiopathic pancreatitis accepted genetic reevaluation; 28 patients had a genetic mutation that totally or partly could explain their pancreatitis, nine of these had two, and 11 patients had HP. CONCLUSION: The prevalence of CP, especially in women, increased over time. Genetic causes that partly or totally could explain the CP were found in 54.90% (95% CI (40.45-68.62)) of those with idiopathic CP, as a minimum estimation 1.9% (95% CI (1.00-3.47)) of the total cohort had HP.

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49 1G [ T, F508del, S549 N, I507del, S549R, 2184delA, G551D, G85E, N1303 K, R560T, R117H, R347H, R347P, R334 W, 2789 ? Login to comment

[hide] Impact of gene patents and licensing practices on ... Genet Med. 2010 Apr;12(4 Suppl):S194-211. Chandrasekharan S, Heaney C, James T, Conover C, Cook-Deegan R
Impact of gene patents and licensing practices on access to genetic testing for cystic fibrosis.
Genet Med. 2010 Apr;12(4 Suppl):S194-211., [PMID:20393308]

Abstract [show]
Cystic fibrosis is one of the most commonly tested autosomal recessive disorders in the United States. Clinical cystic fibrosis is associated with mutations in the CFTR gene, of which the most common mutation among Caucasians, DeltaF508, was identified in 1989. The University of Michigan, Johns Hopkins University, and the Hospital for Sick Children, where much of the initial research occurred, hold key patents on cystic fibrosis genetic sequences, mutations, and methods for detecting them. Several patents, including the one that covers detection of the DeltaF508 mutation, are jointly held by the University of Michigan and the Hospital for Sick Children in Toronto, with Michigan administering patent licensing in the United States. The University of Michigan broadly licenses the DeltaF508 patent for genetic testing with >60 providers of genetic testing to date. Genetic testing is now used in newborn screening, diagnosis, and for carrier screening. Interviews with key researchers and intellectual property managers, a survey of laboratories' prices for cystic fibrosis genetic testing, a review of literature on cystic fibrosis tests' cost-effectiveness, and a review of the developing market for cystic fibrosis testing provide no evidence that patents have significantly hindered access to genetic tests for cystic fibrosis or prevented financially cost-effective screening. Current licensing practices for cystic fibrosis genetic testing seem to facilitate both academic research and commercial testing. More than 1000 different CFTR mutations have been identified, and research continues to determine their clinical significance. Patents have been nonexclusively licensed for diagnostic use and have been variably licensed for gene transfer and other therapeutic applications. The Cystic Fibrosis Foundation has been engaged in licensing decisions, making cystic fibrosis a model of collaborative and cooperative patenting and licensing practice.

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182 The ACMG specifically indicated that "Asian-Americans and Native Americans without significant Caucasian admixture should be informed of Table 1 Recommended core mutation panel for cystic fibrosis carrier screening in the general population Standard mutation panel R560T, ⌬F508a , R553Xb , R1162X, ⌬I507, 2184delA, G542X, G551Db , W1282X, N1303K, 621ϩ1G⌬T, R117H, 1717-1G⌬A, A455E, G85E, R334W, R347P, 711ϩ1G⌬T, 1898ϩ1G⌬A, 3849ϩ10kbC⌬T, 2789ϩ5G⌬A, 3659delC, and 3120ϩ1G⌬A Additional testable mutations I506Vc , I507Vc , F508Cc , and 5T/ 7T/9Td a University of Michigan/HSC Patent No. US 5,776,677. b Johns Hopkins University, Patent No. US 5,407,796. c Benign variants. Login to comment

[hide] Carrier screening for cystic fibrosis. Obstet Gynecol Clin North Am. 2010 Mar;37(1):47-59, Table of Contents. Dungan JS
Carrier screening for cystic fibrosis.
Obstet Gynecol Clin North Am. 2010 Mar;37(1):47-59, Table of Contents., [PMID:20494257]

Abstract [show]
Cystic fibrosis is the first genetic disorder for which universal screening of preconceptional or prenatal patients became a component of standard prenatal care. The molecular genetics and mutation profile of the CFTR gene are complex, with a wide range of phenotypic consequences. Carrier screening can facilitate risk assessment for prospective parents to have an affected offspring, although there remains a small residual risk for carrying a mutation even with a negative screening result. There are ethnic differences with respect to disease incidence and effectiveness of carrier testing, which may complicate counseling.

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102 However, in instances of a positive family history of affected individuals, but with no known mutation, further Table 2 Mutation panel recommended by ACOG and ACMG (listed in order of decreasing frequency in non-Hispanic Caucasian population) F508 del delI507 R347P R1162X G542X R553X 71111G>T 2184delA G551D R117H R560T 189811G>A 62111G>T 3849110kbC>T 3569delC R334W W1282X 1717À1G>T A455E 312011G>T N1303K 278915G>A G85E Data from Watson MS, Cutting GR, Desnick RJ, et al. Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel. Login to comment

[hide] Cystic fibrosis newborn screening: using experienc... J Inherit Metab Dis. 2010 Oct;33(Suppl 2):S255-61. Epub 2010 Jun 3. Hale JE, Parad RB, Dorkin HL, Gerstle R, Lapey A, O'Sullivan BP, Spencer T, Yee W, Comeau AM
Cystic fibrosis newborn screening: using experience to optimize the screening algorithm.
J Inherit Metab Dis. 2010 Oct;33(Suppl 2):S255-61. Epub 2010 Jun 3., [PMID:20521170]

Abstract [show]
Newborn screening (NBS) for cystic fibrosis (CF) offers the opportunity for early diagnosis and improved outcomes in patients with CF and has been universally available in the state of Massachusetts since 1999 using an immunoreactive trypsinogen (IRT)-DNA algorithm. Ideally, CF NBS is incorporated as part of an integrated NBS system that allows for comprehensive and coordinated education, laboratory screening, clinical follow-up, and evaluation so that evidence-based data can be used to maximize quality improvements and optimize the screening algorithm. The New England Newborn Screening Program (NENSP) retrospectively analyzed Massachusetts's CF newborn screening data that yielded decisions to eliminate a screen-positive category, maintain the IRT cutoff value that prompts the second tier DNA testing, and communicate CF relative risk to primary care providers (PCPs) based on categorization of positive CF NBS results.

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77 Discussion In addition to providing the laboratory services necessary for implementation of CF NBS, effective newborn screen- Table 4 Summary of positive screens identified by Massachusetts failsafe cystic fibrosis (CF) newborn screening (NBS) protocol Top 0.2% [IRT] Top 0.1% [IRT] Total Monitored by CF center 3 1 4 Borderline sweat testa 1 1 2 Negative sweat test 364 136 485 Not sweat testedb 91 62 153 IRT Immunoreactive trypsinogen a Infants do not have CF and are not monitored by a CF center b The 153 infants who did not have a documented sweat test either expired prior to a sweat test (36%), had a QNS sweat test and did not return for repeat sweat test (8%), had parents who refused sweat test (2%), or are lost to follow up (54%) Table 5 Infants followed at a cystic fibrosis (CF) center identified by Massachusetts failsafe CF newobrn screening (NBS) protocol Infant [IRT] (ng/ml) Mutations on NBS panel (n) Sweat [Cl- ] MEq/L Extended genotype results Comments A 141 16 41, 83 G85E/R117C Would be identified by current mutation panel B 503 16 103 Negative for 86 CFTR mutations C 274 16 92 Negative for 86 CFTR mutations D 159 39 38 P67L trans to 5t Does not meet 2008 CFF consensus guidelines for CF but is positive for CRMS CFF Cystic Fibrosis Foundation, CRMS cystic fibrosis transmembrane conductance regulator-related metabolic syndrome (Borowitz 2009) ing programs should collect and monitor outcomes for quality assurance purposes. Login to comment

[hide] Identification of the second CFTR mutation in pati... Asian J Androl. 2010 Nov;12(6):819-26. Epub 2010 Jul 26. Giuliani R, Antonucci I, Torrente I, Grammatico P, Palka G, Stuppia L
Identification of the second CFTR mutation in patients with congenital bilateral absence of vas deferens undergoing ART protocols.
Asian J Androl. 2010 Nov;12(6):819-26. Epub 2010 Jul 26., [PMID:20657600]

Abstract [show]
Congenital bilateral absence of vas deferens (CBAVD) is a manifestation of the mildest form of cystic fibrosis (CF) and is characterized by obstructive azoospermia in otherwise healthy patients. Owing to the availability of assisted reproductive technology, CBAVD patients can father children. These fathers are at risk of transmitting a mutated allele of the CF transmembrane conductance regulator (CFTR) gene, responsible for CF, to their offspring. The identification of mutations in both CFTR alleles in CBAVD patients is a crucial requirement for calculating the risk of producing a child with full-blown CF if the female partner is a healthy CF carrier. However, in the majority of CBAVD patients, conventional mutation screening is not able to detect mutations in both CFTR alleles, and this difficulty hampers the execution of correct genetic counselling. To obtain information about the most represented CFTR mutations in CBAVD patients, we analysed 23 CBAVD patients, 15 of whom had a single CFTR mutation after screening for 36 mutations and the 5T allele. The search for the second CFTR mutation in these cases was performed by using a triplex approach: (i) first, a reverse dot-blot analysis was performed to detect mutations with regional impact; (ii) next, multiple ligation-dependent probe amplification assays were conducted to search for large rearrangements; and (iii) finally, denaturing high-performance liquid chromatography was used to search for point mutations in the entire coding region. Using these approaches, the second CFTR mutation was detected in six patients, which increased the final detection rate to 60.8%.

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58 INNO-LiPA CFTR19 INNO-LiPA CFTR17 INNO-LiPA CFTR Italian regional [delta]F508 621+1G>T 1259insA G542X 3849+10kbC>T 4016insT N1303K 2183AA>G 4382delA W1282X 394delTT 852del22 G551D 2789+5G> A R1162X D579G 1717-1G>A 3659delC G1244E R553X R117H G1349D CFTRdele2,3 (21 kb) R334W I502T [delta]I507 R347P L1065P 711+1G>T G85E R1158X 3272-26A>G 3905insT 1078delT T338I R560T A455E S549R(A>C) 1898+1G>A S1251N 2143delA 711+5G>A 991del5 I148T E60X D1152H 3199del6 3120+1G>A 2184delA 1898+3A>G, R1070Q Q552X Poli-T tract variations R1066H R347H 621+3A>G R334Q E217G Abbreviation: CFTR, cystic fibrosis transmembrane conductance regulator. Login to comment

[hide] A new complex allele of the CFTR gene partially ex... Genet Med. 2010 Sep;12(9):548-55. Lucarelli M, Narzi L, Pierandrei S, Bruno SM, Stamato A, d'Avanzo M, Strom R, Quattrucci S
A new complex allele of the CFTR gene partially explains the variable phenotype of the L997F mutation.
Genet Med. 2010 Sep;12(9):548-55., [PMID:20706124]

Abstract [show]
PURPOSE: To evaluate the role of complex alleles, with two or more mutations in cis position, of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in the definition of the genotype-phenotype relationship in cystic fibrosis (CF), and to evaluate the functional significance of the highly controversial L997F CFTR mutation. METHODS: We evaluated the diagnosis of CF or CFTR-related disorders in 12 unrelated subjects with highly variable phenotypes. According to a first CFTR mutational analysis, subjects appeared to be compound heterozygotes for a classic mutation and the L997F mutation. A further CFTR mutational analysis was conducted by means of a protocol of extended sequencing, particularly suited to the detection of complex alleles. RESULTS: We detected a new [R117L; L997F] CFTR complex allele in the four subjects with the highest sweat test values and CF. The eight subjects without the complex allele showed the most varied biochemical and clinical outcome and were diagnosed as having mild CF, CFTR-related disorders, or even no disease. CONCLUSIONS: The new complex allele partially explains the variable phenotype in CF subjects with the L997F mutation. CFTR complex alleles are likely to have a role in the definition of the genotype-phenotype relationship in CF. Whenever apparently identical CFTR-mutated genotypes are found in subjects with divergent phenotypes, an extensive mutational search is mandatory.

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58 G85E (c.254GϾA), S549R(AϾC) (c.1645AϾC), and L320V (c.958TϾ G) are missense mutations located in the first membrane spanning domain-first transmembrane segment, in the first nucleotide-binding domain, and in the first membrane spanning domain- fifth transmembrane segment, respectively. Login to comment
59 F508del, W1282X, G85E,35 and S549R(AϾC)36 are considered classic CFTR mutations that cause a severe CF form when in homozygosity or compound heterozygosity with another classic mutation. Login to comment
77 If one considers only the first two mutations found, the six subjects with apparently the same F508del/L997F genotype (Table 1, Subjects 1-6) showed highly varying sweat test values, ranging from 22 to 90 mEq/L, as did the two subjects (Subjects 7 and 8) with apparently the same G85E/L997F genotype, who had sweat test values of 102 mEq/L and 21 mEq/L, respectively. Login to comment
90 By contrast, the agreement between the presence of the complex allele and clinical outcome was not as good when we compared the two subjects with the same G85E mutation on one allele. Login to comment

[hide] Type of CFTR mutation determines risk of pancreati... Gastroenterology. 2011 Jan;140(1):153-61. Epub 2010 Nov 9. Ooi CY, Dorfman R, Cipolli M, Gonska T, Castellani C, Keenan K, Freedman SD, Zielenski J, Berthiaume Y, Corey M, Schibli S, Tullis E, Durie PR
Type of CFTR mutation determines risk of pancreatitis in patients with cystic fibrosis.
Gastroenterology. 2011 Jan;140(1):153-61. Epub 2010 Nov 9., [PMID:20923678]

Abstract [show]
BACKGROUND & AIMS: Different mutations in the cystic fibrosis gene (CFTR) are associated with different functional status of the exocrine pancreas. We investigated whether CFTR genotypes determine the risk of pancreatitis in patients with cystic fibrosis (CF). METHODS: Patients with pancreatic-sufficient CF were identified from 2 CF population-based databases (N = 277; 62 with pancreatitis and 215 without pancreatitis); patients' genotypes and clinical characteristics were analyzed. The loss of pancreatic function associated with each CFTR genotype was determined based on the pancreatic insufficiency prevalence (PIP) score. RESULTS: Patients with pancreatitis were more likely to have genotypes associated with mild (70%) than moderate-severe (30%) PIP scores (P = .004). The cumulative proportion of patients who developed pancreatitis through to the age of 50 years was significantly greater for genotypes associated with mild (50%) than moderate-severe (27%) PIP scores (P = .006). The genotype associated with mild PIP scores had a hazard ratio of 2.4 for pancreatitis (95% confidence interval, 1.3-4.5; P = .006). Patients with pancreatitis were diagnosed with CF at an older median age than those without pancreatitis (14.9 years [interquartile range, 9.5-27.7] vs 9.3 years [interquartile range, 1.5-21.4]; P = .003) and had lower mean levels of sweat chloride than patients without pancreatitis (74.5 +/- 26.2 mmol/L vs 82.8 +/- 25.2 mmol/L; P = .03). CONCLUSIONS: Specific CFTR genotypes are significantly associated with pancreatitis. Patients with genotypes associated with mild phenotypic effects have a greater risk of developing pancreatitis than patients with genotypes associated with moderate-severe phenotypes. This observation provides further insight into the complex pathogenesis of pancreatitis.

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55 PIP Scores for Common, Well-Defined CFTR Mutations Mutation Canadian Consortium for CF Genetic Studies Verona CF Centre Mutation classTotal PI Total PIϩPS PIP score Total PI Total PIϩPS PIP score 621ϩ1GϾT 96 96 1.00 4 4 1.00 I-III 711ϩ1GϾT 36 36 1.00 1 1 1.00 I-III R553X 24 24 1.00 9 9 1.00 I-III I507del 11 11 1.00 12 12 1.00 I-III G542X 74 75 0.99 22 22 1.00 I-III F508del 1276 1324 0.96 181 188 0.96 I-III 1717-1GϾA 20 21 0.95 23 24 0.96 I-III W1282X 19 20 0.95 2 2 1.00 I-III N1303K 45 48 0.94 30 31 0.97 I-III R1162X 12 13 0.92 21 22 0.95 I-III G551D 59 67 0.88 0 0 - I-III G85E 16 22 0.73 4 5 0.80 I-III A455E 18 37 0.49 0 0 - IV-V 2789ϩ5GϾA 6 16 0.38 3 11 0.27 IV-V R334W 1 10 0.10 0 0 - IV-V 3849ϩ10kbCϾT 2 22 0.09 0 1 0.00 IV-V R117H 1 25 0.04 0 0 - IV-V NOTE. Login to comment

[hide] Mutations that permit residual CFTR function delay... Respir Res. 2010 Oct 8;11:140. Green DM, McDougal KE, Blackman SM, Sosnay PR, Henderson LB, Naughton KM, Collaco JM, Cutting GR
Mutations that permit residual CFTR function delay acquisition of multiple respiratory pathogens in CF patients.
Respir Res. 2010 Oct 8;11:140., [PMID:20932301]

Abstract [show]
BACKGROUND: Lung infection by various organisms is a characteristic feature of cystic fibrosis (CF). CFTR genotype effects acquisition of Pseudomonas aeruginosa (Pa), however the effect on acquisition of other infectious organisms that frequently precede Pa is relatively unknown. Understanding the role of CFTR in the acquisition of organisms first detected in patients may help guide symptomatic and molecular-based treatment for CF. METHODS: Lung infection, defined as a single positive respiratory tract culture, was assessed for 13 organisms in 1,381 individuals with CF. Subjects were divided by predicted CFTR function: 'Residual': carrying at least one partial function CFTR mutation (class IV or V) and 'Minimal' those who do not carry a partial function mutation. Kaplan-Meier estimates were created to assess CFTR effect on age of acquisition for each organism. Cox proportional hazard models were performed to control for possible cofactors. A separate Cox regression was used to determine whether defining infection with Pa, mucoid Pa or Aspergillus (Asp) using alternative criteria affected the results. The influence of severity of lung disease at the time of acquisition was evaluated using stratified Cox regression methods by lung disease categories. RESULTS: Subjects with 'Minimal' CFTR function had a higher hazard than patients with 'Residual' function for acquisition of 9 of 13 organisms studied (HR ranging from 1.7 to 3.78 based on the organism studied). Subjects with minimal CFTR function acquired infection at a younger age than those with residual function for 12 of 13 organisms (p-values ranging: < 0.001 to 0.017). Minimal CFTR function also associated with younger age of infection when 3 alternative definitions of infection with Pa, mucoid Pa or Asp were employed. Risk of infection is correlated with CFTR function for 8 of 9 organisms in patients with good lung function (>90%ile) but only 1 of 9 organisms in those with poorer lung function (<50%ile). CONCLUSIONS: Residual CFTR function correlates with later onset of respiratory tract infection by a wide spectrum of organisms frequently cultured from CF patients. The protective effect conferred by residual CFTR function is diminished in CF patients with more advanced lung disease.

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74 For Pa, the hazard ratio Table 1 Classification of CFTR alleles Category Mutation Specific mutations Class I Defective Protein Synthesis (nonsense, frameshift, aberrant splicing) 1078delT, 1154 insTC, 1525-2A > G, 1717-1G > A, 1898+1G > A, 2184delA, 2184 insA, 3007delG, 3120+1G > A, 3659delC, 3876delA, 3905insT, 394delTT, 4010del4, 4016insT, 4326delTC, 4374+1G > T, 441delA, 556delA, 621+1G > T, 621-1G > T, 711+1G > T, 875+1G > C, E1104X, E585X, E60X, E822X, G542X, G551D/R553X, Q493X, Q552X, Q814X, R1066C, R1162X, R553X, V520F, W1282X, Y1092X Class II Abnormal Processing and Trafficking A559T, D979A, ΔF508, ΔI507, G480C, G85E, N1303K, S549I, S549N, S549R Class III Defective Channel Regulation/Gating G1244E, G1349D, G551D, G551S, G85E, H199R, I1072T, I48T, L1077P, R560T, S1255P, S549 (R75Q) Class IV Decreased Channel Conductance A800G, D1152H, D1154G, D614G, delM1140, E822K, G314E, G576A, G622D, G85E, H620Q, I1139V, I1234V, L1335P, M1137V, P67L, R117C, R117P, R117H, R334W, R347H, R347P, R347P/ R347H, R792G, S1251N, V232D Class V Reduced Synthesis and/or Trafficking 2789+5G > A, 3120G > A, 3272-26A > G, 3849+10kbC > T, 5T variant, 621+3A > G, 711+3A > G, A445E, A455E, IVS8 poly T, P574H was increased 3 fold for those with 'Minimal` function when compared to those with 'Residual` function. Login to comment

[hide] Low abundance of sweat duct Cl- channel CFTR in bo... Am J Physiol Regul Integr Comp Physiol. 2011 Mar;300(3):R605-15. Epub 2011 Jan 12. Brown MB, Haack KK, Pollack BP, Millard-Stafford M, McCarty NA
Low abundance of sweat duct Cl- channel CFTR in both healthy and cystic fibrosis athletes with exceptionally salty sweat during exercise.
Am J Physiol Regul Integr Comp Physiol. 2011 Mar;300(3):R605-15. Epub 2011 Jan 12., [PMID:21228336]

Abstract [show]
To understand potential mechanisms explaining interindividual variability observed in human sweat sodium concentration ([Na(+)]), we investigated the relationship among [Na(+)] of thermoregulatory sweat, plasma membrane expression of Na(+) and Cl(-) transport proteins in biopsied human eccrine sweat ducts, and basal levels of vasopressin (AVP) and aldosterone. Lower ductal luminal membrane expression of the Cl(-) channel cystic fibrosis transmembrane conductance regulator (CFTR) was observed in immunofluorescent staining of sweat glands from healthy young adults identified as exceptionally "salty sweaters" (SS) (n = 6, P < 0.05) and from patients with cystic fibrosis (CF) (n = 6, P < 0.005) compared with ducts from healthy young adults with "typical" sweat [Na(+)] (control, n = 6). Genetic testing of healthy subjects did not reveal any heterozygotes ("carriers") for any of the 39 most common disease-causing CFTR mutations in the United States. SS had higher baseline plasma [AVP] compared with control (P = 0.029). Immunostaining to investigate a potential relationship between higher plasma [AVP] (and sweat [Na(+)]) and ductal membrane aquaporin-5 revealed for all groups a relatively sparse and location-dependent ductal expression of the water channel with localization primarily to the secretory coil. Availability of CFTR for NaCl transport across the ductal membrane appears related to the significant physiological variability observed in sweat salt concentration in apparently healthy humans. At present, a heritable link between healthy salty sweaters and the most prevalent disease-causing CFTR mutations cannot be established.

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114 Mutations tested in this panel were ⌬F508, R334W, S549N, 3659delC, ⌬I507, I347P, A559T, S1255X, 1898ϩ1GϾA, R347H, N1303K, 1898ϩ5GϾT, 3876delA, A455E, 394delTT, 2183GGϾA, 3905insT, 3120ϩ1GϾA, V520F, 2184delA, G85E, Y1092X, 711ϩ1GϾT, 2307insA, Y122X, S549R, M1101K, 1078delT, 2789ϩ5GϾA, G551D, G542X, 621ϩ1GϾT, R560T, W1282X, 1717-1 GϾA, 3849 ϩ 10KbCϾT, R553X, R117H, and R1162X. Login to comment
119 Mutations tested in this panel were ⌬F508, R334W, S549N, 3659delC, ⌬I507, I347P, A559T, S1255X, 1898ϩ1GϾA, R347H, N1303K, 1898ϩ5GϾT, 3876delA, A455E, 394delTT, 2183GGϾA, 3905insT, 3120ϩ1GϾA, V520F, 2184delA, G85E, Y1092X, 711ϩ1GϾT, 2307insA, Y122X, S549R, M1101K, 1078delT, 2789ϩ5GϾA, G551D, G542X, 621ϩ1GϾT, R560T, W1282X, 1717-1 GϾA, 3849 ϩ 10KbCϾT, R553X, R117H, and R1162X. Login to comment

[hide] An overview of international literature from cysti... J Cyst Fibros. 2011 Mar;10(2):71-85. Epub 2011 Jan 22. Salvatore D, Buzzetti R, Baldo E, Forneris MP, Lucidi V, Manunza D, Marinelli I, Messore B, Neri AS, Raia V, Furnari ML, Mastella G
An overview of international literature from cystic fibrosis registries. Part 3. Disease incidence, genotype/phenotype correlation, microbiology, pregnancy, clinical complications, lung transplantation, and miscellanea.
J Cyst Fibros. 2011 Mar;10(2):71-85. Epub 2011 Jan 22., [PMID:21257352]

Abstract [show]
This is the third article related to a review of the literature based on data from national cystic fibrosis (CF) patient registries up to June 2008 and covering a total of 115 published studies. It focuses on several topics: CF incidence, genotype/phenotype correlation, microbiology, pregnancy/paternity, clinical complications, lung transplantation, and others. Seventy seven papers meeting the inclusion criteria were found to be related to the topics listed above. Another seven studies, already evaluated in previous papers of this series, were recalled for specific topics. Incidence is described by several studies, results being quite different from one country to another and quite inhomogeneous among regions within the same country. Studies on genetics address the genotype/phenotype correlation and look for a predictive value of CFTR mutations in terms of clinical outcome, with controversial results. Papers on microbiology describe the clinical relevance of different pathogens and their role in the progress of CF lung disease. A few articles give information on the features of CF women undergoing a pregnancy and try to identify the ones associated with a better outcome. Studies on clinical complications discuss prevalence and the role of haemoptysis, pneumothorax, CF related diabetes, ABPA and cancer. Papers on lung transplantation focus on models able to improve the selection criteria for transplantation candidates and the factors linked to post transplantation survival. Finally, several studies deal with a number of interesting topics related to CF epidemiology: clinical trial methodology, quality of care comparison among countries and centers, relationship between diagnosis and age/gender, and evaluation of pharmacological therapy. On the whole, CF Registries have already contributed to important advances in the knowledge of the natural history of CF, establishing the foundations for future improvement in CF research and care.

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1219 Valle et al. [6] reported an estimated CF incidence of 1:11,252 in the population of Ecuador and one of the highest incidences of G85E in the world (8.9%). Login to comment

[hide] Distribution of CFTR mutations in Eastern Hungaria... J Cyst Fibros. 2011 May;10(3):217-20. doi: 10.1016/j.jcf.2010.12.009. Epub 2011 Feb 4. Ivady G, Madar L, Nagy B, Gonczi F, Ajzner E, Dzsudzsak E, Dvorakova L, Gombos E, Kappelmayer J, Macek M Jr, Balogh I
Distribution of CFTR mutations in Eastern Hungarians: relevance to genetic testing and to the introduction of newborn screening for cystic fibrosis.
J Cyst Fibros. 2011 May;10(3):217-20. doi: 10.1016/j.jcf.2010.12.009. Epub 2011 Feb 4., [PMID:21296036]

Abstract [show]
BACKGROUND: The aim of this study was characterization of an updated distribution of CFTR mutations in a representative cohort of 40 CF patients with the classical form of the disease drawn from Eastern Hungary. Due to the homogeneity of the Hungarian population our data are generally applicable to other regions of the country, including the sizeable diaspora. METHODS: We utilized the recommended "cascade" CFTR mutation screening approach, initially using a commercial assay, followed by examination of the common "Slavic" deletion CFTRdele2,3(21kb). Subsequently, the entire CFTR coding region of the CFTR gene was sequenced in patients with yet unidentified mutations. RESULTS: The Elucigene CF29(Tm) v2 assay detected 81.25% of all CF causing mutations. An addition of the CFTRdele2,3(21kb) increased the mutation detection rate to 86.25%. DNA sequencing enabled us to identify mutations on 79/80 CF alleles. Mutations [CFTRdele2,3(21kb), p.Gln685ThrfsX4 (2184insA) were found at an unusually high frequency, each comprising 5.00% of all CF alleles. CONCLUSION: We have identified common CF causing mutations in the Hungarian population with the most common mutations (p.Phe508del, p.Asn1303Lys, CFTRdele2,3(21kb), 2184insA, p.Gly542X, and p.Leu101X), comprising over 93.75% of all CF alleles. Obtained data are applicable to the improvement of DNA diagnostics in Hungary and beyond, and are the necessary prerequisite for the introduction of a nationwide "two tier" CF newborn screening program.

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34 3528delC), p.Phe316LeufsX12 (c.948delT), p.Ile507del (c.1519_1521delATC), p.Arg347Pro (c.1040 G NC), p.Arg553X (c.1657 C NT), p.Glu60X (c.178 GNT), c.2988+1 GNA, c.2657+5 GNA, c.1766+1 GNA, c.579+1 GNT, p.Gly85Glu (c.254 GNA), c.p.Lys684AsnfsX38 (c.2052delA), and p.Arg560Thr (c.1679 GNC). Login to comment

[hide] Optimal DNA tier for the IRT/DNA algorithm determi... J Cyst Fibros. 2011 Jul;10(4):278-81. Epub 2011 Mar 8. Baker MW, Groose M, Hoffman G, Rock M, Levy H, Farrell PM
Optimal DNA tier for the IRT/DNA algorithm determined by CFTR mutation results over 14 years of newborn screening.
J Cyst Fibros. 2011 Jul;10(4):278-81. Epub 2011 Mar 8., [PMID:21388895]

Abstract [show]
BACKGROUND: There has been great variation and uncertainty about how many and what CFTR mutations to include in cystic fibrosis (CF) newborn screening algorithms, and very little research on this topic using large populations of newborns. METHODS: We reviewed Wisconsin screening results for 1994-2008 to identify an ideal panel. RESULTS: Upon analyzing approximately 1 million screening results, we found it optimal to use a 23 CFTR mutation panel as a second tier when an immunoreactive trypsinogen (IRT)/DNA algorithm was applied for CF screening. This panel in association with a 96th percentile IRT cutoff gave a sensitivity of 97.3%, but restricting the DNA tier to F508del was associated with 90% (P<.0001). CONCLUSIONS: Although CFTR panel selection has been challenging, our data show that a 23 mutation method optimizes sensitivity and is advantageous. The IRT cutoff value, however, is actually more critical than DNA in determining CF newborn screening sensitivity.

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75 CFTR mutationa Proportion of allele Frequency of allele (%) Cumulative detection (%)b F508del 137/214 64.02 92.52 3849+10KbCNT 6/214 2.80 92.52c G542X 5/214 2.34 94.39 N1303K 4/214 1.87 98.13 R117H 4/214 1.87 99.07 R553X 3/214 1.40 99.07 1717-1GNA 2/214 0.93 99.07 G551D 1/214 0.47 100 R347P 1/214 0.47 100 A455E 1/214 0.47 100 W1282X 1/214 0.47 100 621+1GNT 1/214 0.47 100 a The other 11 mutations in ACMG 23 mutation panel are G85E, 711+1GNT, R334W, I507del, R560T, 1898+1GNA, 2184delA, 2789+5GNA, 3120+1GNA, R1162X and 3659delC. Login to comment

[hide] Preconceptional identification of cystic fibrosis ... J Cyst Fibros. 2011 May;10(3):207-11. doi: 10.1016/j.jcf.2011.02.006. Epub 2011 Mar 22. Coiana A, Faa' V, Carta D, Puddu R, Cao A, Rosatelli MC
Preconceptional identification of cystic fibrosis carriers in the Sardinian population: A pilot screening program.
J Cyst Fibros. 2011 May;10(3):207-11. doi: 10.1016/j.jcf.2011.02.006. Epub 2011 Mar 22., [PMID:21429822]

Abstract [show]
BACKGROUND: In Sardinia the mutational spectrum of CFTR gene is well defined. A mutation detection rate of 94% can be achieved by screening for 15 CFTR mutations with a frequency higher than 0.5%. The efficiency of this molecular test suggests that Sardinians may represent a suitable population for a preconceptional screening. METHODS: Five hundred couples of Sardinia descent were screened for 38 mutations using a semi-automated reverse-dot blot and PCR-gel electrophoresis assays. This mutation panel included the 15 most frequent CF alleles in Sardinia. RESULTS: We identified 38 CF carriers, revealing an overall frequency of 1/25 (4%). The most common CF allele was the p.Thr338Ile (T338I) (65%), followed by the p.Phe508del (F508del) (22.5%). We also identified one couple at risk and an asymptomatic female homozygote for the p.Thr338Ile allele. CONCLUSIONS: In spite of the low number of the couples tested, the results herein reported demonstrate the efficacy and efficiency of the preconceptional screening program and the high participation rate of the Sardinian population (99%).

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88 Mutation nomenclaturea Alleles (%) T338I (p.Thr338Ile) 26 (65.0) F508del (p.Phe508del) 9 (22.5) N1303K (p.Asn1303Lys) 1 (2.5) 2183AANG (c.2051_2052delAAinsG) 1 (2.5) 621+1GNT (c.489+1GNT) 1 (2.5) exon 2 del (c.54-5811_164+2187del8108ins182) 1 (2.5) R347P (p.Arg347Pro) 1 (2.5) The 3849+10kbCNT (c.3717+12191CNT), G85E (p.Gly85Glu), 2789+5GNA (c.2657+5GNA), W1282X (p.Trp1282X), G1244E (p.Gly1244Glu), 711+5GNA (c.579+5GNA), 711+1GNT (c.579+1GNA), 4016insT (p.Ser1297PhefsX5), G542X (p.Gly542X), 1717-1GNA (c.1585-1GNA), R553X (p.Arg553X), Q552X (p.Gln552X), G551D (p.Gly551Asp), S549R (ANC) (p.Ser549Arg), I507del (p.Ile507del), F508C (p.Phe508Cys), I502T (p.Ile502Thr), 1706del17 (p.Gln525LeufsX37), 1677delTA (p.Tyr515X), R117H (p.Arg117His), D1152H (p.Asp1152His), L1065P (p.Leu1065Pro), R1066H (p.Arg1066His), L1077P (p.Leu1077Pro), 4382delA (p.Glu1418ArgfsX14), R1162X (p.Arg1162X), R1158X (p.Arg1158X), 1259 insA (p.Gln378AlafsX4), 852del22 (p.Gly241GlufsX13), S912X (p.Ser912X), and 991del5bp (p.Asn287LysfsX19) mutations included in the CF panel were not detected in the population tested. Login to comment

[hide] Implementation of the first worldwide quality assu... Clin Chim Acta. 2011 Jul 15;412(15-16):1376-81. Epub 2011 Apr 14. Earley MC, Laxova A, Farrell PM, Driscoll-Dunn R, Cordovado S, Mogayzel PJ Jr, Konstan MW, Hannon WH
Implementation of the first worldwide quality assurance program for cystic fibrosis multiple mutation detection in population-based screening.
Clin Chim Acta. 2011 Jul 15;412(15-16):1376-81. Epub 2011 Apr 14., 2011-07-15 [PMID:21514289]

Abstract [show]
BACKGROUND: CDC's Newborn Screening Quality Assurance Program collaborated with several U.S. Cystic Fibrosis Care Centers to collect specimens for development of a molecular CFTR proficiency testing program using dried-blood spots for newborn screening laboratories. METHODS: Adult and adolescent patients or carriers donated whole blood that was aliquoted onto filter paper cards. Five blind-coded specimens were sent to participating newborn screening laboratories quarterly. Proficiency testing results were evaluated based on presumptive clinical assessment. Individual evaluations and summary reports were sent to each participating laboratory and technical consultations were offered if incorrect assessments were reported. RESULTS: The current CDC repository contains specimens with 39 different CFTR mutations. Up to 45 laboratories have participated in the program. Three years of data showed that correct assessments were reported 97.7% of the time overall when both mutations could be determined. Incorrect assessments that could have lead to a missed case occurred 0.9% of the time, and no information was reported 1.1% of the time due to sample failure. CONCLUSIONS: Results show that laboratories using molecular assays to detect CFTR mutations are performing satisfactorily. The programmatic results presented demonstrate the importance and complexity of providing proficiency testing for DNA-based assays.

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129 Allele Allele Allele Allele p.Gly85Glu G85E (0.26) p.Arg117His R117H (0.54) c.489+1 GNT 621+1 GNT (1.3) p.Phe508del F508del (66.31) p.Arg347Pro R347P (0.36) p.lle507del I507del (0.90) p.Gly551Asp G551D (1.93) c.2052delA 2184delA (0.15) c.1585-1 GNA 1717-1 GNA (0.44) p.Gly542X G542X (2.64) c.3528delC 3659delC (0.28) p.Asn1303Lys N1303K (1.27) p.Arg553X R553X (1.21) p.Arg560Thr R560T (0.30) p.Arg1162X R1162X (0.30) c.2657+5 GNA 2789+5 GNA (0.38) c.3717+12191 CNT 3849+10kbCNT (0.85) c.2988+1 GNA 3120+1 GNA (0.86) p.Trp1282X W1282X (2.20) p.Ala455Glu A455E (0.26) c.1766+1 GNA 1898+1 GNA (0.13) c.579+1 GNT 711+1 GNT (0.35) p.Arg334Trp R334W (0.37) c.54-5940 _273+10250del21kb CFTR dele2,3 p.Ser549Asn S549N (0.14) c.1584 GNA 1716 G→A c.2051_2052delAAinsG 2183AANG (0.1) c.3140-26ANG 3272-26ANG c.262_263delTT 394delTT p.Arg1066Cys R1066C (0.03) p.Arg1066His R1066H c.1022_1023insTC 1154insTC c.2989-1 GNA 3121-1 GNA c.(?_2989)_(3139_? Login to comment

[hide] Characterization of a novel isolated deletion of t... Clin Biochem. 2011 Jul;44(10-11):799-803. Epub 2011 Apr 22. Tomaiuolo AC, Sirleto P, Centrone C, Surace C, Alghisi F, Petrocchi S, Lombardo A, Rossi M, Torricelli F, Lucidi V, Angioni A
Characterization of a novel isolated deletion of the exon 3 within the CFTR gene: Relevance for phenotypic expression and genetic counseling.
Clin Biochem. 2011 Jul;44(10-11):799-803. Epub 2011 Apr 22., [PMID:21536020]

Abstract [show]
OBJECTIVES: To characterize a novel deletion of exon 3 of CFTR gene and to evaluate the implications in Cystic Fibrosis (CF) care and genetic counseling. DESIGN AND METHODS: We performed a wide mutational analysis of CFTR gene, using reverse dot blot, Multiplex Ligation-dependent Probe Amplification (MLPA) assay and Real Time Quantitative PCR, in a carrier male and two CF patients with the F508del mutation. RESULTS: We found a novel isolate 538bp deletion of exon 3, described as 328del538, giving rise to a nonsense codon 60bp at the 3' end of the new coding sequence or, alternatively, a novel splice site at the breakpoints. CONCLUSIONS: The 328del538 is a rare lesion with the characteristics of a complete, but moderate, phenotypic expression. Its finding underlines the importance of improving the detection of mutations using different methods.

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95 Up to 46 mutations have been described in exon 3, most of them are rare, excluding G85E that is relatively Fig. 2. Login to comment

[hide] Clinical outcomes in infants with cystic fibrosis ... Pediatr Pulmonol. 2011 Apr 29. doi: 10.1002/ppul.21475. Ren CL, Desai H, Platt M, Dixon M
Clinical outcomes in infants with cystic fibrosis transmembrane conductance regulator (CFTR) related metabolic syndrome.
Pediatr Pulmonol. 2011 Apr 29. doi: 10.1002/ppul.21475., 2011-04-29 [PMID:21538969]

Abstract [show]
An unavoidable outcome of cystic fibrosis newborn screening (CF NBS) programs is the detection of infants with an indeterminate diagnosis. The United States CF Foundation recently proposed the term cystic fibrosis transmembrane conductance regulator related metabolic syndrome (CRMS) to describe infants with elevated immunoreactive trypsinogen (IRT) on NBS who do not meet diagnostic criteria for CF. The objective of this study was to describe the clinical outcomes of infants with CRMS identified through an IRT/DNA algorithm. We reviewed the records of all infants with CRMS diagnosed at our CF Center from 2002 to 2010. We identified 12 infants, and compared them to 27 infants diagnosed with CF by NBS. Compared to CF patients, CRMS patients were more likely to be pancreatic sufficient as assessed by fecal elastase measurement (100% vs. 8%, P < 0.01). Their weight for age percentile was normal from birth. A positive oropharyngeal (OP) culture for Pseudomonas aeruginosa (Pa) was found in 25% of CRMS patients. One patient with the F508del/R117H/7T genotype was reassigned the diagnosis of CF after he had a positive OP culture for Pa, and his follow up sweat Cl at 1 year of life was 73 mmol/L. CF patients were more likely to receive oral antibiotics and be hospitalized for pulmonary symptoms. Our results indicate that CRMS patients can develop signs of CF disease, but have a milder clinical course than CF infants. Close initial monitoring of these patients is warranted. Pediatr. Pulmonol. (c) 2011 Wiley-Liss, Inc.

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60 Infants in both groups received treatment with inhaled tobramycin if they had a positive Pa OP culture, and treatment in both groups was associated with eradication of TABLE 1-CFTR Gene Mutation Panel Used by New York CF NBS Program F508del I50e7del G542X G551D W1282X N1303K R553X 621þ1G>T R117H 1717-1G>A A455E R560T R1162X G85E R334W R347P 711þ1G>T 1898þ1G>A 2184delA 1078delT 3849þ10kbC>T 2789þ5G>A 3659delC I148T 3120þ1G>A 3876delA V520F S549R S549N 3849þ4 A-G 3905insT R347H Reflex testing for 5T polymorphism is performed if R117H is detected. Login to comment
61 Infants in both groups received treatment with inhaled tobramycin if they had a positive Pa OP culture, and treatment in both groups was associated with eradication of TABLE 1- CFTR Gene Mutation Panel Used by New York CF NBS Program F508del I50e7del G542X G551D W1282X N1303K R553X 621þ1G>T R117H 1717-1G>A A455E R560T R1162X G85E R334W R347P 711þ1G>T 1898þ1G>A 2184delA 1078delT 3849þ10kbC>T 2789þ5G>A 3659delC I148T 3120þ1G>A 3876delA V520F S549R S549N 3849þ4 A-G 3905insT R347H Reflex testing for 5T polymorphism is performed if R117H is detected. Login to comment

[hide] Ubiquitin-mediated proteasomal degradation of ABC ... J Pharm Sci. 2011 Sep;100(9):3602-19. doi: 10.1002/jps.22615. Epub 2011 May 12. Nakagawa H, Toyoda Y, Wakabayashi-Nakao K, Tamaki H, Osumi M, Ishikawa T
Ubiquitin-mediated proteasomal degradation of ABC transporters: a new aspect of genetic polymorphisms and clinical impacts.
J Pharm Sci. 2011 Sep;100(9):3602-19. doi: 10.1002/jps.22615. Epub 2011 May 12., [PMID:21567408]

Abstract [show]
The interindividual variation in the rate of drug metabolism and disposition has been known for many years. Pharmacogenomics dealing with heredity and response to drugs is a part of science that attempts to explain variability of drug responses and to search for the genetic basis of such variations or differences. Genetic polymorphisms of drug metabolizing enzymes and drug transporters have been found to play a significant role in the patients' responses to medication. Accumulating evidence demonstrates that certain nonsynonymous polymorphisms have great impacts on the protein stability and degradation, as well as the function of drug metabolizing enzymes and transporters. The aim of this review article is to address a new aspect of protein quality control in the endoplasmic reticulum and to present examples regarding the impact of nonsynonymous single-nucleotide polymorphisms on the protein stability of thiopurine S-methyltransferase as well as ATP-binding cassette (ABC) transporters including ABCC4, cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), ABCC11, and ABCG2. Furthermore, we will discuss the molecular mechanisms underlying posttranslational modifications (intramolecular and intermolecular disulfide bond formation and N-linked glycosylation) and ubiquitin-mediated proteasomal degradation of ABCG2, one of the major drug transporter proteins in humans.

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155 Effect of Mutations and Nonsynonymous SNPs on Protein Trafficking, Maturation, or ERAD of ABC Transporters Protein AA Mutation/SNP Effect on Protein Reference ABCA1 W590S Mutation Functional defect 115 R587W Mutation Impaired glycol processing 115 Q597R Mutation Impaired glycol processing, ERAD 115,116 Y1532C Mutation Altered protein trafficking 117 R1925Q Mutation Altered protein trafficking 118 ABCA3 R43L Mutation Altered protein trafficking 119 L101P Mutation Altered protein trafficking 119 R280C Mutation Altered protein trafficking 119 ABCA4 L541P Mutation Mislocalization 120 R602W Mutation Mislocalization 120 A1038V Mutation Mislocalization 120 C1490Y Mutation Mislocalization 120 ABCB1a G268V Mutation ERAD 121 G341C Mutation ERAD 121 I1196S Mutation Reduced glycosylation 122 ABCB4 I541F Mutation Accumulation in ER 123 ABCB11a E135K Mutation Reduced level of mature protein 124 L198P Mutation Reduced level of mature protein 124 E297G Mutation Reduced level of mature protein 124 L413W Mutation Reduced level of mature protein 124 R432T Mutation Reduced level of mature protein 124 D482G Mutation Immature protein in ER 124,125 N490D Mutation Reduced level of mature protein 124 A570T Mutation Reduced level of mature protein 124 T655I Mutation Reduced level of mature protein 124 Y818F SNP Moderate reduction of protein 124 G982R Mutation Retention in ER 125 R1153C Mutation ERAD 125 R1286Q Mutation Retention in ER 125 ABCC2a R768W Mutation Impaired protein trafficking 126 I1173F Mutation Impaired protein maturation 127 R1392 Mutation Impaired protein maturation 128 M1393 Mutation Impaired protein maturation 129 ABCC4a E757K SNP Altered protein trafficking 23 ABCC7 F508 Mutation Misfolding, ERAD 36-39,130 G85E Mutation Impaired protein maturation 130-132 G91R Mutation Impaired protein maturation 130-132 N1303K Mutation Impaired protein maturation 130-132 ABCC8 WT Wild type Ubiquitin-proteasome degradation 133 A116P Mutation Ubiquitin-proteasome degradation 133 V187D Mutation Ubiquitin-proteasome degradation 133 F1388 Mutation Impaired protein trafficking 134 L1544P Mutation Impaired protein trafficking 135,136 ABCC11a G180R SNP Ubiquitin-proteasome degradation 50 27 Mutation Ubiquitin-proteasome degradation 50 ABCG2a V12M SNP Altered protein localization 96 Q141K SNP Ubiquitin-proteasome degradation 102 F208S SNP Ubiquitin-proteasome degradation 78,99 S441N SNP Ubiquitin-proteasome degradation 78,99 Mutations of ABCA1, ABCA3, ABCA4, ABCB4, ABCB11, ABCC2, ABCC7 (CFTR), and ABCC8 are associated with Tangier disease, fatal surfactant deficiency, Stargardt disease, progressive familial intrahepatic cholestasis type 3 (PFIC-3), progressive familial intrahepatic cholestasis type 2 (PFIC-2), Dubin-Johnson syndrome, cystic fibrosis, and familial hyperinsulinism, respectively. Login to comment

[hide] Defective CFTR expression and function are detecta... PLoS One. 2011;6(7):e22212. Epub 2011 Jul 21. Sorio C, Buffelli M, Angiari C, Ettorre M, Johansson J, Vezzalini M, Viviani L, Ricciardi M, Verze G, Assael BM, Melotti P
Defective CFTR expression and function are detectable in blood monocytes: development of a new blood test for cystic fibrosis.
PLoS One. 2011;6(7):e22212. Epub 2011 Jul 21., [PMID:21811577]

Abstract [show]
BACKGROUND: Evaluation of cystic fibrosis transmembrane conductance regulator (CFTR) functional activity to assess new therapies and define diagnosis of cystic fibrosis (CF) is cumbersome. It is known that leukocytes express detectable levels of CFTR but the molecule has not been characterized in these cells. In this study we aim at setting up and validating a blood test to evaluate CFTR expression and function in leukocytes. DESCRIPTION: Western blot, PCR, immunofluorescence and cell membrane depolarization analysis by single-cell fluorescence imaging, using the potential-sensitive DiSBAC(2)(3) probe were utilized. Expression of PKA phosphorylated, cell membrane-localized CFTR was detected in non-CF monocytes, being undetectable or present in truncated form in monocytes derived from CF patients presenting with nonsense mutations. CFTR agonist administration induced membrane depolarization in monocytes isolated from non-CF donors (31 subjects) and, to a lesser extent, obligate CFTR heterozygous carriers (HTZ: 15 subjects), but it failed in monocytes from CF patients (44 subjects). We propose an index, which values in CF patients are significantly (p<0.001) lower than in the other two groups. Nasal Potential Difference, measured in selected subjects had concordant results with monocytes assay (Kappa statistic 0.93, 95%CI: 0.80-1.00). RESULTS AND SIGNIFICANCE: CFTR is detectable and is functional in human monocytes. We also showed that CFTR-associated activity can be evaluated in 5 ml of peripheral blood and devise an index potentially applicable for diagnostic purposes and both basic and translational research: from drug development to evaluation of functional outcomes in clinical trials.

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202 Case Gender Age at diagnosis (years) CFTR genotype* Age (years) Sweat Cl- mEq/L** FEV1 % mean values 2009 Pa PI NPD results*** CF-index 1 F 0 3132delTG 1497delGG 34 129 75 yes yes nd 222,10 2 F 0 R1162X R1162X 43 144 52 yes yes nd 229,65 3 M 0 R1162X R1162X 10 102 59 no yes 1,02 210,18 4 M 0 R1162X R1162X 25 115 81 no yes 1,07 267,11 5 M 7 G542X 711+5 G.A 24 105 59 yes yes nd 25,84 6 M 1 CFTRdele1 G542X 36 107 22 yes yes nd 2113,92 7 M 0 G542X G542X 16 110 71 yes yes 0,97 280,20 8 F 1 Q552X CFTRdele17a-18 35 99 72 yes yes 2,08 2219,81 9 M 16 R1162X 3849+10 Kb C.T 42 74 43 yes no 1,02 271,47 10 M 0 R1162X R1162X 32 105 45 yes yes 1,43 2114,67 11 M 1 F508del F508del 16 86 71 no yes nd 260,04 12 F 0 F508del F508del 16 88 118 no yes nd 248,20 13 M 0 F508del F508del 33 118 51 yes yes nd 265,49 14 M 7 F508del F508del 37 89 37 yes yes nd 2359,82 15 F 0 F508del F508del 27 118 71 yes yes nd 267,26 16 F 8 1717-1 G.A F508del 38 140 74 yes yes nd 2136,80 17 F 0 R1158X F508del 32 95 60 yes yes 1,77 228,31 18 M 7 G542X F508del 39 110 46 yes yes nd 247,52 19 M 0 Q39X F508del 17 101 79 no yes 1,11 264,20 20 F 1 R1162X F508del 41 188 60 no yes 0,94 296,73 21 M 13 3849+10 Kb C.T F508del 24 76 78 yes no 4,67 26,33 22 M 0 W1282X 621+1G.T 33 119 77 yes yes 1,27 242,74 23 F 4 R553X 2789+5 G.A 31 92 44 yes no 7,4 260,94 24 F 11 F508del R553X 39 116 55 yes yes nd 2113,67 25 M 12 F508del 3849+10 Kb C.T 27 51 71 yes no 1,12 298,84 26 F 0 F508del G542X 19 109 109 yes yes nd 2173,24 27 F 0 F508del R1162X 32 94 86 yes yes 1,34 270,16 28 F 0 F508del W57X (TAG) 27 99 78 yes yes 1,21 269,33 29 M 0 F508del Q552X 24 94 41 yes yes 1,50 272,75 30 M 20 F508del 3849+10 Kb C.T 43 58 60 no no 1,13 2112,56 31 M 0 F508del R1162X 12 99 65 no yes 2,14 280,92 32 M 4 F508del 3849+10 Kb C.T 17 60 100 no no nd 2121,31 33 F 1 F508del 1717-1 G.A 26 105 73 yes yes 2,05 255,66 34 F 11 F508del 3849+10 Kb C.T 40 85 59 yes no nd 2152,23 35 F 4 F508del 1717-1 G.A 44 130 97 yes yes nd 2116,56 36 M 13 F508del 3849+10 Kb C.T 43 70 65 yes no CF 265,10 37 F 19 F508del unknown 29 95 100 no no nd 240,53 38 M 6 F508del unknown 15 92 87 yes no nd 270,17 39 F 0 G542X N1303K 34 108 97 yes yes nd 296,14 40 M 50 G1249R IVS8 T5TG12 50 61 74 no no nd 2199,15 41 F 10 2183 AA.G IVS8 T5TG15/T7TG10 45 79 29 yes no 1,9 286,27 42 F 1 G85E unknown 43 120 107 yes no nd 249,21 43 F 0 3272-26 A.G I507del 21 113 88 no no nd 236,79 44 M 8 F508del D1152H 10 77 107 no no nd 210,85 *Cystic Fibrosis mutation database reference: http://www3.genet.sickkids.on.ca/cftr/app. Login to comment

[hide] Pathology of pancreatic and intestinal disorders i... J R Soc Med. 1998;91 Suppl 34:40-9. Wilschanski M, Durie PR
Pathology of pancreatic and intestinal disorders in cystic fibrosis.
J R Soc Med. 1998;91 Suppl 34:40-9., [PMID:9709387]

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152 A small number of more Table 1 Classification of cystic fibrosis gene mutation as severe, mild or indeterminate with respect to pancreatic function Severe Mild Variable (classes 1, I/ or 111) (classes IV or V) (classes IV or V) AF508 R117H G85E 1148T R334W 2789+5G-*A G480C R347P G551D A455E R560T P574H N1303K 3849+1 Okb C-+T G542X G551S W1282X P5748 621 +1 G-T R352Q 1717-1G-T T3381 556delA Adapted from Ref 20 with permission recently described mutations [G85E and 278+5G-÷AI are less clearly determinant with respect to the pancreatic sufficient and pancreatic insufficient phenotypes. Login to comment

[hide] Relation between mutations of the cystic fibrosis ... N Engl J Med. 1998 Sep 3;339(10):653-8. Cohn JA, Friedman KJ, Noone PG, Knowles MR, Silverman LM, Jowell PS
Relation between mutations of the cystic fibrosis gene and idiopathic pancreatitis.
N Engl J Med. 1998 Sep 3;339(10):653-8., 1998-09-03 [PMID:9725922]

Abstract [show]
BACKGROUND: It is unknown whether genetic factors predispose patients to idiopathic pancreatitis. In patients with cystic fibrosis, mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene typically cause pulmonary and pancreatic insufficiency while rarely causing pancreatitis. We examined whether idiopathic pancreatitis is associated with CFTR mutations in persons who do not have lung disease of cystic fibrosis. METHODS: We studied 27 patients (mean age at diagnosis, 36 years), 22 of whom were female, who had been referred for an evaluation of idiopathic pancreatitis. DNA was tested for 17 CFTR mutations and for the 5T allele in intron 8 of the CFTR gene. The 5T allele reduces the level of functional CFTR and is associated with an inherited form of infertility in males. Patients with two abnormal CFTR alleles were further evaluated for unrecognized cystic fibrosis-related lung disease, and both base-line and CFTR-mediated ion transport were measured in the nasal mucosa. RESULTS: Ten patients with idiopathic chronic pancreatitis (37 percent) had at least one abnormal CFTR allele. Eight CFTR mutations were detected (prevalence ratio, 11:1; 95 percent confidence interval, 5 to 23; P<0.001). In three patients both alleles were affected (prevalence ratio, 80:1; 95 percent confidence interval, 17 to 379; P<0.001). These three patients did not have lung disease typical of cystic fibrosis on the basis of sweat testing, spirometry, or base-line nasal potential-difference measurements. Nonetheless, each had abnormal nasal cyclic AMP-mediated chloride transport. CONCLUSION: In a group of patients referred for evaluation of idiopathic pancreatitis, there was a strong association between mutations in the CFTR gene and pancreatitis. The abnormal CFTR genotypes in these patients with pancreatitis resemble those associated with male infertility.

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35 DNA was also tested for the G85E mutation.21 The length of the sequence of thymidines in intron 8 of the CFTR gene was determined with three allele-specific polymerase chain reactions per sample. Login to comment

[hide] Nasal potential difference in congenital bilateral... Am J Respir Crit Care Med. 1998 Sep;158(3):896-901. Pradal U, Castellani C, Delmarco A, Mastella G
Nasal potential difference in congenital bilateral absence of the vas deferens.
Am J Respir Crit Care Med. 1998 Sep;158(3):896-901., [PMID:9731023]

Abstract [show]
Congenital bilateral absence of the vas deferens (CBAVD) is supposed to be due to defective activity of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) in the genital tract. With the aim of studying CFTR activity in vivo we measured nasal potential difference (NPD) in a group of CBAVD subjects, who were then compared with normal control subjects and CF patients. Sodium transport, measured under basal conditions and after amiloride superinfusion, was normal in almost all CBAVD patients, who had NPD values similar to those of normal control subjects. Chloride transport was studied by measuring NPD during perfusion with a chloride-free solution and isoproterenol. Under these circumstances CBAVD patients as a whole showed normal chloride secretion. However, three subjects with CBAVD had abnormal NPD values. They had either elevated sweat chloride concentrations together with symptoms of mild CF, or compound heterozygosity (DeltaF508/R117H). In conclusion the group of CBAVD patients as a whole presented normal bioelectric properties of nasal epithelium, suggesting normal CFTR activity. In a small subgroup NPD was abnormal, suggesting a diagnosis of CF, later confirmed by elevated sweat chloride concentrations or positive DNA testing. We suggest that CBAVD patients with altered NPD should undergo further clinical follow-up in order to detect possible late complications of CF.

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39 ⌬F508, R117H, R1162X, 2183AA→G, N1303K, 3849 ϩ 10KbC→T, G542X, 1717-1G→A, R553X, Q552X, G85E, 711 ϩ 5G→A, 3132delTG and 2789 ϩ 5G→A were tested using for R117H two specifically designed primers which create a CFoI restriction site when the mutation is absent, and for all the other mutations a reverse dot blot assay (19). Login to comment

[hide] Screening of CFTR mutations in an isolated populat... Eur J Hum Genet. 1998 Mar-Apr;6(2):181-4. Chiba-Falek O, Nissim-Rafinia M, Argaman Z, Genem A, Moran I, Kerem E, Kerem B
Screening of CFTR mutations in an isolated population: identification of carriers and patients.
Eur J Hum Genet. 1998 Mar-Apr;6(2):181-4., [PMID:9781064]

Abstract [show]
One important application of the identification of disease-causing mutations is carrier screening in the general population. Such a project requires a simple accurate test by which a large proportion of the mutations can be identified. This study describes screening for CFTR mutations in an isolated Israeli Arab village. Two mutations, G85E and delta F508, accounted for all the CF alleles of these patients. The screening program tested for these two mutations, as well as the 5T allele, which has recently been shown to down-regulate the CFTR expression and cause variable phenotype. The screened population comprised 497 students from one school, which all the children of the village attend. The results revealed high carrier frequency, 8.5%, for the two CFTR mutations, G85E and delta F508, and a carrier frequency of 12% for the 5T allele. Two compound heterozygotes for the CFTR mutations, delta F508/G85E and G85E/5T, were identified. Both of these students had not been diagnosed previously as having CF since their disease presentation was not typical of CF. The CF incidence in this village was found to be extremely high, 1:72 life births. The screening results were reported to the physicians of the village to be used, upon request, for genetic counselling. This study emphasizes the importance of such programs for the identification of non-classical patients and for carrier detection.

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3 Two mutations, G85E and ∆F508, accounted for all the CF alleles of these patients. Login to comment
6 The results revealed high carrier frequency, 8.5%, for the two CFTR mutations, G85E and ∆F508, and a carrier frequency of 12% for the 5T allele. Login to comment
7 Two compound heterozygotes for the CFTR mutations, ∆F508/G85E and G85E/5T, were identified. Login to comment
33 Mutation Analysis Analysis of CFTR mutations was performed by polymerase chain reaction (PCR) using oligonucleotide primers for the amplification of exons 3, 10 and 98 to identify the mutations G85E, ∆F508 and 5T respectively. Login to comment
34 Mutation detection was performed as described elsewhere: G85E,3 ∆F5089 and 5T.7 Results and Discussion The screened population descended from one original family, which had founded the village near Jerusalem in the 16th century. Login to comment
37 The group studied was tested for the ∆F508 and the G85E mutations which accounted for all the CFTR mutations carried by the CF patients known in this village.10 Of the 497 students, we found that 41 (8.3%) were carriers of the G85E mutation and one (0.2%) was a carrier of the ∆F508 mutation. Login to comment
40 Therefore, we recalculated the carrier frequency according to nuclear families. This analysis revealed that 22 (7.7%) of the nuclear families had 1-4 carriers of the G85E mutation and one family (0.4%) had a ∆F508 carrier. Login to comment
47 Thus, the overall CF carrier frequency (for the ∆F508, G85E and the 5T allele) is very high, > 20%, indicating that CF disease is a major health problem in this village. Login to comment
49 One of the students was compound heterozygous for the G85E and the ∆F508 mutations. Login to comment
54 This child had a brother who had died from CF at 6 years of age. Another student was initially found in the screening program to be heterozygous for the G85E mutation. Login to comment
69 In the course of the screening program two additional CF patients were identified (∆F508/G85E and G85E/5T as mentioned above). Login to comment
73 This strong deviation from the Hardy-Weinberg calculation reflects the extent of consanguineous marriage12 which is characteristic of Arab villages in Israel.13 We also screened 89 relatives of the CF patients for the G85E and ∆F508 mutations, aiming to identify additional carriers and patients. Login to comment
76 of Carrier families with Carrier Mutation carriers frequency (%) carriers frequency (%) G85E 41 8.3 22 7.7 ∆F508 1 0.2 1 0.4 Total 42 8.5 23 8.1 5Ta 24 12 21 13 aThe size of the group which was screened for the 5T allele was n=203 for the students and n=161 for the nuclear families. Login to comment
78 Forty carried the G85E mutation and four carried the ∆F508 mutation. Login to comment
81 The screening results of the ∆F508 and the G85E mutations were reported to the physicians of the village. Login to comment

[hide] Validation of double gradient denaturing gradient ... Clin Chem. 1999 Jan;45(1):35-40. Cremonesi L, Carrera P, Fumagalli A, Lucchiari S, Cardillo E, Ferrari M, Righetti SC, Zunino F, Righetti PG, Gelfi C
Validation of double gradient denaturing gradient gel electrophoresis through multigenic retrospective analysis.
Clin Chem. 1999 Jan;45(1):35-40., [PMID:9895335]

Abstract [show]
Among established techniques for the identification of either known or new mutations, denaturing gradient gel electrophoresis (DGGE) is one of the most effective. However, conventional DGGE is affected by major drawbacks that limit its routine application: the different denaturant gradient ranges and migration times required for different DNA fragments. We developed a modified version of DGGE for high-throughput mutational analysis, double gradient DGGE (DG-DGGE), by superimposing a porous gradient over the denaturant gradient, which maintains the zone-sharpening effect even during lengthy analyses. Because of this innovation, DG-DGGE achieves the double goals of retaining full effectiveness in the detection of mutations while allowing identical run time conditions for all fragments analyzed. Here we use retrospective analysis of a large number of well-characterized mutations and polymorphisms, spanning all predicted melting domains and the whole genomic sequence of three different genes--the cystic fibrosis transmembrane conductance regulator (CFTR), the beta-globin, and the p53 genes--to demonstrate that DG-DGGE may be applied to the rapid scanning of any sequence variation.

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31 Mutations and polymorphisms analyzed in the CFTR gene. Position Denaturant gradient Mutation Exon 1 40-90% 125G/Ca,b M1V (A3G at 133) 175insT 182delT Exon 3 10-60% W57G (T3G at 301) 356G/Aa G85E (G3A at 386) Exon 4 20-70% R117H (G3A at 482) 541delC 621ϩ1G3T I148T (T3C at 575) Exon 5 20-70% E193K (G3A at 709) Intron 5 20-70% 711ϩ3A3G Exon 7 20-70% 1078delT R334W (C3T at 1132) T338I (C3T at 1145) R347P (G3C at 1172)b R347H (G3A at 1172) R352Q (G3A at 1187) Exon 10 20-70% M470V (1540A/G)a ⌬F508 (del 3 bp at 1652) Intron 10 10-60% 1717-1G3A Exon 11 10-60% G542X (G3T at 1756) 1784delG R553X (C3T at 1789) Exon 12 10-60% D579G (A3G at 1868) E585X (G3T at 1885) Intron 12 10-60% 1898ϩ3A3G Exon 13 30-80% 2183AA3G E730X (G3T at 2320) L732X (T3G at 2327) 2347delG Exon 14a 10-60% T854T (2694T/G)a V868V (2736G/A)a Intron 14b 30-80% 2789ϩ5G3A Exon 15 20-70% M952I (G3C at 2988)b Exon 17a 20-70% L997F (G3C at 3123)b Exon 17b 20-70% F1052V (T3G at 3286) R1066C (C3T at 3328) R1066H (G3A at 3329) A1067T (G3A at 3331) Exon 18 20-70% D1152H (G3C at 3586)b Exon 19 30-80% R1158X (C3T at 3604) Exon 20 20-70% S1251N (G3A at 3384) W1282X (G3A at 3978) Exon 21 20-70% N1303K (C3G at 4041)b Exon 22 30-80% G1349D (G3A at 4178) 4382delA Exon 24 30-80% Y1424Y (4404C/T)a a Polymorphism. Login to comment

[hide] Clinical and genetic risk factors for cystic fibro... Pediatrics. 1999 Jan;103(1):52-7. Wilschanski M, Rivlin J, Cohen S, Augarten A, Blau H, Aviram M, Bentur L, Springer C, Vila Y, Branski D, Kerem B, Kerem E
Clinical and genetic risk factors for cystic fibrosis-related liver disease.
Pediatrics. 1999 Jan;103(1):52-7., [PMID:9917439]

Abstract [show]
OBJECTIVE: The aim of this study was to define the role of possible risk factors for the development of cystic fibrosis (CF)-related liver disease and to analyze the association between liver disease and the different genotypes present in the Israeli CF patient population. PATIENTS AND METHODS: All patients followed at the seven CF centers in Israel were included in this study. Liver disease was determined by persistently elevated serum liver enzymes and/or bilirubin, and/or significant ultrasonographic changes suggestive of chronic liver disease. The following clinical parameters were evaluated: ethnic origin, age at assessment of liver function, sex, history of meconium ileus, pancreatic function, history of distal intestinal obstruction syndrome, pulmonary function, and cystic fibrosis transmembrane conductance regulator mutation analysis. RESULTS: Of the 288 patients screened, 80 (28%) had liver disease. Of the 256 patients with pancreatic insufficiency, 80 (31%) had liver disease compared with none of the 32 patients with pancreatic sufficiency. Genotype-phenotype correlation was performed on 207 patients carrying identified mutations that were previously classified according to phenotype severity. Liver disease was found in 56 (32%) of 173 patients carrying mutations associated with a severe phenotype and in 6 (38%) of 16 patients carrying at least one mutation associated with a variable genotype (G85E and/or 5T allele). None of the 18 patients carrying the 3849+10kb C->T mutation had liver disease. Prevalence of liver disease increased with age. No correlation was found between liver disease and severity of lung disease, nutritional status, history of meconium ileus, or distal intestinal obstruction syndrome. CONCLUSION: CF patients who have pancreatic insufficiency and carry mutations associated with a severe or a variable genotype are at increased risk to develop liver disease.

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51 These mutations were shown to be associated with PI, early age at diagnosis, higher sweat chloride level, and lower current age.3,4 Patients with the milder genotype carried the mutation 3849ϩ10kb C-ϾT, which was shown to be associated with increased incidence of PS, later age at diagnosis, higher current age, normal or intermediate range sweat chloride levels, and male fertility.19,20 Patients with a variable genotype were homozygous or compound heterozygous for the G85E mutation, or compound heterozygous for the 5T allele that may present with either severe or milder phenotype.6,7 Unclassified genotypes were mutations which, because of the small number of patients, have not been sufficiently characterized. Login to comment
114 However, patients carrying the variable genotype (G85E or 5T,6,7 ), which is also associated with high rate of PS, had a Fig 1. Login to comment
117 Classification of Identified Genotype According to Severity of Disease Severe n Milder n Variable n Unclassified n ⌬F508/⌬F508 52 3849 ϩ 10kbC 3 T/⌬F508 7 ⌬F508/G85E 1 S549R/S549R 1 W1282X/W1282X 30 3849 ϩ 10kbC 3 T/405 ϩ 1G3A 3 G85E/G85E 5 S549R/G542X 2 ⌬F508/W1282X 39 3849 ϩ 10 kbC 3 T/W1282X 7 G85E/5T 1 S549R/W1282X 1 ⌬F508/G542X 10 3849 ϩ 10kbC 3 T/G85E 1 ⌬F508/5T 1 ⌬F508/W1089X 1 W1282X/G542X 12 W1282X/5T 2 Y1092X/Y1092X 1 W1282X/N1303K 7 W1282X/5T 1 Q359K-T360K/? Login to comment

[hide] Detection of CFTR mutations using temporal tempera... Electrophoresis. 2004 Aug;25(15):2593-601. Wong LJ, Alper OM
Detection of CFTR mutations using temporal temperature gradient gel electrophoresis.
Electrophoresis. 2004 Aug;25(15):2593-601., [PMID:15300780]

Abstract [show]
Cystic fibrosis (CF), caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, is one of the most common autosomal recessive diseases with variable incidences and mutation spectra among different ethnic groups. Current commercially available mutation panels designed for the analysis of known recurrent mutations have a detection rate between 38 to 95%, depending upon the ethnic background of the patient. We describe the application of a novel mutation detection method, temporal temperature gradient gel electrophoresis (TTGE), to the study of the molecular genetics of Hispanic CF patients. TTGE effectively identified numerous rare and novel mutations and polymorphisms. One interesting observation is that the majority of the novel mutations are splice site, frame shift, or nonsense mutations that cause severe clinical phenotypes. Our data demonstrate that screening of the 27 exons and intron/exon junctions of the CFTR gene by TTGE greatly improves the molecular diagnosis of Hispanic CF patients.

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96 Detection of known mutations and polymorphisms by TTGE Base substitution Mutation Exon or intron Homozygote or heterozygote Polymorphism or mutation # Alleles detected 1 c.386G.A p.G85E 3 Heterozygote Mutation 2 2 c.575T.C p.I148T 4 Heterozygote Mutation 2 3 c.406-1G.A Splice Int 4 Heterozygote Mutation 9 4 c.71111G.T Splice Int 5 Heterozygote Mutation 1 5 c.1059_1069del 3bp p.F311del 7 Heterozygote Mutation 2 6 c.1132C.T p.R334W 7 Heterozygote Mutation 2 7 c.1652_1655del 3bp p.F508del 10 Heterozygote Mutation 94 8 Homozygote Mutation 12 c.1540A/G p.M470V 10 Heterozygote Polymorphism 15 9 Homozygote Polymorphism 4 c.1756G.T p.G542X 11 Heterozygote Mutation 13 10 c.1784G.A p.G551D 11 Heterozygote Mutation 1 11 c.1778G.A p.S549N 11 Heterozygote Mutation 4 12 c.1789C.T p.R553X 11 Homozygote Mutation 2 13 c.1807G.A p.A559T 11 Heterozygote Mutation 2 14 c.189811G.A Splice Int 12 Heterozygote Mutation 1 15 c.1949del84bp Frameshift 13 Heterozygote Mutation 3 16 c.278915G.A Splice Int 14b Heterozygote Mutation 2 17 c.312011G.A Splice Int 16 Heterozygote Mutation 9 18 c.3171delC Frameshift 17a Heterozygote Mutation 1 19 c.3398G.A p.W1089X 17b Heterozygote Mutation 1 20 c.3425G.A p.W1098X 17b Heterozygote Mutation 1 21 c.3616C.T p.R1162X 19 Heterozygote Mutation 2 22 c.3791delC Frameshift 19 Heterozygote Mutation 1 23 c.3821delT Frameshift 19 Heterozygote Mutation 1 24 c.3876delA Frameshift 20 Heterozygote Mutation 4 25 c.3905insT Frameshift 20 Heterozygote Mutation 1 26 c.4041C.G p.N1303K 21 Heterozygote Mutation 2 Total 194 The translation starts at c.133 of CFTR CDNA sequence in GenBank Acc. Login to comment

[hide] Spectrum of mutations in the CFTR gene in cystic f... Ann Hum Genet. 2007 Mar;71(Pt 2):194-201. Alonso MJ, Heine-Suner D, Calvo M, Rosell J, Gimenez J, Ramos MD, Telleria JJ, Palacio A, Estivill X, Casals T
Spectrum of mutations in the CFTR gene in cystic fibrosis patients of Spanish ancestry.
Ann Hum Genet. 2007 Mar;71(Pt 2):194-201., [PMID:17331079]

Abstract [show]
We analyzed 1,954 Spanish cystic fibrosis (CF) alleles in order to define the molecular spectrum of mutations in the CFTR gene in Spanish CF patients. Commercial panels showed a limited detection power, leading to the identification of only 76% of alleles. Two scanning techniques, denaturing gradient gel electrophoresis (DGGE) and single strand conformation polymorphism/hetroduplex (SSCP/HD), were carried out to detect CFTR sequence changes. In addition, intragenic markers IVS8CA, IVS8-6(T)n and IVS17bTA were also analyzed. Twelve mutations showed frequencies above 1%, p.F508del being the most frequent mutation (51%). We found that eighteen mutations need to be studied to achieve a detection level of 80%. Fifty-one mutations (42%) were observed once. In total, 121 disease-causing mutations were identified, accounting for 96% (1,877 out of 1,954) of CF alleles. Specific geographic distributions for the most common mutations, p.F508del, p.G542X, c.1811 + 1.6kbA > G and c.1609delCA, were confirmed. Furthermore, two other relatively common mutations (p.V232D and c.2789 + 5G > A) showed uneven geographic distributions. This updated information on the spectrum of CF mutations in Spain will be useful for improving genetic testing, as well as to facilitate counselling in people of Spanish ancestry. In addition, this study contributes to defining the molecular spectrum of CF in Europe, and corroborates the high molecular mutation heterogeneity of Mediterranean populations.

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45 (%) p.F508del # E.10 1009 (51.74) p.G542X # E.11 150 (7.69) p.N1303K # E.21 57 (2.92) c.1811 + 1.6kbA > G I.11 36 (1.84) p.R334W # E.7 35 (1.79) p.L206W E.6a 32 (1.64) c.711 + 1G > T # I.5 31 (1.58) p.Q890X E.15 28 (1.43) p.R1162X # E.19 25 (1.28) c.2789 + 5G > A # I.14b 24 (1.23) p.R1066C E.17b 23 (1.18) p.I507del # E.10 21 (1.07) c.1609delCA E.10 18 (0.92) c.712-1G > T I.5 18 (0.92) c.3272-26A > G I.17a 18 (0.92) c.2183AA > G # E.13 16 (0.82) p.G85E # E.3 15 (0.77) c.2869insG E.15 15 (0.77) p.W1282X # E.20 15 (0.77) p.V232D E.6a 14 (0.71) p.A1006E * E.17a 12 (0.61) c.2184insA E.13 11 (0.56) p.K710X E.13 11 (0.56) TOTAL (n = 23) 1,634 (83.72) * , the complex allele [p.A1006E; p.V562I; IVS8-6(5T)] #, CF mutations identified with the Celera Diagnosis Cystic Fibrosis v2 genotyping assay and the Inno-Lipa CFTR12, CFTR17 + Tn Samples with microsatellite haplotypes 16/45-46-47 (IVS8CA/IVS17bTA) were submitted to direct analysis of the c.1811 + 1.6kbA > G mutation, which was found mainly associated with the 16-46 haplotype. Login to comment
105 Our impression is that Table 3 Common CF mutations identified in this study and in several Latin American populations Mutation This study Hispanic1 Mexico2 Colombia3 Brazil4 Argentina5 Chile6 p.F508del 51.7 51.6 40.7 41.8 48.4 58.6 45.0 p.G542X 7.7 4.0 6.2 3.8 8.8 4.1 7.0 p.N1303K 2.9 0.8 2.0 0.5 2.5 2.7 - c.1811 + 1,6kbA > G 1.8 - - 6.5 - 0.9 - p.R334W 1.8 1.6 - 0.5 2.5 1.1 2.0 p.L206W 1.6 - - - 0.6 - - c.711 + 1G > T 1.6 - - - - - - p.Q890X 1.4 - - - - - - p.R1162X 1.3 0.8 - 1.1 2.5 0.4 2.0 c.2789 + 5G > A 1.2 - - 0.5 0.3 0.7 - p.R1066C 1.2 1.6 - 0.5 - 0.2 - p.I507del 1.0 - 2.6 - - 0.7 - c.2183AA > G 0.8 - 1.0 - 0.2 - p.G85E 0.7 0.8 0.5 - 1.3 0.7 - p.W1282X 0.7 0.8 - 1.1 1.3 2.7 5.0 c.3849 + 10kbC > T 0.4 4.0 0.5 - - 0.9 3.0 p.S549N - 2.4 2.6 - - - - c.3120 + 1G > A - 1.6 - 0.5 - - - c.3876delA - 5.6 - - - - - c.406-1G > A - 1.6 1.5 - - - - c.935delA - 1.6 1.0 - - - - p.R75X - 0.8 1.5 - - - - c.2055del9 - - 1.0 - - - - p.I506T - - 1.0 - - - - c.3199del6 - - 1.0 - - - - p.S549R 0.4 - - 2.2 - 0.2 - c.1717-1G > A 0.2 - - - 0.3 1.1 - p.G551D 0.2 0.8 0.5 - - - 1.0 p.R553X 0.4 - 0.5 - 0.6 0.2 1.0 No. Login to comment

[hide] Outcomes of a cystic fibrosis carrier testing clin... Med J Aust. 2009 Nov 2;191(9):499-501. Christie LM, Ingrey AJ, Turner GM, Proos AL, Watts GE
Outcomes of a cystic fibrosis carrier testing clinic for couples.
Med J Aust. 2009 Nov 2;191(9):499-501., 2009-11-02 [PMID:19883345]

Abstract [show]
OBJECTIVE: To review the outcomes of offering carrier testing for cystic fibrosis (CF) to couples considering pregnancy, and to women in early pregnancy and their partners. METHODS: An after-hours clinic was established in Newcastle for discussion of issues related to prenatal testing. Couples were offered CF carrier testing by extracting DNA from a mouthwash sample. An expanded one-step model was used with both partners being tested initially for the p.F508del cystic fibrosis transmembrane conductance regulator gene (CFTR) mutation. If one partner was a p.F508del carrier, the other partner was tested for an additional 28 CFTR mutations. RESULTS: Of 1000 individuals who were offered CF carrier testing, none declined. No re-collections of mouthwash samples were required, and results were available within 14 days. There were 730 individuals who had no family history of CF (73%); 27 were carriers (4%; 95% CI, 2.4%-5.3%), and there were two high-risk couples where both partners were carriers of p.F508del. There were 270 individuals who had an affected family member with CF or a child identified as a CF carrier through newborn screening; 126 were carriers (46%; 95% CI, 40.6%-52.8%), and there were two high-risk couples - one couple where both partners were carriers of p.F508del, and another couple where the woman was homozygous for p.F508del and the man was a p.F508del carrier. The information on carrier status led the four high-risk couples to change their reproductive decisions to avoid having a child with CF. CONCLUSION: CF carrier testing for couples using an expanded one-step model will detect about 80% of high-risk couples and enables various reproductive choices. We believe that all couples considering pregnancy, and women in early pregnancy and their partners, should be offered CF carrier testing.

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72 This provides each individual with information on their carrier status, and accurate residual risks of 1 CFTR mutations tested for in individuals whose partner was a carrier of p.F508del* p.F508del p.F316leufsX p.I507del p.R347P p.G542X p.S1251N p.G551D p.E60X p.N1303K p.W1282X c.1585-1G>A p.D1152H p.R553X c.2988+1G>A c.489+G>T c.2657+5G>A p.R117H c.1766+1G>A p.R1162X c.579+1G>A c.3717+10kbC>T p.G85E p.R334W p.K684fs p.A455E p.I148T p.K684fs p.R560T p.T1176fs CFTR = gene encoding cystic fibrosis transmembrane conductance regulator. Login to comment

[hide] Genetic, epidemiological, and clinical aspects of ... Am J Gastroenterol. 2010 Aug;105(8):1876-83. Epub 2010 May 25. Joergensen MT, Brusgaard K, Cruger DG, Gerdes AM, Schaffalitzky de Muckadell OB
Genetic, epidemiological, and clinical aspects of hereditary pancreatitis: a population-based cohort study in Denmark.
Am J Gastroenterol. 2010 Aug;105(8):1876-83. Epub 2010 May 25., [PMID:20502448]

Abstract [show]
OBJECTIVES: In a population-based, well-defined group of patients first regarded as having pancreatitis of unknown origin (PUO), we identified, described, and compared the clinical and genetic aspects of patients with hereditary pancreatitis (HP) and with cystic fibrosis transmembrane conductance regulator gene (CFTR) and serine protease inhibitor Kazal type 1 gene (SPINK1) mutations with patients who retained the diagnosis of true idiopathic pancreatitis (tIP) after genetic testing for HP, SPINK1, and CFTR mutations. METHODS: Patients with PUO were identified in the Danish National Registry of Patients or were referred by clinicians. DNA from blood was analyzed for cationic trypsinogen (PRSS1), SPINK1, and CFTR mutations. Considering the diagnosis of HP, a pedigree was drawn for each patient. RESULTS: A genetic mutation was found in 40% of 122 patients with PUO. After testing first-degree relatives of the 18 initially identified HP patients, 38 HP patients in total were identified, and 28 patients had SPINK1-CFTR mutations. Among HP patients, no p.N29I mutations were found and the p.A16V mutation was more frequent than previously reported, 45 and 32% had exocrine and endocrine insufficiency, respectively, and among tIP patients 9 and 12%, respectively. Pancreatic cancer was diagnosed in 5% of the HP families. CONCLUSIONS: The genotype of the Danish population with HP differs from that of previously described cohorts. The occurrence of exocrine and endocrine insufficiency is higher among patients with HP than in patients with SPINK1-CFTR mutations and tIP, and more HP families develop pancreatic cancer. Genetic testing thus helps to predict the prognosis of the pancreatitis.

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57 The samples were also tested for 33 CFTR mutations, and all 6 classeswererepresented:394delTT,p.R553X,621+1G>T,p.R1162X, 1717-1G>A,3659delC,p.G542X,2183AA>G,p.W1282X,1078delT, 711+1G>T, F508del, p.S549N, I507del, p.S549R, 2184delA, p.G551D, p.G85E, p.N1303K, p.R560T, p.R117H, p.R347H, p.R347P, p.R334W, 2789+5G>A, 3849+10kbC>T, p.A445E, 3120+1G>A, p.V520F,1898+1G>A,3876delA,3905insT,andIVS8-5T.DNAwas amplified by multiplex PCR (Hybaid 4 A62, Middlesex, UK). Login to comment

[hide] Cystic fibrosis in Chilean patients: Analysis of 3... J Cyst Fibros. 2011 Jan;10(1):66-70. Epub 2010 Oct 30. Lay-Son G, Puga A, Astudillo P, Repetto GM
Cystic fibrosis in Chilean patients: Analysis of 36 common CFTR gene mutations.
J Cyst Fibros. 2011 Jan;10(1):66-70. Epub 2010 Oct 30., [PMID:21036675]

Abstract [show]
BACKGROUND: CFTR gene mutations have worldwide differences in prevalence and data on Chilean patients is scarce. METHODS: We studied 36 of the most common CFTR mutations in Chilean patients from the CF National Program [Programa Nacional de Fibrosis Quistica (PNFQ)] of the Ministry of Health of Chile. RESULTS: Two hundred and eighty-nine patients were studied. Fourteen different mutations were identified with an overall allele detection rate of 42.0%. Mutations with frequencies greater than 1% were p.F508del (30.3% of alleles), p.R334W (3.3%), p.G542X (2.4%), c.3849+10Kb C>T (1.7%), and p.R553X (1.2%). A north to south geographical gradient was observed in the overall rate of detection. CONCLUSIONS: Southern European CFTR mutations predominate in the Chilean population, but a high percentage of alleles remain unknown. Geographical heterogeneity could be explained in part by admixture. Complementary analyses are necessary to allow for effective genetic counselling and improve cost-effectiveness of screening and diagnostic tests.

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81 Mutation This study Rios et al. [4] Molina et al. [5] Repetto et al. [6] Perez et al. [13] CFGAC [2] (n=578) (%) (n=72) (%) (n=36) (%) (n=100) (%) (n=4102) (%) (n=43,849) (%) Chile Chile Chile Chile Latin-Americaa Worldwide Unknown 58.0 66.6 61.1 34.0 36.7 22.7 p.F508del 30.6 29.2 30.6 45.0 47.1 66.0 p.R334W 3.1 - - 2.0 0.8 0.1 p.G542X 2.4 0 8.3 7.0 5.0 2.4 c.3849+10Kb CNT 1.7 - - 3.0 0.3 0.2 p.R553X 1.2 4.2 0 1.0 0.4 0.7 p.R1162X 0.9 - - 2.0 1.0 0.3 p.1078delT 0.5 - - 0 b0.1 0.1 p.G85E 0.5 - - - 0.8 0.2 p.W1282X 0.2 - - 5.0 1.0 1.2 c.3120+1 GNA 0.2 - - - 0.3 - c.711+1 GNT 0.2 - - - 0.1 0.1 p.R117H 0.2 - - 0 b0.1 0.3 p.A455E 0.2 - - 0 0 0.1 p.I148T 0.2 - - - - - p.G551D 0 0 0 1.0 0.1 1.6 p.N1303K 0 0 0 0 1.8 1.3 c.621+1 GNT 0 - - 0 0.2 0.7 c.1717-1 GNA 0 - - 0 0.3 0.6 p.I507del 0 - - 0 0.2 0.2 p.R347P 0 - - 0 0 0.2 c.2789+5 GNA 0 - - - 0.2 0.1 c.1898+1 GNA 0 - - - 0.1 0.1 c.2184delA 0 - - - b0.1 0.1 p.S549N 0 - 0 - 0.1 0.1 c.3659delC 0 - - 0 0.1 0.1 p.R560T 0 - - - 0 0.1 c.1811+1.6Kb ANG 0 - - - 0.4 - c.2183AANG 0 - - 0 0.1 - p.S549R 0 - - - 0.1 - c.3272-26 ANG 0 - - - 0.1 - c.3199del6 0 - - - b0.1 - p.E60X 0 - - 0 0 - c.3905insT 0 - - - 0 - p.S1251N 0 - - 0 - - CFTRdele2,3 0 - - - - - p.R347H 0 - - - - - p.V520F 0 - - - - - p.Q552X 0 - - - - - c.394delTT 0 - - - - - c.711+1 GNA 0 - - - - - c.2143delT 0 - - - - - c.3876delA 0 - - - - - a Data from Chilean patients published in Rios et al., Molina et al., and Repetto et al. [4-6] included in this publication were excluded in this table to avoid repetition. Login to comment

[hide] Membrane-integration characteristics of two ABC tr... J Mol Biol. 2009 Apr 17;387(5):1153-64. Epub 2009 Feb 21. Enquist K, Fransson M, Boekel C, Bengtsson I, Geiger K, Lang L, Pettersson A, Johansson S, von Heijne G, Nilsson I
Membrane-integration characteristics of two ABC transporters, CFTR and P-glycoprotein.
J Mol Biol. 2009 Apr 17;387(5):1153-64. Epub 2009 Feb 21., [PMID:19236881]

Abstract [show]
To what extent do corresponding transmembrane helices in related integral membrane proteins have different membrane-insertion characteristics? Here, we compare, side-by-side, the membrane insertion characteristics of the 12 transmembrane helices in the adenosine triphosphate-binding cassette (ABC) transporters, P-glycoprotein (P-gp) and the cystic fibrosis transmembrane conductance regulator (CFTR). Our results show that 10 of the 12 CFTR transmembrane segments can insert independently into the ER membrane. In contrast, only three of the P-gp transmembrane segments are independently stable in the membrane, while the majority depend on the presence of neighboring loops and/or transmembrane segments for efficient insertion. Membrane-insertion characteristics can thus vary widely between related proteins.

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264 Structural cues involved in endoplasmic reticulum degradation of G85E and G91R mutant cystic fibrosis transmembrane conductance regulator. J. Clin. Login to comment
261 Structural cues involved in endoplasmic reticulum degradation of G85E and G91R mutant cystic fibrosis transmembrane conductance regulator. J. Clin. Login to comment

[hide] Arsenite regulates Cystic Fibrosis Transmembrane C... Cell Physiol Biochem. 2005;16(1-3):109-18. Maitra R, Hamilton JW
Arsenite regulates Cystic Fibrosis Transmembrane Conductance Regulator and P-glycoprotein: evidence of pathway independence.
Cell Physiol Biochem. 2005;16(1-3):109-18., [PMID:16121039]

Abstract [show]
In the past, people have argued for and against the theory of reciprocal regulation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and P-glycoprotein (Pgp). Data have indicated that this may occur in vitro during drug-induced selection of cells, and in vivo during development. Much of this debate has been caused by a severe lack of mechanistic details involved in such regulation. Our past data indicate that certain Pgp modulators can affect CFTR expression and function. The goal of this study was to investigate the effects of trivalent arsenic (arsenite), a known transcriptional activator of Pgp, on CFTR expression. In vitro analyses in T-84 cells that express basal levels of Pgp and CFTR were conducted using a variety of molecular techniques. Expressions of both genes were altered following treatment with arsenite in a dose- and time-dependent fashion. CFTR expression was suppressed almost three-fold by arsenite, along with a concomitant increase in P-glycoprotein expression. We also report that a member of the MAPK-family, the ERK-mediated signaling cascade is implicated in suppression of CFTR expression following treatment with arsenite. However, this particular pathway is not involved in regulation of P-glycoprotein expression in T-84 cells following treatment with arsenite. Thus, the regulatory pathways that control functional expression of CFTR and P-glycoprotein following arsenite treatment in T-84 cells are distinct and independent.

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234 J Biol Chem 2000;275:24970-24976 19 Xiong X, Bragin A, Widdicombe JH, Cohn J, Skach WR: Structural cues involved in endoplasmic reticulum degradation of G85E and G91R mutant cystic fibrosis transmembrane conductance regulator. Login to comment

[hide] Report on the p.Ser489X (p.Ser489*) CFTR mutation,... Genet Med. 2012 Oct;14(10):883-6. doi: 10.1038/gim.2012.57. Epub 2012 May 24. De Bie I, Agatep R, Scott P, Ruchon A
Report on the p.Ser489X (p.Ser489*) CFTR mutation, a variant with severe associated phenotype and high prevalence in a Quebec French-Canadian cystic fibrosis patient population.
Genet Med. 2012 Oct;14(10):883-6. doi: 10.1038/gim.2012.57. Epub 2012 May 24., [PMID:22627569]

Abstract [show]
Purpose:This study reports on the phenotype of cystic fibrosis patients identified to be carriers of the p.Ser489X (p.Ser489*; c.1466C>A) cystic fibrosis transmembrane conductance regulator (CFTR) mutation, a variant rarely described in the cystic fibrosis literature, as well as on its allelic frequency in a French-Canadian cystic fibrosis patient cohort.Methods:Reported phenotypes and allelic frequency of this variant were collected based on the data from a large French-Canadian cystic fibrosis patient cohort.Results:Cystic fibrosis patients found to carry the p.Ser489X variant generally presented with classic gastrointestinal manifestations of this condition in infancy. The allelic frequency of this variant was calculated to be 0.7% for this population.Conclusion:The p.Ser489X CFTR variant is a severe disease-causing CFTR allele that is relatively frequent in the French-Canadian cystic fibrosis patient population, warranting its inclusion into CFTR molecular testing panel for this population.Genet Med 2012:14(10):883-886.

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31 All but one of these CF patients ascertained in our French-Canadian cohort were compound heterozygotes for the p.Ser489X variant and another severe disease-causing CFTR allele, namely p.Phe508del, 621+1G>T (c.489+1G>T), 711+1G>T (c.579+1G>T), p.Ile1023_Val1024del (3199del6; c.3067_3072delATAGTG), or p.Gly85Glu (p.G85E; c.254G>A) (Table 1). Login to comment
42 CFTR genotype Age at diagnosis Reported symptoms Note 1 p.[Ser489X];[Ser489X] Newborn Volvulus, meconium ileus, positive trypsinogen 2 p.[Phe508del];[Ser489X] 3 Months Meconium ileus and positive sweat test 3 (Sib of 2) p.[Phe508del];[Ser489X] 1 Week Meconium ileus 4 p.[Phe508del];[Ser489X] 2 Weeks Meconium ileus 5 p.[Phe508del];[Ser489X] 5 Months Positive sweat test, intestinal obstruction, and steatorrhea 6 p.[Phe508del];[Ser489X] 15 Months Positive sweat test, no additional symptoms reported 7 p.[Phe508del];[Ser489X] Unknown Unknown Tested at 7 years of age 8 p.[Phe508del];[Ser489X] Unknown Unknown Tested at 9 years of age 9 p.[Phe508del];[Ser489X] Unknown Unknown Diagnosis made in infancy; molecular testing performed as an adult (26 years old) 10 [621+1G>T];p.[Ser489X] 2 Months Growth restriction, failure to thrive 11 [711+1G>T];p.[Ser489X] 3 Months Positive sweat test, no additional symptoms reported 12 p.[Ile1023_Val1024del]; [Ser489X] Unknown Positive sweat test, no additional symptoms reported Tested at 13 years of age 13 p.[Gly85Glu];[Ser489X] Unknown Unknown Diagnosis made in infancy, molecular testing performed as an adult (18 years old) Genetics in medicine | Volume 14 | Number 10 | October 2012 p.Ser489X, a high-prevalence CFTR variant in a cystic fibrosis patient population | DE BIE et al brief report been mostly characterized in a CF mouse model,7,8 and has only scarcely been reported in the human CF literature.4,9-11 This variant results in premature chain termination and is predicted to behave as a functional class I allele, causing reduced CFTR protein synthesis. Login to comment
30 All but one of these CF patients ascertained in our French-Canadian cohort were compound heterozygotes for the p.Ser489X variant and another severe disease-causing CFTR allele, namely p.Phe508del, 621+1G>T (c.489+1G>T), 711+1G>T (c.579+1G>T), p.Ile1023_Val1024del (3199del6; c.3067_3072delATAGTG), or p.Gly85Glu (p.G85E; c.254G>A) (Table 1). Login to comment
41 CFTR genotype Age at diagnosis Reported symptoms Note 1 p.[Ser489X];[Ser489X] Newborn Volvulus, meconium ileus, positive trypsinogen 2 p.[Phe508del];[Ser489X] 3 Months Meconium ileus and positive sweat test 3 (Sib of 2) p.[Phe508del];[Ser489X] 1 Week Meconium ileus 4 p.[Phe508del];[Ser489X] 2 Weeks Meconium ileus 5 p.[Phe508del];[Ser489X] 5 Months Positive sweat test, intestinal obstruction, and steatorrhea 6 p.[Phe508del];[Ser489X] 15 Months Positive sweat test, no additional symptoms reported 7 p.[Phe508del];[Ser489X] Unknown Unknown Tested at 7 years of age 8 p.[Phe508del];[Ser489X] Unknown Unknown Tested at 9 years of age 9 p.[Phe508del];[Ser489X] Unknown Unknown Diagnosis made in infancy; molecular testing performed as an adult (26 years old) 10 [621+1G>T];p.[Ser489X] 2 Months Growth restriction, failure to thrive 11 [711+1G>T];p.[Ser489X] 3 Months Positive sweat test, no additional symptoms reported 12 p.[Ile1023_Val1024del]; [Ser489X] Unknown Positive sweat test, no additional symptoms reported Tested at 13 years of age 13 p.[Gly85Glu];[Ser489X] Unknown Unknown Diagnosis made in infancy, molecular testing performed as an adult (18 years old) Genetics in medicine | Volume 14 | Number 10 | October 2012 p.Ser489X, a high-prevalence CFTR variant in a cystic fibrosis patient population | DE BIE et al brief report been mostly characterized in a CF mouse model,7,8 and has only scarcely been reported in the human CF literature.4,9-11 This variant results in premature chain termination and is predicted to behave as a functional class I allele, causing reduced CFTR protein synthesis. Login to comment
55 Frequencies of CFTR disease-causing alleles in the French-Canadian cystic fibrosis patient population CFTR variant All variants p.Phe508del 621+1G>T 711+1G>T p.Ala455Glu p.Ser489X p.Arg334Trp 3199del6 p.Gly542X p.Gly85Glu p.Ile507del FC SLSJ FC SLSJ FC SLSJ FC SLSJ FC SLSJ FC SLSJ FC SLSJ FC SLSJ FC SLSJ FC SLSJ FC SLSJ Number of CF chromosomes 1712 172 1321 116 143 29 61 1 51 13 12 0 9 0 8 2 6 1 5 1 4 1 % Of total CF chromosomes 100.00 100.00 77.16 67.44 8.35 16.86 3.56 0.58 2.98 7.56 0.70 0.00 0.53 0.00 0.47 1.16 0.35 0.58 0.29 0.58 0.23 0.58 CF, cystic fibrosis; FC, French-Canadian; SLSJ, Saguenay-Lac-St-Jean. Login to comment

[hide] Hispanic Infants with cystic fibrosis show low CFT... J Genet Couns. 2012 Oct;21(5):671-5. doi: 10.1007/s10897-012-9481-2. Epub 2012 Feb 4. Watts KD, Layne B, Harris A, McColley SA
Hispanic Infants with cystic fibrosis show low CFTR mutation detection rates in the Illinois newborn screening program.
J Genet Couns. 2012 Oct;21(5):671-5. doi: 10.1007/s10897-012-9481-2. Epub 2012 Feb 4., [PMID:22311127]

Abstract [show]
States develop specific protocols for cystic fibrosis (CF) newborn screening to reflect the population served. We hypothesized that mutation distribution and detection rates would differ between Hispanic and non-Hispanic CF patients diagnosed by IL newborn screen with more Hispanic infants carrying mutations not detected by the state panel. Data from CF cases diagnosed via newborn screen in IL between 3/1/2008 and 10/31/2010 were reviewed. More Hispanic infants with CF had one or more undefined mutations after screening, in comparison to non-Hispanic Caucasian patients (40% vs. 9.5%; p < 0.002). The risk of having a positive diagnosis of CF with only one mutation noted by positive newborn screen increases 2-fold in Hispanic Caucasian versus non-Hispanic Caucasian infants (5% vs. 2.4%). Health care providers must be aware of the limitations of CF newborn screening to ensure appropriate counseling and prompt referral for a positive newborn screen, even when zero or one mutations are identified.

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39 Mutation Frequency Table 1 shows the mutations found in Illinois patients diagnosed with CF after a positive NBS and compares these to mutations documented in Hispanic Caucasian Table 1 CFTR mutation frequency detected by Illinois newborn screen Mutation IL Newborn Screen CFF Patient Registry Total alleles Non-Hispanic Caucasian Hispanic Caucasian African American Ethnicity/Race Missing Hispanic Caucasian ΔF508 63.9% 71.6% 36.7% 33.3% 58.3% 44.7% R117H 7.7% 10.1% 3.3% - - 0.3% G542X 1.9% 2.0% - - 4.2% 4.1% 3120+1G>A 1.9% 0.7% 3.3% 33.3% - 0.7% ΔI507 1.4% 0.7% - - 8.3% 1.3% G551D 1.4% 2.0% - - - 0.5% 3659delC 1.4% 1.3% 3.3% - - 0.1% 3849+10 kbC>T 1.4% - 6.7% 16.7% - 1.0% ΔF311 1.4% - 6.7% - 4.2% 0.03% 1288insT 0.5% - 3.3% - - 0% 621+1G>T 0.5% - 3.3% - - 0.4% G85E 1.0% - 3.3% - 4.2% 0.3% 2184delA 0.5% - 3.3% - - 0.2% S549N 0.5% - 3.3% - - 0.7% R334W 1.0% 0.7% - 16.7% - 1.0% N1303K 1.0% - - - 8.3% 1.6% Other 4.4% 6.2%a 0% 0% 0% 12.8%b Unknown 8.2% 4.7% 23.5% 0% 12.5% 15.7% a R347P, 1898+1G>A, 2789+5G>A, 3272-26A>G, 3876delA, CFTRdel2,3, W1282X occurred in non-Hispanic Caucasian patients only with an allele frequency of 0.5% of the entire IL NBS population b In the 2004 CFF Patient Registry 12.8% of alleles are not included in the above table because they occur in less than 1% of the population. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... J Cyst Fibros. 2012 Sep;11(5):355-62. doi: 10.1016/j.jcf.2012.05.001. Epub 2012 Jun 2. Ooi CY, Durie PR
Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in pancreatitis.
J Cyst Fibros. 2012 Sep;11(5):355-62. doi: 10.1016/j.jcf.2012.05.001. Epub 2012 Jun 2., [PMID:22658665]

Abstract [show]
BACKGROUND: The pancreas is one of the primary organs affected by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. While exocrine pancreatic insufficiency is a well-recognized complication of cystic fibrosis (CF), symptomatic pancreatitis is often under-recognized. RESULTS: The aim of this review is to provide a general overview of CFTR mutation-associated pancreatitis, which affects patients with pancreatic sufficient CF, CFTR-related pancreatitis, and idiopathic pancreatitis. The current hypothesis regarding the role of CFTR dysfunction in the pathogenesis of pancreatitis, and concepts on genotype-phenotype correlations between CFTR and symptomatic pancreatitis will be reviewed. Symptomatic pancreatitis occurs in 20% of pancreatic sufficient CF patients. In order to evaluate genotype-phenotype correlations, the Pancreatic Insufficiency Prevalence (PIP) score was developed and validated to determine severity in a large number of CFTR mutations. Specific CFTR genotypes are significantly associated with pancreatitis. Patients who carry genotypes with mild phenotypic effects have a greater risk of developing pancreatitis than patients carrying genotypes with moderate-severe phenotypic consequences at any given time. CONCLUSIONS: The genotype-phenotype correlation in pancreatitis is unique compared to other organ manifestations but still consistent with the complex monogenic nature of CF. Paradoxically, genotypes associated with otherwise mild phenotypic effects have a greater risk for causing pancreatitis; compared with genotypes associated with moderate to severe disease phenotypes. Greater understanding into the underlying mechanisms of disease is much needed. The emergence of CFTR-assist therapies may potentially play a future role in the treatment of CFTR-mutation associated pancreatitis.

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847 Total PI Total PI+PS PIP score 621+1G>T 96 96 1.00 Classes I - III 711+1G>T 36 36 1.00 Classes I - III R553X 24 24 1.00 Classes I - III I507del 34 34 1.00 Classes I - III G542X 74 75 0.99 Classes I - III F508del 1276 1324 0.96 Classes I - III 1717-1G>A 20 21 0.95 Classes I - III W1282X 19 20 0.95 Classes I - III N1303K 45 48 0.94 Classes I - III R1162X 12 13 0.92 Classes I - III G551D 59 67 0.88 Classes I - III G85E 16 22 0.73 Classes I - III A455E 18 37 0.49 Classes IV - V 2789+5G>A 6 16 0.38 Classes IV - V R334W 1 10 0.10 Classes IV - V 3849+10kbC>T 2 22 0.09 Classes IV - V R117H 1 25 0.04 Classes IV - V Mutation Canadian Consortium for CF Genetic Studies Mutation class The PIP score for a specific mutation is the ratio between the pancreatic insufficient patients carrying the mutation (Total PI) and all pancreatic insufficient and sufficient patients (Total PI+PS) carrying the same mutation in a homozygous state or heterozygous in a combination with a severe mutation such as F508del, G551D or a Class I mutation. Login to comment
855 CFTR mutation Total PI Total PI + PS PIP score CFTR mutation Total PI Total PI + PS PIP score 621+1G>T 96 96 1.00 G542X 74 75 0.99 711+1G>T 36 36 1.00 F508del 1276 1324 0.96 I507del 34 34 1.00 1717-1G>A 20 21 0.95 R553X 24 24 1.00 W1282X 19 20 0.95 Q493X 11 11 1.00 N1303K 45 48 0.94 S489X 11 11 1.00 R1162X 12 13 0.92 1154insTC 10 10 1.00 Y1092X 12 13 0.92 3659delC 9 9 1.00 I148T 10 11 0.91 CFTRdele2 7 7 1.00 V520F 9 10 0.90 4016insT 7 7 1.00 G551D 59 67 0.88 E60X 7 7 1.00 L1077P 5 6 0.83 R560T 7 7 1.00 R1066C 5 6 0.83 R1158X 7 7 1.00 2184insA 9 12 0.75 3905insT 6 6 1.00 2143delT 3 4 0.75 I148T;3199del6 5 5 1.00 1161delC 3 4 0.75 2183AA>G 5 5 1.00 3120+1G>A 3 4 0.75 1898+1G>A 5 5 1.00 S549N 3 4 0.75 2347delG 4 4 1.00 G85E 16 22 0.73 Q1313X 3 3 1.00 R117C 2 3 0.67 Q220X 3 3 1.00 M1101K 19 30 0.63 2184delA 3 3 1.00 P574H 3 5 0.60 1078delT 3 3 1.00 474del13BP 1 2 0.50 L1254X 3 3 1.00 R352Q 1 2 0.50 E585X 3 3 1.00 Q1291H 1 2 0.50 3876delA 2 2 1.00 A455E 18 37 0.49 S4X 2 2 1.00 R347P 6 15 0.40 R1070Q 2 2 1.00 2789+5G>A 6 16 0.38 F508C 2 2 1.00 L206W 6 18 0.33 DELI507 2 2 1.00 IVS8-5T 4 16 0.25 Q1411X 2 2 1.00 3272-26A>G 1 4 0.25 365-366insT 2 2 1.00 R334W 1 10 0.10 R709X 2 2 1.00 3849+10kbC>T 2 22 0.09 1138insG 2 2 1.00 P67L 1 14 0.07 CFTRdele2-4 2 2 1.00 R117H 1 25 0.04 3007delG 2 2 1.00 R347H 0 5 0.00 Q814X 2 2 1.00 G178R 0 3 0.00 394delTT 2 2 1.00 E116K 0 2 0.00 406-1G>A 2 2 1.00 875+1G>C 0 2 0.00 R75X 2 2 1.00 V232D 0 2 0.00 CFTRdel2-3 2 2 1.00 D579G 0 2 0.00 E193X 2 2 1.00 L1335P 0 2 0.00 185+1G>T 2 2 1.00 Mild mutations (based on PIP scores) are shaded in gray. Login to comment

[hide] Newborn screening for cystic fibrosis: Polish 4 ye... Eur J Hum Genet. 2012 Aug 15. doi: 10.1038/ejhg.2012.180. Sobczynska-Tomaszewska A, Oltarzewski M, Czerska K, Wertheim-Tysarowska K, Sands D, Walkowiak J, Bal J, Mazurczak T
Newborn screening for cystic fibrosis: Polish 4 years' experience with CFTR sequencing strategy.
Eur J Hum Genet. 2012 Aug 15. doi: 10.1038/ejhg.2012.180., [PMID:22892530]

Abstract [show]
Newborn screening for cystic fibrosis (NBS CF) in Poland was started in September 2006. Summary from 4 years' experience is presented in this study. The immunoreactive trypsin/DNA sequencing strategy was implemented. The group of 1 212 487 newborns were screened for cystic fibrosis during the programme. We identified a total of 221 CF cases during this period, including, 4 CF cases were reported to be omitted by NBS CF. Disease incidence in Poland based on the programme results was estimated as 1/4394 and carrier frequency as 1/33. The frequency of the F508del was similar (62%) to population data previously reported. This strategy allowed us to identify 29 affected infants with rare genotypes. The frequency of some mutations (eg, 2184insA, K710X) was assessed in Poland for the first time. Thus, sequencing assay seems to be accurate method for screening programme using blood spots in the Polish population.European Journal of Human Genetics advance online publication, 15 August 2012; doi:10.1038/ejhg.2012.180.

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57 Mutations D537N and P731L have not been Period of NBS CF Method The most frequent mutations in Polish population under analysis September 2006 - December 2007 Estonia Asper Biotech assay E60X, G85E, 394delTT, R117H, R117P, R117L, I148T, 621G>A, 711+1G>T, 711+5G>A, 1078delT, R334W, R347H, R347P, R347L, IVS8-T, A455E, I507del, F508del, 1717-1G>A, G542X, p.G551D, Q552X, R553X, R553G, R560T, R560K, 1898+1G>A, 1898+1G>T, 1898+1G>C, 2143delT, 2184delA, 2183AA>G, 2789+5G>A, 3120+1G>A, 3199del6, 3272-26A>G, R1162X, 3659delC, 3849+10kbC>T, 3905insT, S1235R, S1251N, W1282X, W1282C, N1303K, CFTRdele2,3 January 2007 - June 2009 Sanger sequencing of exons: 4, 7, 10, 11, 13, 21, fragment of intron 19 F508del, CFTRdele2,3, 3849+10kbC>T, R117H+IVS8-T*, R334W, R347P, 1717-1G>A, G542X, R553X, K710X, 2184insA, 2143delT, 2183AA>G, N1303K July 2009 - currently Sanger sequencing of exons: 7, 10, 11, 13, 17b, 20, 21, fragment of intron 19 F508del, CFTRdele2,3, 3849+10kbC>T, R334W, R347P, 1717-1G>A, G542X, R553X, K710X, 2184insA, 2143delT, 2183AA>G, N1303K, 3272-26A>G**, W1282X** * removed from DNA analysis since July 2009 , **added into DNA analysis since July 2009 Figure 1 NBS CF in Poland. Login to comment

[hide] Improving test properties for neonatal cystic fibr... J Inherit Metab Dis. 2012 Jul;35(4):635-40. Cornel MC, Gille JJ, Loeber JG, Vernooij-van Langen AM, Dankert-Roelse J, Bolhuis PA
Improving test properties for neonatal cystic fibrosis screening in the Netherlands before the nationwide start by May 1st 2011.
J Inherit Metab Dis. 2012 Jul;35(4):635-40., [PMID:22302635]

Abstract [show]
When new technical possibilities arise in health care, often attunement is needed between different actors from the perspectives of research, health care providers, patients, ethics and policy. For cystic fibrosis (CF) such a process of attunement in the Netherlands started in a committee of the Health Council on neonatal screening in 2005. In the balancing of pros and cons according to Wilson and Jungner criteria, the advantages for the CF patient were considered clear, even though CF remains a severe health problem with treatment. Nevertheless, screening was not started then, mainly since the specificity of the tests available at that time was considered too low. Many healthy infants would have been referred for sweat testing and much uncertainty would arise in their parents. Also the limited sensitivity for immigrants and the detection of less severe phenotypes and carriers were considered problematic. The Health Council recommended a pilot screening project which was subsequently performed in some provinces, leading to a 4-step protocol: IRT, PAP, screening for a CFTR mutation panel, and sequencing of the CFTR gene. This would lead to the identification of 23 cases of classical CF, two infants with less severe forms and 12 carriers per year in the Netherlands. Thus many CF patients can be diagnosed early, while limiting the number of referrals, the number of infants with less severe forms diagnosed and the number of carriers identified. Technical solutions were found to limit the ethical problems. A nationwide program using this four step protocol started by 1 May 2011.

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69 This protocol was expected to identify 25 CF patients on an annual basis, additional to four infants already diagnosed because of meconium ileus (Health Council of 1 Using the LiPA test (INNO-LiPA CFTR 19 en INNO-LiPA CFTR 17+Tn; Innogenetics, Gent, Belgium) the following CFTR mutations can be detected: exon 2-3del (21 kb), 394delTT, E60X, G85E, R117H, 621+1G>T, 711+1G>T, 711+5G>A, 1078delT, R334W, R347P, A455E, I507del, F508del, 1717-1G>A, G542X, G551D, Q552X, R553X, R560T, 1898+1G>A, 2143delT, 2183AA>G, 2184delA, 2789+5G>A, 3120+1G>A, 3199del6, 3272-26A>G, 3659delC, R1162X, 3849+10kbC>T, 3905insT, S1251N, W1282X en N1303K. Login to comment
70 This test also identifies the CFTR polymorphism Tn in intron 8 which is important in cases where the mutation R117H is detected. Login to comment

[hide] Rapid detection of the ACMG/ACOG-recommended 23 CF... J Biomol Tech. 2012 Apr;23(1):24-30. Elliott AM, Radecki J, Moghis B, Li X, Kammesheidt A
Rapid detection of the ACMG/ACOG-recommended 23 CFTR disease-causing mutations using ion torrent semiconductor sequencing.
J Biomol Tech. 2012 Apr;23(1):24-30., [PMID:22468138]

Abstract [show]
Cystic fibrosis (CF) is one of the most frequently diagnosed autosomal-recessive diseases in the Caucasian population. For general-population CF carrier screening, the American College of Medical Genetics (ACMG)/American College of Obstetricians and Gynecologists (ACOG) have recommended a core panel of 23 mutations that will identify 49-98% of carriers, depending on ethnic background. Using a genotyping technology that can rapidly identify disease-causing mutations is important for high-throughput general-population carrier screening, confirming clinical diagnosis, determining treatment options, and prenatal diagnosis. Here, we describe a proof-of-concept study to determine whether the Ion Torrent Personal Genome Machine (PGM) sequencer platform can reliably identify all ACMG/ACOG 23 CF transmembrane conductance regulator (CFTR) mutations. A WT CF specimen along with mutant DNA specimens representing all 23 CFTR mutations were sequenced bidirectionally on the Ion Torrent 314 chip to determine the accuracy of the PGM for CFTR variant detection. We were able to reliably identify all of the targeted mutations except for 2184delA, which lies in a difficult, 7-mer homopolymer tract. Based on our study, we believe PGM sequencing may be a suitable technology for identifying CFTR mutations in the future. However, as a result of the elevated rate of base-calling errors within homopolymer stretches, mutations within such regions currently need to be evaluated carefully using an alternative method.

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26 Amplicons were then pooled together in equimolar concentrations and purified using the T A B L E 1 Data Generation from Three PGM Runs Run Total number of reads Total bases (Mbp) AQ17 total bases (Mbp) AQ17 avg. read length CF WT 101,211 8.5 6.5 68 CF 23 pooled mutants 222,247 18.6 12.52 64 CF mutant 135,000 11.7 8.8 72 T A B L E 2 CFTR Variant Coverage, Mutant Read Percentage, and Base-Call Accuracy from a WT Library Using PGM Sequencing Variant cDNA position Coverage Mutant read % Accuracy/base G85E c.254G Ͼ A 408 0 99.5 R117H c.350G Ͼ A 3627 0 99.9 621 ϩ 1G Ͼ T c.489 ϩ 1G Ͼ T 245 0 99.6 711 ϩ 1G Ͼ T c.579 ϩ 1G Ͼ T 2660 0 99.9 R334W c.1000C Ͼ T 5419 0 99.7 R347P c.1040G Ͼ C 3562 0 99.4 A455E c.1364C Ͼ A 10,340 0 99.9 ⌬I507 c.1519_1521delATC 6507 0 98.6 ⌬F508 c.1521_1523delCTT 6507 0 99.4 1717-1G Ͼ A c.1585-1G Ͼ A 2086 0 99.2 G542X c.1624G Ͼ T 854 0 97.8 G551D c.1652G Ͼ A 3901 0 99 R553X c.1657C Ͼ T 3915 0 99.9 R560T c.1679G Ͼ C 3924 0 99.6 1898 ϩ 1G Ͼ A c.1766 ϩ 1G Ͼ A 1793 0 97.6 2184delAa c.2052delA 2001 35% 63.6 2789 ϩ 5G Ͼ A c.2657 ϩ 5G Ͼ A 293 0 100 3120 ϩ 1G Ͼ A c.2988 ϩ 1G Ͼ A 2408 0 100 R1162X c.3484C Ͼ T 9610 0 98.1 3659delC c.3528delC 9271 0 100 3849 ϩ 10kbC Ͼ T c.3717 ϩ 12191C Ͼ T 10,157 0 99.9 W1282X c.3846G Ͼ A 4789 0 95.6 N1303K c.3909C Ͼ G 3236 0 99.5 a The 2184delA variant lies in a homopolymer stretch of seven adenines and is not detected accurately as a result of homopolymer-length sequencing errors. Login to comment
67 For this data set, the PGM 314 chip output was 18.6 Mbp, with ϳ67% aligning to the CFTR T A B L E 3 PGM CFTR Variant Coverage and Mutant Read Percentage from a Pooled Mutant Library Representing All 23 ACMG/ACOG Mutations Variant cDNA position Coverage Mutant read % Predicted read % Genotype G85E c.254G Ͼ A 93 33 50 Het R117H c.350G Ͼ A 6228 39 50 Het 621 ϩ 1G Ͼ T c.489 ϩ 1G Ͼ T 1243 46 50 Het 711 ϩ 1G Ͼ T c.579 ϩ 1G Ͼ T 1352 29 50 Het R334W c.1000C Ͼ T 13,284 8 25 Het R347P c.1040G Ͼ C 9454 27 25 Het A455E c.1364C Ͼ A 19,527 43 50 Het ⌬I507 c.1519_1521delATC 15,587 14 25 Het ⌬F508 c.1521_1523delCTT 15,587 68 50 Homo 1717-1G Ͼ A c.1585-1G Ͼ A 3584 36 50 Het G542X c.1624G Ͼ T 610 41 50 Het G551D c.1652G Ͼ A 6714 16 17 Het R553X c.1657C Ͼ T 6670 15 17 Het R560T c.1679G Ͼ C 6395 22 17 Het 1898 ϩ 1G Ͼ A c.1766 ϩ 1G Ͼ A 3293 49 50 Het 2184delAa c.2052delA 2256 63 50 Het 2789 ϩ 5G Ͼ A c.2657 ϩ 5G Ͼ A 1765 54 50 Het 3120 ϩ 1G Ͼ A c.2988 ϩ 1G Ͼ A 7447 40 50 Het R1162X c.3484C Ͼ T 19,060 54 50 Het 3659delC c.3528delC 28,321 30 50 Het 3849 ϩ 10kbC Ͼ T c.3717 ϩ 12191C Ͼ T 27,102 46 50 Het W1282X c.3846G Ͼ A 9219 48 50 Het N1303K c.3909C Ͼ G 4842 49 50 Het a The 2184delA variant lies in a homopolymer stretch of seven adenines and is not accurately detected as a result of homopolymer-length sequencing errors. Login to comment
70 Variant G85E had relatively low coverage of 93 reads. Login to comment
71 The G85E region consistently resulted in low read counts for all PGM runs that we conducted. Login to comment
73 Low read counts in relation to the other variants were also observed for G85E using the Illumina GAIIx platform. Login to comment
86 Using samples characterized previously, we analyzed the PGM`s data out- T A B L E 4 PGM CFTR Variant Coverage and Mutant Read Percentage from an Individual Harboring Two Disease-Causing CFTR Mutations Variant cDNA position Coverage Mutant read % G85E c.254G Ͼ A 237 0 R117H c.350G Ͼ A 3774 0 621 ϩ 1G Ͼ T c.489 ϩ 1G Ͼ T 936 0 711 ϩ 1G Ͼ T c.579 ϩ 1G Ͼ T 2018 0 R334W c.1000C Ͼ T 10,899 0 R347P c.1040G Ͼ C 7720 0 A455E c.1364C Ͼ A 14,525 0 ⌬I507 c.1519_1521delATC 8855 0 ⌬F508 c.1521_1523delCTT 8855 47 1717-1G Ͼ A c.1585-1G Ͼ A 2216 0 G542X c.1624G Ͼ T 2035 41 G551D c.1652G Ͼ A 4581 0 R553X c.1657C Ͼ T 4545 0 R560T c.1679G Ͼ C 4774 0 1898 ϩ 1G Ͼ A c.1766 ϩ 1G Ͼ A 2702 0 2184delAa c.2052delA 2837 18.5 2789 ϩ 5G Ͼ A c.2657 ϩ 5G Ͼ A 860 0 3120 ϩ 1G Ͼ A c.2988 ϩ 1G Ͼ A 4347 0 R1162X c.3484C Ͼ T 12,039 0 3659delC c.3528delC 7169 0 3849 ϩ 10kbC Ͼ T c.3717 ϩ 12191C Ͼ T 11,588 0 W1282X c.3846G Ͼ A 6187 0 N1303K c.3909C Ͼ G 4479 0 a The 2184delA variant lies in a homopolymer stretch of seven adenines and is not accurately detected as a result of homopolymer-length sequencing errors. Login to comment
100 However, the G85E variant, which was under-represented consistently in the PGM data, also had relatively low coverage when sequenced on the Illumina platform. Login to comment
101 In this instance, the complexity of the G85E genomic region makes it a difficult target to sequence. Login to comment
66 For this data set, the PGM 314 chip output was 18.6 Mbp, with b03;67% aligning to the CFTR T A B L E 3 PGM CFTR Variant Coverage and Mutant Read Percentage from a Pooled Mutant Library Representing All 23 ACMG/ACOG Mutations Variant cDNA position Coverage Mutant read % Predicted read % Genotype G85E c.254G b0e; A 93 33 50 Het R117H c.350G b0e; A 6228 39 50 Het 621 af9; 1G b0e; T c.489 af9; 1G b0e; T 1243 46 50 Het 711 af9; 1G b0e; T c.579 af9; 1G b0e; T 1352 29 50 Het R334W c.1000C b0e; T 13,284 8 25 Het R347P c.1040G b0e; C 9454 27 25 Het A455E c.1364C b0e; A 19,527 43 50 Het èc;I507 c.1519_1521delATC 15,587 14 25 Het èc;F508 c.1521_1523delCTT 15,587 68 50 Homo 1717-1G b0e; A c.1585-1G b0e; A 3584 36 50 Het G542X c.1624G b0e; T 610 41 50 Het G551D c.1652G b0e; A 6714 16 17 Het R553X c.1657C b0e; T 6670 15 17 Het R560T c.1679G b0e; C 6395 22 17 Het 1898 af9; 1G b0e; A c.1766 af9; 1G b0e; A 3293 49 50 Het 2184delAa c.2052delA 2256 63 50 Het 2789 af9; 5G b0e; A c.2657 af9; 5G b0e; A 1765 54 50 Het 3120 af9; 1G b0e; A c.2988 af9; 1G b0e; A 7447 40 50 Het R1162X c.3484C b0e; T 19,060 54 50 Het 3659delC c.3528delC 28,321 30 50 Het 3849 af9; 10kbC b0e; T c.3717 af9; 12191C b0e; T 27,102 46 50 Het W1282X c.3846G b0e; A 9219 48 50 Het N1303K c.3909C b0e; G 4842 49 50 Het a The 2184delA variant lies in a homopolymer stretch of seven adenines and is not accurately detected as a result of homopolymer-length sequencing errors. Login to comment
69 Variant G85E had relatively low coverage of 93 reads. Login to comment
72 Low read counts in relation to the other variants were also observed for G85E using the Illumina GAIIx platform. Login to comment
85 Using samples characterized previously, we analyzed the PGM`s data out- T A B L E 4 PGM CFTR Variant Coverage and Mutant Read Percentage from an Individual Harboring Two Disease-Causing CFTR Mutations Variant cDNA position Coverage Mutant read % G85E c.254G b0e; A 237 0 R117H c.350G b0e; A 3774 0 621 af9; 1G b0e; T c.489 af9; 1G b0e; T 936 0 711 af9; 1G b0e; T c.579 af9; 1G b0e; T 2018 0 R334W c.1000C b0e; T 10,899 0 R347P c.1040G b0e; C 7720 0 A455E c.1364C b0e; A 14,525 0 èc;I507 c.1519_1521delATC 8855 0 èc;F508 c.1521_1523delCTT 8855 47 1717-1G b0e; A c.1585-1G b0e; A 2216 0 G542X c.1624G b0e; T 2035 41 G551D c.1652G b0e; A 4581 0 R553X c.1657C b0e; T 4545 0 R560T c.1679G b0e; C 4774 0 1898 af9; 1G b0e; A c.1766 af9; 1G b0e; A 2702 0 2184delAa c.2052delA 2837 18.5 2789 af9; 5G b0e; A c.2657 af9; 5G b0e; A 860 0 3120 af9; 1G b0e; A c.2988 af9; 1G b0e; A 4347 0 R1162X c.3484C b0e; T 12,039 0 3659delC c.3528delC 7169 0 3849 af9; 10kbC b0e; T c.3717 af9; 12191C b0e; T 11,588 0 W1282X c.3846G b0e; A 6187 0 N1303K c.3909C b0e; G 4479 0 a The 2184delA variant lies in a homopolymer stretch of seven adenines and is not accurately detected as a result of homopolymer-length sequencing errors. Login to comment
99 However, the G85E variant, which was under-represented consistently in the PGM data, also had relatively low coverage when sequenced on the Illumina platform. Login to comment

[hide] CFTR, SPINK1, CTRC and PRSS1 variants in chronic p... Gut. 2012 Mar 17. Rosendahl J, Landt O, Bernadova J, Kovacs P, Teich N, Bodeker H, Keim V, Ruffert C, Mossner J, Kage A, Stumvoll M, Groneberg D, Kruger R, Luck W, Treiber M, Becker M, Witt H
CFTR, SPINK1, CTRC and PRSS1 variants in chronic pancreatitis: is the role of mutated CFTR overestimated?
Gut. 2012 Mar 17., [PMID:22427236]

Abstract [show]
OBJECTIVE: In chronic pancreatitis (CP), alterations in several genes have so far been described, but only small cohorts have been extensively investigated for all predisposing genes. DESIGN: 660 patients with idiopathic or hereditary CP and up to 1758 controls were enrolled. PRSS1, SPINK1 and CTRC were analysed by DNA sequencing, and cystic fibrosis transmembrane conductance regulator (CFTR) by melting curve analysis. RESULTS: Frequencies of CFTR variants p.R75Q, p.I148T, 5T-allele and p.E528E were comparable in patients and controls. We identified 103 CFTR variants, which represents a 2.7-fold risk increase (p<0.0001). Severe cystic fibrosis (CF)-causing variants increased the risk of developing CP 2.9-fold, and mild CF-causing variants 4.5-fold (p<0.0001 for both). Combined CF-causing variants increased CP risk 3.4-fold (p<0.0001), while non-CF-causing variants displayed a 1.5-fold over-representation in patients (p=0.14). CFTR compound heterozygous status with variant classes CF-causing severe and mild represented an OR of 16.1 (p<0.0001). Notably, only 9/660 (1.4%) patients were compound heterozygotes in this category. Trans-heterozygosity increased CP risk, with an OR of 38.7, with 43/660 (6.5%) patients and 3/1667 (0.2%) controls being trans-heterozygous (p<0.0001). CONCLUSIONS: Accumulation of CFTR variants in CP is less pronounced than reported previously, with ORs between 2.7 and 4.5. Only CF-causing variants reached statistical significance. Compound and trans-heterozygosity is an overt risk factor for the development of CP, but the number of CFTR compound heterozygotes in particular is rather low. In summary, the study demonstrates the complexity of genetic interactions in CP and a minor influence of CFTR alterations in CP development.

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72 The following CFTR variants were analysed with specific FRET probes: p.E60X, p.R75Q, p.G85E, p.R117H, p.I148T, c.621 +1G>T (IVS4+1G>T), c.711+1G>T (IVS5+1G>T), c.1078delT, p.R334W, p.R347P, 9-13TG, 5-9T, p.A455E, p.M470V, p.F508del, c.1716G>A (p.E528E), c.1717-1G>A (IVS10-1G>A), p.G542X, p.S549N, p.R553X, p.R560T, c.1898+1G>A (IVS12 +1G>A), c.2143delT, c.2183AA>G, c.2562T>G, c.2657+5G>A (IVS14B+5G>A), p.L997F, p.I1005R, p.Y1092X, p.D1152H, p.R1162X, c.3659delC, p.S1235R, p.S1251N, p.W1282X, p.N1303K, and c.4389G>A. Login to comment
69 The following CFTR variants were analysed with specific FRET probes: p.E60X, p.R75Q, p.G85E, p.R117H, p.I148T, c.621 +1G>T (IVS4+1G>T), c.711+1G>T (IVS5+1G>T), c.1078delT, p.R334W, p.R347P, 9-13TG, 5-9T, p.A455E, p.M470V, p.F508del, c.1716G>A (p.E528E), c.1717-1G>A (IVS10-1G>A), p.G542X, p.S549N, p.R553X, p.R560T, c.1898+1G>A (IVS12 +1G>A), c.2143delT, c.2183AA>G, c.2562T>G, c.2657+5G>A (IVS14B+5G>A), p.L997F, p.I1005R, p.Y1092X, p.D1152H, p.R1162X, c.3659delC, p.S1235R, p.S1251N, p.W1282X, p.N1303K, and c.4389G>A. Login to comment

[hide] CFTR mutation analysis and haplotype associations ... Mol Genet Metab. 2012 Feb;105(2):249-54. doi: 10.1016/j.ymgme.2011.10.013. Epub 2011 Oct 26. Cordovado SK, Hendrix M, Greene CN, Mochal S, Earley MC, Farrell PM, Kharrazi M, Hannon WH, Mueller PW
CFTR mutation analysis and haplotype associations in CF patients.
Mol Genet Metab. 2012 Feb;105(2):249-54. doi: 10.1016/j.ymgme.2011.10.013. Epub 2011 Oct 26., [PMID:22137130]

Abstract [show]
Most newborn screening (NBS) laboratories use second-tier molecular tests for cystic fibrosis (CF) using dried blood spots (DBS). The Centers for Disease Control and Prevention's NBS Quality Assurance Program offers proficiency testing (PT) in DBS for CF transmembrane conductance regulator (CFTR) gene mutation detection. Extensive molecular characterization on 76 CF patients, family members or screen positive newborns was performed for quality assurance. The coding, regulatory regions and portions of all introns were sequenced and large insertions/deletions were characterized as well as two intronic di-nucleotide microsatellites. For CF patient samples, at least two mutations were identified/verified and four specimens contained three likely CF-associated mutations. Thirty-four sequence variations in 152 chromosomes were identified, five of which were not previously reported. Twenty-seven of these variants were used to predict haplotypes from the major haplotype block defined by HapMap data that spans the promoter through intron 19. Chromosomes containing the F508del (p.Phe508del), G542X (p.Gly542X) and N1303K (p.Asn1303Lys) mutations shared a common haplotype subgroup, consistent with a common ancient European founder. Understanding the haplotype background of CF-associated mutations in the U.S. population provides a framework for future phenotype/genotype studies and will assist in determining a likely cis/trans phase of the mutations without need for parent studies.

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104 Mutation N alleles c.966T>G(5'flanking) c.234T>A(5'flanking)a c.-8G>C(5'UTR) c.-4G>C(Exon1) c.274-179G>A(Intron3) c.743+40A>G(Intron6) c.744-31TTGA(5_7)(Intron6) c.869+11C>T(Intron7) c.869+88T>A(Intron7) c.1209+43T>G(Intron9) IVS8CA(15-23)(Intron9) TG(10-13)_T(5-9)(Intron9) c.1393-61A>G(Intron10) M470V(Exon11) F508del(Exon11) c.1766+152T>A(Intron13) c.1767-231T>C(Intron13) c.1767-136T>C(Intron13) c.1767-132A>G(Intron13) c.2562T>G(Exon15) c.2604A>G(Exon15) c.2619+86_2619+87del(Intron15) c.2619+106T>A(Intron15) c.2909-92G>A(Intron17) IVS17bCA(11-17)(Intron20) c.3368-140A>C(Intron20) c.3469-65C>A(Intron21) F508del 32 TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- GA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- A5- 55- 55- 55- 66- 66- 66- 66- 66- 66- 66- 66- 66- 66- 55- 55- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TC- TT- TT- TT- TC- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TG- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- T17- 10_9- G- F508del- TA- 13C F508del 29 G23- 10_9- G- F508del- TA- 13C F508del 1 G21- 10_9- G- GG- G-F508del- TA- 13C F508del 1 G17- 10_9- G- F508del- A- G- delTA- 17- C- A N1303K 6 G542X 6 3849+10kbC→T 1 del Ex17a, b, Ex18 1 GG- GG- GG- 23- 10_9- GG-F508- T- TA- 13- C A455E 1 G22- 10_9- G- F508- T- TA- 13- C 621+1G→T 5 G21- 10_9- G- GG- GG- F508C- TA- 13- C 711+1G→T 3 3272-26A→G 2 3659delC 2 R347P 2 G16- 11_7- A- A-F508- TA- 13C del Ex 2, 3 2 del Ex 17a,17b 2 Normal 1 R334W 2 G17- 11_7- A- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA-AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- A-AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- AA- F508- TA- 13C 2183AA→G 2 G16- 10_7- F508- TATA- TATA- TATA- TATA- TATA- TATA- 13C del Ex 2 1 G16- 11_7- F508- 14C 1288insTA 1 G16- 12_7- F508- 13C Normal 1 G16- 12_7- F508- 13C R1162X 1 G17- 10_7- F508- 13C del Ex 2,3 1 G16- 11_7- F508- A17- C del Ex 17a,17b 1 GA- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT- TT-16- 11_7- F508- 14- C G85E 1 G16- 11_7- F508- 15C 1898+1G→A 1 G16- 11_7- F508- G13- C no mut detected 1 GT- TT- T16- 10_7- F508- 13C no mut detected 1 G16- 10_7- F508- 17A W1282X 2 G17- 10_7- F508- 17A W1282X 4 GC- CC- C17- 10_7- F508- delTA- 17- A Q39X 1 I507del 1 3849+10kbC→T 1 R560T 2 1717-1G→A 2 G551D 3 G16- 10_7- F508- delTA- 17- A G551D 2 1154insTC 1 G16- 10_7- F508- delTA- 17- 1717- 17A 1717-1G→A 1 2789+5G→A 1 GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- G17- 10_7- F508- AdelTA- A R1066C 1 GG- 17- 10_7- F508- delTA- A R1066H 1 GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- GG- G17- 9_7- F508- delTAC R553X 3 GG- GG- CA- AA- AA- AA- A17- 12_7- F508- delTA- 11- C 3121-1G→A 1 C17- 12_7- F508- delTA- 11- C R334W 1 G17- 12_7- F508- TA- 13- C (TG)13T5b 1 G17- 13_5- F508- delTA- 13- C CC- CC- CC- CC- CC- CC- CC- CC- CC- CC- CC- CC- CC- CC- CC- CC- CC- R117H 1 CA- 6C- TT- 15- 12_5- AG- F508- T- TT- AT- ATA- TG- 13A- C R117H1 1 CA- 6C- TT- 16- 12_5- AG- F508- T- TT- AT- ATA- TG- 13A- C 1717-1G→A 1 R117Hb 1 GA- 6C- TT- 16- 10_7- AA- F508- A- TC- AG- AdelTA- TG- 13A- C 144c a Variation found in a sample where the haplotype could not be predicted. Login to comment

[hide] Frequency of the hyperactive W493R ENaC variant in... J Cyst Fibros. 2012 Jan;11(1):53-5. Epub 2011 Sep 13. Handschick M, Hedtfeld S, Tummler B
Frequency of the hyperactive W493R ENaC variant in carriers of a CFTR mutation.
J Cyst Fibros. 2012 Jan;11(1):53-5. Epub 2011 Sep 13., [PMID:21917531]

Abstract [show]
BACKGROUND: The basic defect of the autosomal recessive disorder cystic fibrosis (CF) manifests in chloride hyposecretion and sodium hyperabsorption. CF-like disease has been reported in a heterozygous carrier of F508del CFTR and the hyperactive variant p.W493R-SCNN1A of the epithelial sodium channel (ENaC). METHODS: The hypothesis that heterozygosity for p.W493R-SCNN1A and one loss-of-function CFTR mutation causes or predisposes to CF or CF-like disease was tested in 441 parents of a child with CF. RESULTS: p.W493R-SCNN1A was detected in three female carriers of F508del CFTR who did not show any symptoms of respiratory or intestinal disease that could be interpreted as the manifestation of CF or CFTR-related disorder. Frequency of p.W493R was lower in CF parents than in Caucasian control subjects. CONCLUSIONS: A hyperactive ENaC does not necessarily cause CF-like disease in a CF gene carrier, but its low frequency in CF parents suggests that it is a risk factor.

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53 A. Caucasians a F508del 378 2184delA 2 CFTRdele2,3(21 kb) 4 2789+5 G-A 1 R117H 1 I1005R 1 405+1 G-A 1 L1077P 1 H199Y 1 Y1092X 1 L206W 1 3601-111 G-C 1 R347P 3 3849+10 kb C-T 1 Q414X 1 3850-3 T-G 1 G551D 4 W1282X 1 R553X 8 N1303K 2 1717-1 G-A 1 4374+1 G-T 1 2143delT 1 Unknown 9 B. Turks K68N 1 1525-1 G-A 1 G85E 1 F508del 2 E92K 1 1677delTA 1 CFTRdele2(ins186) 2 2184delA 1 CFTRdele2,3(21 kb) 2 3601-2 A-G 1 435insA 1 Unknown 1 a The subjects were born in Austria (N=9 subjects), Belgium (2), France (4), Germany (374), Greece (4), Italy (12), The Netherlands (7), Poland (2), Spain (5), Sweden (2) and United Kingdom (5). Login to comment

[hide] Extensive molecular analysis of patients bearing C... J Mol Diagn. 2012 Jan;14(1):81-9. Epub 2011 Oct 20. Amato F, Bellia C, Cardillo G, Castaldo G, Ciaccio M, Elce A, Lembo F, Tomaiuolo R
Extensive molecular analysis of patients bearing CFTR-related disorders.
J Mol Diagn. 2012 Jan;14(1):81-9. Epub 2011 Oct 20., [PMID:22020151]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR)-related disorders (CFTR-RDs) may present with pancreatic sufficiency, normal sweat test results, and better outcome. The detection rate of mutations is lower in CFTR-RD than in classic CF: mutations may be located in genes encoding proteins that interact with CFTR or support channel activity. We tested the whole CFTR coding regions in 99 CFTR-RD patients, looking for gene mutations in solute carrier (SLC) 26A and in epithelial Na channel (ENaC) in 33 patients who had unidentified mutations. CFTR analysis revealed 28 mutations, some of which are rare. Of these mutations, RT-PCR demonstrated that the novel 1525-1delG impairs exon 10 splicing; by using minigene analysis, we excluded the splicing effect of three other novel intronic variants. Analysis of SLC26A genes revealed several variants, some of which are novel, that did not affect mRNA expression. Other mutations occurred in the ENaC genes encoding the ENaC subunits, but their frequency did not significantly differ between patients and controls. Our data, although obtained on a preliminary cohort of CFTR-RD patients, exclude a role of mutations in SLC26A and in SCNN genes in the pathogenesis of such disease; we confirm that CFTR analysis has a relevant role in CFTR-RD patients; and it appears mandatory to use CFTR scanning techniques and approaches to reveal the effect of novel mutations.

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69 Allele Frequency and CFTR Mutations in Patients Bearing CFTR-RDs Mutation (traditional name) HGVS nomenclature15 CBAVD (118 alleles)* RP (42 alleles)* DB (38 alleles)* Total (198 alleles)* TG12-T5-470V 34 (28.8) 2 (4.8) 10 (26.3) 46 (23.2) F508del c.1521_1523del 19 (16.1) 7 (16.7) 4 (10.5) 30 (15.2) 3195del6 c.3063_3069del 9 (7.6) 0 0 9 (4.5) N1303K c.3909CϾG 3 (2.5) 1 (2.4) 4 (10.5) 8 (4.0) G542X c.1624GϾT 4 (3.4) 1 (2.4) 1 (2.6) 6 (3.0) D1152H c.3454GϾC 1 (0.8) 2 (4.8) 2 (5.3) 5 (2.5) G85E c.254GϾA 2 (1.7) 3 (7.1) 0 5 (2.5) 1525-1delG c.1394de 3 (2.5) 1 (2.4) 0 4 (3.0) 4016insT c.3885insT 2 (1.7) 1 (2.4) 0 3 (1.5) 2789ϩ5GϾA c.2657ϩ5GϾA 0 3 (7.1) 0 3 (1.5) Q1476X c.4426CϾT 3 (2.5) 0 0 3 (1.5) 2183AAϾG c.2051_2052delinsG 1 (0.8) 1 (2.4) 0 2 (1.0) R553X c.1657CϾT 1 (0.8) 1 (2.4) 0 2 (1.0) L568F c.1704GϾT 2 (1.7) 0 0 2 (1.0) R1158X c.3472CϾT 2 (1.7) 0 0 2 (1.0) V920M c.2758GϾA 1 (0.8) 0 1 (2.6) 2 (1.0) 711ϩ1GϾT c.579ϩ1GϾT 0 1 (2.4) 0 1 (0.5) D614G c.1841AϾG 1 (0.8) 0 0 1 (0.5) 2184insA c.2052del 0 1 (2.4) 0 1 (0.5) 621ϩ1GϾT c.489ϩ1GϾT 1 (0.8) 0 0 1 (0.5) R1438W c.4312CϾT 0 1 (2.4) 0 1 (0.5) E193X c.577GϾT 0 1 (2.4) 0 1 (0.5) G1244E c.3731GϾA 1 (0.8) 0 0 1 (0.5) K68E c.202AϾG 1 (0.8) 0 0 1 (0.5) R347P c.1040GϾC 1 (0.8) 0 0 1 (0.5) 621ϩ3AϾG c.489ϩ3AϾG 1 (0.8) 0 0 1 (0.5) L997F c.2991GϾC 0 1 (2.4) 0 1 (0.5) F508C c.1523TϾG 1 (0.8) 0 0 1 (0.5) Total 94 (79.7) 28 (66.7) 22 (57.9) 144 (72.7) Undetected 24 (20.3) 14 (33.3) 16 (42.1) 54 (27.3) *Data are given as number (percentage). Login to comment
86 Six mutations were present in Ͼ2.0% of chromosomes (namely, 3195del6, N1303K, G542X, D1152H, 1525-1delG, and G85E). Login to comment

[hide] Measurements of CFTR-Mediated Cl(-) Secretion in H... PLoS One. 2012;7(10):e47708. doi: 10.1371/journal.pone.0047708. Epub 2012 Oct 17. Sousa M, Servidoni MF, Vinagre AM, Ramalho AS, Bonadia LC, Felicio V, Ribeiro MA, Uliyakina I, Marson FA, Kmit A, Cardoso SR, Ribeiro JD, Bertuzzo CS, Sousa L, Kunzelmann K, Ribeiro AF, Amaral MD
Measurements of CFTR-Mediated Cl(-) Secretion in Human Rectal Biopsies Constitute a Robust Biomarker for Cystic Fibrosis Diagnosis and Prognosis.
PLoS One. 2012;7(10):e47708. doi: 10.1371/journal.pone.0047708. Epub 2012 Oct 17., [PMID:23082198]

Abstract [show]
BACKGROUND: Cystic Fibrosis (CF) is caused by approximately 1,900 mutations in the CF transmembrane conductance regulator (CFTR) gene encoding for a cAMP-regulated chloride (Cl(-)) channel expressed in several epithelia. Clinical features are dominated by respiratory symptoms, but there is variable organ involvement thus causing diagnostic dilemmas, especially for non-classic cases. METHODOLOGY/PRINCIPAL FINDINGS: To further establish measurement of CFTR function as a sensitive and robust biomarker for diagnosis and prognosis of CF, we herein assessed cholinergic and cAMP-CFTR-mediated Cl(-) secretion in 524 freshly excised rectal biopsies from 118 individuals, including patients with confirmed CF clinical diagnosis (n = 51), individuals with clinical CF suspicion (n = 49) and age-matched non-CF controls (n = 18). Conclusive measurements were obtained for 96% of cases. Patients with "Classic CF", presenting earlier onset of symptoms, pancreatic insufficiency, severe lung disease and low Shwachman-Kulczycki scores were found to lack CFTR-mediated Cl(-) secretion (<5%). Individuals with milder CF disease presented residual CFTR-mediated Cl(-) secretion (10-57%) and non-CF controls show CFTR-mediated Cl(-) secretion >/=30-35% and data evidenced good correlations with various clinical parameters. Finally, comparison of these values with those in "CF suspicion" individuals allowed to confirm CF in 16/49 individuals (33%) and exclude it in 28/49 (57%). Statistical discriminant analyses showed that colonic measurements of CFTR-mediated Cl(-) secretion are the best discriminator among Classic/Non-Classic CF and non-CF groups. CONCLUSIONS/SIGNIFICANCE: Determination of CFTR-mediated Cl(-) secretion in rectal biopsies is demonstrated here to be a sensitive, reproducible and robust predictive biomarker for the diagnosis and prognosis of CF. The method also has very high potential for (pre-)clinical trials of CFTR-modulator therapies.

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188 Interestingly, and corroborating such prediction among these four, there is a 19-year old patient with the F508del/G85E genotype, moderate PI (FEE = 103.89 mg/g) and ,12% of CFTR function (Fig.S2-C, Table S1) who recently (last 1K yr) started to progress from a relatively mild to moderate-severe lung disease, with five pulmonary exacerbations plus surgery to remove nasal polyps associated with a strong sinusitis. Login to comment
209 Rectal biopsies from (A) Non-CF individual showing large cholinergic (carbachol, CCH, 100 mM, basolateral) and cAMP-dependent (3-isobutyl-1-methylxantine, IBMX, 100 mM, and forskolin, Fsk, 2 mM, basolateral) Chloride (Cl2 ) secretion (lumen-negative responses); (B) CF patient homozygous for F508del-CFTR mutation with absence of Cl2 secretion (only lumen-positive responses, reflecting potassium (K+ ) secretion, were observed); (C) CF patient (genotype: F508del/G85E-CFTR) showing very little (,12%) cAMP-dependent Cl2 secretion (biphasic responses observed upon co-cholinergic stimulation with CCH); and (D) CF patient (genotype: 3120+1G.A/L206W-CFTR) presenting larger CFTR residual function (,57%) and milder phenotype than in (C). Login to comment
208 Rectal biopsies from (A) Non-CF individual showing large cholinergic (carbachol, CCH, 100 mM, basolateral) and cAMP-dependent (3-isobutyl-1-methylxantine, IBMX, 100 mM, and forskolin, Fsk, 2 mM, basolateral) Chloride (Cl2 ) secretion (lumen-negative responses); (B) CF patient homozygous for F508del-CFTR mutation with absence of Cl2 secretion (only lumen-positive responses, reflecting potassium (K+ ) secretion, were observed); (C) CF patient (genotype: F508del/G85E-CFTR) showing very little (,12%) cAMP-dependent Cl2 secretion (biphasic responses observed upon co-cholinergic stimulation with CCH); and (D) CF patient (genotype: 3120+1G.A/L206W-CFTR) presenting larger CFTR residual function (,57%) and milder phenotype than in (C). Login to comment

[hide] Alteration of CFTR transmembrane span integration ... Mol Biol Cell. 2011 Dec;22(23):4461-71. Epub 2011 Oct 12. Patrick AE, Karamyshev AL, Millen L, Thomas PJ
Alteration of CFTR transmembrane span integration by disease-causing mutations.
Mol Biol Cell. 2011 Dec;22(23):4461-71. Epub 2011 Oct 12., [PMID:21998193]

Abstract [show]
Many missense mutations in the cystic fibrosis transmembrane conductance regulator protein (CFTR) result in its misfolding, endoplasmic reticulum (ER) accumulation, and, thus, cystic fibrosis. A number of these mutations are located in the predicted CFTR transmembrane (TM) spans and have been projected to alter span integration. However, the boundaries of the spans have not been precisely defined experimentally. In this study, the ER luminal integration profiles of TM1 and TM2 were determined using the ER glycosylation machinery, and the effects of the CF-causing mutations G85E and G91R thereon were assessed. The mutations either destabilize the integrated conformation or alter the TM1 ER integration profile. G85E misfolding is based in TM1 destabilization by glutamic acid and loss of glycine and correlates with the temperature-insensitive ER accumulation of immature full-length CFTR harboring the mutation. By contrast, temperature-dependent misfolding owing to the G91R mutation depends on the introduction of the basic side chain rather than the loss of the glycine. This work demonstrates that CF-causing mutations predicted to have similar effects on CFTR structure actually result in disparate molecular perturbations that underlie ER accumulation and the pathology of CF.

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3 In this study, the ER luminal integration profiles of TM1 and TM2 were determined using the ER glycosylation machinery, and the effects of the CF-causing mutations G85E and G91R thereon were assessed. Login to comment
5 G85E misfolding is based in TM1 destabilization by glutamic acid and loss of glycine and correlates with the temperature-insensitive ER accumulation of immature full-length CFTR harboring the mutation. Login to comment
36 The CF-causing mutants G91R and G85E are in the original predicted TM1 span, residues 81-102 (Riordan et al., 1989). Login to comment
66 In this study, the span boundaries or integration profiles of TM1 and TM2 were determined and the effects of the CF-causing mutations G85E and G91R assessed. Login to comment
67 The G91R and G85E mutations Figure 1: Predicted TM1 and TM2 spans. Login to comment
69 The positions of the CF-causing mutants G85E and G91R are indicated by triangles. Login to comment
71 This work further elucidates CFTR membrane-spanning structures and provides mechanistic insight into the molecular pathology of the G85E and G91R CF-causing mutations. Login to comment
76 Moreover, when the CF-causing mutations G85E and G91R were analyzed using these algorithms, variable effects of mutations were predicted, including shortening, no affect, no TM, or shifting TM1 boundaries (Supplemental Table S1). Login to comment
114 Core glycosylation analysis of WT, G91R, and G85E CFTR containing the artificial ECL1 site and lacking the ECL4 sites was performed by deletion of residues between the glycosylation site and TM1 (A) or between the glycosylation site and TM2 (B). Login to comment
117 Schematics of the experimentally identified ER integration profiles in WT (C) or G91R (D) and G85E (E) mutants. Login to comment
130 Determining the ER integration profiles of CF-causing mutants To assess effects of the CF-causing mutants G91R and G85E on the TM1 ER integration profile, these mutations were analyzed using the ECL1 site core glycosylation assay. Login to comment
131 The G85E and G91R mutations introduce an ionizable group into or near the predicted TM1 span and might be reasonably expected to alter its ER integration profile (Xiong et al., 1997). Login to comment
133 The G91R and G85E mutations in CFTR containing the natural ECL4 sites or the ECL1 site resulted in misfolding and accumulation in the ER (Supplemental Figure S3). Login to comment
134 G85E reduced CFTR maturation and degradation in constructs containing either the natural ECL4 sites or the artificial ECL1 site (Supplemental Figure S4). Login to comment
136 Thus, in the same manner as WT ECL1, glycosylation analysis was used to characterize the TM1 integration profiles for the G91R and G85E mutants. Login to comment
141 Cystic fibrosis-causing mutant G85E dramatically alters the TM1 integration profile in the ER membrane The effect of the G85E mutation on the TM1 ER integration profile was examined by introducing it into the ECL1 core glycosylation assay. Login to comment
143 In the presence of G85E, the unmodified ELC1 site is not glycosylated (Figure 4A). Login to comment
146 This aberrant pattern is in stark contrast to WT TM1 and is consistent with multiple conformations and/or profiles of G85E TM1 in the ER membrane, with the two extreme positions defined by the addition of two residues and the deletion of 14 residues. Login to comment
149 However, the R104 TM1 integration profile edge is dramatically shifted from WT, indicating a significant disruption in the G85E TM1. Login to comment
150 Comparison of the experimental integration profile edge to predicted TM1 boundaries reveals that it is surprisingly close to several of the original predicted WT TM1 boundaries (Figure 1) and the Kyte and Doolittle scale-predicted G85E TM1 (Supplemental Table S1). Login to comment
151 G85E alters the position of TM1 in the membrane The positioning of G85E TM1 in the membrane as monitored by OST accessibility is altered as compared with WT. Login to comment
154 CFTR has a cysteine at position 76, which is within the experimentally derived WT, but not G85E, TM1 integration profile. Login to comment
163 The reaction of C76 with AMS in the presence of G91R and G85E was also tested. Login to comment
165 In contrast, G85E C76 reacts with AMS, indicating that this position is no longer protected by the membrane (Figure 5, B and C). Login to comment
166 This result is consistent with glycosylation scanning results and an initial positioning of TM1 in the presence of G85E that is more C-terminal than for either WT or G91R. Login to comment
168 Role of the ionizable side chain in altered G91R and G85E TM1 ER integration profiles In the G91R and G85E mutants, an ionizable side chain replaces the glycine Cα hydrogen. Login to comment
174 This integration profile is the same as the G91R TM1integration profile and one of the two extreme G85E TM1 integration profiles. Login to comment
175 The two-residue boundary shifts are consistent with the substitution for glycine causing a small but consistent decreased distance between the ECL1 site and the ER membrane. It is striking that the G85A TM1 glycosylation pattern does not indicate multiple profiles for TM1, indicating that the aberrant pattern of G85E TM1 results from introduction of the glutamate side chain (Figure 6A). Login to comment
176 Role of the ionizable side chain in trafficking of G91R and G85E The data from the glycosylation assay demonstrate that the G85E mutant splits the integration profile of TM1, whereas the G91R, G85A, and G91A mutants do not. Login to comment
182 In stark contrast to G91A, G85A accumulates in the ER, suggesting that both introduction of charge and loss of glycine at position 85 contribute to G85E ER accumulation. Login to comment
187 It is striking that the G85E mutant exhibited temperature-insensitive ER accumulation. Login to comment
188 This observation cannot be accounted for by lower protein expression of G85E, as band B is not measurably altered. Login to comment
189 Of importance, G85E temperature-insensitive ER accumulation correlates with the G85E- perturbed TM1 integration profile with an edge at R104. Login to comment
194 This study determined the TM1 and TM2 ER luminal integration profile edges and CF-causing mutant G91R and G85E effects on TM1, using the mammalian ER luminal core glycosylation machinery. Login to comment
204 (B) A CFTR construct containing C76 with G91R or G85E was exposed to AMS and examined for the presence of an interaction by gel shift. Login to comment
205 G85E has a gel shift, indicating that C76 is exposed to cytosol. Login to comment
206 (C) Schematics of G91R and G85E mutant CFTR three-TM constructs with the relative positions of C76 and the mutants labeled. Login to comment
235 HeLa cell trafficking of WT, ΔF508, G85E, G85A, G91R, and G91A mutant CFTR at 37°C was analyzed by Western blot analysis (A). Login to comment
253 The core glycosylation experiments demonstrate that G85E is within TM1 and causes at least two TM1 positions with distinct ER integration profiles. Login to comment
254 There are several alternate integration profiles within the monitored G85E constructs, for which multiple potential explanations exist. Login to comment
255 One is that G85E samples distinct conformations during integration, and specific conformational distributions between the different constructs are being monitored. Login to comment
256 The mutation G85E might also lead to different interactions between the TM span and the translocation machinery, similar to interactions mediated by an acidic residue within TM8 of CFTR (Pitonzo et al., 2009). Login to comment
257 Polar residues have also been shown to drive associations between TM spans (Choma et al., 2000; Zhou et al., 2000, 2001); therefore G85E could result in altered interactions between TM1 and other TM spans that are reflected in the glycosylation pattern. Login to comment
258 This would indicate that the perturbed pattern for G85E could be due to more than a simple alteration of the 12-residue rule and that perturbations associated with introduction of an acidic residue may be occurring. Login to comment
259 However, positioning of the G85E TM1 in the membrane monitored by cysteine exposure was found to be more C-terminal in a three-TM span construct that lacked the other nine CFTR TM spans. Login to comment
260 Thus the simplest interpretation is that the integration profile defect is consistent with a distinct placement or destabilization of G85E TM1 in the membrane. Login to comment
261 Of interest, the most C-terminal extreme G85E TM1 boundary is within several residues of the original predicted WT TM1 span boundary (Figure 1) and a boundary predicted by TopPred KD (Supplemental Table S1). Login to comment
263 Thus G85E destabilization is an early-folding defect potentially recognizable in the ER before translation is complete. Login to comment
265 Consequently, G85E misfolding results from both introduction of an ionizable group and glycine loss, which respectively correlate with temperature-insensitive and temperature-sensitive accumulation in the ER. Login to comment
268 This complex has been implicated in the recognition and removal of improperly folded CFTR, including G85E mutant CFTR (Sun et al., 2006; Younger et al., 2006; Wang et al., 2008). Login to comment
270 Previous work demonstrated that the G85E and G91R mutations also disrupt later steps in CFTR folding, particularly interdomain interactions, which were proposed to underlie mutant recognition by ER quality control machinery (Xiong et al., 1997). Login to comment
271 The results presented here demonstrate that G85E dramatically alters the integration profile of TM1. Login to comment
274 Yet the experimental evidence presented here distinguishes G91R from G85E with respect to perturbations from the ionizable side chain, the role of glycine, and temperature sensitivity. Login to comment
277 It is striking that the corrector compound 4 exhibited mutant-specific effects, partially rescuing the G91R but not G85E CFTR (Grove et al., 2009). Login to comment
286 The mutations G91R, G91A, G85E, and G85A were introduced into CFTR constructs containing the natural, artificial, and deletion mutants on the artificial site. Login to comment

[hide] Cystic fibrosis mutations for p.F508del compound h... Clin Genet. 2012 Dec;82(6):546-551. doi: 10.1111/j.1399-0004.2011.01804.x. Epub 2011 Nov 29. Sebro R, Levy H, Schneck K, Dimmock D, Raby B, Cannon C, Broeckel U, Risch N
Cystic fibrosis mutations for p.F508del compound heterozygotes predict sweat chloride levels and pancreatic sufficiency.
Clin Genet. 2012 Dec;82(6):546-551. doi: 10.1111/j.1399-0004.2011.01804.x. Epub 2011 Nov 29., [PMID:22035343]

Abstract [show]
Sebro R, Levy H, Schneck K, Dimmock D, Raby BA, Cannon CL, Broeckel U, Risch NJ. Cystic fibrosis mutations for p.F508del compound heterozygotes predict sweat chloride levels and pancreatic sufficiency. Cystic fibrosis (CF) is a monogenetic disease with a complex phenotype. Over 1500 mutations in the CFTR gene have been identified; however, the p.F508del mutation is most common. There has been limited correlation between the CFTR mutation genotype and the disease phenotypes. We evaluated the non-p.F508del mutation of 108 p.F508del compound heterozygotes using the biological classification method, Grantham and Sorting Intolerant from Tolerant (SIFT) scores to assess whether these scoring systems correlated with sweat chloride levels, pancreatic sufficiency, predicted FEV(1) , and risk of infection with Pseudomonas aeruginosa in the last year. Mutations predicted to be 'mild' by the biological classification method are associated with more normal sweat chloride levels (p < 0.001), pancreatic sufficiency (p < 0.001) and decreased risk of infection with Pseudomonas in the last year (p = 0.014). Lower Grantham scores are associated with more normal sweat chloride levels (p < 0.001), and pancreatic sufficiency (p = 0.014). Higher SIFT scores are associated with more normal sweat chloride levels (p < 0.001) and pancreatic sufficiency (p = 0.011). There was no association between pulmonary function measured by predicted FEV(1) and the biological classification (p = 0.98), Grantham (p = 0.28) or SIFT scores (p = 0.62), which suggests the pulmonary disease related to CF may involve other modifier genes and environmental factors.

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64 CFTR mutation classification for compound heterozygotesa Mutations n (%) Biological classification Grantham score SIFT Q493X 3 (3) Ib - - G542X 21 (20) Ib,c,e - - R553X 4 (4) Ib,e - - Y1092X 2 (2) Ib - - R1158X 1 (1) NA - - W1282X 9 (9) Ib,e - - G85E 4 (4) IIIb 98 0.01 R117H 4 (4) IVb,c 29 0.60 R334W 1 (1) IVb 101 0.02 R347P 1 (1) IVb 103 0.05 R352Q 1 (1) NA 43 0.35 G551D 20 (19) IIIb,c 94 0.00 R560T 3 (3) IIIb 71 0.00 D1270N 1 (1) NA 23 0.01 N1303K 6 (6) IIg 94 0.00 I507del 3 (3) IId - - 394delTT 1 (1) NAc - - 621+1G>T 7 (7) Ib,f - - 711+1G>T 2 (2) Ib - - 1717-1G>A 5 (5) Ib,c,e,f - - 1898+1G>A 2 (2) NA - - 2789+5G>A 3 (3) Vb - - 3659delC 1 (1) Ib - - 3849+10kbC>T 2 (2) Vb,c,f - - 3905insT 1 (1) Ib - - NA, not applicable; SIFT, Sorting Intolerant from Tolerant. a The following mutations biological classification scores could not be verified: 1898+G-A, 394delTT, D1270N, R352Q, and R1158X. Login to comment

[hide] The D1152H cystic fibrosis mutation in prenatal ca... J Med Screen. 2011;18(4):169-72. Epub 2011 Dec 7. Peleg L, Karpati M, Bronstein S, Berkenstadt M, Frydman M, Yonath H, Pras E
The D1152H cystic fibrosis mutation in prenatal carrier screening, patients and prenatal diagnosis.
J Med Screen. 2011;18(4):169-72. Epub 2011 Dec 7., [PMID:22156145]

Abstract [show]
OBJECTIVE: To assess the frequency of the D1152H mutation in the CFTR gene in normal individuals, in cystic fibrosis (CF) patients and in the setting of prenatal diagnosis. SETTING: A database analysis of sequential screening results seen at the Sheba Medical Center, Israel, between 2001 and 2010. METHODS: We retrospectively analyzed the frequency of D1152H in a large cohort of healthy individuals who were screened as part of a routine prenatal care programme, in individuals referred due to CF-related symptoms and in the setting of prenatal diagnosis. RESULTS: We found one asymptomatic homozygous female and 195 D1152H carriers among 49,940 healthy individuals screened, establishing a carrier rate of 1:255 for this mutation. We detected D1152H in nine of 103 individuals referred due to CF-related symptoms. Four suffered from respiratory symptoms and five from congenital bilateral absence of the vas deferens (CBAVD). During this period D1152H was detected in three pregnancies, two of which were aborted. CONCLUSION: The increased frequency of D1152H in individuals referred due to CF-related symptoms compared with healthy individuals included in the CF carrier screening programme (P < 0.001) clearly indicates that it is a disease-causing mutation.

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180 of mutations Group of mutations 2001 Ashkenazi Jews 7 Group A Non-Ashkenazi Jews 11 Group A þ B Georgian Jews 12 Group A þ B þ T360K/Q359K 9.2004-7.2005 Yemenite Jews 12 Groups A þ B þ I1234V Iraqi Jews 12 Groups A þ B þY1092X 8.2005-12.2007 Iraqi Jews 14 Groups A þ B þY1092X þ 3121-1G-A 1.2008-2010 14 mutations for all 14 Groups A þ B þ C Georgian Jews 15 Groups A þ B þ C þ T360K/Q359K Arabic population 19 Groups A þ B þ C þ D Group A: G542X, W1282X, N1303K, F508del, 3849 þ 10KbC-T, 1717-1G-A, D1152H Group B: W1089X, G85E, 405 þ 1G-A, S549R(T-G) Group C: Y1092X, 3121-1G-A, I1234V Group D: 4010delTATT, S549I, 3120 þ 1Kbdel18.6Kb, 2183AA-G, R75X Between 2005-2008 the Iraqi population was screened for an additional mutation 2751 þ 1insT. Login to comment

[hide] The K+ channel opener 1-EBIO potentiates residual ... PLoS One. 2011;6(8):e24445. Epub 2011 Aug 31. Roth EK, Hirtz S, Duerr J, Wenning D, Eichler I, Seydewitz HH, Amaral MD, Mall MA
The K+ channel opener 1-EBIO potentiates residual function of mutant CFTR in rectal biopsies from cystic fibrosis patients.
PLoS One. 2011;6(8):e24445. Epub 2011 Aug 31., [PMID:21909392]

Abstract [show]
BACKGROUND: The identification of strategies to improve mutant CFTR function remains a key priority in the development of new treatments for cystic fibrosis (CF). Previous studies demonstrated that the K(+) channel opener 1-ethyl-2-benzimidazolone (1-EBIO) potentiates CFTR-mediated Cl(-) secretion in cultured cells and mouse colon. However, the effects of 1-EBIO on wild-type and mutant CFTR function in native human colonic tissues remain unknown. METHODS: We studied the effects of 1-EBIO on CFTR-mediated Cl(-) secretion in rectal biopsies from 47 CF patients carrying a wide spectrum of CFTR mutations and 57 age-matched controls. Rectal tissues were mounted in perfused micro-Ussing chambers and the effects of 1-EBIO were compared in control tissues, CF tissues expressing residual CFTR function and CF tissues with no detectable Cl(-) secretion. RESULTS: Studies in control tissues demonstrate that 1-EBIO activated CFTR-mediated Cl(-) secretion in the absence of cAMP-mediated stimulation and potentiated cAMP-induced Cl(-) secretion by 39.2+/-6.7% (P<0.001) via activation of basolateral Ca(2)(+)-activated and clotrimazole-sensitive KCNN4 K(+) channels. In CF specimens, 1-EBIO potentiated cAMP-induced Cl(-) secretion in tissues with residual CFTR function by 44.4+/-11.5% (P<0.001), but had no effect on tissues lacking CFTR-mediated Cl(-) conductance. CONCLUSIONS: We conclude that 1-EBIO potentiates Cl(-)secretion in native CF tissues expressing CFTR mutants with residual Cl(-) channel function by activation of basolateral KCNN4 K(+) channels that increase the driving force for luminal Cl(-) exit. This mechanism may augment effects of CFTR correctors and potentiators that increase the number and/or activity of mutant CFTR channels at the cell surface and suggests KCNN4 as a therapeutic target for CF.

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46 CFabsent CFresidual CFTR genotype Number of individuals CFTR genotype Number of individuals F508del/F508del 10 F508del/Y161C 1 F508del/W57X 1 F508del/V232D 1 F508del/G85E 3 F508del/R334W 2 F508del/120del23 1 F508del/T338I 1 F508del/182delT 1 F508del/I1234V 1 F508del/G542X 1 F508del/3272-26 A.G 1 F508del/A561E 1 F508del/3849+10 kb C.T 1 F508del/Y1092X 1 F508del/4005 +5727 A.G 1 F508del/N1303K 1 F508del/G576A 1 F508del/1525-1 G.A 2 N1303K/R334W 1 F508del/Q39X 1 F1052V/M1137R 1 F508del/Q552X 1 1898+3 A.G/ 1898+3 A.G 1 G85E/G85E 1 R334W/3199del6 1 Q552X/R1162X 1 R334W/X 1 A561E/A561E 2 dele2,3/X 1 R764X/1717-1 G.A 1 R1158X/2183AA.G 1 R1158X/R560T 1 doi:10.1371/journal.pone.0024445.t001 luminal and basolateral surfaces of the epithelium were perfused continuously with a solution of the following composition (mmol/ L): NaCl 145, KH2PO4 0.4, K2HPO4 1.6, D-glucose 5, MgCl2 1, Ca-gluconate 1.3, pH 7.4, at 37uC. Login to comment

[hide] The use of DHPLC (Denaturing High Performance Liqu... J Prenat Med. 2010 Jul;4(3):45-8. Mesoraca A, Di Natale M, Cima A, Di Giacomo G, Sarti M, Barone MA, Bizzoco D, Cignini P, Mobili L, D'emidio L, Giorlandino C
The use of DHPLC (Denaturing High Performance Liquid Chromatography) in II level screening of the CFTR gene in Prenatal Diagnosis.
J Prenat Med. 2010 Jul;4(3):45-8., [PMID:22439061]

Abstract [show]
OBJECTIVE: The aim of the study is to evaluate the role of Denaturing High Performance Liquid Chromatography (DHPLC) in the second level screening of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. METHODS: A 9-month prospective study, between June 2008 and March 2009 at Artemisia Fetal Medical Centre, included 3829 samples of amniotic fluid collected from women undergoing mid-trimester amniocentesis.The genetic diagnosis of CF was based on research of the main mutations of the CFTR gene on fetal DNA extracted from the amniocytes, (first level screening) using different commercial diagnostic systems. A second level screening using DHPLC, on the amniotic fluid and on a blood sample from the couple, was offered in case of fetuses heterozygous at first level screening. RESULTS: Of 3829 fetuses, 134 were found to be positive, 129 heterozygous and 5 affected. Of the 129 couples, following appropriate genetic counselling, 53 requested a second level screening. Through the use of DHPLC, 44 couples were found to be negative, and in nine couples, nine rare mutations were identified. CONCLUSIONS: The first level screening can be useful to evidence up to 75% of the CF mutations. The second level screening can identify a further 10% of mutant alleles. DHPLC was found to be a reliable and specific method for the rapid identification of the rare CFTR mutations which were not revealed in initial first level screening.

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100 48 Journal of Prenatal Medicine 2010; 4 (3): 45-50 Table III Mutations found with II level screening through DHPLC Mutations of mutated alleles DF508 29 W1282X 3 N1303K 8 1717-1G®A 2 3659delC 1 G85E 1 2789 +5G®A 2 R553X 2 R1162X 1 R117H 1 G542X 3 Total 53Table I Mutations found through I level screeningMutations analysed with I level screening through OLA CFTR Mutations Position on the CFTR gene DF508 Exon 10 3849+10KbC®T Intron 19 R334W Exon 7 W1282X Exon 10 V520F Exon 10 3905insT Exon 20 N1303K Exon 21 3876delA Exon 20 1717-1G®A Exon 11 3659delC Exon 19 DI507 Exon 10 A455E Exon 9 G85E Exon 3 2789 +5G®A Exon 14 / Intron 14 2183AA®G Exon 13 1898+1G®A Exon 12 / Intron 12 R347P Exon 7 R347H Exon 7 R560T Exon 11 1078delT Exon 7 R553X Exon 11 711+1G®T Exon 5 / Intron 5 G551D Exon 11 R1162X Exon 19 S549R Exon 11 R117H Exon 4 S549N Exon 11 621+1G®T Exon 4 G542X Exon 11 394delTT Exon 3 3120+1G®ðA Exon 16/ Intron 16 2184delA Exon 13 Table II Mutations found through I level screening Mutations Positions on CFTR gene R1066C Exon 17 b L1065P Exon 17 b A1006E Exon 19 R75Q Exon 3 D537E Exon 11 W1134X Exon 18 W1145X Exon 18 L1077P Exon 17b C524X Exon 11 Total 9 The use of DHPLC (Denaturing High Performance Liquid Chromatography) in II level screening of the CFTR gene in Prenatal Diagnosis Journal of Prenatal Medicine 2010; 4 (3): 45-50 49 tion was to provide the couple with adequate counselling in order to better understand the genotype-phenotype correlation in the various associations of mutations. Login to comment

[hide] An immunocytochemical assay to detect human CFTR e... Mol Cell Probes. 2009 Dec;23(6):272-80. Epub 2009 Jul 15. Davidson H, Wilson A, Gray RD, Horsley A, Pringle IA, McLachlan G, Nairn AC, Stearns C, Gibson J, Holder E, Jones L, Doherty A, Coles R, Sumner-Jones SG, Wasowicz M, Manvell M, Griesenbach U, Hyde SC, Gill DR, Davies J, Collie DD, Alton EW, Porteous DJ, Boyd AC
An immunocytochemical assay to detect human CFTR expression following gene transfer.
Mol Cell Probes. 2009 Dec;23(6):272-80. Epub 2009 Jul 15., [PMID:19615439]

Abstract [show]
BACKGROUND: To assess gene therapy treatment for cystic fibrosis (CF) in clinical trials it is essential to develop robust assays that can accurately detect transgene expression in human airway epithelial cells. Our aim was to develop a reproducible immunocytochemical assay for human CFTR protein which can measure both endogenous CFTR levels and augmented CFTR expression after gene delivery. METHODS: We characterised an antibody (G449) which satisfied the criteria for use in clinical trials. We optimised our immunocytochemistry method and identified G449 dilutions at which endogenous CFTR levels were negligible in CF samples, thus enhancing detection of transgenic CFTR protein. After developing a transfection technique for brushed human nasal epithelial cells, we transfected non-CF and CF cells with a clinically relevant CpG-free plasmid encoding human CFTR. RESULTS: The optimised immunocytochemistry method gave improved discrimination between CF and non-CF samples. Transfection of a CFTR expression vector into primary nasal epithelial cells resulted in detectable RNA and protein expression. CFTR protein was present in 0.05-10% of non-CF cells and 0.02-0.8% of CF cells. CONCLUSION: We have developed a sensitive, clinically relevant immunocytochemical assay for CFTR protein and have used it to detect transgene-expressed CFTR in transfected human primary airway epithelial cells.

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215 CF genotypes for nasal brushings were 15 DF508/DF508, 2 DF508/ND, 1 DF508/A455E, 1 DF508/G551D, 1 G85E/ND, 1 ND/ND (where 'ND` indicates that none of the 31 most common mutations was detected). Login to comment
217 CF genotypes for bronchial brushings were 5 DF508/DF508, 1 G85E/ND and 1 ND/ND. Login to comment
218 CF genotypes for nasal brushings were 15 DF508/DF508, 2 DF508/ND, 1 DF508/A455E, 1 DF508/G551D, 1 G85E/ND, 1 ND/ND (where 'ND` indicates that none of the 31 most common mutations was detected). Login to comment
220 CF genotypes for bronchial brushings were 5 DF508/DF508, 1 G85E/ND and 1 ND/ND. Login to comment

[hide] CFTR H609R mutation in Ecuadorian patients with cy... J Cyst Fibros. 2009 Jul;8(4):280-1. Epub 2009 May 19. Moya-Quiles MR, Glover G, Mondejar-Lopez P, Pastor-Vivero MD, Fernandez-Sanchez A, Sanchez-Solis M
CFTR H609R mutation in Ecuadorian patients with cystic fibrosis.
J Cyst Fibros. 2009 Jul;8(4):280-1. Epub 2009 May 19., [PMID:19457724]

Abstract [show]
Mutation epidemiology in each ethnic group is important for cystic fibrosis diagnosis and genetic counselling. To date, little has been reported on the prevalence of cystic fibrosis in the Ecuadorian population where the mutation distribution appears to differ from that of Europe. We present a series of four Ecuadorian patients homozygous for the H609R mutation in the CFTR gene. This is the first report of detection of this mutation in the Ecuadorian population. Taking advantage of the homozygous status of the patients, an evaluation of the most important clinical parameters is presented. From the diagnostic point of view, the information provided by our study is of relevance in designing an appropriate strategy for genetic testing of patients in Ecuador and in European countries where immigration from Ecuador is common.

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14 There are two other reports of mutations on the CFTR gene in CF patients from Ecuador; in the first, the estimated Ecuadorian CF incidence was 1:11,252 and mutations were, in order of frequency, F508del (37.1%), G85E (8.9%), G542X (2.4%), N1303K (2.4%), G551D (1.6%) and R334W (0.8%), with a detection rate of 53.22% of the total CF chromosomes studied [7]. Login to comment
15 In the second report, a compilation of data from CFTR gene analysis in Latin American CF patients, four mutations were found: F508del (31.37%), G542X (1.96%), G85E (1.96%) and N1303K (1.96%), with 63.7% of Ecuadorian CF mutations remaining unidentified [6]. Login to comment
28 Genotypes G85E/G85E and G85E/S549R were found in the other two patients. Login to comment
13 There are two other reports of mutations on the CFTR gene in CF patients from Ecuador; in the first, the estimated Ecuadorian CF incidence was 1:11,252 and mutations were, in order of frequency, F508del (37.1%), G85E (8.9%), G542X (2.4%), N1303K (2.4%), G551D (1.6%) and R334W (0.8%), with a detection rate of 53.22% of the total CF chromosomes studied [7]. Login to comment
26 Genotypes G85E/G85E and G85E/S549R were found in the other two patients. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... J Hepatol. 2009 Jan;50(1):150-7. Epub 2008 Oct 7. Henckaerts L, Jaspers M, Van Steenbergen W, Vliegen L, Fevery J, Nuytten H, Roskams T, Rutgeerts P, Cassiman JJ, Vermeire S, Cuppens H
Cystic fibrosis transmembrane conductance regulator gene polymorphisms in patients with primary sclerosing cholangitis.
J Hepatol. 2009 Jan;50(1):150-7. Epub 2008 Oct 7., [PMID:18992954]

Abstract [show]
BACKGROUND/AIMS: Primary sclerosing cholangitis (PSC) is a progressive cholestatic disease commonly associated with inflammatory bowel disease (IBD) and characterized by fibrosing inflammatory destruction of bile ducts. The histological features in the liver of PSC patients are similar to those observed in cystic fibrosis (CF). Our aim was to study whether variants in the CFTR gene are associated with the occurrence and/or evolution of PSC. METHODS: PSC patients (n=140) were genotyped for F508del, the TGmTn variants, and four additional polymorphic loci (1001+11 C>T, M470V, T854T and Q1463Q), and compared to 136 matched healthy controls. RESULTS: The 1540G-allele, encoding V470, was less frequent in PSC (52%) than in controls (64%, p=0.003), and was associated with protection against PSC in individuals without IBD (OR 0.25, 95% CI 0.12-0.52, p=0.0002). Also TG11-T7 was less frequent in PSC (53%) than in controls (61%, p=0.04), this haplotype was associated with reduced risk for PSC (OR 0.34, 95% CI 0.17-0.70, p=0.003) in individuals without IBD. CONCLUSIONS: In this cohort of PSC patients, several CFTR-variants affecting the functional properties of the CFTR protein seem to offer protection against the development of PSC, confirming our hypothesis that CFTR might be implicated in the pathogenesis of PSC.

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91 There was Table 4 Summary of the 37 CFTR variants studied in the exploratory phase INNO-LiPA CFTR 19 INNO-LiPA CFTR17+Tn Update F508del 621+1GfiT G542X 3849+10kbCfiT N1303K 2183AAfiG W1282X 394delTT G551D 2789+5GfiA 1717-1GfiA R1162X R553X 3659delC CFTRdele2,3(21kb) R117H I507del R334W 711+1GfiT R347P 3272-26AfiG G85E 3905insT 1078delT R560T A455E 1898+1GfiA 2143delT S1251N E60X I148T 2184delA 3199del6 711+5GfiA 3120+1GfiA Tn Q552X Fig. 1. Login to comment

[hide] Cystic fibrosis carrier frequency and estimated pr... J Cyst Fibros. 2008 Sep;7(5):440-3. Epub 2008 Feb 1. Ratbi I, Genin E, Legendre M, Le Floch A, Costa C, Cherkaoui-Deqqaqi S, Goossens M, Sefiani A, Girodon E
Cystic fibrosis carrier frequency and estimated prevalence of the disease in Morocco.
J Cyst Fibros. 2008 Sep;7(5):440-3. Epub 2008 Feb 1., [PMID:18243066]

Abstract [show]
BACKGROUND: The epidemiology of cystic fibrosis (CF) is poorly known in North African populations, in particular in Morocco and the CF carrier frequency in the general Moroccan population has never been evaluated. METHODS: To estimate the prevalence of CF mutations in Morocco, blood samples from 150 healthy Moroccans were tested for frequent CFTR mutations and the intron 8 polyT variant. RESULTS: Two subjects were heterozygous for F508del and eight others for the (T)5 variant. CONCLUSION: These findings indicate that the Moroccan population is at risk for CF and CFTR-related disorders. CF prevalence could be in the range of that found in European populations. Wider studies are necessary to identify the clinical pattern and accurately determine the prevalence and molecular basis of CF in Morocco.

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27 We screened for 32 CFTR gene mutations (G85E, 394delTT, R117H, 621+1GNT, 711+1GNT, R334W, R347P, R347H, 1078delT, A455E, I507del, F508del, V520F, 1717-1GNA, G542X, G551D, R553X, R560T, S549R(TNG), S549N, 1898+1GNA, 2183AANG, 2184delA, 2789+5GNA, 3120 + 1G NA, R1162X, 3659delC, 3849 + 10kbC NT, W1282X, 3905insT, 3876delA, N1303K) and the (T)5 splicing variant of intron 8, using a commercial kit (CF v3 Genotyping Assay, Abbott, Rungis, France). Login to comment

[hide] Validation of high-resolution DNA melting analysis... J Mol Diagn. 2008 Sep;10(5):424-34. Epub 2008 Aug 7. Audrezet MP, Dabricot A, Le Marechal C, Ferec C
Validation of high-resolution DNA melting analysis for mutation scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.
J Mol Diagn. 2008 Sep;10(5):424-34. Epub 2008 Aug 7., [PMID:18687795]

Abstract [show]
High-resolution melting analysis of polymerase chain reaction products for mutation scanning, which began in the early 2000s, is based on monitoring of the fluorescence released during the melting of double-stranded DNA labeled with specifically developed saturation dye, such as LC-Green. We report here the validation of this method to scan 98% of the coding sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We designed 32 pairs of primers to amplify and analyze the 27 exons of the gene. Thanks to the addition of a small GC-clamp at the 5' ends of the primers, one single melting domain and one identical annealing temperature were obtained to co-amplify all of the fragments. A total of 307 DNA samples, extracted by the salt precipitation method, carrying 221 mutations and 21 polymorphisms, plus 20 control samples free from variations (confirmed by denaturing high-performance liquid chromatography analysis), was used. With the conditions described in this study, 100% of samples that carry heterozygous mutations and 60% of those with homozygous mutations were identified. The study of a cohort of 136 idiopathic chronic pancreatitis patients enabled us to prospectively evaluate this technique. Thus, high-resolution melting analysis is a robust and sensitive single-tube technique for screening mutations in a gene and promises to become the gold standard over denaturing high-performance liquid chromatography, particularly for highly mutated genes such as CFTR, and appears suitable for use in reference diagnostic laboratories.

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171 Results of CFTR Analysis by HRM on 136 Samples of Patients with Idiopathic Chronic Pancreatitis (ICP) Exon Number of positive samples Mutations identified Variants identified New positive controls 1 14 14 125GϾC 2 1 1 R31C 3 9 1 G85E 7 R75Q 1 R74W 4 4 1 R117G 1 I148T R117G 1 R117H 1 A120T 5 1 1 L188P L188P 6a 5 1 V201M 1 A221A A221A 3 875ϩ40 AϾG 6b 27 1 M284T 26 1001ϩ11CϾT M284T 7 1 1 L320V L320V 8 0 0 9 1 1 D443Y 10 16 8 F508del 8 E528E 11 1 1 G542X 12 6 4 G576A 1 Y577Y L568F 1 L568F 13 7 1 S737F 4 R668C S737F 1 V754M L644L 1 L644L 14a 53 52 T854T T854TϩI853I 1 T854TϩI853I 14b 0 0 15 3 1 L967S T908S 1 T908S 1 S945L 16 0 0 17a 10 7 L997F 1 3271ϩ18CϾT 3271 ϩ 3AϾG 1 3271 ϩ 3 AϾG 1 Y1014C 17b 3 1 L1096L L1096L 1 H1054DϩG1069R 1 3272-33AϾG H1054DϩG1069R 3272-33AϾG 18 2 1 D1152H E1124del 1 E1124del 19 5 5 S1235R poly 20 7 1 W1282X 5 P1290P 1 D1270N 21 2 1 N1303K 1 T1299T 22 0 0 23 1 0 4374ϩ13 AϾG 24 43 40 Q1463Q 2 Y1424Y 1 Q1463QϩY1024Y ing domain of a gene brings an excellent sensitivity for heterozygote detection that is very close to 100%. Login to comment

[hide] Consensus on the use and interpretation of cystic ... J Cyst Fibros. 2008 May;7(3):179-96. Castellani C, Cuppens H, Macek M Jr, Cassiman JJ, Kerem E, Durie P, Tullis E, Assael BM, Bombieri C, Brown A, Casals T, Claustres M, Cutting GR, Dequeker E, Dodge J, Doull I, Farrell P, Ferec C, Girodon E, Johannesson M, Kerem B, Knowles M, Munck A, Pignatti PF, Radojkovic D, Rizzotti P, Schwarz M, Stuhrmann M, Tzetis M, Zielenski J, Elborn JS
Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice.
J Cyst Fibros. 2008 May;7(3):179-96., [PMID:18456578]

Abstract [show]
It is often challenging for the clinician interested in cystic fibrosis (CF) to interpret molecular genetic results, and to integrate them in the diagnostic process. The limitations of genotyping technology, the choice of mutations to be tested, and the clinical context in which the test is administered can all influence how genetic information is interpreted. This paper describes the conclusions of a consensus conference to address the use and interpretation of CF mutation analysis in clinical settings. Although the diagnosis of CF is usually straightforward, care needs to be exercised in the use and interpretation of genetic tests: genotype information is not the final arbiter of a clinical diagnosis of CF or CF transmembrane conductance regulator (CFTR) protein related disorders. The diagnosis of these conditions is primarily based on the clinical presentation, and is supported by evaluation of CFTR function (sweat testing, nasal potential difference) and genetic analysis. None of these features are sufficient on their own to make a diagnosis of CF or CFTR-related disorders. Broad genotype/phenotype associations are useful in epidemiological studies, but CFTR genotype does not accurately predict individual outcome. The use of CFTR genotype for prediction of prognosis in people with CF at the time of their diagnosis is not recommended. The importance of communication between clinicians and medical genetic laboratories is emphasized. The results of testing and their implications should be reported in a manner understandable to the clinicians caring for CF patients.

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1236 Table 1 Geographical distribution of the most common mutations E60X Southern European S549N Indian CFTR Slavic - Eastern European G551D United Kingdom, Central Europe R75X Southern European, US-Hispanic Q552X Southern European, Italian 394delTT Nordic - Baltic sea region R553X Central European G85E Southern Europe A559T African-American 406-1GNA US-Hispanic R560T Northern Irish R117H European-derived populations 1811+1.6kbANG Spanish, US-Hispanic R117C Northern European 1898+1GNA United Kingdom, Central Europe 621+1GNT Southern European 1898+5GNT East Asian populations 711+1GNT French, French Canadian 2143delT Slavic - Eastern European 711+5GNA US-Hispanic 2183delAANG Southern Europe, Middle Eastern, Iranian, Latin American L206W Spanish and US-Hispanic 2184delA European-derived populations V232D Spanish and US-Hispanic 2789+5GNA European-derived populations 1078delT French Brittany Q890X Southern European R334W Southern European, Latin American 3120+1GNA African, Arabian, African-American, Southern Europe 1161delC Indian 3272-26ANG European-derived populations R347P European-derived, Latin America 3659delC Scandinavian R347H Turkish 3849+10kbCNT Ashkenazi-Jewish, Southern European, Middle Eastern, Iranian, Indian A455E Dutch R1066C Southern European 1609delCA Spanish, US-Hispanic Y1092X (CNA) Southern European I506T Southern European, Spanish M1101K US-Hutterite I507del European-derived populations 3905insT Swiss F508del European-derived populations D1152H European-derived populations 1677delTA Southern European, Middle Eastern R1158X Southern European 1717-GNA European-derived populations R1162X Italian, Amerindian, Latin America V520F Irish S1251N European-derived populations G542X Southern European, Mediterranean W1282X Ashkenazi-Jewish, Middle Eastern S549R(TNG) Middle Eastern N1303K Southern European, Middle Eastern Legend: these alleles occur with a frequency superior to 0.1% in selected populations. Login to comment
1239 Table 1 Geographical distribution of the most common mutations E60X Southern European S549N Indian CFTR Slavic - Eastern European G551D United Kingdom, Central Europe R75X Southern European, US-Hispanic Q552X Southern European, Italian 394delTT Nordic - Baltic sea region R553X Central European G85E Southern Europe A559T African-American 406-1GNA US-Hispanic R560T Northern Irish R117H European-derived populations 1811+1.6kbANG Spanish, US-Hispanic R117C Northern European 1898+1GNA United Kingdom, Central Europe 621+1GNT Southern European 1898+5GNT East Asian populations 711+1GNT French, French Canadian 2143delT Slavic - Eastern European 711+5GNA US-Hispanic 2183delAANG Southern Europe, Middle Eastern, Iranian, Latin American L206W Spanish and US-Hispanic 2184delA European-derived populations V232D Spanish and US-Hispanic 2789+5GNA European-derived populations 1078delT French Brittany Q890X Southern European R334W Southern European, Latin American 3120+1GNA African, Arabian, African-American, Southern Europe 1161delC Indian 3272-26ANG European-derived populations R347P European-derived, Latin America 3659delC Scandinavian R347H Turkish 3849+10kbCNT Ashkenazi-Jewish, Southern European, Middle Eastern, Iranian, Indian A455E Dutch R1066C Southern European 1609delCA Spanish, US-Hispanic Y1092X (CNA) Southern European I506T Southern European, Spanish M1101K US-Hutterite I507del European-derived populations 3905insT Swiss F508del European-derived populations D1152H European-derived populations 1677delTA Southern European, Middle Eastern R1158X Southern European 1717-GNA European-derived populations R1162X Italian, Amerindian, Latin America V520F Irish S1251N European-derived populations G542X Southern European, Mediterranean W1282X Ashkenazi-Jewish, Middle Eastern S549R(TNG) Middle Eastern N1303K Southern European, Middle Eastern Legend: these alleles occur with a frequency superior to 0.1% in selected populations. Login to comment

[hide] High incidence of the CFTR mutations 3272-26A-->G ... J Cyst Fibros. 2007 Nov 30;6(6):371-5. Epub 2007 May 3. Storm K, Moens E, Vits L, De Vlieger H, Delaere G, D'Hollander M, Wuyts W, Biervliet M, Van Schil L, Desager K, Nothen MM
High incidence of the CFTR mutations 3272-26A-->G and L927P in Belgian cystic fibrosis patients, and identification of three new CFTR mutations (186-2A-->G, E588V, and 1671insTATCA).
J Cyst Fibros. 2007 Nov 30;6(6):371-5. Epub 2007 May 3., [PMID:17481968]

Abstract [show]
We have analyzed 143 unrelated Belgian patients with a positive diagnosis of cystic fibrosis (CF) for mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. An initial screening for 29 CFTR mutations led to mutation identification in 89.9% of the tested chromosomes. Subsequently an extensive analysis of the CFTR gene was performed by denaturating gradient gel electrophoresis (DGGE) in those patients with at least one unknown mutation after preliminary screening. In addition to 10 previously reported mutations we identified 2 new mutations 186-2A-->G and E588V. A third new mutation 1671insTATCA was identified during routine screening for DeltaF508. Two mutations were detected with a higher frequency than expected: 3272-26A-->G, which is the second most common mutation after DeltaF508 in our CF population with a frequency of 3.8%, and L927P (2.4%). The clinical data is presented for the mutations 186-2A-->G, E588V, 3272-26A-->G and L927P. The mutation data are useful for the Belgian population to supplement the initial screening set of mutations.

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32 The Inno Lipa™ CFTR17 assay contains normal and mutant probes for 17 other CFTR mutations (394delTT, G85E, 621+1G→T, R117H, 1078delT, R347P, R334W, E60X, 711+5G→A, 2789 + 5G→ A, R1162X, 3659delC, 3849 + 10kbC→ T, 2143delT, A455E, 2183AA→G, 2184delA) (Innogenetics). Login to comment

[hide] Highly preferential association of NonF508del CF m... J Cyst Fibros. 2007 Jan;6(1):15-22. Epub 2006 Jun 19. Ciminelli BM, Bonizzato A, Bombieri C, Pompei F, Gabaldo M, Ciccacci C, Begnini A, Holubova A, Zorzi P, Piskackova T, Macek M Jr, Castellani C, Modiano G, Pignatti PF
Highly preferential association of NonF508del CF mutations with the M470 allele.
J Cyst Fibros. 2007 Jan;6(1):15-22. Epub 2006 Jun 19., [PMID:16784904]

Abstract [show]
BACKGROUND: On the basis of previous findings on random individuals, we hypothesized a preferential association of CF causing mutations with the M allele of the M470V polymorphic site of the CFTR gene. METHODS: We have determined the M/V-CF mutation haplotype in a series of 201 North East Italian and 73 Czech CF patients who were not F508del homozygotes, as F508del was already known to be fully associated with the M allele. RESULTS: Out of 358 not F508del CF genes, 84 carried the V allele and 274 the less common M allele. In the N-E Italian population, MM subjects have a risk of carrying a CF causing mutation 6.9x greater than VV subjects when F508del is excluded and 15.4x when F508del is included. In the Czech population a similar, although less pronounced, association is observed. CONCLUSIONS: Besides the possible biological significance of this association, the possibility of exploiting it for a pilot screening program has been explored in a local North East Italian population for which CF patients were characterized for their CF mutation. General M470V genotyping followed by common CF mutation screening limited to couples in which each partner carries at least one M allele would need testing only 39% of the couples, which contribute 89% of the total risk, with a cost benefit.

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91 Among NonF CF mutations, G85E, R553X and N1303K were found both with the M and the Vallele within the Italian sample; 1898+1 G>A and 3849+10 Kb C>T were found both with the M and the V allele within the Czech Republic; and G551D was found in Italy with the V allele (1/1) and in the Czech Republic with the M allele (12/12). Login to comment

[hide] Mutational spectrum of cystic fibrosis patients fr... Mol Genet Metab. 2006 Apr;87(4):370-5. Epub 2006 Jan 19. Ramirez AM, Ramos MD, Jimenez J, Ghio A, de Botelli MM, Rezzonico CA, Marques I, Pereyro S, Casals T, de Kremer RD
Mutational spectrum of cystic fibrosis patients from Cordoba province and its zone of influence: implications of molecular diagnosis in Argentina.
Mol Genet Metab. 2006 Apr;87(4):370-5. Epub 2006 Jan 19., [PMID:16423550]

Abstract [show]
Cystic Fibrosis (CF) is an autosomal recessive disorder affecting 1/2000-4000 newborns in Caucasian populations. This lethal disease mainly affects respiratory and digestive organs as well as fertility in man. So far, the CF prevalence and mutational spectrum have showed specificity among populations and regions, making it necessary to establish them in each one. In this study, we present the spectrum and frequency of CFTR gene mutations in CF patients from Cordoba (a province with 3.1 millions inhabitants in the middle of Argentina) and its zone of influence, to offer an accurate genetic testing. The study includes 78 families in which 98 patients fulfilled clinical criteria to CF diagnosis. The strategy for the molecular diagnosis comprised analysis of 21 common mutations, microsatellite haplotypes and the complete CFTR gene analysis using scanning techniques followed by sequencing of the abnormal migration patterns. Our first step led us to the identification of 10 mutations that represented 76% of alleles. Another four mutations (p.R1066C, c.1811 + 1.6 kbA > G, c.711 + 1G > T, and p.G85E) were found based on the microsatellite haplotype-mutation association. Finally, 14 mutations were characterized after the CFTR gene scanning, three of them are not previously described (p.G27R, c.622-2A > G, and p.W277R). In summary, we have identified 27 mutations accounting for 94.23% of CF alleles. This characteristic mutational spectrum highlights the 14 most frequent mutations (>1%) in the Cordoba region.

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8 Another four mutations (p.R1066C, c.1811 + 1.6kbA > G, c.711 + 1G > T, and p.G85E) were found based on the microsatellite haplotype-mutation association. Login to comment
62 According to the obtained haplotypes, the following mutations were studied: c.2869insG, CFTRdele2.3 (g.24291_29180del21kb), p.R1066C, c.711+1G>T, c.1811+1.6kbA>G, and p.G85E, the last four mutations were detected in seven patients. Login to comment
85 Haplotype (n D 20) No. of chromosomes (n D 64)a Mutations associated (No. of chromosomes) 23-31 14 p.F508del 17-31 7 p.F508del 17-7 7 p.R1066C (3), p.W277R, c.2789 + 5G > A, c.3120 + 1G > A, c.3849 + 10KbC > T 16-7 6 c.3272-26A > G (2), p.G27R, c.622-2A > G, unknown (2) 16-32 5 p.S589I (2), unknown (3) 16-30 3 IVS8-5T (2), unknown 23-33 2 p.G542X, p.R1283M 23-32 2 p.G542X 23-30 2 p.F508del, p.N1303K 24-31 2 p.N1303K 16-24 2 p.G85E 16-31 3 c.1898 + 1G > A, p.W1089X, unknown 16-46 2 c.1811 + 1.6KbA > G 16-25 1 c.711 + 1G > T 16-33 1 Unknown 16-44 1 c.1898 + 1G > A 16-45 1 p.Y913C 16-47 1 c.4005 + 1G > A 17-30 1 Unknown 23-7 1 [c.3199_3204delATAGTG; p.I148T] Table 2 Frequency of the mutations in the 78 CF Argentinean patients of Córdoba region a IdentiWed novel mutations. Login to comment
86 Mutation Exon/Intron CF alleles % p.F508del Exon 10 94 60.26 p.N1303K Exon 21 8 5.13 p.G542X Exon 11 7 4.49 p.R334W Exon 7 3 1.93 p.R1066C Exon 17b 3 1.93 c.2789 + 5G > A Intron 14b 3 1.93 p.G85E Exon 3 2 1.28 c.3659del C Exon 19 2 1.28 c.1811 + 1.6kbA > G Intron 11 2 1.28 c.1898 + 1G > A Intron 12 2 1.28 c.3272-26A > G Intron 17a 2 1.28 p.S589I Exon 12 2 1.28 p.R553X Exon 11 2 1.28 IVS8-5T Intron 8 2 1.28 c.3849 + 10kb C > T Intron 19 1 0.64 c.621 + 1G > T Intron 4 1 0.64 p.R1162X Exon 19 1 0.64 c.711 + 1G > T Intron 5 1 0.64 c.3120 + 1G > A Intron 16 1 0.64 p.Y913C Exon 15 1 0.64 c.4005 + 1G > A Intron 20 1 0.64 p.W1089X Exon 17b 1 0.64 p.R1283M Exon 20 1 0.64 [p.I148T;c.3199_3204del ATAGTG] Exon 4, Exon 17a 1 0.64 p.G27Ra Exon 2 1 0.64 p.W277Ra Exon 6b 1 0.64 c.622-2A > Ga Intron4 1 0.64 Unknown allele - 9 5.77 Wrst year of life he required several internments, for hydroelectric desequilibrium and persistent pulmonary infections causing failure to thrive. Login to comment
89 Fourteen mutations have a frequency higher than 1%, p.F508del (60.26%), p.N1303K (5.13%), p.G542X (4.49%), and three mutations, p.R334W, p.R1066C, c.2789 + 5G> A (1.93%), and another eight, p.G85E, c.3659delC, c.1811 + 1.6kbA > G, c.1898 + 1G > A, c.3272-26A > G, p.S589I, p.R553X, and 5T (1.28%). Login to comment

[hide] Cytogenetic analysis of azoospermic patients: kary... Eur J Obstet Gynecol Reprod Biol. 2006 Feb 1;124(2):197-203. Epub 2005 Sep 12. Stipoljev F, Vujisic S, Parazajder J, Hafner D, Jezek D, Sertic J
Cytogenetic analysis of azoospermic patients: karyotype comparison of peripheral blood lymphocytes and testicular tissue.
Eur J Obstet Gynecol Reprod Biol. 2006 Feb 1;124(2):197-203. Epub 2005 Sep 12., [PMID:16157443]

Abstract [show]
OBJECTIVE: The objective was to compare the results of a complete chromosomal, genetic and histological investigation in 13 azoospermic men with the results of the intracytoplasmic sperm injection (ICSI) procedure. STUDY DESIGN: Peripheral blood samples were used for the measurement of follicle-stimulating hormone (FSH) levels, chromosomal analysis, microdeletions in the azoospermia factor (AZF) region of the Y chromosome and cystic fibrosis transmembrane conductance regulator (CFTR) mutation analysis. Testicular tissue was used for histological scoring and cytogenetic evaluation. RESULTS: Peripheral blood cytogenetic analysis revealed a normal male karyotype in all cases. Chromosomal analysis from testicular tissue revealed a mosaicism for the terminal deletion of chromosome 22 with a breakpoint site at 22q13 in one patient with congenital bilateral absence of the vas deferens (CBAVD). Deletions in the AZFa, ATFb, and AZFc regions were not detected. The CFTR mutational analysis showed normal results in all patients. CONCLUSIONS: Cytogenetic evaluation of testicular tissue should be performed in non-obstructive and obstructive azoospermic patients as well as in patients with multiple failed IVF and recurrent spontaneous abortion.

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60 DNA samples were screened for the most common CFTR gene mutations and polythymidine tract variant within the intron 8 by: INNO-LIPA CFTR17 + Tn; exon 3: 394delTT, G85E, E60X; exon/intron 4: 621 + 1G ! Login to comment

[hide] A haplotype framework for cystic fibrosis mutation... J Mol Diagn. 2006 Feb;8(1):119-27. Elahi E, Khodadad A, Kupershmidt I, Ghasemi F, Alinasab B, Naghizadeh R, Eason RG, Amini M, Esmaili M, Esmaeili Dooki MR, Sanati MH, Davis RW, Ronaghi M, Thorstenson YR
A haplotype framework for cystic fibrosis mutations in Iran.
J Mol Diagn. 2006 Feb;8(1):119-27., [PMID:16436643]

Abstract [show]
This is the first comprehensive profile of cystic fibrosis transmembrane conductance regulator (CFTR) mutations and their corresponding haplotypes in the Iranian population. All of the 27 CFTR exons of 60 unrelated Iranian CF patients were sequenced to identify disease-causing mutations. Eleven core haplotypes of CFTR were identified by genotyping six high-frequency simple nucleotide polymorphisms. The carrier frequency of 2.5 in 100 (1 in 40) was estimated from the frequency of heterozygous patients and suggests that contrary to popular belief, cystic fibrosis may be a common, under-diagnosed disease in Iran. A heterogeneous mutation spectrum was observed at the CFTR locus in 60 cystic fibrosis (CF) patients from Iran. Twenty putative disease-causing mutations were identified on 64 (53%) of the 120 chromosomes. The five most common Iranian mutations together represented 37% of the expected mutated alleles. The most frequent mutation, DeltaF508 (p.F508del), represented only 16% of the expected mutated alleles. The next most frequent mutations were c.1677del2 (p.515fs) at 7.5%, c.4041C>G (p.N1303K) at 5.6%, c.2183AA>G (p.684fs) at 5%, and c.3661A>T (p.K1177X) at 2.5%. Three of the five most frequent Iranian mutations are not included in a commonly used panel of CF mutations, underscoring the importance of identifying geographic-specific mutations in this population.

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94 The mutation p.G85E has been shown to reduce gene activity by altering the tertiary structure of the CFTR channel;31 p.R334W leads to loss of conductivity of the large conductance channel of CFTR;32 p.T338I is a mutation in the sixth transmembrane segment of the MSD1 domain; p.L467F and p.I506T are mutations in the first nucleotide binding domain (NBD1); and p.N1303K is a mutation in the second nucleotide binding domain (NBD2) of the CFTR protein. Login to comment
111 of Patients Total alleles* Associated haplotype Global distributionHom Het Exon 1 c.134TϾC M1T 1 1 Rare Exon 3 c.386GϾA G85E 1 1 Global Exon 4 c.460GϾC D110H 1 1 H2 Europe Exon 7 c.1132CϾT R334W 1 1 H2 Global Exon 7 c.1145CϾT T338I 1 1 Europe Intron 9 c.1525-1GϾA Mis-splicing 1 1 H8 Pakistan Exon 10 c.1529CϾG S466X 1 2 H4 Germany Exon 10 c.1531CϾT L467F 1 1 Rare Exon 10 c.1649TϾC I506T 1 2 H8 Lebanon Exon 10 c.1652del3† ⌬F508 6 7 19 H5 Global Exon 10 c.1677delTA 515fs 4 1 9 H1 Europe Exon 11 c.1756GϾT G542X 1 1 H5 Global Exon 12 c.1821CϾA Y563X 2 2 Europe Exon 13 c.2183AAϾG 684fs 3 6 H3 Europe Exon 17a c.3170CϾT P1013L 1 1 Turkey Exon 19 c.3616CϾT R1162X 2 2 H2 Germany Exon 19 c.3661AϾT K1177X 1 1 3 H2 Bahrain Intron 20 c.4005ϩ1GϾA Mis-splicing 1 2 H2 Europe Exon 21 c.4041CϾG N1303K 3 1 7 H5 Global Exon 23 c.4363CϾT Q1412X 1 1 Rare *A total of 64 (53%) of the 120 expected alleles were observed. Login to comment

[hide] Spectrum of mutations in CFTR in Finland: 18 years... J Cyst Fibros. 2005 Dec;4(4):233-7. Epub 2005 Jul 26. Kinnunen S, Bonache S, Casals T, Monto S, Savilahti E, Kere J, Jarvela I
Spectrum of mutations in CFTR in Finland: 18 years follow-up study and identification of two novel mutations.
J Cyst Fibros. 2005 Dec;4(4):233-7. Epub 2005 Jul 26., [PMID:16051530]

Abstract [show]
BACKGROUND: The incidence of cystic fibrosis (CF) is low in the isolated Finnish population and the Finnish CF mutation spectrum has differed from many European countries. METHODS: We have analyzed the mutation spectrum and the geographical distribution of CF mutations in Finland covering the last 18 years (1987-2004). RESULTS: A total of 14 mutations were identified; two of them new, 774insT and S589T (G>C at 1,898). The overall coverage of mutations was 97% (99/102 chromosomes). The most frequent mutations were F508del and 394delTT, found in 36% (37/102) and 35% (36/102) of the CF chromosomes respectively. Of the rare mutations, a mutation of presumable Slavic origin, CFTRdele2.3 (21 kb), was enriched in a rural isolate with a frequency of 5,9% (6/102), and a mutation that possibly indicates Swedish influence, 3659delC, was scattered throughout the country with a similar frequency of 5,9% (6/102). G542X, R1162X, R117H, 3732delA, 1,898 + 3A >C, S1196X, S945L, W57R, 774insT and S589T were each identified in a number of chromosomes from one to three. CONCLUSIONS: Our observations of the Finnish CF mutation spectrum fit well with the characteristics of Finland as a population of multiple local founder effects.

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36 The InnoLipa assay recognizes 36 mutations: E60X (c.178G>T, p.Glu60X), G85E (c.254G>A, p.Gly85- Glu), 394delTT, R117H (c.350G>A, p.Arg117His), I148T (c.443T>C, p.Ile148Thr), 621+1G>T (c.489+1G>T), 711+1G>T (c.579+1G>T), 711+5G>A (c.579+5G>A), 1078delT (c.948delT, p.Phe316fs), R334W (c.1000C>T, p.Arg334Trp), R347P (c.1040G>C, p.Arg347Pro), A455E (c.1364C>A, p.Ala455Glu), I507del (c.1519_1521delATC, p.Ile507del), F508del, 1717À1G>A (c.1585À1G>A), G542X, G551D (c.1652G >A, p.Gly551Asp), Q552X (c.1654C > T, p.Gln552X), R553X (c.1657C > T, p.Arg553X), R560T (1679G>vC, p.Arg560Thr), 1898+ 1G > A (c.1766 + 1G > A), 2143delT (c.2012delT, p.Leu671fs), 2183AA > G (c.2051_2052delAAinsG, p.Lys684fs), 2184delA (c.2052delA, p.Lys684fs), 2789+ 5G>A (c.2657+5G>A), 3120+1G>A (c.2988+1G>A), 3199del6 (c.3067_3072del, p.Ile1023_Val1024del), 3272À 26A > G (c.3140 À26A > G), R1162X (c.3484C > T, p.Arg1162X), 3849+10kbCYT, 3659delC (c.3528delC, p.Lys1177fs), S1251N (c.3752G > A, p.Ser1251Asn), 3905insT (c.3773dupT, p.Leu1258fs), W1282X (c.3846G> A, p.Trp1282X), N1303K (c.3909C>G, p.Asn1303Lys), CFTRdele2,3(21kb) and Tn-polymorphism on intron 8. Login to comment
37 The InnoLipa assay recognizes 36 mutations: E60X (c.178G>T, p.Glu60X), G85E (c.254G>A, p.Gly85Glu), 394delTT, R117H (c.350G>A, p.Arg117His), I148T (c.443T>C, p.Ile148Thr), 621+1G>T (c.489+1G>T), 711+1G>T (c.579+1G>T), 711+5G>A (c.579+5G>A), 1078delT (c.948delT, p.Phe316fs), R334W (c.1000C>T, p.Arg334Trp), R347P (c.1040G>C, p.Arg347Pro), A455E (c.1364C>A, p.Ala455Glu), I507del (c.1519_1521delATC, p.Ile507del), F508del, 1717 1G>A (c.1585 1G>A), G542X, G551D (c.1652G >A, p.Gly551Asp), Q552X (c.1654C > T, p.Gln552X), R553X (c.1657C > T, p.Arg553X), R560T (1679G>vC, p.Arg560Thr), 1898+ 1G > A (c.1766 + 1G > A), 2143delT (c.2012delT, p.Leu671fs), 2183AA > G (c.2051_2052delAAinsG, p.Lys684fs), 2184delA (c.2052delA, p.Lys684fs), 2789+ 5G>A (c.2657+5G>A), 3120+1G>A (c.2988+1G>A), 3199del6 (c.3067_3072del, p.Ile1023_Val1024del), 3272 26A > G (c.3140 26A > G), R1162X (c.3484C > T, p.Arg1162X), 3849+10kbCYT, 3659delC (c.3528delC, p.Lys1177fs), S1251N (c.3752G > A, p.Ser1251Asn), 3905insT (c.3773dupT, p.Leu1258fs), W1282X (c.3846G> A, p.Trp1282X), N1303K (c.3909C>G, p.Asn1303Lys), CFTRdele2,3(21kb) and Tn-polymorphism on intron 8. Login to comment

[hide] Hyperechogenic fetal bowel: counseling difficultie... Eur J Med Genet. 2005 Oct-Dec;48(4):421-5. Marcus-Soekarman D, Offermans J, Van den Ouweland AM, Mulder AL, Muntjewerff N, Vossen M, Kleijer W, Schrander-Stumpel C, Dooijes D
Hyperechogenic fetal bowel: counseling difficulties.
Eur J Med Genet. 2005 Oct-Dec;48(4):421-5., [PMID:16378926]

Abstract [show]
The detection of echodense fetal bowel on ultrasound examination in the second trimester of pregnancy justifies invasive procedures such as amniocentesis to detect an underlying cause. We present a case in which initial tests identified only one mutation in the cystic fibrosis transmembrane regulator (CFTR)-gene of the fetus, the family history being negative for CF. Strongly reduced intestinal enzyme activities suggested intestinal obstruction and further increased the estimated risk for CF. After the 24th gestational week, a second mutation was found, confirming cystic fibrosis in this child. Problems in counseling in this particular case are discussed.

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67 Routine CFTR-mutation analysis, using Table 1 CFTR-mutations screened for in the first step E60X 2143delT G542X G85E 2183AA-G G551D 394delTT 2184delA Q552X 621 + 1G-T 2789 + 5G-A R553X R117H 3849 + 10kbC-T R560T 711 + 5G-A R1162X S1251N 1078delT 3659delC 390insT R334W delta I507 W1282X R347P delta F508 N1303K A455E 1717-1G-A a panel of 29 CFTR-mutations, detects only 41.6% of CFTR-mutations in the Turkish population [1]. Login to comment

[hide] Genotyping microarray for the detection of more th... J Mol Diagn. 2005 Aug;7(3):375-87. Schrijver I, Oitmaa E, Metspalu A, Gardner P
Genotyping microarray for the detection of more than 200 CFTR mutations in ethnically diverse populations.
J Mol Diagn. 2005 Aug;7(3):375-87., [PMID:16049310]

Abstract [show]
Cystic fibrosis (CF), which is due to mutations in the cystic fibrosis transmembrane conductance regulator gene, is a common life-shortening disease. Although CF occurs with the highest incidence in Caucasians, it also occurs in other ethnicities with variable frequency. Recent national guidelines suggest that all couples contemplating pregnancy should be informed of molecular screening for CF carrier status for purposes of genetic counseling. Commercially available CF carrier screening panels offer a limited panel of mutations, however, making them insufficiently sensitive for certain groups within an ethnically diverse population. This discrepancy is even more pronounced when such carrier screening panels are used for diagnostic purposes. By means of arrayed primer extension technology, we have designed a genotyping microarray with 204 probe sites for CF transmembrane conductance regulator gene mutation detection. The arrayed primer extension array, based on a platform technology for disease detection with multiple applications, is a robust, cost-effective, and easily modifiable assay suitable for CF carrier screening and disease detection.

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51 Complete List of Mutations Detectable with the CF APEX Assay CFTR location Amino acid change Nucleotide change 1 E 1 Frameshift 175delC 2 E 2,3 Frameshift del E2, E3 3 E 2 W19C 189 GϾT 4 E 2 Q39X 247 CϾT 5 IVS 2 Possible splicing defect 296 ϩ 12 TϾC 6 E 3 Frameshift 359insT 7 E 3 Frameshift 394delTT 8 E 3 W57X (TAG) 302GϾA 9 E 3 W57X (TGA) 303GϾA 10 E 3 E60X 310GϾT 11 E 3 P67L 332CϾT 12 E 3 R74Q 353GϾA 13 E 3 R75X 355CϾT 14 E 3 G85E 386GϾA 15 E 3 G91R 403GϾA 16 IVS 3 Splicing defect 405 ϩ 1GϾA 17 IVS 3 Possible splicing defect 405 ϩ 3AϾC 18 IVS 3 Splicing defect 406 - 1GϾA 19 E 4 E92X 406GϾT 20 E 4 E92K 406GϾA 21 E 4 Q98R 425AϾG 22 E 4 Q98P 425AϾC 23 E 4 Frameshift 444delA 24 E 4 Frameshift 457TATϾG 25 E 4 R117C 481CϾT 26 E 4 R117H 482GϾA 27 E 4 R117P 482GϾC 28 E 4 R117L 482GϾT 29 E 4 Y122X 498TϾA 30 E 4 Frameshift 574delA 31 E 4 I148T 575TϾC 32 E 4 Splicing defect 621GϾA 33 IVS 4 Splicing defect 621 ϩ 1GϾT 34 IVS 4 Splicing defect 621 ϩ 3AϾG 35 E 5 Frameshift 624delT 36 E 5 Frameshift 663delT 37 E 5 G178R 664GϾA 38 E 5 Q179K 667CϾA 39 IVS 5 Splicing defect 711 ϩ 1GϾT 40 IVS 5 Splicing defect 711 ϩ 1GϾA 41 IVS 5 Splicing defect 712 - 1GϾT 42 E 6a H199Y 727CϾT 43 E 6a P205S 745CϾT 44 E 6a L206W 749TϾG 45 E 6a Q220X 790CϾT 46 E 6b Frameshift 935delA 47 E 6b Frameshift 936delTA 48 E 6b N287Y 991AϾT 49 IVS 6b Splicing defect 1002 - 3TϾG 50 E 7 ⌬F311 3-bp del between nucleotides 1059 and 1069 51 E 7 Frameshift 1078delT 52 E 7 Frameshift 1119delA 53 E 7 G330X 1120GϾT 54 E 7 R334W 1132CϾT 55 E 7 I336K 1139TϾA 56 E 7 T338I 1145CϾT 57 E 7 Frameshift 1154insTC 58 E 7 Frameshift 1161delC 59 E 7 L346P 1169TϾC 60 E 7 R347H 1172GϾA 61 E 7 R347P 1172GϾC 62 E 7 R347L 1172GϾT 63 E 7 R352Q 1187GϾA 64 E 7 Q359K/T360K 1207CϾA and 1211CϾA 65 E 7 S364P 1222TϾC 66 E 8 Frameshift 1259insA 67 E 8 W401X (TAG) 1334GϾA 68 E 8 W401X (TGA) 1335GϾA 69 IVS 8 Splicing changes 1342 - 6 poly(T) variants 5T/7T/9T 70 IVS 8 Splicing defect 1342 - 2AϾC Table 1. Continued CFTR location Amino acid change Nucleotide change 71 E 9 A455E 1496CϾA 72 E 9 Frameshift 1504delG 73 E 10 G480C 1570GϾT 74 E 10 Q493X 1609CϾT 75 E 10 Frameshift 1609delCA 76 E 10 ⌬I507 3-bp del between nucleotides 1648 and 1653 77 E 10 ⌬F508 3-bp del between nucleotides 1652 and 1655 78 E 10 Frameshift 1677delTA 79 E 10 V520F 1690GϾT 80 E 10 C524X 1704CϾA 81 IVS 10 Possible splicing defect 1717 - 8GϾA 82 IVS 10 Splicing defect 1717 - 1GϾA 83 E 11 G542X 1756GϾT 84 E 11 G551D 1784GϾA 85 E 11 Frameshift 1784delG 86 E 11 S549R (AϾC) 1777AϾC 87 E 11 S549I 1778GϾT 88 E 11 S549N 1778GϾA 89 E 11 S549R (TϾG) 1779TϾG 90 E 11 Q552X 1786CϾT 91 E 11 R553X 1789CϾT 92 E 11 R553G 1789CϾG 93 E 11 R553Q 1790GϾA 94 E 11 L558S 1805TϾC 95 E 11 A559T 1807GϾA 96 E 11 R560T 1811GϾC 97 E 11 R560K 1811GϾA 98 IVS 11 Splicing defect 1811 ϩ 1.6 kb AϾG 99 IVS 11 Splicing defect 1812 - 1GϾA 100 E 12 Y563D 1819TϾG 101 E 12 Y563N 1819TϾA 102 E 12 Frameshift 1833delT 103 E 12 D572N 1846GϾA 104 E 12 P574H 1853CϾA 105 E 12 T582R 1877CϾG 106 E 12 E585X 1885GϾT 107 IVS 12 Splicing defect 1898 ϩ 5GϾT 108 IVS 12 Splicing defect 1898 ϩ 1GϾA 109 IVS 12 Splicing defect 1898 ϩ 1GϾC 110 IVS 12 Splicing defect 1898 ϩ 1GϾT 111 E 13 Frameshift 1924del7 112 E 13 del of 28 amino acids 1949del84 113 E 13 I618T 1985TϾC 114 E 13 Frameshift 2183AAϾG 115 E 13 Frameshift 2043delG 116 E 13 Frameshift 2055del9ϾA 117 E 13 D648V 2075TϾA 118 E 13 Frameshift 2105-2117 del13insAGAA 119 E 13 Frameshift 2108delA 120 E 13 R668C 2134CϾT 121 E 13 Frameshift 2143delT 122 E 13 Frameshift 2176insC 123 E 13 Frameshift 2184delA 124 E 13 Frameshift 2184insA 125 E 13 Q685X 2185CϾT 126 E 13 R709X 2257CϾT 127 E 13 K710X 2260AϾT 128 E 13 Frameshift 2307insA 129 E 13 V754M 2392GϾA 130 E 13 R764X 2422CϾT 131 E 14a W846X 2670GϾA 132 E 14a Frameshift 2734delGinsAT 133 E 14b Frameshift 2766del8 134 IVS 14b Splicing defect 2789 ϩ 5GϾA 135 IVS 14b Splicing defect 2790 - 2AϾG 136 E 15 Q890X 2800CϾT 137 E 15 Frameshift 2869insG 138 E 15 S945L 2966CϾT 139 E 15 Frameshift 2991del32 140 E 16 Splicing defect 3120GϾA interrogation: ACCAACATGTTTTCTTTGATCTTAC 3121-2A3G,T S; 5Ј-ACCAACATGTTTTCTTTGATCTTAC A GTTGTTATTAATTGTGATTGGAGCTATAG-3Ј; CAACAA- TAATTAACACTAACCTCGA 3121-2A3G,T AS. Login to comment
73 Genomic DNA Samples Used for Mutation Evaluation on the APEX Array Mutations validated with native DNA CFTRdel 2,3 (21 kb) 394delTT G85E R75X 574delA Y122X R117C R117H 621 ϩ 1GϾT 621 ϩ 3AϾG 711 ϩ 1GϾT I336K R334W R347P IVS8-5T IVS8-7T IVS8-9T A455E ⌬F508 ⌬I507 1677delTA 1717 - 1GϾA G542X G551D R553X R560T S549N 1898 ϩ 1GϾA 1898 ϩ 1GϾC 2183AAϾG 2043delG R668C 2143delT 2184delA 2184insA 2789 ϩ 5GϾA S945L 3120 ϩ 1GϾA I1005R 3272 - 26AϾG R1066C G1069R Y1092X (CϾA) 3500 - 2AϾT R1158X R1162X 3659delC S1235R 3849 ϩ 10 kb CϾT W1282X primer. Login to comment

[hide] Diagnostic testing by CFTR gene mutation analysis ... J Mol Diagn. 2005 May;7(2):289-99. Schrijver I, Ramalingam S, Sankaran R, Swanson S, Dunlop CL, Keiles S, Moss RB, Oehlert J, Gardner P, Wassman ER, Kammesheidt A
Diagnostic testing by CFTR gene mutation analysis in a large group of Hispanics: novel mutations and assessment of a population-specific mutation spectrum.
J Mol Diagn. 2005 May;7(2):289-99., [PMID:15858154]

Abstract [show]
Characterization of CFTR mutations in the U.S. Hispanic population is vital to early diagnosis, genetic counseling, patient-specific treatment, and the understanding of cystic fibrosis (CF) pathogenesis. The mutation spectrum in Hispanics, however, remains poorly defined. A group of 257 self-identified Hispanics with clinical manifestations consistent with CF were studied by temporal temperature gradient electrophoresis and/or DNA sequencing. A total of 183 mutations were identified, including 14 different amino acid-changing novel variants. A significant proportion (78/85) of the different mutations identified would not have been detected by the ACMG/ACOG-recommended 25-mutation screening panel. Over one third of the mutations (27/85) occurred with a relative frequency >1%, which illustrates that the identified mutations are not all rare. This is supported by a comparison with other large CFTR studies. These results underscore the disparity in mutation identification between Caucasians and Hispanics and show utility for comprehensive diagnostic CFTR mutation analysis in this population.

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187 CFTR Sequence Variants Identified in Five Comprehensive CFTR Studies in US Hispanics CFTR mutations Alleles Relative mutation frequency (%) (of 317) deltaF508 123 38.80 3876delA 15 4.70 G542X 12 3.80 406 - 1GϾA 8 2.50 3849 ϩ 10kbCϾT 5 1.60 R75X 4 1.30 935delA 4 1.30 S549N 4 1.30 W1204X 4 1.30 R334W 4 1.30 2055del9ϾA 3 1 R74W 3 1 H199Y 3 1 L206W 3 1 663delT 3 1 3120 ϩ 1GϾA 3 1 L997F 3 1 I1027T 3 1 R1066C 3 1 W1089X 3 1 D1270N 3 1 2105del13insAGAAA 3 1 Q98R 2 Ͻ1 E116K 2 Ͻ1 I148T 2 Ͻ1 R668C 2 Ͻ1 P205S 2 Ͻ1 V232D 2 Ͻ1 S492F 2 Ͻ1 T501A 2 Ͻ1 1949del84 2 Ͻ1 Q890X 2 Ͻ1 3271delGG 2 Ͻ1 3272 - 26AϾG 2 Ͻ1 G1244E 2 Ͻ1 D1445N 2 Ͻ1 R553X 2 Ͻ1 E588V 2 Ͻ1 1717 - 8GϾA 2 Ͻ1 A1009T 2 Ͻ1 S1235R 2 Ͻ1 G85E 1 Ͻ1 296 ϩ 28AϾG 1 Ͻ1 406 - 6TϾC 1 Ͻ1 V11I 1 Ͻ1 Q179K 1 Ͻ1 V201 mol/L 1 Ͻ1 874insTACA 1 Ͻ1 I285F 1 Ͻ1 deltaF311 1 Ͻ1 F311L 1 Ͻ1 L320V 1 Ͻ1 T351S 1 Ͻ1 R352W 1 Ͻ1 1248 ϩ 1GϾA 1 Ͻ1 1249 - 29delAT 1 Ͻ1 1288insTA 1 Ͻ1 1341 ϩ 80GϾA 1 Ͻ1 1429del7 1 Ͻ1 1525 - 42GϾA 1 Ͻ1 P439S 1 Ͻ1 1717 - 1GϾA 1 Ͻ1 1811 ϩ 1GϾA 1 Ͻ1 deltaI507 1 Ͻ1 G551D 1 Ͻ1 A559T 1 Ͻ1 Y563N 1 Ͻ1 (Table continues) In this study, we used temporal temperature gradient gel electrophoresis (TTGE) and direct DNA sequencing to increase the sensitivity of mutation detection in U.S. Hispanics, and to determine whether additional mutations are recurrent. Login to comment

[hide] A novel method for creating artificial mutant samp... J Mol Diagn. 2005 May;7(2):247-51. Jarvis M, Iyer RK, Williams LO, Noll WW, Thomas K, Telatar M, Grody WW
A novel method for creating artificial mutant samples for performance evaluation and quality control in clinical molecular genetics.
J Mol Diagn. 2005 May;7(2):247-51., [PMID:15858148]

Abstract [show]
The lack of readily available, patient-derived materials for molecular genetic testing of many heterozygous or rare disorders creates a major impediment for laboratory proficiency and quality control procedures. The paucity of clinically derived mutation-positive samples could be surmounted if it were possible to construct artificial samples containing mutations of interest that would sufficiently resemble natural human samples. Such samples could then function as acceptable and realistic performance evaluation challenges and quality control reagents for recipient laboratories. Using the cystic fibrosis gene (CFTR) as a prototype, we have devised and executed experiments designed to generate unique DNA samples that could be used for these purposes. We used site-directed mutagenesis to generate mutations of interest in plasmid DNA derived from common bacterial artificial chromosome sources containing the cystic fibrosis transmembrane conductance receptor gene. CFTR mutations G85E and 1078delT were chosen to represent mutations in the original American College of Medical Genetics-recommended population-screening panel of 25 mutations. DNA samples containing predetermined concentrations and ratios of wild-type and mutated plasmids, bacterial artificial chromosomes of interest, and nonhuman genomic carrier DNA were characterized and tested in-house and in a group of nine pilot testing laboratories using a variety of technical platforms. The results indicate that these constructs, containing CFTR mutations in heterozygous and homozygous states, can serve as valid and accessible materials for quality assurance, including performance evaluation, proficiency testing, and assay quality control.

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5 CFTR mutations G85E and 1078delT were chosen to represent mutations in the original American College of Medical Genetics-recommended population- screening panel of 25 mutations. Login to comment
37 It is ϳ250 kb in size and contains 27 exons.8 Several mutations were chosen for preliminary targeting experiments, including G85E (exon 3), N1303K (exon 21), and 1078delT (exon 7). Login to comment
39 The study described here focused on the G85E and 1078delT target sequences because they are rarer and harder to obtain from natural sources than N1303K; the latter was used as a marker primarily to ensure that our CFTR constructs potentially encompassed all possible mutations in the original ACMG panel. Login to comment
60 Because it was the major DNA component, we determined that when salmon sperm DNA was used as template for PCR analysis using any of our usual oligonucleotide primer sets, no amplified products of the anticipated sizes were observed by either an in-house amplification restriction digestion method or a commercial CFTR hybridization assay (Roche Diagnostics, Indianapolis, IN).10 Samples were formulated and analyzed for each of the following five genotypes: wild-type (homozygous normal), homozygous G85E, homozygous 1078delT, heterozygous G85E, and heterozygous 1078delT. Login to comment
62 Results of in-house pilot testing of constructed heterozygous and homozygous products for CFTR mutations G85E and 1078delT using a commercial reverse hybridization strip system (Roche Linear Array CF Gold 1.0). Login to comment
64 The observed genotypes are: lane 1, negative for tested mutations; lane 2, G85E homozygote; lane 3, 1078delT homozygote; lane 4, G85E heterozygote; lane 5, 1078delT heterozygote; lane 6, negative for tested mutations. Login to comment
93 Pilot Testing Results Sample Laboratory analysis (correct results/total results) Unable to analyze Normal (w.t.) 7/8 1 G85E, heterozygous 8/9 G85E, homozygous 8/9 1078delT, heterozygous 8/9 1078delT, homozygous 7/9 Table 2. Login to comment

[hide] Complete gene scanning by temperature gradient cap... J Mol Diagn. 2005 Feb;7(1):111-20. Chou LS, Gedge F, Lyon E
Complete gene scanning by temperature gradient capillary electrophoresis using the cystic fibrosis transmembrane conductance regulator gene as a model.
J Mol Diagn. 2005 Feb;7(1):111-20., [PMID:15681482]

Abstract [show]
Many inherited diseases involve large genes with many different mutations. Identifying a wide spectrum of mutations requires an efficient gene-scanning method. By differentiating thermodynamic stability and mobility of heteroduplexes from heterozygous samples, temperature gradient capillary electrophoresis (TGCE) was used to scan the entire coding region of the cystic fibrosis transmembrane conductance regulator gene. An initial panel (29 different mutations) showed 100% agreement between TGCE scanning and previously genotyped results for heterozygous samples. Different peak patterns were observed for single base substitutions and base insertions/deletions. Subsequently, 12 deidentified clinical samples genotyped as wild type for 32 mutations were scanned for the entire 27 exons. Results were 100% concordance with the bidirectional sequence analysis. Ten samples had nucleotide variations including a reported base insertion in intron 14b (2789 + 2insA) resulting in a possible mRNA splicing defect, and an unreported missense mutation in exon 20 (3991 G/A) with unknown clinical significance. This methodology does not require labeled primers or probes for detection and separation through a temperature gradient eliminates laborious temperature optimization required for other technologies. TGCE automation and high-throughput capability can be implemented in a clinical environment for mutation scanning with high sensitivity, thus reducing sequencing cost and effort.

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75 Mutation Samples with Known Genotypes Scanned by TGCE* Exon Mutation† Amplicon size (bp) Location of mutation from 5Ј end (bp) Base change Detection‡ 3 G85E 234 124 G to A 1/1 3 394delTT 234 132 del TT 1/1 4 R117H 270 83 G to T 2/2 4 I148T 270 176 T to C 3/3 Intron 4 621 ϩ 1 G/T 270 233 G to T 1/1 5 663delT/663delT 186 75 del T 0/1 Intron 5 711 ϩ 1 G/T 186 124 G to T 1/1 7 R334W 345 208 C to T 1/1 7 R347P 345 248 G to C 1/1 9 A455E 263 155 C to A 2/2 10 I506V 292 168 A to G 1/1 10 ⌬I507 292 171 del ATC 2/2 10 ⌬F508 292 174 del TTT 2/2 10 ⌬F508/⌬F508 292 174 del TTT 0/1 10 F508C 292 175 T to G 1/1 10 V520F 292 210 G to T 1/1 Intron 10 1717-1 G/A 175 50 G to A 1/1 11 G542X 175 90 G to T 2/2 11 G542X/G542X 175 90 G to T 0/1 11 G551D 175 118 G to A 3/3 11 R553X 175 123 C to T 3/3 11 R560T 175 145 G to C 2/2 13 2184delA 834 356 del A 1/1 Intron 14b 2789 ϩ 5G/A 192 102 G to A 1/1 Intron 16 3120 ϩ 1G/A 216 111 G to A 1/1 19 R1162X 322 68 C to T 1/1 19 3659delC 322 111 del C 1/1 20 W1282X 206 154 G to A 1/1 21 N1303K 250 175 C to G 2/2 Total exon/intron Overall accuracy 17 93% *Samples were compared with their respective wild-type control (confirmed by sequencing). Login to comment

[hide] Rapid screening for 31 mutations and polymorphisms... Methods Mol Med. 2005;114:147-71. Dunbar SA, Jacobson JW
Rapid screening for 31 mutations and polymorphisms in the cystic fibrosis transmembrane conductance regulator gene by Lminex xMAP suspension array.
Methods Mol Med. 2005;114:147-71., [PMID:16156102]

Abstract [show]
A suspension array hybridization assay is described for the detection of 31 mutations and polymorphisms in the cystic fibrosis transmembrane conductance regulator (CFTR) gene using Luminex xMAP technology. The Luminex xMAP system allows simultaneous detection of up to 100 different targets in a single multiplexed reaction. Included in the method are the procedures for design of oligonucleotide capture probes and PCR amplification primers, coupling oligonucleotide capture probes to carboxylated microspheres, hybridization of coupled microspheres to oligonucleotide targets, production of targets from DNA samples by multiplexed PCR amplification, and detection of PCR-amplified targets by direct hybridization to probe-coupled microspheres. Mutation screening with the system is rapid, requires relatively few sample manipulations, and provides adequate resolution to reliably genotype the 25 CFTR mutations and 6 CFTR polymorphisms contained in the ACMG/ACOG/NIH-recommended core mutation panel for general population CF carrier screening.

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25 A 635-nm 10-mW red diode laser excites the two fluo- 148 Dunbar and Jacobson xMAP™ 149 Table 1 Recommended Core Mutation Panel for General Population Cystic Fibrosis (CF) Carrier Screening Standard mutation panel ΔF508 ΔI507 G542X G551D W1282X N1303K R553X 621+1G→T R117H 1717-1G→A A455E R560T R1162X G85E R334W R347P 711+1G→T 1898+1G→A 2184delA 1078delT 3849+10kbC→T 2789+5G→A 3659delC 1148T 3120+1G→A Reflex tests I506Va I507Va F508Ca 5T/7T/9Tb a Benign variants. Login to comment
94 Methods The methods described below include: (1) design of oligonucleotide capture probes, (2) design of PCR amplification primers and multiplexed PCR reactions, (3) preparation of the probe-conjugated microsphere sets, (4) verification 150 Dunbar and Jacobson xMAPTM Table 2 Oligonucleotide Capture Probesa Target Microsphere Probe sequence Modificationb Sequence 5' → 3' set Standard mutation panel 1c I507 & F508 5'-AmMC12 AACACCAAAGATGATATTTT 006 2B DI507 5'-AmMC12 ACACCAAAGATATTTTCTT 008 3B DF508 5'-AmMC12 AAACACCAATGATATTTTC 015 4B W1282 5'-AmMC12 CAACAGTGGAGGAAAGCC 012 5B W1282X 5'-AmMC12 CAACAGTGAAGGAAAGCC 020 6 1717-1G 5'-AmMC12 TTGGTAATAGGACATCTCCA 017 7 1717-1GÆA 5'-AmMC12 TTGGTAATAAGACATCTCCA 019 8B G542 5'-AmMC12 TATAGTTCTTGGAGAAGGTGGA 026 9B G542X 5'-AmMC12 TATAGTTCTTTGAGAAGGTGGA 028 10C G551 & R553 5'-AmMC12 AGTGGAGGTCAACGAGCAA 038 11B G551D 5'-AmMC12 GTGGAGATCAACGAGCAA 030 12C R553X 5'-AmMC12 GTGGAGGTCAATGAGCAA 032 13 R560 5'-AmMC12 CTTTAGCAAGGTGAATAACT 035 14 R560T 5'-AmMC12 CTTTAGCAACGTGAATAACT 039 15 R117 5'-AmMC12 AGGAGGAACGCTCTATCGCG 042 16 R117H 5'-AmMC12 AGGAGGAACACTCTATCGCG 025 17B I148 5'-AmMC12 CTTCATCACATTGGAATGCAGA 034 18B I148T 5'-AmMC12 CTTCATCACACTGGAATGCAGA 045 19C 621+1G 5'-AmMC12 TTTATAAGAAGGTAATACTTCCT 046 20E 621+1G→T 5'-AmMC12 ATTTATAAGAAGTTAATACTTCCTT 048 21 N1303 5'-AmMC12 GGGATCCAAGTTTTTTCTAA 051 22 N1303K 5'-AmMC12 GGGATCCAACTTTTTTCTAA 052 23B 1078T 5'-AmMC12 CACCACAAAGAACCCTGA 054 24C 1078delT 5'-AmMC12 ACACCACAAGAACCCTGA 061 25 R334 5'-AmMC12 ATATTTTCCGGAGGATGATT 063 26 R334W 5'-AmMC12 ATATTTTCCAGAGGATGATT 064 27B R347 5'-AmMC12 ACCGCCATGCGCAGAACAA 067 28B R347P 5'-AmMC12 ACCGCCATGGGCAGAACAA 053 29C 711+1G 5'-AmMC12 ATTTGATGAAGTATGTACCTAT 059 30C 711+1G→T 5'-AmMC12 ATTTGATGAATTATGTACCTAT 071 31 G85 5'-AmMC12 TGTTCTATGGAATCTTTTTA 066 32B G85E 5'-AmMC12 ATGTTCTATGAAATCTTTTTA 073 33 3849+10kbC 5'-AmMC12 GTCTTACTCGCCATTTTAAT 077 34 3849+10kbC→T 5'-AmMC12 GTCTTACTCACCATTTTAAT 075 35 A455 5'-AmMC12 CCAGCAACCGCCAACAACTG 011 36D A455E 5'-AmMC12 TCCAGCAACCTCCAACAACTG 036 37 R1162 5'-AmMC12 TAAAGACTCGGCTCACAGAT 060 38 R1162X 5'-AmMC12 TAAAGACTCAGCTCACAGAT 068 39B 3659C 5'-AmMC12 TTGACTTGGTAGGTTTAC 022 40C 3659delC 5'-AmMC12 TTGACTTGTAGGTTTACC 079 41B 2789+5G 5'-AmMC12 TGGAAAGTGAGTATTCCATGTC 074 42D 2789+5G→A 5'-AmMC12 TTGGAAAGTGAATATTCCATGTC 014 43E 2184A 5'-AmMC12 GAAACAAAAAAACAATC 007 44E 2184delA 5'-AmMC12 AGAAACAAAAAACAATC 018 45B 1898+1G 5'-AmMC12 TATTTGAAAGGTATGTTCTTTG 013 (Continued) of microsphere coupling, (5) direct hybridization of biotinylated PCR amplification products to the multiplexed probe-coupled microsphere sets, and (6) results and data analysis. Login to comment
106 Table 3 Reverse Complementary Oligonucleotide Targetsa Target Target sequence Modification Sequence 5' → 3' Standard mutation panel C1b I507 & F508 5'-Biotin AAAATATCATCTTTGGTGTT C2 ΔI507 5'-Biotin AAAGAAAATATCTTTGGTGT C3 ΔF508 5'-Biotin AGAAAATATCATTGGTGTTT C4 W1282 5'-Biotin GGCTTTCCTCCACTGTTGC C5 W1282X 5'-Biotin GGCTTTCCTTCACTGTTGC C6 1717-1G 5'-Biotin TGGAGATGTCCTATTACCAA C7 1717-1G→A 5'-Biotin TGGAGATGTCTTATTACCAA C8 G542 5'-Biotin CCACCTTCTCCAAGAACTAT C9 G542X 5'-Biotin CCACCTTCTCAAAGAACTAT C10 G551 & R553 5'-Biotin CTTGCTCGTTGACCTCCACT C11 G551D 5'-Biotin CTTGCTCGTTGATCTCCACT C12 R553X 5'-Biotin CTTGCTCATTGACCTCCACT C13 R560 5'-Biotin AGTTATTCACCTTGATAAAG C14 R560T 5'-Biotin AGTTATTCACGTTGCTAAAG C15 R117 5'-Biotin CGCGATAGAGCGTTCCTCCT C16 R117H 5'-Biotin CGCGATAGAGTGTTCCTCCT C17 I148 5'-Biotin CTGCATTCCAATGTGATGAA C18 I148T 5'-Biotin CTGCATTCCAGTGTGATGAA C19 621+1G 5'-Biotin GGAAGTATTACCTTCTTATA C20 621+1G→T 5'-Biotin GGAAGTATTAACTTCTTATA C21 N1303 5'-Biotin TTAGAAAAAACTTGGATCCC C22 N1303K 5'-Biotin TTAGAAAAAAGTTGGATCCC C23 1078T 5'-Biotin CTCAGGGTTCTTTGTGGTGT C24 1078delT 5'-Biotin TCTCAGGGTTCTTGTGGTGT C25 R334 5'-Biotin AATCATCCTCCGGAAAATAT C26 R334W 5'-Biotin AATCATCCTCTGGAAAATAT C27 R347 5'-Biotin ATTGTTCTGCGCATGGCGGT C28 R347P 5'-Biotin ATTGTTCTGCCCATGGCGGT C29 711+1G 5'-Biotin TAGGTACATACTTCATCAAA C30 711+1G→T 5'-Biotin TAGGTACATAATTCATCAAA C31 G85 5'-Biotin TAAAAAGATTCCATAGAACA C32 G85E 5'-Biotin TAAAAAGATTTCATAGAACA C33 3849+10kbC 5'-Biotin ATTAAAATGGCGAGTAAGAC C34 3849+10kbC→T 5'-Biotin ATTAAAATGGTGAGTAAGAC C35 A455 5'-Biotin CAGTTGTTGGCGGTTGCTGG C36 A455E 5'-Biotin CAGTTGTTGGAGGTTGCTGG C37 R1162 5'-Biotin ATCTGTGAGCCGAGTCTTTA C38 R1162X 5'-Biotin ATCTGTGAGCTGAGTCTTTA (Continued) Rapid CF Screening by xMAPTM 153 Table 3 (Continued) Target Target sequence Modification Sequence 5' → 3' C39 3659C 5'-Biotin GGTAAACCTACCAAGTCAAC C40 3659delC 5'-Biotin AGGTAAACCTACAAGTCAAC C41 2789+5G 5'-Biotin ACATGGAATACTCACTTTCC C42 2789+5G→A 5'-Biotin ACATGGAATATTCACTTTCC C43 2184A 5'-Biotin AAGATTGTTTTTTTGTTTCT C44 2184delA 5'-Biotin AAGATTGTTTTTTGTTTCTG C45 1898+1G 5'-Biotin AAAGAACATACCTTTCAAAT C46 1898+1G→A 5'-Biotin AAAGAACATATCTTTCAAAT C47 3120+1G 5'-Biotin TTTTTACATACCTGGATGAA C48 3120+1G→A 5'-Biotin TTTTTACATATCTGGATGAA Reflex panel CR2 I506V 5'-Biotin GAAAATGTCATCTTTGGTGT CR3 I507V 5'-Biotin GAAAATATCGTCTTTGGTGT CR4 F508C 5'-Biotin AAAATATCATCTGTGGTGTT CR5 5T 5'-Biotin TCCCTGTTAAAAACACACAC CR6 7T 5'-Biotin CCCTGTTAAAAAAACACACA CR7 9T 5'-Biotin CCTGTTAAAAAAAAACACAC a The position and sequence of the mutation or variation is indicated in bold type. b Target C1 (I507 & F508) is also used in the reflex panel. Login to comment
114 Using a small target DNA (approx 100-300 bp) minimizes the potential for steric hindrance to affect the xMAPTM Table 4 PCR Amplification Primers Size CFTR target Mutation(s) Primer 5' Modification Sequence 5' → 3' (bp) Exon 10 ΔI507, ΔF508, BE10U 5'-Biotin TTCTGTTCTCAGTTTTCCTGG 107 I506V, I507V, E10D None TTGGCATGCTTTGATGACG F508C Exon 20 W1282X E20U None TTGAGACTACTGAACACTGAAGG 126 BE20D 5'-Biotin TTCTGGCTAAGTCCTTTTGC Intron 10 1717-1G→A E11U None TCAGATTGAGCATACTAAAAGTGAC 89 BE11D2 5'-Biotin GAACTATATTGTCTTTCTCTGCAAAC Exon 11 G542X, G551D, E11U2 None AAGTTTGCAGAGAAAGACAATATAG 135 R553X, R560T BE11D 5'-Biotin GAATGACATTTACAGCAAATGC Exon 4 R117H E4U None TTTGTAGGAAGTCACCAAAGC 145 BE4D2 5'-Biotin GAGCAGTGTCCTCACAATAAAGAG Exon 4/intron 4 I148T, E4U2 None CTTCTCTTTATTGTGAGGACACTGC 169 621+1G→T BE4D 5'-Biotin ATGACATTAAAACATGTACGATACAG Exon 21 N1303K BE21U 5'-Biotin TGCTATAGAAAGTATTTATTTTTTCTGG 106 E21D None AGCCTTACCTCATCTGCAAC Exon 7 1078delT, BE7U 5'-Biotin GAACAGAACTGAAACTGACTCG 199 R334W, R347P E7D3 None CAGGGAAATTGCCGAGTG Intron 5 711+1G→T I5U None CAACTTGTTAGTCTCCTTTCC 99 BI5D2 5'-Biotin AGTTGTATAATTTATAACAATAGTGC Exon 3 G85E E3U None CTGGCTTCAAAGAAAAATCC 117 BE3D2 5'-Biotin TGAATGTACAAATGAGATCCTTACC Chromosome 7 3849+10kbC→T BC7U 5'-Biotin GACTTGTCATCTTGATTTCTGG 148 C7D None TTTGGTGCTAGCTGTAATTGC Exon 9 A455E BE9U 5'-Biotin TCACTTCTTGGTACTCCTGTCC 105 E9D None CAAAAGAACTACCTTGCCTGC Exon 19-I R1162X BE19U 5'-Biotin ATTGTGAAATTGTCTGCCATTC 167 E19Da None CAATAATCATAACTTTCGAGAGTTG Exon 19-II 3659delC BE19U2 5'-Biotin TTTAAGTTCATTGACATGCCAAC 91 E19Da None CAATAATCATAACTTTCGAGAGTTG Intron 14B 2789+5G→A I14BU None GTGTCTTGTTCCATTCCAGG 147 BI14BD 5'-Biotin TGGATTACAATACATACAAACATAGTGG Exon 13 2184delA E13U None AGATGCTCCTGTCTCCTGG 126 BE13D 5'-Biotin TGCACAATGGAAAATTTTCGTATAG Intron 12 1898+1G→A I12U None TTAGACTCTCCTTTTGGATACC 110 BI12D 5'-Biotin GTCTTTCTTTTATTTTAGCATGAGC Intron 16 3120+1G→A I16U None ATGACCTTCTGCCTCTTACC 118 BI16D 5'-Biotin ATGAAAACAAAATCACATTTGC Intron 8 5T/7T/9T I8U None TAATGGATCATGGGCCATGTGC 212 BI8D 5'-Biotin ACTGAAGAAGAGGCTGTCATCACC CFTR, cystic fibrosis transmembrane conductance regulator gene. Login to comment
119 Coriell Cell Repositories, NA12960 ΔI507/R347P Patient sample G551D/R347P Coriell Cell Repositories, NA12785 621+1G→T/711+1G→T Coriell Cell Repositories, NA11280 621+1G→T/G85E Coriell Cell Repositories, NA11282 3849+10kbC→T/3849+10kbC→T Coriell Cell Repositories, NA11860 A455E/normal Patient sample 621+1G→T/A455E Coriell Cell Repositories, NA11290 R1162X/normal Coriell Cell Repositories, NA12585 ΔF508/3659delC Coriell Cell Repositories, NA11275 2789+5G→A/2789+5G→A Coriell Cell Repositories, NA11859 2184delA/normal Patient sample 1898+1G→A/normal Patient sample 621+1G→T/3120+1G→A Coriell Cell Repositories, NA07441 3120+1G→A/3120+1G→A Patient sample F508C/normal Coriell Cell Repositories, NA13033 I506V/normal Coriell Cell Repositories, NA13032 R347H/normal Patient sample ΔF508/3120G→A Patient sample S549N/normal Patient sample S549R/normal Patient sample CFTR, cystic fibrosis transmembrane conductance regulator gene. Login to comment

[hide] High heterogeneity of CFTR mutations and unexpecte... J Cyst Fibros. 2004 Dec;3(4):265-72. des Georges M, Guittard C, Altieri JP, Templin C, Sarles J, Sarda P, Claustres M
High heterogeneity of CFTR mutations and unexpected low incidence of cystic fibrosis in the Mediterranean France.
J Cyst Fibros. 2004 Dec;3(4):265-72., [PMID:15698946]

Abstract [show]
In this report, we present updated spectrum and frequency of mutations of the CFTR gene that are responsible for cystic fibrosis (CF) in Languedoc-Roussillon (L-R), the southwestern part of France. A total of 75 different mutations were identified by DGGE in 215 families, accounting for 97.6% of CF genes and generating 88 different mutational genotypes. The frequency of p.F508del was 60.23% in L-R versus 67.18% in the whole country and only five other mutations (p.G542X, p.N1303K, p.R334W, c.1717-1G>A, c.711+1G>T) had a frequency higher than 1%. The mutations were scattered over 20 exons or their border. This sample representing only 5.7% of French CF patients contributed to 24% of CFTR mutations reported in France. This is one of the highest molecular allelic heterogeneity reported so far in CF. We also present the result of a neonatal screening program based on a two-tiered approach "IRT/20 mutations/IRT" analysis on blood spots, implemented in France with the aim to improve survival and quality of life of patients diagnosed before clinical onset. This 18-month pilot project showed an unexpected low incidence of CF (1/8885) in South of France, with only six CF children detected among 43,489 neonates born in L-R, and 13 among 125,339 neonates born in Provence-Alpes-Cote-d'Azur (PACA).

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68 of chromosomes (frequency %) p.M1V 1 1 (0.23) p.M1K 1 1 (0.23) c.300delA 3 1 (0.23) p.P67L 3 1 (0.23) c.359insT 3 1 (0.23) p.G85E 3 3 (0.70) c.394delTT 3 1 (0.23) p.Q98R 4 1 (0.23) p.R117H 4 2 (0.47) p.Y122X 4 2 (0.47) p.Y161N 4 1 (0.23) c.621+1GNT intron 4 1 (0.23) c.621+2TNG intron 4 1 (0.23) p.I175V 5 2 (0.47) c.711+1GNT intron 5 5 (1.16) p.L206W 6 3 (0.70) p.Q220X 6 1 (0.23) p.L227R 6 1 (0.23) c.1078delT 7 2 (0.47) p.R334W 7 7 (1.63) p.R347P 7 2 (0.47) c.1215delG 7 1 (0.23) c.T5 intron 8 1 (0.23) p.D443Y 9 1 (0.23) p.I506T 10 1 (0.23) p.I507del 10 4 (0.93) p.F508del 10 259 (60.23) p.F508C 10 1 (0.23) c.1677delTA 10 1 (0.23) c.1717-8GNA intron 10 1 (0.23) c.1717-1GNA intron 10 6 (1.40) p.G542X 11 23 (5.35) p.S549R 11 1 (0.23) p.G551D 11 2 (0.47) p.R553X 11 1 (0.23) c1811+1.6kbANG intron 11 4 (0.93) c.1812-1GNA intron 11 1 (0.23) p.T582I 12 1 (0.23) p.E585X 12 2 (0,47) c.1898+1GNA intron 12 1 (0.23) [c.1898+5GNA ;p.E725K] intron 12 1 (0.23) c.1898+73TNG intron 12 1 (0.23) c.2183AANG 13 4 (0.93) c.2184insA 13 1 (0.23) p.K710X 13 4 (0.93) c.2423delG 13 1 (0.23) p.S776X 13 1 (0.23) c.2493ins8 13 1 (0.23) p.R792X 13 1 (0.23) p.K830X 13 1 (0.23) p.D836Y 14a 1 (0.23) p.W846X1 14a 1 (0.23) c.2711delT 14a 1 (0.23) c.2789+5GNA intron 14b 3 (0.70) p.S945L 15 3 (0.70) p.D993Y 16 1 (0.23) c.3129del4 17a 1 (0.23) c.3195del6 17a 1 (0.23) c.3272-26ANG intron 17a 1 (0.23) [c.3395insA ;pI148T] 17b/4 1 (0,23) p.Y1092X 17b 3 (0.70) Table 1 (continued) Mutation Location exon/intron No. Login to comment
131 The panel of 30 mutations (c.1717-1GNA, p.G542X, p.W1282X, p.N1303K, p.F508del, c.3849+10kbCNT, c.621+1GNT, p.R553X, p.G551D, p.R117H, p.R1162X, p.R334W, p.A455E, c.2183AANG, c.3659delC, c.1078delT, p.I507del, p.R347P, p.S1251N, p.E60X, p.Y1092X, c.394delTT, c.1811+1.6kbANG, c.3272-26ANG, c.2789+5GNA, c.3120+1GNA, c.711+ 1GNT, p.G85E, p.Y122X, p.W846X) should account for 83.32% of the CF alleles in L-R and 84.25% in the whole country. Login to comment

[hide] Microsphere bead arrays and sequence validation of... J Mol Diagn. 2004 Nov;6(4):348-55. Hadd AG, Laosinchai-Wolf W, Novak CR, Badgett MR, Isgur LA, Goldrick M, Walkerpeach CR
Microsphere bead arrays and sequence validation of 5/7/9T genotypes for multiplex screening of cystic fibrosis polymorphisms.
J Mol Diagn. 2004 Nov;6(4):348-55., [PMID:15507674]

Abstract [show]
The development of simple and rapid methods for the detection of the common genetic mutations associated with cystic fibrosis (CF) requires access to positive-control samples including the 5/7/9T variants of intron 8. We used PCR and a simple multiplex bead-array assay to identify 5/7/9T control samples from 29 commercially available DNA samples. Unpurified PCR products were directly hybridized to color-coded beads containing allele-specific capture probes for 5/7/9T detection. The performance of the assay was investigated using reverse-complement oligonucleotides, individual PCR products, and multiplex PCR products for 5/7/9T detection within a complex CFTR screening assay. Samples were genotyped by grouping the relative signal intensities from each capture probe. Of 29 commercially available DNA samples analyzed, 2 5T/7T, 2 5T/9T, 9 7T/9T, 11 7T/7T, and 5 9T/9T genotypes were identified. The genotype within each sample group was confirmed by DNA sequencing. The assay was compatible with the analysis of 10 to 1000 ng of genomic DNA isolated from whole blood and allowed for the separate identification of primary CFTR mutations from reflex variants. The correct identification of positive controls demonstrated the utility of a simple bead-array assay and provided accessible samples for assay optimization and for routine quality control in the clinical laboratory.

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197 Intron 8 Genotype by Coriell Number, Characterized CF Mutation and Allele Fraction for 5/7/9T Intron 8 genotype Coriell sample Characterized mutation Allele fraction by probe 5T 7T 9T 7T/7T NA09947 Normal 0.04 0.93 0.03 NA11277 ⌬I507/normal 0.06 0.90 0.04 NA11761 G551D/R553X 0.06 0.92 0.02 NA11859 2789ϩ5GϾA/2789ϩ5GϾA 0.02 0.96 0.02 NA11860 3849ϩ10kbCϾT/3849ϩ10kbCϾT 0.03 0.94 0.03 NA12444 1717-1GϾT/normal 0.06 0.87 0.07 NA12585 R1162X/normal 0.07 0.86 0.08 NA12785 R347P/G551D 0.04 0.92 0.05 NA12960 R334W/normal 0.06 0.92 0.02 NA12961 V520F/normal 0.06 0.89 0.05 NA13033 F508C/normal 0.03 0.93 0.04 9T/9T NA01531 ⌬F508/⌬F508 0.14 0.04 0.82 NA11281 621ϩ1GϾT/⌬F508 0.14 0.04 0.82 NA11283 A455E/⌬F508 0.13 0.05 0.82 NA11290 A455E/621ϩ1GϾT 0.12 0.01 0.87 NA11496 G542X/G542X 0.14 0.05 0.81 5T/7T NA11723 W1282X/normal 0.53 0.44 0.03 NA13032 I506V/normal 0.58 0.39 0.03 5T/9T NA11279 129GϾC/⌬F508 0.51 0.00 0.49 NA13591 R117H/⌬F508 0.52 0.00 0.48 7T/9T NA07441 3120ϩ1GϾA/621ϩ1GϾA 0.08 0.41 0.51 NA07552 R553X/⌬F508 0.09 0.36 0.55 NA07830 556dA/⌬F508 0.11 0.37 0.52 NA11275 3659dC/⌬F508 0.10 0.37 0.53 NA11278 Q493X/⌬F508 0.09 0.38 0.53 NA11280 711ϩ1GϾT/621ϩ1GϾA 0.09 0.37 0.54 NA11282 G85E/621ϩ1GϾA 0.07 0.39 0.53 NA11284 R560T/⌬F508 0.08 0.39 0.52 NA11472 N1303K/G1349D 0.08 0.39 0.54 Figure 3. Login to comment

[hide] CFTR Cl- channel function in native human colon co... Gastroenterology. 2004 Oct;127(4):1085-95. Hirtz S, Gonska T, Seydewitz HH, Thomas J, Greiner P, Kuehr J, Brandis M, Eichler I, Rocha H, Lopes AI, Barreto C, Ramalho A, Amaral MD, Kunzelmann K, Mall M
CFTR Cl- channel function in native human colon correlates with the genotype and phenotype in cystic fibrosis.
Gastroenterology. 2004 Oct;127(4):1085-95., [PMID:15480987]

Abstract [show]
BACKGROUND & AIMS: Cystic fibrosis (CF) is caused by over 1000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and presents with a widely variable phenotype. Genotype-phenotype studies identified CFTR mutations that were associated with pancreatic sufficiency (PS). Residual Cl- channel function was shown for selected PS mutations in heterologous cells. However, the functional consequences of most CFTR mutations in native epithelia are not well established. METHODS: To elucidate the relationships between epithelial CFTR function, CFTR genotype, and patient phenotype, we measured cyclic adenosine monophosphate (cAMP)-mediated Cl- secretion in rectal biopsy specimens from 45 CF patients who had at least 1 non-DeltaF508 mutation carrying a wide spectrum of CFTR mutations. We compared CFTR genotypes and clinical manifestations of CF patients who expressed residual CFTR-mediated Cl- secretion with patients in whom Cl- secretion was absent. RESULTS: Residual anion secretion was detected in 40% of CF patients, and was associated with later disease onset (P < 0.0001), higher frequency of PS (P < 0.0001), and less severe lung disease (P < 0.05). Clinical outcomes correlated with the magnitude of residual CFTR activity, which was in the range of approximately 12%-54% of controls. CONCLUSIONS: Specific CFTR mutations confer residual CFTR function to rectal epithelia, which is related closely to a mild disease phenotype. Quantification of rectal CFTR-mediated Cl- secretion may be a sensitive test to predict the prognosis of CF disease and identify CF patients who would benefit from therapeutic strategies that would increase residual CFTR activity.

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78 Relationship Between the CFTR Genotype and Cl- Channel Function in Native Rectal Epithelia CFTR genotype Number of individuals Sweat Cl-concentration (mmol/L)a cAMP-mediated response Carbachol-induced plateau response or maximal lumen-negative response Isc-cAMP (␮A/cm2) Cl- secretion (% of control) Isc-carbachol (␮A/cm2) Cl- secretion (% of control) Cl- secretion absent R1162X/Q552X 1 71 17.1 0 0.7 0 W1282X/3121-2AϾG 1 112 1.9 0 0.6 0 1898 ϩ 1G Ͼ T/1609delCA 2b 114, 118 25.4, 13.4 0, 0 0, 0.7 0, 0 ⌬F508/Q39X 2b 127, 129 2.6, 4.4 0, 0 1.7, 3.7 0, 0 ⌬F508/G542X 1 102 29.0 0 6.6 0 ⌬F508/R553X 3 112, 102, 109 13.1, 4.5, 23.8 0, 0, 0 1.5, 4.4, 1.0 0, 0, 0 ⌬F508/E585X 1 115 1.4 0 1.1 0 ⌬F508/Q637X 1 100 2.9 0 1.2 0 ⌬F508/Y1092X 1 119 0.0 0 -0.3 0 ⌬F508/120del23c 1 72 20.1 0 3.3 0 ⌬F508/182delT 1 116 10.8 0 5.2 0 ⌬F508/3905insT 2 88, 96 8.4, 5.6 0, 0 2.3, -1.1 0, 1 ⌬F508/V520F 1 68 1.2 0 1.7 0 ⌬F508/A561E 3 113, 146, 100 17.0, 17.0, 16.0 0, 0, 0 2.1, 1.5, 3.7 0, 0, 0 ⌬F508/R1066C 1 138 0.0 0 0.0 0 ⌬F508/N1303K 3 100, 117, 94 1.7, 4.1, 1.5 0, 0, 0 -0.6, 2.2, 0.8 0, 0, 0 A561E/A561E 2 101, 116 6.6, 2.0 0, 0 7.3, 3.3 0, 0 Residual Cl- secretiond G542X/I148N 1 75 -50.1 54 -22.2 12 1898 ϩ 3A Ͼ G/1898 ϩ 3A Ͼ G 1 82 -36.8 39 -12.9 7 ⌬F508/3272-26A Ͼ G 1 116 -17.8 19 -27.2 14 ⌬F508/S108F 1 118 -15.8 17 -12.3 7 ⌬F508/R117H 1 90 -35.9 38 -207.7 109 ⌬F508/Y161Cc 1 44 -35.1 37 -45.9 25 ⌬F508/P205S 1 80 -23.3 25 -10.4 5 ⌬F508/V232D 1 120 -16.9 18 -26.9 14 ⌬F508/R334W 1 92 -22.1 23 -21.1 11 ⌬F508/R334W 1 101 -24.5 26 -37.4 20 ⌬F508/T338I 1 73 -44.4 47 -79.4 42 ⌬F508/G576A 1 40 -16.9 18 -115.5 61 ⌬F508/I1234V 1 113 -13.6 15 -8.6 5 G576A/G85E 1 95 -26.1 28 -61.6 32 F1052V/M1137R 1 47 -36.7 39 -146.6 77 M1101K/M1101K 1 94 -11.1 12 -4.8 3 S1159F/S1159F 1 67 -47.9 51 -38.7 21 N1303K/R334W 1 91 -30.3 32 -47.7 25 NOTE. CFTR Cl- channel function was determined in rectal epithelia from Cl- secretory responses induced by IBMX/forskolin (Isc-cAMP) and after co-activation with carbachol (Isc-carbachol). Login to comment
101 Functional Classification and Protein Location of CFTR Mutations Mutation type Severe mutations (protein location) Mild mutations (protein location) Missense V520F, A561E (NBD1) G85E (MSD1, TM1) R1066C (MSD2, CL4) S108F, R117H (MSD1, EL1) N1303K (NBD2) I148N, Y161Ca (MSD1, CL1) P205S (MSD1, TM3) V232D (MSD1, TM4) R334W, T338I (MSD1, TM6) G576A (NBD1) I1234V (NBD2) F1052V, M1101K (MSD2, CL4) M1137R (MSD2, TM12) S1159F (pre-NBD2) Splice 1898 ϩ 1G Ͼ T (R domain) 1898 ϩ 3A Ͼ G (R domain) 3121-2A Ͼ G (MSD2, TM9) 3272-26A Ͼ G (MSD2, TM10) Single amino acid deletion ⌬F508 (NBD1) Nonsense Q39X (N-terminus) G542X, Q552X, R553X, E585X (NBD1) Q637X (R domain) Y1092X (MSD2, CL4) R1162X (pre-NBD2) W1282X (NBD2) Frameshift 120del23a 182delT (N-terminus) 1609delCA (NBD1) 3905insT (NBD2) NOTE. Severe mutation, Cl- secretion absent; mild mutation, residual cAMP-mediated Cl- secretion. Login to comment

[hide] Cystic fibrosis at the Reunion Island (France): sp... J Cyst Fibros. 2004 Aug;3(3):185-8. Dugueperoux I, Bellis G, Lesure JF, Renouil M, Flodrops H, De Braekeleer M
Cystic fibrosis at the Reunion Island (France): spectrum of mutations and genotype-phenotype for the Y122X mutation.
J Cyst Fibros. 2004 Aug;3(3):185-8., [PMID:15463906]

Abstract [show]
BACKGROUND: The Reunion Island is a French administrative department located in the Indian Ocean between the islands of Madagascar and Mauritius. Its population is known to be at a high risk of cystic fibrosis (CF). METHODS: Data concerning all CF patients born at the Reunion Island was extracted from the French CF Registry. Twenty-eight DeltaF508/DeltaF508, 17 Y122X/DeltaF508, and 11 Y122X/Y122X were included in a genotype-phenotype study. RESULTS: The detection rate of the CFTR mutations was 83% among the CF patients born at the Reunion Island. Three CFTR mutations accounted for 75% of the detected CF alleles at the Reunion Island (DeltaF508, Y122X, and 3120 + 1G-->A.). The DeltaF508/DeltaF508, DeltaF508/Y122X, and Y122X/Y122X genotypes accounted for 60.2% of the CF patients. Patients carrying at least one Y122X mutation were pancreatic insufficient, had high sweat chloride values and significantly lower anthropometric measures. The mean anthropometric values in all three groups were lower that in the whole CF population followed in "continental" France. This may reflect the poor compliance and even the refusal of treatment noted by the clinicians. CONCLUSIONS: The distribution of CFTR mutations could be explained by the history of the Reunion Island: admixture of French settlers, African and Asian populations, founder effect and isolation followed by genetic drift. The Y122X allele appears to be associated with a severe phenotype.

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77 G 1 (1.49) D993Y 1 (0.68) DeltaF508/ G551D 1 (1.49) G149R 1 (0.68) DeltaF508/1161delC 1 (1.49) G85E 1 (0.68) Y122X/3120 + 1G ! Login to comment

[hide] Pancreatitis in hispanic patients with cystic fibr... Clin Gastroenterol Hepatol. 2004 Jun;2(6):504-9. Maisonneuve P, Campbell P 3rd, Durie P, Lowenfels AB
Pancreatitis in hispanic patients with cystic fibrosis carrying the R334W mutation.
Clin Gastroenterol Hepatol. 2004 Jun;2(6):504-9., [PMID:15181620]

Abstract [show]
BACKGROUND & AIMS: Cystic fibrosis (CF) results from abnormal production of sticky mucus, which obstructs many organs. In most cases, the pancreas is severely compromised, but 10%-15% of patients with CF have pancreas sufficiency (PS) and are subject to develop pancreatitis. The aim of this study is to determine which specific genotypes lead to the development of pancreatitis in patients with CF. METHODS: We used prospective data collected by the Cystic Fibrosis Foundation and performed a nested case-control study with all patients who reported at least 1 episode of pancreatitis constituting the cases. We used logistic regression to assess the association between pancreatitis and genotype and the Kaplan-Meier method to estimate the cumulative incidence of pancreatitis for selected genotypes. RESULTS: Three hundred sixty-four of 17,871 genotyped patients with CF (2.0%) reported at least 1 episode of pancreatitis. Only 0.9% of 12,997 patients with genotypes generally associated with pancreas insufficiency reported pancreatitis against 11.9% of 868 patients carrying at least 1 mild CF mutation generally associated with PS. The greatest rate of pancreatitis (19.0%) was observed for patients carrying an R334W mutation: 48% of these 79 patients were Hispanic and 13 patients were living in Puerto Rico. CONCLUSIONS: Of all patients with CF, those carrying an R334W mutation have the greatest risk for developing pancreatitis. This mutation is found mostly in Hispanic patients with CF living in Puerto Rico. There are no current data to determine whether asymptomatic carriers of the R334W mutation are at greater risk for developing pancreatitis or whether this mutation is frequent in Hispanics with idiopathic pancreatitis.

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26 Of Ͼ1000 identified mutations in the CFTR gene, only 25 proven disease-causing mutant alleles (⌬F508, G551D, G542X, R553X, W1282X, R347P, NI303K, R560T, ⌬I507, 1717-1GϾA, A455E, 3120ϩ1GϾA, 621ϩ1GϾT, R117H, 711ϩ1GϾT, R1162X, 3849ϩ10kbCϾT, 2789ϩ5GϾT, R334W, G85E, 1078delT, 1898ϩ1GϾT, 2184delA, 3659delC, and I148T) are recommended by the American College of Medical Genetics for routine diagnostic and carrier testing.16 Most of these are routinely recorded in the CFF registry, but rarer mutations can be recorded if identified by more comprehensive testing. Login to comment
29 The remaining patients were grouped as "genotype incompletely determined" if they carried either mutations for which pancreas function is not well determined (G85E, 1898ϩ1GϾA, and 1078delT) or other or unrecognized mutations.17 Data regarding pancreatitis and genotype were extracted directly from the CFF registry database and collected by using the standard CF registry questionnaire.18 We performed a nested case-control study. Login to comment
42 The greatest frequency of pancreatitis was reported in patients carrying at least 1 R334W mutation (19.0%), followed by patients with a 2789ϩ5GϾA mutation (14.9%), R117H mutation (11.7%), R347P mutation (11.6%), 3849ϩ10kbCϾT mutation (9.0%), A455E mutation (8.3%), or G85E mutation (8.0%; Table 1). Login to comment
47 This sex difference was unique for R334W and was not apparent for patients with other pancreatitis-related genotypes (R347P, R117H, G85E, 2789ϩ5GϾA, or 3849ϩ10kbCϾT) or for patients with pancreatitis in the PI group. Login to comment
71 ePatients with either genotype for which pancreas function is not well determined (G85E, 1898ϩ1GϾA, 1078delT) or with other or unrecognized mutations. Login to comment
85 Characteristics of 15 Patients With CF Carrying an R334W Mutation Who Developed Pancreatitis Genotype Sex Race Ethnicity Age at CF (yr) CF diagnosis Age (yr)a R334W/⌬F508 Female White Non-Hispanic 4 Respiratory symptoms 38 R334W/⌬F508 Male White Non-Hispanic 23 Respiratory symptoms, nasal polyps 51 R334W/⌬F508 Female White Non-Hispanic 7 Respiratory symptoms 28 R334W/⌬F508 Female White Non-Hispanic 16 Electrolyte imbalance, respiratory symptoms 34 R334W/⌬F508 Male White Hispanic 13 Respiratory symptoms, electrolyte imbalance, failure to thrive Dead 18 R334W/⌬I507 Female White Hispanic 29 Respiratory symptoms, steatorrhea 31 R334W/G542X Female White Hispanic 7 Respiratory symptoms 10 R334W/R553X Female White Hispanic 10 Respiratory symptoms 21 R334W/G85E Female White Hispanic 0 Respiratory symptoms 8 R334W/G85E Female White Hispanic 4 Failure to thrive 10 R334W/R334W Male White Hispanic 4 Meconium ileus 10 R334W/R334W Female White Hispanic 0 Respiratory symptoms 41 R334W/? Login to comment

[hide] Glucose intolerance in children with cystic fibros... J Pediatr. 2003 Feb;142(2):128-32. Solomon MP, Wilson DC, Corey M, Kalnins D, Zielenski J, Tsui LC, Pencharz P, Durie P, Sweezey NB
Glucose intolerance in children with cystic fibrosis.
J Pediatr. 2003 Feb;142(2):128-32., [PMID:12584532]

Abstract [show]
OBJECTIVE: To evaluate the relations among glucose intolerance, genotype, and exocrine pancreatic status in patients with cystic fibrosis (CF). STUDY DESIGN: Data on 335 patients <18 years of age were from the Toronto CF database. A modified oral glucose tolerance test was given to 94 patients 10 to 18 years of age without recognized CF-related diabetes. CF transmembrane conductance regulator mutations and exocrine pancreatic status were determined for all patients. RESULTS: CF-related diabetes was clinically recognized in 9 of 335 (2.7%) patients <18 years of age, all of whom were pancreatic insufficient, and 8 of 9 had severe (classes I through III) mutations on both alleles. The ninth patient had unidentified mutations. Although all patients given the oral glucose tolerance test were asymptomatic and had normal fasting blood glucose, 16 of 94 (17%) had impaired glucose tolerance and 4 of 94 (4.3%) had CF-related diabetes without fasting hyperglycemia. Abnormal glucose tolerance was associated exclusively with severe mutations and exocrine pancreatic insufficiency. Glycosylated hemoglobin (HbA(1)C) levels did not correlate with glucose tolerance results. CONCLUSIONS: Screening of pancreatic-insufficient, adolescent patients with CF identified more with abnormal oral glucose tolerance than was suspected clinically and is recommended as a routine practice. HbA(1)C was not useful in screening for CF-related glucose intolerance.

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118 of patients with IGT 2 10 2 0 0 1/1 16 No of patients with CFRD without FH 0 4 0 0 0 0 4 *Genotype class based on mutation with ∆F508: Class I, 621+1G→T, G542X, 441delA, R553X, W1282X, 3120+1G→A, 4016insT, 1154insTC, I1027T; Class II, ∆F508; Class III, G551D, G85E, S549N, L1077P, H199R; Class IV, Class V, 3849+10kbC→T, 5T; Unknown, G85E/-, ∆F508/-; Other, G551D/R506T, W1282X/W1282X. Login to comment

[hide] Genotype and phenotype correlations in patients wi... Gastroenterology. 2002 Dec;123(6):1857-64. Durno C, Corey M, Zielenski J, Tullis E, Tsui LC, Durie P
Genotype and phenotype correlations in patients with cystic fibrosis and pancreatitis.
Gastroenterology. 2002 Dec;123(6):1857-64., [PMID:12454843]

Abstract [show]
BACKGROUND & AIMS: Pancreatitis is known to occur in some patients with cystic fibrosis (CF), but the prevalence, natural history, and genotypic basis are unclear. We examined a well-defined cohort of patients with CF to answer these questions. METHODS: Patients with CF were identified from a computerized database (1966-1996). Chart audit identified all patients with CF and pancreatitis. RESULTS: Among 1075 patients with CF, 937 (87%) were pancreatic insufficient at diagnosis, 28 (3%) were pancreatic sufficient but developed pancreatic insufficiency after diagnosis, and 110 (10%) have remained pancreatic sufficient. No patients with pancreatic insufficiency developed pancreatitis. Nineteen patients (17.3%) with pancreatic sufficiency experienced one or more attacks of pancreatitis. The mean age at diagnosis of pancreatitis was 22.7 +/- 10.3 years (range, 10-35 years), and pancreatitis was recognized before the diagnosis of CF in 6 patients (32%). The diagnosis of CF in pancreatic-sufficient patients, with and without pancreatitis, was established at a significantly older age than in those with pancreatic insufficiency (P < 0.0001). Genotyped patients with pancreatic insufficiency carried 2 severe mutant alleles. All genotyped patients with pancreatic sufficiency and pancreatitis carried at least one mild mutation. No specific genotype was predictive of pancreatitis. CONCLUSIONS: Patients with CF with pancreatic sufficiency carry at least one mild mutant allele and are at a significant risk of developing pancreatitis. Symptoms of pancreatitis may precede the diagnosis of CF. Pancreatitis is associated with an otherwise mild CF phenotype.

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105 CFTR Genotypes Among CF Patients With PS With and Without Pancreatitis Two mutations (n) ⌬F508/R117H (9) ⌬F508/(5T) (6) ⌬F508/3272-26A 3 G (4) ⌬F508/R347H (2) ⌬F508/P574H (2) ⌬F508/875 ϩ 1G Ͼ C (2) ⌬F508/3849 ϩ 10kb C 3 T (1) ⌬F508/A455E (1) ⌬F508/D614G (1) ⌬F508/G85E (1) ⌬F508/R347P (1) ⌬F508/S1251N (1) ⌬F508/⌬F508a (1) ⌬F508/3120G Ͼ A (1) ⌬F508/G551Da (1) G542X/R117H (1) R560T/L206W (1) R117H/R117H (1) R31L/P67L (1) 1461ins4 (AGAT)/G85E (1) G551D/(5T) (1) R1066C/3849 ϩ 10kb C Ͼ T (1) G551D/3849 ϩ 10kb C Ͼ T (1) R334W/R334W (1) R334W/681delC (1) W1282X/3489 ϩ 10kb C Ͼ T (1) One mutation (n) ⌬F508/- (18) L1077P/- (1) W1282X/- (1) M1137V/- (1) G551D/- (1) R347H/- (1) Q30X1/- (1) G1244E/- (1) R117H/- (1) 621 ϩ 2G621 ϩ 1G 3 T/- (1) NOTE. Login to comment

[hide] Spectrum of CFTR mutations in cystic fibrosis and ... Hum Mutat. 2000;16(2):143-56. Claustres M, Guittard C, Bozon D, Chevalier F, Verlingue C, Ferec C, Girodon E, Cazeneuve C, Bienvenu T, Lalau G, Dumur V, Feldmann D, Bieth E, Blayau M, Clavel C, Creveaux I, Malinge MC, Monnier N, Malzac P, Mittre H, Chomel JC, Bonnefont JP, Iron A, Chery M, Georges MD
Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France.
Hum Mutat. 2000;16(2):143-56., [PMID:10923036]

Abstract [show]
We have collated the results of cystic fibrosis (CF) mutation analysis conducted in 19 laboratories in France. We have analyzed 7, 420 CF alleles, demonstrating a total of 310 different mutations including 24 not reported previously, accounting for 93.56% of CF genes. The most common were F508del (67.18%; range 61-80), G542X (2.86%; range 1-6.7%), N1303K (2.10%; range 0.75-4.6%), and 1717-1G>A (1.31%; range 0-2.8%). Only 11 mutations had relative frequencies >0. 4%, 140 mutations were found on a small number of CF alleles (from 29 to two), and 154 were unique. These data show a clear geographical and/or ethnic variation in the distribution of the most common CF mutations. This spectrum of CF mutations, the largest ever reported in one country, has generated 481 different genotypes. We also investigated a cohort of 800 French men with congenital bilateral absence of the vas deferens (CBAVD) and identified a total of 137 different CFTR mutations. Screening for the most common CF defects in addition to assessment for IVS8-5T allowed us to detect two mutations in 47.63% and one in 24.63% of CBAVD patients. In a subset of 327 CBAVD men who were more extensively investigated through the scanning of coding/flanking sequences, 516 of 654 (78. 90%) alleles were identified, with 15.90% and 70.95% of patients carrying one or two mutations, respectively, and only 13.15% without any detectable CFTR abnormality. The distribution of genotypes, classified according to the expected effect of their mutations on CFTR protein, clearly differed between both populations. CF patients had two severe mutations (87.77%) or one severe and one mild/variable mutation (11.33%), whereas CBAVD men had either a severe and a mild/variable (87.89%) or two mild/variable (11.57%) mutations.

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102 Distribution of 310 CF Mutations in France With Respect to Relative Frequencies (Total Number of CF Chromosomes = 7,420) Group Mutations Number of alleles % Cum. % A F508del 4,985 67.18 G542X 212 2.86 N1303K 156 2.10 73.45 1717-1G>A 97 1.31 B G551D 73 0.98 2789+5G>A 72 0.97 W1282X 68 0.91 R553X 66 0.89 I507del 52 0.70 1078delT 49 0.66 7.47 2183AA>G 48 0.64 711+1G>T 33 0.44 R1162X 33 0.44 Y1092X 30 0.40 3849+10kbC>T 30 0.40 C 12 mutationsa 29 to 15 (239) 0.39-0.20 19 mutationsb 14 to 8 (190) 0.19-0.10 11 mutationsc 7 to 6 (71) 0.09-0.08 11 mutationsd 5 (55) 0.06 10.57 15 mutationse 4 (60) 0.05 23 mutationsf 3 (69) 0.04 50 mutationsg 2 (100) 0.02 D 154 mutationsh 1 (154) 0.01 2.07 6,942 93.56 a 3659delC, R347P, 3272-26A>G, R334W, W846X, 621+1G>T, G85E, R1066C, L206W, 394delTT, 4055+1G>A, R347H. Login to comment

[hide] A comparison of fluorescent SSCP and denaturing HP... Hum Mutat. 2000;15(6):556-64. Ellis LA, Taylor CF, Taylor GR
A comparison of fluorescent SSCP and denaturing HPLC for high throughput mutation scanning.
Hum Mutat. 2000;15(6):556-64., [PMID:10862085]

Abstract [show]
We examined 67 different mutations in 16 different amplicons in a comparison of mutation detection by fluorescent single strand conformation polymorphism (F-SSCP) and by denaturing HPLC (DHPLC). F-SSCP was used to analyze fluorescent amplicons with internal size standards and automated fragment analysis (GeneScan, PE Applied Biosystems, Foster City, CA). In DHPLC, unlabelled amplicons were analyzed by reverse phase HPLC with fragment detection by absorbance at 260nm. Both methods had high sensitivity (95-100%) and specificity (100%). Overall, F-SSCP with external temperature control was the more sensitive method, but DHPLC was particularly useful for the rapid analysis of novel fragments.

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97 Comparison of F-SSCP and DHPLC Using a Panel of ABCC7 Mutations Gel condition Location Location 49:1 49:1 49:1 49:1 MDE MDE MDE Capillary DHPLC °C from 5' (bp) from 3' (bp) 15 20 25 35 20 25 35 35 N/A Exon 3 (320bp) E60X 128 192 + + + + + + + + - P67L 150 170 + + + - + + + - + R75X 173 147 + + + + + + + + + R75Q 174 146 + + + - + + + + + G85E 204 116 + + + - + + + + + L88S 213 107 + + + + + + + + + Exon 4 (400bp) 441delA 135 265 + + + + + + + + + D110H 154 246 + + + + + + - + + R117H/H 176 224 + + + + + + + + N/A R117R/H 176 224 + + + + + + + + + L137H 236 164 + + + + + + + + + I148T 261 139 + + + + + + + + + 621+1 (G>T) 309 91 + + + + + + + + + Exon 7 (360bp) R334W 180 180 + + + + + + + - + 1058delC 105 255 + + + + + + + + + 1078delT 125 235 + + + - + + + + + 1138insG 226 134 - + + - + + + + + 1154insTC 202 158 + + + + + + + + + 1161delC 209 151 + + + + + + + + + R347H 220 140 + + + + + + - + + R347P 220 140 + + + - + + + - + A349V 226 134 + + + + + + + + + W356X 248 112 + + + + + + + + + Exon 10 (365bp) M470V 143 222 + + + + + + + + + Q493X 212 153 + + + + + + - + - DelF508 255 110 + + + + + + + + - Del I507 253 112 + + + + + + + + + V520F 293 72 + + - + + - + - + Exon 11 (190bp) 1717-1 (G>A) 54 136 + + + - + + - + + G542X 94 96 + + + - + + - + + S549N 116 74 + + + + + + + + - S549R 117 73 + + + + - - - + + G551D 122 68 + - - - + + + - + R553X 127 63 + + + + + + + + + G551D/R553X + + + + + + + + + R560T 149 41 + + + - - - - - + R560K 149 41 + + + - + + + - + 1811+1 (G>C) 150 40 + + + + + + + + + Exon 12 (250bp) 1898+1(G>A) 167 83 + + + + + + - + + Exon 13a (290bp) C590W 87 203 + + - - + - - + + Exon 13b (405bp) 2184insA 148 257 + + + + + + + - + R709X 220 185 - + - - - - - - + V754M 453 52 + + + + + + + - - Exon 13c (345bp) V754M 65 280 + + + + + + - - + R785X 158 187 + + - - + + - - + Exon 19 (370bp) 3601-17 (T>C) 29 341 - + + - + + + - + R1162X 61 309 + + - - + - - + + 3659delC 105 265 - - - + + + + + + Y1182X 123 247 - + + - + + + - + Exon 20 (370bp) W1282X 186 184 + + + + + + + + + % detected 90 96 86 66 94 88 74 72 90 remainder were detected using DGGE. Login to comment

[hide] Calnexin family members as modulators of genetic d... Semin Cell Dev Biol. 1999 Oct;10(5):473-80. Chevet E, Jakob CA, Thomas DY, Bergeron JJ
Calnexin family members as modulators of genetic diseases.
Semin Cell Dev Biol. 1999 Oct;10(5):473-80., [PMID:10597630]

Abstract [show]
The endoplasmic reticulum (ER) is an intracellular compartment devoted to the synthesis, segregation and folding of soluble and membrane secretory proteins. Some mutations in these proteins lead to their incorrect or incomplete folding in the ER. The ER has a quality control system which detects misfolded proteins and then specifies their fate. Some mutated proteins are retained in the ER wherein they accumulate (Russell bodies for misfolded immunoglobulin heavy chains, the PiZZ for alpha 1-antitrypsin), others are retrotranslocated from the ER and degraded by the cytosolic proteasomal system, and yet other proteins are eventually secreted (in AZC-treated cells). In this review we summarize the role of ER resident proteins in quality control of mutated secretory proteins.

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85 Potential involvement of calnexin and calreticulin in genetically-inherited diseases Protein Mutation Disease Calnexin Calreticulin Degradation ␣1-antitrypsin null Hong-Kong Emphysema q ref. 54 ᎐ q ref. 34 Ž .Liver disease Z-allele Emphysema q ref. 2 ᎐ q ref. 2 Ž .ref. 2 Lungrliver disease CFTR ⌬F508 ref. 51 Cystic fibrosis q ref. 51 ᎐ ᎐ G85E ref. 55 ND ND G91R ref. 55 ND ND Myeloperoxidase Y173C ref. 56 Decreased q ref. 57 q ref. 57 q ref. 56 W569R ref. 56 microbicidal response Factor VIII R2307Q ref. 58 Haemophilia q ref. 37 q ref. 37 Thyroglobulin Congenital q ref. 59 q ref. 59 ᎐ hyperthyroid goiter Figure 4. Login to comment
119 ref. 2 Lungrliver disease CFTR DF508 ref. 51 Cystic fibrosis q ref. 51 ] ] G85E ref. 55 ND ND G91R ref. 55 ND ND Myeloperoxidase Y173C ref. 56 Decreased q ref. 57 q ref. 57 q ref. 56 W569R ref. 56 microbicidal response Factor VIII R2307Q ref. 58 Haemophilia q ref. 37 q ref. 37 Thyroglobulin Congenital q ref. 59 q ref. 59 ] hyperthyroid goiter Figure 4. Login to comment

[hide] The molecular basis of disease variability among c... Genomics. 1998 Nov 1;53(3):276-83. Chiba-Falek O, Kerem E, Shoshani T, Aviram M, Augarten A, Bentur L, Tal A, Tullis E, Rahat A, Kerem B
The molecular basis of disease variability among cystic fibrosis patients carrying the 3849+10 kb C-->T mutation.
Genomics. 1998 Nov 1;53(3):276-83., [PMID:9799593]

Abstract [show]
Disease severity varies among cystic fibrosis (CF) patients carrying the same CFTR genotype. Here we studied the mechanism underlying disease variability in individuals carrying a splicing CFTR mutation, 3849+10 kb C-->T. This mutation was shown to produce both correctly and aberrantly spliced CFTR transcripts containing an additional cryptic exon. Semiquantitative nondifferential RT-PCR showed considerable variability in the level (0-28%) of aberrantly spliced RNA transcribed from the 3849+10 kb C-->T mutation in nasal epithelium from 10 patients. A significant inverse correlation was found between the level of the aberrantly spliced CFTR transcripts and pulmonary function, expressed as FEV1 (r = 0.92, P < 0.0001). Patients with normal pulmonary function (FEV1 > 80% predicted) had lower levels of aberrantly spliced CFTR RNA (0 to 3%) than those with FEV1 < 80%, (9 to 28% aberrantly spliced RNA). Only aberrantly spliced CFTR RNA was detected in the lung of a patient with severe lung disease who underwent lung transplantation. Our results show that the severity of CF lung disease correlates with insufficiency of normal CFTR RNA. Thus, the regulation of alternative splice site selection may be an important mechanism underlying partial penetrance in CF. Further understanding of this regulation will contribute to potential therapy for patients carrying splicing mutations in human disease genes.

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39 These patients were compound heterozygous for the 3849ϩ10 kb C3T mutation and one of the following CFTR mutations: 4 patients carried the ⌬F508 mutation, 2 carried the W1282X mutation, 3 carried the 405ϩ1 G3A mutation, and 1 carried the G85E mutation (Welsh et al., 1995) (Table 1). Login to comment
108 Sex Other mutation Current age (years) Age at diagnosis (years) Sweat chloride (meq/L) Pancreatic status FVC (% predicted) FEV1 (% predicted) Aberrant transcript (% of total) 1 F ⌬F508 25 8 84 PS 92 88 0 2 F W1282X 13 13 95 PS 100 90 3 Ϯ 1 3 M G85E 11 0.3 54 PS-ϾPI 92 68 9 Ϯ 0.5 4 M ⌬F508 11 11 40 PS 51 44 22 Ϯ 1 5a M 405ϩ1 G3A 31, 32 10 63 PS 52, 40 30, 25 23 Ϯ 2, 28 Ϯ 2 6 M W1282X 19 13 97 PS 73 64 17 Ϯ 1 7b M 405ϩ1 G3A 22 5 112 PS 84 65 12 Ϯ 1 8b M 405ϩ1 G3A 21 4 74 PS 44 37 21 Ϯ 2 9b M ⌬F508 18 16 40 PS 78 46 26 Ϯ 1 10b F ⌬F508 10 8 37 PS 104 99 2 Ϯ 0.5 11c F Unknown 32 25 56 PS 25 18 46 Ϯ 2 a This patient was analyzed twice, at a year`s interval. Login to comment
38 These patients were compound heterozygous for the 3849110 kb C3T mutation and one of the following CFTR mutations: 4 patients carried the DF508 mutation, 2 carried the W1282X mutation, 3 carried the 40511 G3A mutation, and 1 carried the G85E mutation (Welsh et al., 1995) (Table 1). Login to comment

[hide] 3849 + 10 kb C --> T splicing mutation in Hispanic... Mol Genet Metab. 1998 Jul;64(3):213-6. Liang MH, Wertz KK, Bowman CM, Hsu E, Shapiro B, Wong LJ
3849 + 10 kb C --> T splicing mutation in Hispanic CF patients.
Mol Genet Metab. 1998 Jul;64(3):213-6., [PMID:9719631]

Abstract [show]
Seven patients compound heterozygous for the 3849 + 10kb C --> T mutation in the CFTR gene were found among the 152 patients attending the CHLA CF Clinic. The frequency of this mutation accounts for 2.3 and 3.9% of thetotal and Hispanic CF alleles of CHLA patients. These are significantly higher than the 0.6% of the general CF population. The average age of diagnosis of this group of Hispanics is 3.1 years, which is much younger than that reported for CF patients of other ethnicities with the same mutation. Both pancreatic sufficient and pancreatic insufficient patients were observed. It is concluded that the 3849 + 10kb C --> T mutation is associated with a variable but potentially mild type of CF.

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32 RESULTS AND DISCUSSION Seven of the 152 patients were compound heterozygous for the 3849 ϩ 10 kb C 3 T mutation with four ⌬F508, one G85E, and two unidentified mutations on the other chromosome. Login to comment
41 : 1 2 3 4 5 6 7 Sex (M/F) F F M M M F M Genotype 3849 ϩ 10kb/ unknown 3849 ϩ 10kb/ unknown 3849 ϩ 10kb/ G85E 3849 ϩ 10kb/ ⌬F508 3849 ϩ 10kb/ ⌬F508 3849 ϩ 10kb/ ⌬F508 3849 ϩ 10kb/ ⌬F508 Ethnicity Hispanic Hispanic Hispanic Hispanic Caucasian Hispanic Caucasian Age/age @ diagnosis (years, months) 6.5/3 10/3,11 23/1 7/11 month 44/16.5 10/6,8 46.5/27 Sweat chloridea (mmol/liter) 52 72 53 86 84 55 47 FEV1 b (%)/ Unavailable 67/6 29/17 Unavailable 20/38.5 96/6.5 51/42.5 age (years) Unavailable 64/8 33/21 Unavailable 16/40.5 94/10 46/46 FVCc (%)/ Unavailable 86/6 54/17 Unavailable 42/38.5 110/6.5 71/42.5 age (years) Unavailable 90/8 71/21 Unavailable 41/40.5 103/10 71/46 FEV1/FVCd Unavailable 0.78/6 0.54/17 Unavailable 0.48/38.5 0.87/6.5 0.72/42.5 age (years) Unavailable 0.71/8 0.46/21 Unavailable 0.39/40.5 0.91/10 0.65/46 Pancreatic statuse PI PI PS PS PS PS PS Height (%)/Weight (%)f 35/25/2 70/50/4 Ͻ5/Ͻ5/17 45/25/14 months 35/Ͻ5/29 75/75/6, 5 55/Ͻ5/42, 7 Age (years, months) 10/Ͻ5/6 35/55/8, 9 Ͻ5/Ͻ5/22 Ͼ95/Ͼ95/3, 3 35/Ͼ5/40, 7 80/85/10, 2 55/Ͻ5/45 Microbiological colonizationg a/mucoid a/mucoid, c a/mucoid a a/mucoid, c a, b a/mucoid Other clinical features Lobar atelectasis Lobectomy (2.5 years) Bronchiectasis Pancrease MT16e Malnutrition Chronic cough Bronchiectasis Ultrase 12e On lung transplant list No known children Pneumonia Pancreasee stopped Hyponatremia Dehydration Lung transplant (43 years) Two children Pneumonia Nasal polyps Chronic cough Sinus Surgery Repeat pancreatitis Pneumonia Decreased sperm count Nasal polyps Gallbladder removed (25 years) Appendectomy (teenage) No known children a Sweat test, concentrations of Cl in mmol/L. Login to comment
72 The clinical phenotype of a rare compound heterozygote, 3849 ϩ 10 kb C 3 T/G85E has never been described before. Login to comment
73 The missense mutation, G85E, changes the uncharged small glycine residue at position 85 in the first membrane spanning domain of the CFTR to a negatively charged glutamic acid (14). Login to comment
76 Our patient with 3849 ϩ 10kb C 3 T/ G85E genotype was diagnosed early (at 1 year of age). Login to comment
79 Patients with G85E mutation reportedly demonstrated phenotypic heterogeneity (14,15). Login to comment
31 GM982710 213 1096-7192/98 $25.00 Copyright (c) 1998 by Academic Press All rights of reproduction in any form reserved. RESULTS AND DISCUSSION Seven of the 152 patients were compound heterozygous for the 3849 1 10 kb C 3 T mutation with four DF508, one G85E, and two unidentified mutations on the other chromosome. Login to comment
40 : 1 2 3 4 5 6 7 Sex (M/F) F F M M M F M Genotype 3849 1 10kb/ unknown 3849 1 10kb/ unknown 3849 1 10kb/ G85E 3849 1 10kb/ DF508 3849 1 10kb/ DF508 3849 1 10kb/ DF508 3849 1 10kb/ DF508 Ethnicity Hispanic Hispanic Hispanic Hispanic Caucasian Hispanic Caucasian Age/age @ diagnosis (years, months) 6.5/3 10/3,11 23/1 7/11 month 44/16.5 10/6,8 46.5/27 Sweat chloridea (mmol/liter) 52 72 53 86 84 55 47 FEV1 b (%)/ Unavailable 67/6 29/17 Unavailable 20/38.5 96/6.5 51/42.5 age (years) Unavailable 64/8 33/21 Unavailable 16/40.5 94/10 46/46 FVCc (%)/ Unavailable 86/6 54/17 Unavailable 42/38.5 110/6.5 71/42.5 age (years) Unavailable 90/8 71/21 Unavailable 41/40.5 103/10 71/46 FEV1/FVCd Unavailable 0.78/6 0.54/17 Unavailable 0.48/38.5 0.87/6.5 0.72/42.5 age (years) Unavailable 0.71/8 0.46/21 Unavailable 0.39/40.5 0.91/10 0.65/46 Pancreatic statuse PI PI PS PS PS PS PS Height (%)/Weight (%)f 35/25/2 70/50/4 ,5/,5/17 45/25/14 months 35/,5/29 75/75/6, 5 55/,5/42, 7 Age (years, months) 10/,5/6 35/55/8, 9 ,5/,5/22 .95/.95/3, 3 35/.5/40, 7 80/85/10, 2 55/,5/45 Microbiological colonizationg a/mucoid a/mucoid, c a/mucoid a a/mucoid, c a, b a/mucoid Other clinical features Lobar atelectasis Lobectomy (2.5 years) Bronchiectasis Pancrease MT16e Malnutrition Chronic cough Bronchiectasis Ultrase 12e On lung transplant list No known children Pneumonia Pancreasee stopped Hyponatremia Dehydration Lung transplant (43 years) Two children Pneumonia Nasal polyps Chronic cough Sinus Surgery Repeat pancreatitis Pneumonia Decreased sperm count Nasal polyps Gallbladder removed (25 years) Appendectomy (teenage) No known children a Sweat test, concentrations of Cl in mmol/L. Login to comment
71 The clinical phenotype of a rare compound heterozygote, 3849 1 10 kb C 3 T/G85E has never been described before. Login to comment
75 Our patient with 3849 1 10kb C 3 T/ G85E genotype was diagnosed early (at 1 year of age). Login to comment
78 Patients with G85E mutation reportedly demonstrated phenotypic heterogeneity (14,15). Login to comment

[hide] Is meconium ileus genetically determined or associ... J Med Genet. 1998 Mar;35(3):262-3. De Braekeleer M, Allard C, Leblanc JP, Aubin G, Simard F
Is meconium ileus genetically determined or associated with a more severe evolution of cystic fibrosis?
J Med Genet. 1998 Mar;35(3):262-3., [PMID:9541118]

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16 Although the A455E mutation is Table 1 Distribution of meconium ileus among CFTR genotypes in Saguenay Lac-Saint-Jean No of CF Proportion No of CFpatients Proportion Proportion ofMI Genotypes patients (%) with meconium ileus (%) among genotypes AF508/AF508 52 39.4 5 26.3 9.6 AF508/621+G-*T 34 25.8 9 47.4 26.5 AF508/A455E 14 10.6 0 0.0 0.0 621+1G-*T/A455E 8 6.1 0 0.0 0.0 621+1G-*T/G85E 2 1.5 1 5.3 50.0 621+1G-*T/Y1092X 1 0.8 0 0.0 0.0 AF508/Y1092X 4 3.0 1 5.3 25.0 A455E/R117C 1 0.8 0 0.0 0.0 AF508/I148T 2 1.5 0 0.0 0.0 621+1G-*T/ 4 3.0 0 0.0 0.0 711 +1G-*T 621+1G-4T/S489X 1 0.8 0 0.0 0.0 AF508/Q890X 1 0.8 1 5.3 100.0 621+1G->T/ 6 4.5 2 10.5 33.3 621+1G-sT AF508/unknown 1 0.8 1 5.3 100.0 Unknown/unknown 1 0.8 0 0.0 0.0 Table 2 Main clinicalfindings in patients with meconium ileus With MI Without MI p value No of patients 18 18 Sex (M/IF) 6/12 6/12 No of patients alive 16 17 Mean age (SD) 16.75 (9.7) 16.70 (7.9) p=0.99 Mean birth weight (SD) 3.24 (0.40) 3.02 (0.47) p=O.18 Mean birth height (SD) 50.0 (2.27) 50.0 (2.58) p=0.86 Currentweightcentile (SD) 26.7 (24.5) 14.1 (18.0) p=0.06 Current height centile (SD) 29.9 (25.1) 20.6 (25.6) p=0.33 Sweat chloride concentration (mEq/l) 105.9 (6.5) 101.1 (9.8) p=O.12 Mean FVC (SD) 89.7 (24.4) 93.0 (17.0) p=0.75 Mean FEV (SD) 73.1 (23.9) 75.4 (18.7) p=0.81 Mean Shwachman score (SD) 82.8 (11.8) 79.2 (12.6) p=0.36 Colonisation with Pseudomonas aeruginosa 13 14 p=0.70 Staphyloccoccus aureus 16 17 p=0.55 Haemophilus influenzae 13 14 p=0.70 Pseudomonas maltophilia 4 6 p=0.46 Pseudomonas cepacia 0 1 Pancreatic insufficiency 18 18 DIOS 7 1 p=0.016 Rectal prolapse 1 2 p=0.55 Recurrent abdominal pain 6 1 p=0.035 Diabetes mellitus 5 0 p=0.016 Liver complications 3* 0 p=0.07 Nasal polyposis 6 6 p=1.00 DIOS=distal intestinal obstruction syndrome. Login to comment
54 Phenotypic heterogeneity in CF sibs compound heterozygous for the G85E and 621+1G-*T mutations. Clin Genet 1995;47: 110-11. Login to comment
55 Phenotypic heterogeneity in CF sibs compound heterozygous for the G85E and 621+1G-*T mutations. Clin Genet 1995;47: 110-11. Login to comment

[hide] Correlation of sweat chloride concentration with g... Clin Biochem. 1998 Feb;31(1):33-6. De Braekeleer M, Allard C, Leblanc JP, Aubin G, Simard F
Correlation of sweat chloride concentration with genotypes in cystic fibrosis patients in Saguenay Lac-Saint-Jean, Quebec, Canada.
Clin Biochem. 1998 Feb;31(1):33-6., [PMID:9559222]

Abstract [show]
OBJECTIVES: Saguenay Lac-Saint-Jean, a geographically isolated region of northeastern Quebec has a high incidence of cystic fibrosis (CF) and three mutations only account for 94% of the CF chromosomes. The objective of the present study was to determine whether different mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene had different effects upon the sweat chloride concentration. DESIGN AND METHODS: The sweat chloride concentration of 114 patients was measured by quantitative pilocarpine iontophoresis. RESULTS: CF patients carrying the A455E mutation, usually associated with pancreatic sufficiency, had lower sweat chloride concentrations than those carrying mutations associated with pancreatic insufficiency (delta F508 and 621 + 1G-->T). CONCLUSIONS: Our results confirm that mutations resulting in a reduction of the chloride current at the apical membrane of epithelial cells induce lower sweat chloride values. However, there are differences in the chloride current between genotypes, even if they are composed of mutations apparently having the same functional effect.

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57 In class III, the CFTR protein reaches the cell membrane but is irresponsive to cAMP stimulation (e.g., G551D, N1303K, G85E mutations). Login to comment
59 In class III, the CFTR protein reaches the cell membrane but is irresponsive to cAMP stimulation (e.g., G551D, N1303K, G85E mutations). Login to comment

[hide] Co- and posttranslational translocation mechanisms... J Biol Chem. 1998 Jan 2;273(1):568-76. Lu Y, Xiong X, Helm A, Kimani K, Bragin A, Skach WR
Co- and posttranslational translocation mechanisms direct cystic fibrosis transmembrane conductance regulator N terminus transmembrane assembly.
J Biol Chem. 1998 Jan 2;273(1):568-76., [PMID:9417117]

Abstract [show]
Transmembrane topology of most eukaryotic polytopic proteins is established cotranslationally at the endoplasmic reticulum membrane through the action of alternating signal and stop transfer sequences. Here we demonstrate that the cystic fibrosis transmembrane conductance regulator (CFTR) achieves its N terminus topology through a variation of this mechanism that involves both co- and posttranslational translocation events. Using a series of defined chimeric and truncated proteins expressed in a reticulocyte lysate system, we have identified two topogenic determinants encoded within the first (TM1) and second (TM2) membrane-spanning segments of CFTR. Each sequence independently (i) directed endoplasmic reticulum targeting, (ii) translocated appropriate flanking residues, and (iii) achieved its proper membrane-spanning orientation. Signal sequence activity of TM1, however, was inefficient due to the presence of two charged residues, Glu92 and Lys95, located within its hydrophobic core. As a result, TM1 was able to direct correct topology for less than half of nascent CFTR chains. In contrast to TM1, TM2 signal sequence activity was both efficient and specific. Even in the absence of a functional TM1 signal sequence, TM2 was able to direct CFTR N terminus topology through a ribosome-dependent posttranslational mechanism. Mutating charged residues Glu92 and Lys95 to alanine improved TM1 signal sequence activity as well as the ability of TM1 to independently direct CFTR N terminus topology. Thus, a single functional signal sequence in either the first or second TM segment was sufficient for directing proper CFTR topology. These results identify two distinct and redundant translocation pathways for CFTR N terminus transmembrane assembly and support a model in which TM2 functions to ensure correct topology of CFTR chains that fail to translocate via TM1. This novel arrangement of topogenic information provides an alternative to conventional cotranslational pathways of polytopic protein biogenesis.

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43 Plasmid pSPCFTR(E92A/ K95A) was generated by PCR amplification of pSPCFTR(E92A) (sense primer (SP6 promoter) ATTTAGGTGACACTATAG, and antisense primer TACTGCAGCGGTGACGGCGCCTAA), digestion of the PCR fragment with AvaI/PstI (PstI encoded in antisense oligonucleotides) and ligation of the fragment into an AvaI/PstI digested pSPCFTRK95A vector. Plasmids pSPCFTR(G85E) and pSPCFTR(G91R) are described elsewhere (33). Login to comment
44 Plasmids TM1.P, TM1.P(G85E), TM1.P(G91R), TM1.P(E92A), TM1.P(E95A), and TM1.P(E92A/E95A) were constructed by PCR amplification of WT or corresponding mutant CFTR plasmids (sense primer (SP6 promoter), antisense primer TAGATAGGTCACCATAGAGCGTTCCTCCT) and ligation of HindIII/BstEII-digested PCR fragments into a HindIII/BstEII-digested vector, S.L.ST.gG.P (described in Ref. Login to comment
55 Plasmids encoding G85E together with E115K/E116K, E116K/G126D or E115K/E116K/G126D mutations were made in the identical manner except that pSPCFTR(G85E) was used as the template for the initial 5Ј PCR reactions. Login to comment
82 In contrast to WT chains, no protease-protected fragments were observed for TM1.P(G85E) or TM1.P(G91R) chains, demonstrating that these inherited cystic fibrosis-related mutations abolished the ability of TM1 to direct translocation of the P reporter (lanes 4-6 and 7-9, respectively). Login to comment
95 Plasmids TM1.P, TM1.P(G85E), TM1.P(G91R), TM1.P(E92A), TM1.P(K95A), and TM1.P- (E92A/K95A) were expressed in rabbit reticulocyte lysate supplemented with canine pancreas microsomal membranes (A) or in microinjected Xenopus oocytes (B) as described under "Materials and Methods." Login to comment
103 located); (ii) G85E and G91R mutations essentially abolished TM1 signal sequence activity (Ͻ5% of chains translocated); and (iii) E92A and E92A/K95A mutations improved TM1 signal sequence activity (36% and 70% of chains translocated, respectively). Login to comment
105 This, however, was not the case because greater than 70% of WT as well as G85E and G91R chains achieved their correct N terminus topology in the ER membrane ((33) and Fig. 4). Login to comment
144 Plasmids encoding TM1 mutations, G85E and G91R (33), were also included for comparison. Login to comment
155 When the TM1 mutation G85E was introduced into chains containing E116K/G126D or E115K/E116K/G126D mutations, translocation efficiency was further reduced to 45% and 48% of WT levels, respectively (Fig. 5, A and B). Login to comment
160 Only 55% of truncated E115K/E116K chains achieved correct topology (Fig. 5C), and the G85E mutation had little effect on these chains. Login to comment
191 Furthermore, even chains containing a completely defective TM1 signal sequence (G85E and G91R mutants) were properly oriented in the membrane but only if a functional TM2 signal sequence was present. Login to comment
45 Plasmids TM1.P, TM1.P(G85E), TM1.P(G91R), TM1.P(E92A), TM1.P(E95A), and TM1.P(E92A/E95A) were constructed by PCR amplification of WT or corresponding mutant CFTR plasmids (sense primer (SP6 promoter), antisense primer TAGATAGGTCACCATAGAGCGTTCCTCCT) and ligation of HindIII/BstEII-digested PCR fragments into a HindIII/BstEII-digested vector, S.L.ST.gG.P (described in Ref. 18). Login to comment
56 Plasmids encoding G85E together with E115K/E116K, E116K/G126D or E115K/E116K/G126D mutations were made in the identical manner except that pSPCFTR(G85E) was used as the template for the initial 59 PCR reactions. Login to comment
83 In contrast to WT chains, no protease-protected fragments were observed for TM1.P(G85E) or TM1.P(G91R) chains, demonstrating that these inherited cystic fibrosis-related mutations abolished the ability of TM1 to direct translocation of the P reporter (lanes 4-6 and 7-9, respectively). Login to comment
96 Plasmids TM1.P, TM1.P(G85E), TM1.P(G91R), TM1.P(E92A), TM1.P(K95A), and TM1.P- (E92A/K95A) were expressed in rabbit reticulocyte lysate supplemented with canine pancreas microsomal membranes (A) or in microinjected Xenopus oocytes (B) as described under "Materials and Methods." Login to comment
104 located); (ii) G85E and G91R mutations essentially abolished TM1 signal sequence activity (,5% of chains translocated); and (iii) E92A and E92A/K95A mutations improved TM1 signal sequence activity (36% and 70% of chains translocated, respectively). Login to comment
106 This, however, was not the case because greater than 70% of WT as well as G85E and G91R chains achieved their correct N terminus topology in the ER membrane ((33) and Fig. 4). Login to comment

[hide] Complete identification of cystic fibrosis transme... Clin Genet. 1998 Jan;53(1):44-6. De Braekeleer M, Mari C, Verlingue C, Allard C, Leblanc JP, Simard F, Aubin G, Ferec C
Complete identification of cystic fibrosis transmembrane conductance regulator mutations in the CF population of Saguenay Lac-Saint-Jean (Quebec, Canada).
Clin Genet. 1998 Jan;53(1):44-6., [PMID:9550360]

Abstract [show]
Over the past few years, we have conducted a systematic study of 230 cystic fibrosis (CF) chromosomes in the Saguenay Lac-Saint-Jean (SLSJ) population which has a high CF incidence (1/936 live births). We identified 11 mutations accounting for 100% of the CF chromosomes found in patients born in SLSJ. Our results indicate that denaturing gradient gel electrophoresis (DGGE) is a powerful method of identifying CF mutations. They have also considerable implications for genetic counselling and molecular characterization of doubtful patients. They make carrier screening technically feasible in this population.

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No. Sentence Comment
33 Distributon of CFTR mutations in CF patients born in SLSJ Mutations No. CF chromosomes Proportion(%) AF508 120 621tlG-T 51 A455E 17 Y1092X 3 1148T 2 711+1G+T 2 G85E 1 Q890X 1 s489x 1 R117C 1 R1158X 1 60 25.5 8.5 1.5 1 1 0.5 0.5 0.5 0.5 0.5 Table 1 gives the distribution of the mutations found on the C F chromosomes from patients born in the SLSJ region. Login to comment
43 Distributionof CFtR genotypes in CF patients born in SLSJ Genotypes No. CF patients AFS08/AF508 AF5@/621+lG-T AF508/A455E 621t 1G+T/A455E 621t 1G+T/621 t 1G-T AF508,N109W AF508/1148T 621t1G+T/711 t1G+T 621t 1G+T/G85E 621t1G+T/YlO92X A455E/R117C 621+1G+TjS489X AF508/Q890X AF508/R1158X 37 30 6 5 2 2 2 1 1 1 1 1 1 45 De Braekeleer et al. identify 100% of the CFTR mutations in the CF population born in SLSJ. Login to comment

[hide] High heterogeneity for cystic fibrosis in Spanish ... Hum Genet. 1997 Dec;101(3):365-70. Casals T, Ramos MD, Gimenez J, Larriba S, Nunes V, Estivill X
High heterogeneity for cystic fibrosis in Spanish families: 75 mutations account for 90% of chromosomes.
Hum Genet. 1997 Dec;101(3):365-70., [PMID:9439669]

Abstract [show]
We have analyzed 640 Spanish cystic fibrosis (CF) families for mutations in the CFTR gene by direct mutation analysis, microsatellite haplotypes, denaturing gradient gel electrophoresis, single-strand conformation analysis and direct sequencing. Seventy-five mutations account for 90.2% of CF chromosomes. Among these we have detected seven novel CFTR mutations, including four missense (G85V, T582R, R851L and F1074L), two nonsense (E692X and Q1281X) and one splice site mutation (711+3A-->T). Three variants, two in intronic regions (406-112A/T and 3850-129T/C) and one in the coding region (741C/T) were also identified. Mutations G85V, T582R, R851L, E692X and Q1281X are severe, with lung and pancreatic involvement; 711+3A-->T could be responsible for a pancreatic sufficiency/insufficiency variable phenotype; and F1074L was associated with a mild phenotype. These data demonstrate the highest molecular heterogeneity reported so far in CF, indicating that a wide mutation screening is necessary to characterize 90% of the Spanish CF alleles.

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33 Eight mutations have frequencies 366 Table 1 Seventy-five CFTR mutations identified in 640 Spanish families with cystic fibrosis (CF) Mutation Exon/intron CF alleles % ∆F508 E.10 681 53.20 G542X E.11 108 8.43 N1303K E.21 34 2.65 1811+1.6kbA→Ga I.11 24 1.87 711+1G→T I.5 22 1.71 R1162Xa E.19 21 1.64 R334Wa E.7 21 1.64 R1066C E.17b 14 1.09 1609delCAa E.10 13 1.01 Q890X E.15 13 1.01 G85E E.3 12 0.94 712-1G→Ta I.5 11 0.86 2789+5G→A I.14b 11 0.86 ∆I507 E.10 10 0.78 W1282X E.20 10 0.78 2869insGa E.15 9 0.70 L206W E.6a 7 0.54 R709X E.13 7 0.54 621+1G→T I.4 6 0.47 3272-26A→G I.17a 6 0.47 R347H E.7 5 0.39 2183AA→G E.13 5 0.39 K710X E.13 5 0.39 2176insC E.13 5 0.39 3849+10kbC→T I.19 5 0.39 P205Sa E.6a 4 0.31 1078delT E.7 4 0.31 R553X E.11 4 0.31 G551D E.11 4 0.31 1812-1G→Aa I.11 4 0.31 CFdel#1a E.4-7/11-18 4 0.31 V232D E.6a 3 0.23 936delTAa E.6b 3 0.23 1717-8G→A I.10 3 0.23 1949del84 E.13 3 0.23 W1089X E.17b 3 0.23 R347P E.7 3 0.23 del E.3a E.3 2 0.16 R117H E.4 2 0.16 L558S E.11 2 0.16 A561E E.12 2 0.16 2603delT E.13 2 0.16 Y1092X E.17b 2 0.16 Q1100Pa E.17b 2 0.16 M1101K E.17b 2 0.16 delE.19a E.19 2 0.16 G1244E E.20 2 0.16 P5La E.1 1 0.08 Q30Xa E.2 1 0.08 G85Va E.3 1 0.08 E92Ka E.4 1 0.08 A120Ta E.4 1 0.08 I148T E.4 1 0.08 711+3A→Ta I.5 1 0.08 H199Y E.6a 1 0.08 875+1G→A I.6a 1 0.08 Table 1 (continued) Mutation Exon/intron CF alleles % 1717-1G→A I.10 1 0.08 L571S E.12 1 0.08 T582Ra E.12 1 0.08 E585X E.12 1 0.08 1898+3A→G I.12 1 0.08 G673X E.13 1 0.08 E692Xa E.13 1 0.08 R851X E.14a 1 0.08 R851La E.14a 1 0.08 A1006E E.17a 1 0.08 L1065Ra E.17b 1 0.08 F1074La E.17b 1 0.08 R1158X E.19 1 0.08 3667del4a E.19 1 0.08 3860ins31a E.20 1 0.08 3905insT E.20 1 0.08 4005+1G→A I.20 1 0.08 Q1281Xa E.20 1 0.08 Q1313X E.21 1 0.08 Known mutations (75) 1155 90.23 Unknown mutations 125 9.77 a Mutations discovered by the CF group of the Medical and Molecular Genetics Centre - IRO, Barcelona, Spain that range between 0.5% and 0.9%, representing 6.0% of the CF chromosomes. Login to comment
46 Another mutation that affects the same codon, mutation G85E, is involved in a milder clinical presentation of CF with late-onset pancreatic sufficiency (PS) in 70% of cases (Vazquez et al. 1996). Login to comment

[hide] A missense cystic fibrosis transmembrane conductan... Pediatrics. 1997 Sep;100(3):E5. Kerem E, Nissim-Rafinia M, Argaman Z, Augarten A, Bentur L, Klar A, Yahav Y, Szeinberg A, Hiba O, Branski D, Corey M, Kerem B
A missense cystic fibrosis transmembrane conductance regulator mutation with variable phenotype.
Pediatrics. 1997 Sep;100(3):E5., [PMID:9271620]

Abstract [show]
OBJECTIVE: Cystic fibrosis (CF) has variable clinical presentation. Disease severity is partially associated with the type of mutation. The aim of this study was to report genotype-phenotype analysis of the G85E mutation. PATIENTS: The phenotype of 12 patients (8 were from the same extended family, and 5 of them were siblings from 2 families) carrying at least one copy of the G85E mutation was evaluated and compared with the phenotype of 40 patients carrying the two severe mutations, W1282X and/or DeltaF508 (group 1), and with 20 patients carrying the splicing mutation, 3849+10kb C->T, which was found to be associated with milder disease (group 2). RESULTS: A high phenotypic variability was found among the patients carrying the G85E mutation. This high variability was found among patients carrying the same genotype and among siblings. All the studied chromosomes carrying the G85E mutation had the 7T variant in the polythymidine tract at the branch/acceptor site in intron 8. Of the G85E patients, 25% had pancreatic sufficiency and none had meconium ileus, compared with 0% and 32%, respectively, of patients from group 1, and 80% and 0%, respectively, from group 2. Two patients carrying the G85E mutation had sweat chloride levels <60 mmol/L whereas all the others had typically elevated levels >80 mmol/L. Compared with group 2, patients carrying the G85E mutation were diagnosed at an earlier age and had higher sweat chloride levels, with mean values similar to group 1 but significantly more variable. Forced expiratory volume in 1 second (FEV1) was similar in the three groups, with no differences in the slope or in age-adjusted mean values of FEV1. The levels of transcripts lacking exon 9 transcribed from the G85E allele measured in 3 patients were 55%, 49%, and 35% and their FEV1 values were 82%, 83%, and 50% predicated, respectively. CONCLUSIONS: The G85E mutation shows variable clinical presentation in all clinical parameters. This variability could be seen among patients carrying on the other chromosome the same CFTR mutation, and also among siblings. This variability is not associated with the level of exon 9 skipping. Thus, the G85E mutation cannot be classified either as a severe or as a mild mutation.

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20 Thus, the G85E mutation cannot be classified either as a severe or as a mild mutation. Login to comment
33 Pancreatic status was suggested to be the best parameter in differentiating between the two groups.17 The G85E mutation is a missense mutation result- From the *Department of Pediatrics, Cystic Fibrosis (CF) Clinic, Shaare Zedek Medical Center, Jerusalem, Israel; and the ‡Department of Genetics, Hebrew University, Jerusalem; the §Department of Paediatrics, CF Clinic, Chaim Sheba Medical Center, Tel Hashomer, Israel; the ʈDepartment of Paediatrics, CF Clinic, Rambam Medical Center, Haifa; the ¶Department of Paediatrics, Bikur Cholim Hospital, Jerusalem, Israel; and the #Research Institute, Hospital for Sick Children, Toronto, Canada. Login to comment
39 3 September 1997 ing from a substitution of glutamic acid for glycine at amino acid 85. Login to comment
42 Two patients, 1 with PI and the other with PS and exceptionally mild lung disease, were previously reported.18,19 Thus, for further determination of the correlation between the G85E mutation and disease phenotype a larger cohort of patients was required. Login to comment
63 Hybridization to RT-PCR Products 50 ␮L of each differential RT-PCR reaction were subjected to electrophoresis and were subsequently blotted and hybridized to the exon 3 oligonucleotide G85E-N 5ЈGTTCTATGGAATCTTT 3Ј that identified the normal sequence, washed at 42°C, and to G85E-M 5ЈGTTCTATGAAATCTTT 3Ј that identified the G85E mutation, washed at 40°C. Login to comment
70 To assess the severity of disease presentation of the patients carrying the G85E mutation, we compared their clinical parameters with those of two groups of patients. Login to comment
75 RESULTS Twelve CF patients were found to carry the G85E mutation, 9 were Arab, 8 from the same extended family (Table 1, patients 1 to 4 and 6 to 9), and 3 were Jews from Turkish origin (patients 10 to 12). Login to comment
76 Six patients were homozygous for the G85E mutation and 6 were compound heterozygote for the G85E and the ⌬F508, W1282X or the 3849ϩ10kb C-ϾT mutations. Login to comment
77 All the studied chromosomes carrying the G85E mutation had the 7T variant in the polythymidine tract at the branch/acceptor site in intron 8. Login to comment
78 The clinical parameters of the patients carrying the G85E mutation are presented in Table 1. Login to comment
79 It shows a high variability in the age at diagnosis among patients carrying the G85E. Login to comment
85 One had 3 children homozygous for the G85E mutation (Table 1, patients 1 to 3). Login to comment
89 The second family had 2 children compound heterozygous for G85E and ⌬F508 mutations (Table 1, patients 7 and 8). Login to comment
92 Comparison of the patients carrying the G85E mutation with patients carrying the ⌬F508 and/or W1282X mutations previously associated with PI and the more severe disease (group 1) or with patients carrying the 3849ϩ10kb C-ϾT previously associated with higher frequency of PS and a milder disease (group 2), revealed that the mean age at diagnosis and age at assessment in the group of patients carrying the G85E mutation were not significantly different from those in group 1, but significantly lower than in group 2 (P ϭ .001 for age of diagnosis, P ϭ .04 for age at assessment; Table 2). Login to comment
93 Likewise, mean sweat chloride levels in patients carrying the G85E mutation was similar to group 1, and significantly higher than group 2 (P ϭ .001; Table 2). Login to comment
94 Thus, it seems that patients carrying the G85E mutation have similarly severe disease as patients carrying the ⌬F508 and/or W1282X mutations. Login to comment
95 However, the F test of equal variance was significantly different between these groups: the range of age at diagnosis and sweat chloride levels were much broader for the G85E group than for group 1 (P Ͻ .0001), and sweat chloride was more variable in the G85E mutation than in group 2 (P ϭ .002). Login to comment
97 Clinical Data of CF Patients Carrying the G85E Mutation Patient No. Login to comment
98 Genotype Sex Age at Diagnosis Sweat Chloride meq/l Mode of Presentation Current Age Pancreatic Status FEV1 % Predicted Weight Percentile Sputum Culture 1* G85E/G85E F 9 mo 143 Respir ϩ GI Died 13 y PI NA Ͻ3 Klebsiela 2* G85E/G85E M 2 mo 184 Respir Died 10 y PI NA Ͻ3 H influenzae 3* G85E/G85E M 12 y 54 Liver 13 y PS 82 Ͻ3 H influenzae 4 G85E/G85E F 6 mo 157 Respir ϩ GI Died 6 y PI 20 Ͻ3 P aeruginosa 5 G85E/G85E F 2 mo 90 Respir ϩ GI 4 mo PI NA Ͻ3 H influenzae 6 G85E/G85E F 3 mo 97 Respir ϩ GI 6 yr PS 86 35 H influenzae ϩ Staph 7† G85E/⌬F508 M 6 mo 158 GI Died 11 y PI NA 3 NA 8† G85E/⌬F508 M 10 y 108 Screening 12 y PS 83 80 Staph 9 G85E/⌬F508 M 5 mo NA Respir ϩ GI 18 y PI 50 Ͻ3 P aeruginosa ϩ Staph 10 G85E/W1282X F 7 mo 117 Respir 20 y PI 65 50 P aeruginosa 11 G85E/W1282X F 19 y 82 Pancreatitis ϩ Respir 34 y PI 22 50 P aeruginosa 12 G85E/3849 ϩ 10KB M 5 mo 54 Respir 10 y PI 65 97 P aeruginosa * and † Patients are siblings, respectively. Login to comment
101 Comparison of Clinical Data Between CF Patients Carrying the G85E Mutations and Patients Homozygous or Compound Heterozygous for Two Severe Mutations (W1282X and ⌬F508) and Those Carry a Milder Mutation (3849ϩ10kb C-ϾT) Variable G85E W1282X/W1282X ⌬F508/⌬F508 W1282X/⌬F508 3849 ϩ 10kb C-ϾT n 12 40 21 Age at diagnosis* 3.7 Ϯ 6.3 0.7 Ϯ 1.8࿣ 13.2 Ϯ 7.8 Sweat chloride (mmol/L)* 119 Ϯ 38 111 Ϯ 12࿣ 71 Ϯ 18¶ Died, n 4 2 2 Pancreatic sufficiency, %§ 25 0 79 Meconium ileus, %§ 0 33 0 Current age, y† 12.8 Ϯ 8.5 10.8 Ϯ 7.0 19.8 Ϯ 8.8 Weight (percentile)‡ 28 Ϯ 34 24 Ϯ 24 50 Ϯ 34 Forced expiratory volume in 1 second (% predicted) 55 Ϯ 26 (n ϭ 7) 66 Ϯ 28 (n ϭ 24) 55 Ϯ 21 (n ϭ 19) * P Ͻ .0001, † P Ͻ .001, ‡ P Ͻ .01 analysis of variance for three means. Login to comment
103 ࿣ P Ͻ .0001 F test of equal variance compared with G85E. Login to comment
104 ¶ P Ͻ .01 F test of equal variance compared with G85E. Login to comment
105 Of the patients carrying the G85E mutation, 25% had PS significantly higher than in group 1 (0%), and significantly lower than in group 2 (80%) (P Ͻ .001; Table 2). Login to comment
106 Furthermore, similar to the patients in group 2, none of the patients carrying the G85E mutation had meconium ileus, whereas a third of the patients from group 1 had meconium ileus (P ϭ .002). Login to comment
107 The nutritional status of patients carrying the G85E mutation was poor. Login to comment
109 However, 58% of the patients carrying the G85E mutation were under the third percentile of weight, compared with 25% of the group 1 patients (P ϭ .02), and 20% of group 2 (0.05). Login to comment
114 Levels of Correctly Spliced CFTR RNA Transcribed From the G85E Allele in Respiratory Epithelium We next studied the possibility that the variability in pulmonary function found among our patients carrying the G85E mutation is associated with the level of exon 9 skipping that leads to the translation of a nonfunctional CFTR protein. Login to comment
115 The level of aberrantly spliced CFTR RNA transcribed in respiratory epithelial cells from the G85E allele was analyzed using semiquantitative nondifferential RT-PCR. Login to comment
116 The RT-PCR was performed on respiratory epithelial cells from 3 patients who carried at least one G85E allele (patients 3, 8, and 9). Login to comment
118 The results showed that the level of transcripts lacking exon 9 transcribed from the G85E allele was 55 Ϯ 3% for patient 3, 49 Ϯ 2% for patient 8, and 35 Ϯ 4% for patient 9. Login to comment
121 Thus, the level of aberrantly spliced transcripts could not explain the variable pulmonary function among patients carrying the G85E mutation. Login to comment
122 DISCUSSION This study demonstrates high variability of clinical presentation among patients carrying the G85E mutation. Login to comment
123 Thus, the G85E mutation cannot be classified either as a severe or as a mild mutation. Login to comment
131 The incidence of PS among the patients carrying the G85E mutation (25%), suggests that it cannot be classified either as a PI- or PS-associated mutation. Login to comment
132 Slightly elevated, borderline, or even normal values of sweat chloride were previously reported among patients carrying at least one copy of the splicing mutation 3849ϩ10kb C-ϾT.7,8,15 Two of the patients in our study, carrying the G85E mutation, had borderline sweat chloride levels, whereas the others had typically high levels. Login to comment
133 Furthermore, as shown in Table 2, it seems that the G85E patients present to medical attention at a later age than the patients in the severe group. Login to comment
134 However, again this is attributable to the wide range of the age of diagnosis among the G85E patients, and not attributable to a milder disease course. Login to comment
136 Thus, the results of our analysis show that the G85E mutation is associated with variable disease presentation in all the studied parameters. Login to comment
139 The mechanism causing the variability seen among the patients carrying the G85E mutation is unknown. Login to comment
141 Sequence variations within the CFTR gene that modulate the CF phenotype have been reported in 2 CF patients.29,30 However, in our study 8 patients were from the same extended family, carrying the same G85E allele, as evidenced by extended haplotype analysis using polymorphic markers in the CFTR locus (data not shown). Login to comment
147 In our study all the G85E chromosomes contained the 7T variant. Login to comment
148 In addition, the level of RNA transcripts lacking exon 9, transcribed from the G85E allele, varied among different individuals. Login to comment
153 Further understanding the mechanisms underlying the disease variability found among patients carrying the G85E mutation will contribute to our understanding of the genotype-phenotype association and disease variability in CF. Login to comment

[hide] A cystic fibrosis transmembrane conductance regula... Am J Respir Crit Care Med. 1997 Jun;155(6):1914-20. Kerem E, Rave-Harel N, Augarten A, Madgar I, Nissim-Rafinia M, Yahav Y, Goshen R, Bentur L, Rivlin J, Aviram M, Genem A, Chiba-Falek O, Kraemer MR, Simon A, Branski D, Kerem B
A cystic fibrosis transmembrane conductance regulator splice variant with partial penetrance associated with variable cystic fibrosis presentations.
Am J Respir Crit Care Med. 1997 Jun;155(6):1914-20., [PMID:9196095]

Abstract [show]
Some patients express various features of cystic fibrosis (CF) even though essential characteristics of the disease might be absent. Such patients may suffer from respiratory disease without pancreatic insufficiency and normal sweat chloride levels. Others may present as male infertility because of congenital bilateral aplasia of the vas deferens (CBAVD) with no other signs of CF. The 5T allele, a DNA variant in a noncoding region of the cystic fibrosis transmembrane conductance regulator (CFTR) gene that reduces the level of the normal CFTR transcripts, was found in increased frequency among male patients with CBAVD. The purpose of this study was to investigate the possibility that the 5T allele is associated with dysfunction of organs other than the male reproductive system, leading to CF or atypical CF. Analysis of the 5T allele was performed on 148 subjects (29 with CF, 61 with atypical CF, and 58 with CBAVD) carrying 232 chromosomes with unidentified CFTR mutations, and on 142 non-CF chromosomes from healthy subjects of Ashkenazi origin. The frequency of the 5T allele among chromosomes from patients of Jewish Ashkenazi origin with CF and atypical CF (six of 33; 18%) was significantly higher than the frequency in the normal Ashkenazi population (eight of 142; 6%; p = 0.03). Analysis of the clinical presentation of the five patients with CF and the 12 patients with atypical CF carrying the 5T allele indicated that most patients suffered from respiratory disease presenting as asthma like symptoms, nasal polyposis, chronic sinusitis, chronic bronchitis, or bronchiectasis. Six patients had pancreatic insufficiency, two with meconium ileus. Sweat Cl- levels ranged from normal to elevated. Of the six male patients with respiratory disease who were old enough to be evaluated for fertility status, five were fertile and one had pancreatic insufficiency. Among male patients with CBAVD, 41% suffered from respiratory symptoms. Thus, the 5T allele is a variant with partial penetrance causing disease with an extreme variability of clinical presentation: from normal healthy fertile subjects or male patients with CBAVD to those with atypical or typical clinical phenotype of CF.

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51 In cases in which family members were not available, the assignment was performed by the complete correlation between the 9T allele and the AF508 and the N1303K, and between the 7T allele and the W1282X, G85E, D1152H, and W1089X mutations. Login to comment
70 The frequency of the 5T allele among normal chromosomes was significantly lower than its frequency among chromosomes carried by CF patients and patients with atypical TABLE 1 MUTATIONS AND POLYTHYMIDINE VARIANTS ON THE OTHER CHROMOSOME OF UNRELATED PATIENTS WITH 5T ALLELE Mutation CF and Atypical CF CBAVD Total AF508 4 8 12 W1282X 1 6 7 N1303K 0 2 2 G85E 1 1 2 D1152H 1 0 1 W 1089X 1 0 1 G542X 0 1 1 ST 0 3 3 7T' 7 9 16 9T* 2 2 4 Total 17 32 49 Definition of abbreviations: CF = cystic fibrosis; CBAVD = congenital bilateral aplasia of the vas deferens. Login to comment
105 Haplotype analysis of nine polymorphic sites spanning the CFTR locus from XV2C 5' upstream to the gene to W30 3' downstream to the gene, revealed that as expected, these patients carry the same 5T allele identical by descent determined by the haplotype C,1,1,2,3,1,2,12 and the same G85E allele determined by the haplotype C,1,2,1,6,1,2,5. Login to comment
107 One was the 36-yr-old healthy father (FEVI , 88% predicted) of a child with CF and the G85E/5T genotype with normal sweat Cl- level. Login to comment
108 The second (Patient 13 in Table 3) had nasal and sinopulmonary disease typical of CF and carried the G85E/5T genotype. Login to comment
109 The additional three relatives were infertile male patients with CBAVD who were otherwise healthy and carried the genotypes G85E/5T, 5T/5T, and 5T/unidentified mutation. Login to comment
137 13 Nasopulmonary M/1 7 0.5 52 - - + + + H flu + 40 5T/G85E Chronic cough not S. pneumoniae responding to antiasthma medications. Login to comment

[hide] Analysis of 16 cystic fibrosis mutations in Mexica... Am J Med Genet. 1997 Apr 14;69(4):380-2. Villalobos-Torres C, Rojas-Martinez A, Villareal-Castellanos E, Cantu JM, Sanchez-Anzaldo FJ, Saiki RK, Barrera-Saldana HA
Analysis of 16 cystic fibrosis mutations in Mexican patients.
Am J Med Genet. 1997 Apr 14;69(4):380-2., [PMID:9098486]

Abstract [show]
We carried out molecular analysis of 80 chromosomes from 40 unrelated Mexican patients with a diagnosis of cystic fibrosis. The study was performed in two PCR steps: a preliminary one to identify mutation delta F508, the most frequent cause of cystic fibrosis worldwide, and the second a reverse dot-blot with allele-specific oligonucleotide probes to detect 15 additional common mutations in the Caucasian population. A frequency of 45% for delta F508 was found, making it the most common in our sample of Mexican patients. Another five mutations (G542X, 3,849 + 10 kb C-->T, N1303K, SN549N, and 621 + 1 G-->T) were detected, and those accounted for 11.25%. The remaining mutations (43.75%) were undetectable with the methodology used.

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14 According to data from the Cystic Fibrosis Genetic Analysis Consortium [1994] (CFGAC), the most frequent non-⌬F508 mutations are the following: G542X (2.4%), G551D (1.6%), N1303K (1.3%), W1282X (1.2%), R553X (0.7%), 621 + 1 G→T (0.7%), 1717 - 1 G→T (0.6%), R117H (0.3%), R1162X (0.3%), G85E (0.2%), R347P (0.2%), ⌬I507 (0.2%), and 3849 + 10 kb C→T (0.2%). Login to comment

[hide] CFTR mutations and IVS8-5T variant in newborns wit... J Med Genet. 1997 Apr;34(4):297-301. Castellani C, Bonizzato A, Mastella G
CFTR mutations and IVS8-5T variant in newborns with hypertrypsinaemia and normal sweat test.
J Med Genet. 1997 Apr;34(4):297-301., [PMID:9138152]

Abstract [show]
Neonates positive for immunoreactive trypsinogen assay (IRT) and negative for sweat test have formerly been found to carry the major cystic fibrosis (CF) mutation, delta F508, much more frequently than the general population. Among the 716 IRT positive newborns detected by a three tier (IRT, mutation analysis plus meconium lactase assay, sweat test) CF screening programme in north eastern Italy during the period January 1993 to March 1996, we found 45 carriers, a number significantly higher than the expected 17 (p < 0.001). We speculated that some of these heterozygotes could actually be affected by a very mild form of CF, and carry on the other chromosome an undetected CFTR mutation or a DNA variant, such as the 5-thymidine allele in intron 8 of the CFTR gene (IVS8-5T). This hypothesis was tested in four samples; group A (the 45 carriers mentioned above), group B (51 non-carrier, IRT positive neonates), group C (50 IRT negative neonates), and group D (90 CF adult female carriers). Chromosomes with IVS8-5T were seven (7.78%) in group A, seven (6.86%) in group B, five (5%) in group C, and four in group D (2.22%). The 5T prevalence in group A was significantly higher (p < 0.05) compared to group D; similarly, a higher (p < 0.05) 5T frequency in group A compared to group C was detected by considering the chromosomes free from CFTR mutations. This study is consistent with previous papers in finding among neonates with high trypsin levels a CF carrier frequency significantly higher than that expected. It is also suggested that in at least some babies raised trypsin levels at birth could be a phenotypic expression of compound heterozygosity for a major CF mutation plus IVS8-5T.

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21 Initially we tested for AF508, Rl 162X, and N1303K, estimated by a cohort study7 to cover 61 % of CF chromosomes in our area; from March 1995 10 other mutations were included (2183AAG, 3849+1OKbCT, G542X, 1717-1GA, R553X, Q552X, G85E, 711+5GA, 3132delTG, Cystic Fibrosis Centre, Ospedale Civile Maggiore, Piazzale Stefani, 37126 Verona, Italy C Castellani A Bonizzato G Mastella Correspondence to: Dr Castellani. Login to comment

[hide] Association of domains within the cystic fibrosis ... Biochemistry. 1997 Feb 11;36(6):1287-94. Ostedgaard LS, Rich DP, DeBerg LG, Welsh MJ
Association of domains within the cystic fibrosis transmembrane conductance regulator.
Biochemistry. 1997 Feb 11;36(6):1287-94., [PMID:9063876]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel composed of two membrane-spanning domains (MSD), two nucleotide-binding domains (NBD), and an R domain. To understand how these domains interact, we expressed various constructs of CFTR containing membrane-spanning and/or cytosolic domains either separately or together. We then tested for functional association of these domains using the SPQ halide-efflux assay or physical association using coimmunoprecipitation experiments. Coexpression of the amino-terminal half (MSD1, NBD1, and the R domain) and the carboxy-terminal half (MSD2 and NBD2) of CFTR generated functional Cl- channel activity whereas expression of either alone did not give a signal with the SPQ assay. This result suggests that the two halves associate in the membrane. Using domain-specific antibodies, we found that either half of CFTR could coimmunoprecipitate the other, suggesting a physical association. Coimmunoprecipitation persisted between halves missing the NBDs, the R domain, or the amino-terminal tail. Moreover, constructs from MSD1 containing only the first and second transmembrane sequences and intervening extracellular loop were sufficient for interaction with MSD2. These data suggest that interactions between the two membrane-spanning domains of CFTR may mediate association between the two halves of the protein.

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209 Two other naturally occurring mutations in M1, E92K and G85E (Nunes et al., 1993; Zielenski et al., 1991), also failed to disrupt associations between halves. Login to comment

[hide] The molecular basis of partial penetrance of splic... Am J Hum Genet. 1997 Jan;60(1):87-94. Rave-Harel N, Kerem E, Nissim-Rafinia M, Madjar I, Goshen R, Augarten A, Rahat A, Hurwitz A, Darvasi A, Kerem B
The molecular basis of partial penetrance of splicing mutations in cystic fibrosis.
Am J Hum Genet. 1997 Jan;60(1):87-94., [PMID:8981951]

Abstract [show]
The splicing variant, 5T allele, in intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene was shown to be associated with partial penetrance of the clinical expression. This splicing variant leads to two possible transcripts: one normal and the other aberrantly spliced that lacks exon 9. The aim of this study was to analyze the molecular basis of the partial penetrance in individuals carrying the 5T allele. We analyzed the level of the correctly spliced RNA transcribed from the 5T allele in nasal and epididymal epithelium and correlated it with disease expression. Semiquantitative nondifferential reverse-transcriptase-PCR showed a considerable variability (6%-37%) in the total level of correctly spliced RNA transcribed from the 5T allele in nasal epithelium from 11 patients. A significant nonlinear correlation (r = .82, P = .002) between the level of the normal CFTR transcripts and the severity of lung disease was shown. No individuals with normal lung function and minimal or no lung disease (FEV1 >80% predicted) had <25% of normal transcripts, and individuals with <15% of normal transcripts did not have FEV1 >80%. The level of normal transcripts in epididymal epithelial cells from four infertile males with congenital bilateral absence of the vas deferens was low (6%-24%). In infertile males with normal lung function the level of correctly spliced transcripts in the nasal epithelium was higher than the level in the epididymal epithelium. These results indicate that there is variability in the efficiency of the splicing mechanism, among different individuals and between different organs of the same individual. This variability provides the molecular basis of the partial penetrance of cystic fibrosis disease in patients carrying the 5T allele.

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36 Two of the individuals were homozygous for the ST allele, and 9 were heterozygotes, in 8 of whom an additional CFTR mutation on the other chromosome was identified: 3 carried the G85E mutation (Zielenski et al. Login to comment
38 Three individuals (1474, 658, and 607) either homozygous for the ST allele or compound heterozygous for the ST allele and the G85E mutation were from the same family carrying the same ST allele, as evidenced by their pedigree and by an extended haplotype analysis of the CFTR locus (Kerem et al., in press). Login to comment
51 Biopsies from epithelial epididymis were obtained Table 1 Levels of Normally Spliced RNA Transcribed from the 5T Allele in Nasal and Epididymal Epithelium, and Clinical and Genetic Features NASAL EPITHELIUM EPIDIDYMAL EPITHELIUM (% Normal RNA) (% Normal RNA) PATIENT SEx/AGE DIAGNOSIS GENOTYPE FEV, From ST From Total From 5T From Total 607b M/41 CBAVD ST/ST 88 37 37 24 24 1549 F/19 Healthy ST/ST 83 31 31 ... ... 658b M/36 CBAVD ST/G85E 78 28 14 ... ... 1703 F/33 Healthy ST/G85E 92 62 31 1474b M/17 CF ST/G85E 40 16 8 660 M/29 CBAVD ST/AFS08 91 72 36 20 10 666 M/31 CBAVD ST/AFS08 83 52 26 32 16 662 M/33 CBAVD ST/AFS08 71 24 12 628 M/35 CBAVD ST/N1303K 57 12 6 12 6 642 M/39 CBAVD ST/W1282X 72 12 6 ... ... 610 M/36 CBAVD ST/(unknown mutation) 86 S0 25 ... ... aValues are means of repeated experiments (n = 2-6); variability between experiments was <10% of the mean. Login to comment
72 RT-PCR 2 products hybridized to the exon 3 oligonucleotide G85E-N 5' GTTCTATGGAATCTTT 3', which identified the normal sequence, washed at 420C, and to G85E-M 5' GTTCTATGAAATCTTT 3', which identified the G85E mutation, washed at 40'C. The RT-PCR 3 products were hybridized in the same way as was RT-PCR 1. Login to comment
80 In cases in which family members were not available the assignment was performed by the complete correlation between the 9T allele and the AF508 and N1303K mutations and between the 7T allele and the W1282X and G85E mutations. Login to comment
93 The reaction was performed on a patient heterozygous for the ST allele and the G85E mutation. Login to comment
94 The vertical line represents the G85E site. Login to comment
107 The example is from patient 1474, heterozygous for the ST allele, the G85E mutation, and the M470V polymorphism. Login to comment

[hide] Cystic fibrosis mutation frequencies in upstate Ne... Hum Mutat. 1997;10(6):436-42. Shrimpton AE, Borowitz D, Swender P
Cystic fibrosis mutation frequencies in upstate New York.
Hum Mutat. 1997;10(6):436-42., [PMID:9401006]

Abstract [show]
Upstate New York patients (100) with cystic fibrosis (i.e., 200 CF chromosomes), 72 from the CF center in Syracuse and 28 from a Buffalo CF center, were analyzed for their CF-causing mutations using restriction enzyme digest, single-strand conformation analysis (SSCA), and Heteroduplex (HA) analysis. Polymerase chain reaction (PCR) amplified products from all 27 CFTR exons using primers that included flanking intron junction sequence were investigated. More than 120 known cystic fibrosis transmembrane conductance regulator (CFTR) disease-causing mutations were screened. Four novel CFTR disease-causing mutations were identified (N287Y in exon 6b, 1259insA in exon 8, R1070P in exon 17b, and CF?20kbdel14b-18). A detection rate of 96% of the combined Syracuse and Buffalo population CF chromosomes was obtained.

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84 % Comment 3 G85E 1 1 0.5 4 R117H 1 1 0.5 i4 621 + 1,G>T 1 2 3 1.5 5 711 + 1,G>T 1 1 0.5 6b N287Y 1 1 0.5 Novel 7 1154insTC 2 2 1.0 8 1259insA 1 1 0.5 Novel 9 A455E 1 1 0.5 10 Delta F508 109 39 148 74.0 10 1609delCA 1 1 0.5 Spanish i10 1717-1,G>A 3 3 1.5 11 G542X 2 1 3 1.5 11 G551D 3 3 1.5 11 R553X 4 4 2.0 i12 1898+1,G>A 2 2 1.0 13 2143delT 1 1 0.5 13 2184delA+G>A 1 1 0.5 i14 2789+5,G>A 2 2 1.0 17b R1070P 1 1 0.5 Novel 17b Y1092X(C>A) 2 2 1.0 French Canadian (Rozen et al., 1992) 17b CF?20kbdel 14b-18 1 1 0.5 Novel (Shrimpton and Borowitz, 1997) i19 3849+10kb,C>T 1 1 0.5 20 W1282X 2 2 0.5 Ashkenazi 21 N1303K 3 3 6 3.0 Unknown 4/144 4/56 8/200 4.0 AL. 75 and 81 mMol/L. Login to comment

[hide] Missense mutation R1066C in the second transmembra... Hum Mutat. 1997;10(5):387-92. Casals T, Pacheco P, Barreto C, Gimenez J, Ramos MD, Pereira S, Pinheiro JA, Cobos N, Curvelo A, Vazquez C, Rocha H, Seculi JL, Perez E, Dapena J, Carrilho E, Duarte A, Palacio AM, Nunes V, Lavinha J, Estivill X
Missense mutation R1066C in the second transmembrane domain of CFTR causes a severe cystic fibrosis phenotype: study of 19 heterozygous and 2 homozygous patients.
Hum Mutat. 1997;10(5):387-92., [PMID:9375855]

Abstract [show]
We report the clinical features of 21 unrelated cystic fibrosis (CF) patients from Portugal and Spain, who carry the mutation R1066C in the CFTR gene. The current age of the patients was higher in the R1066C/any mutation group (P < 0.01), as compared to the deltaF508/deltaF508 group. Poor values for lung radiological involvement (Chrispin-Norman) and general status (Shwachman-Kulcycki) were observed in the R1066C/any mutation group (P < 0.005 and P < 0.0004). A slightly, but not significantly worse lung function was found in the R1066C/any mutation group when compared with the deltaF508/deltaF508 patients. No significant differences were detected regarding the age at diagnosis, sweat Cl-values, or percentiles of height and weight between the two groups. Neither were significant differences observed regarding sex, meconium ileus (4.7% vs. 11.1%), dehydration (10.5% vs. 14.7%), or pancreatic insufficiency (PI) (100% vs. 97.8%). The proportion of patients with lung colonization by bacterial pathogens was slightly, but not significantly higher in the R1066C/any mutation group (70.0%), as compared with the deltaF508/deltaF508 group (57.5%). Other clinical complications were significantly more frequent in the R1066C/any mutation patients(P < 0.02) than in the deltaF508/deltaF508 group. The two homozygous R1066C/R1066C patients died at the ages of 3 months and 7 years. The data presented in this study clearly demonstrate that the R1066C mutation is responsible for a severe phenotype similar to that observed in homozygous deltaF508 patients. The poor clinical scores and complications of patients with the R1066C mutation are probably related to their slightly longer survival.

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56 The current clinical data for missense mutations derived from a relatively large number of cases are limited to a few mutations: N1303K (Osborne et al., 1992; CF genotype-phenotype Consortium 1993), R117H (CF genotype-phenotype Consortium 1993), P205S (Chillón et al., 1993), A455E (Gan et al., 1995), L206W (Desgeorges et al., 1995), R334W (Estivill et al., 1995), and G85E (Vázquez et al., 1996). Login to comment
60 (Strong et al., 1991), E92K (Nunes et al., 1993), S549N (Curtis et al., 1993), I175V (Romey et al., 1994), A559T (McDowell et al., 1995), and G85E (Vázquez et al., 1996). Login to comment

[hide] Rapid characterization of the variable length poly... Hum Mutat. 1997;10(2):108-15. Friedman KJ, Heim RA, Knowles MR, Silverman LM
Rapid characterization of the variable length polythymidine tract in the cystic fibrosis (CFTR) gene: association of the 5T allele with selected CFTR mutations and its incidence in atypical sinopulmonary disease.
Hum Mutat. 1997;10(2):108-15., [PMID:9259194]

Abstract [show]
The CFTR intron 8 variable length polythymidine tract modulates the cystic fibrosis (CF) phenotype associated with the mutation R117H. To explore whether other mutations reside on multiple intron 8 backgrounds with discernible impacts on phenotype, we developed an allele-specific PCR assay to characterize this locus. Our approach types samples rapidly without the use or radioisotopes. Polythymidine alleles were identified for mutations either associated with a wide range of clinical phenotypes (R117H, R347P, G85E, D1152H, R334W, 2789 + 5 G > A, 3849 + 10kb C > T), and/or located at hypermutable CpG loci (R117H, 3845 + 10kb C > T, R553X, R334W, S945L and R75Q). R117H was detected in cis with each of three alleles (5T, 7T, 9T) at the intron 8 locus. The novel R117H-9T association was detected in a 10-month African-American male with borderline-to-mildly elevated sweat chloride values (approximately 50-66 mEq/L). All other mutations studied were associated with 7T except 3849 + 10kb C > T, which was detected on both 7T and 9T backgrounds, but not 5T. Three individuals with a delta F508/3849 + 10kb C > T genotype were 9T,9T and had pancreatic sufficiency and normal sweat chloride values, whereas 15 others who carried 3849 + 10kb C > T on a 7T background had variable pancreatic function (sufficient, n = 12, insufficient, n = 3), and variable sweat chloride values (normal, n = 12, elevated, n = 3). Surprisingly, when not associated with known CFTR mutations, 5T was detected with elevated frequency among individuals with sinopulmonary disease of ill-defined etiology, but with some characteristics of variant CF. In summary, the 5T allele was not found in cis with CF-causing mutations besides R117H, but an elevated 5T allele frequency in variant CF patients suggests 5T may be associated with disease in some situations.

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3 Polythymidine alleles were identified for mutations either associated with a wide range of clinical phenotypes (R117H, R347P, G85E, D1152H, R334W, 2789+5 G>A, 3849+10kb C>T), and/or located at hypermutable CpG loci (R117H, 3849+10kb C>T, R553X, R334W, S945L and R75Q). Login to comment
37 We report the development of a rapid, nonisotopic assay that facilitates typing of this locus and utilize it to explore the role of the polythymidine tract alleles in thepathogenesisofCF.Mutationsassociatedwithclini- calheterogeneity(R347P,G85E,D1152H,R334W,and 3849 + 10kb C>T) and/or occurring at hypermutable loci (3849 + 10kb C>T, R334W, S945L, R553X, and R75Q) were analyzed for their association with different intron 8 alleles in CF and atypical patients. Login to comment
39 Mutation screening was performed for R553X (Cutting et al., 1990), R334W (Gasparini et al., 1991), G85E (Zielenski et al., 1991a), S945L (Claustres et al., 1993), 3849 + 10kb C>T (Highsmith et al., 1994), R117H and R347P (Dean et al., 1990), 2789+5G>A (Highsmith et al., 1997), D1152H (Highsmith, per. Login to comment
43 For R553X, G85E, S945L, 3849 + 10KB C>T, and R334W, the digested samples were electrophoresed at 100 V for 2 hr in 4% agarose gels (3:1 Nusieve: SeaKem, FMC Bioproducts, Rockland, ME). Login to comment
94 Association of Selected CFTR Mutations with Intron 8 Polythymidine Alleles Chromosomes In cis with In cis with In cis with Mutation Site CpG locus 5T 7T 9T R75Qa EXON 3 Y 0 8 0 G85E EXON 3 N 0 5 0 R117H EXON 4 Y 8 5 1 R334W EXON 7 Y 0 4 0 R347P EXON 7 N 0 7 0 R553X EXON 11 Y 0 7 0 2789+5 G>A INTRON 14B N 0 5 0 S945L EXON 15 Y 0 3 0 D1152H EXON 18 N 0 7 0 3849+10kb C>T INTRON 19 Y 0 15 2 a Sequence variant. Login to comment

[hide] Thirteen cystic fibrosis patients, 12 compound het... J Med Genet. 1996 Oct;33(10):820-2. Vazquez C, Antinolo G, Casals T, Dapena J, Elorz J, Seculi JL, Sirvent J, Cabanas R, Soler C, Estivill X
Thirteen cystic fibrosis patients, 12 compound heterozygous and one homozygous for the missense mutation G85E: a pancreatic sufficiency/insufficiency mutation with variable clinical presentation.
J Med Genet. 1996 Oct;33(10):820-2., [PMID:8933333]

Abstract [show]
To study the severity of mutation G85E, located in the first membrane spanning domain of the CFTR gene, we studied the clinical features of 13 Spanish patients with cystic fibrosis (CF) carrying this mutation. G85E accounts for about 1% of Spanish CF alleles. One patient was homozygous G85E/G85E and the rest were compound heterozygotes for G85E and other mutations (delta F508 nine patients, delta I507 two patients, and 712-1G > T one patient). The characteristics of the pooled G85E/any mutation group were compared with those of 30 delta F508 homozygotes. Mean age at diagnosis and percentage of ideal height for age were higher in the G85E/any mutation group (4.2 (SD 4.7) v 2.4 (SD 2.3), p < 0.05, and 102.8 (SD 4.7) v 97.8 (SD 4.1), p < 0.01), both probably related to the greater prevalence of pancreatic sufficiency (70% v 0%, p < 0.01). The G85E homozygote was pancreatic sufficient. Sweat sodium levels were slightly higher, and salt loss related problems more frequent, in the G85E/any group. Two of the G85E patients died of respiratory failure aged 6 and 14 years. Striking discordance in the phenotype was observed in two pairs of sibs, one of them dizygotic twins, suggesting that factors, genetic and environmental, other than CFTR genotype are important in determining CF phenotype.

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1 Received 4 December 1995 Revised version accepted for publication 28 May 1996 Abstract To study the severity of mutation G85E, located in the first membrane spanning domain of the CFTR gene, we studied the clinical features of 13 Spanish patients with cystic fibrosis (CF) carrying this mutation. Login to comment
2 G85E accounts for about 1% of Spanish CF alleles. Login to comment
3 One patient was homozygous G85E/G85E and the rest were compound heterozygotes for G85E and other mutations (AF508 nine patients, A1507 two patients, and 712-1G>T one patient). Login to comment
4 The characteristics ofthe pooled G85E/any mutation group were compared with those of30 AF508 homozygotes. Login to comment
5 Mean age at diagnosis and percentage of ideal height for age were higher in the G85E/any mutation group (4.2 (SD 4.7) v 2.4 (SD 2.3), p<0.05, and 102.8 (SD 4.7) v 97.8 (SD 4.1), p<0.01)), both probably related to the greater prevalence of pancreatic sufficiency (70% v 0%, p<0O01). Login to comment
6 The G85E homozygote was pancreatic sufficient. Login to comment
7 Sweat sodium levels were slighdy higher, and salt loss related problems more frequent, in the G85E/any group. Login to comment
8 Two of the G85E patients died ofrespiratory failure aged 6 and 14 years. Login to comment
10 (JMed Genet 1996;33:820-822) Key words: cystic fibrosis; CFTR mutations; phenotype; G85E mutation. Login to comment
17 '8 Other mutations associated with mild CF are some splicing mutations in which low levels of normal transcripts are produced.6 17 We present here the clinical findings in 13 Spanish CF patients carrying the missense mutation G85E, a G to A change at nucleotide position 386 in exon 3, which results in a glutamic acid for glycine substitution at codon 85 in the first MSD of the CFTR. Login to comment
21 The clinical features of patients with a G85E/any mutation genotype were compared with those of 30 AF508 homozygotes under control at Hospital de Cruces. Login to comment
26 The best FEV1 over the last year was chosen as an index of pulmonary group.bmj.comon October 25, 2012 - Published byjmg.bmj.comDownloaded from Thirteen cystic fibrosis patients compound heterozygous or homozygous for G85E mutation Table 1 Clinicalfeatures of 13 CFpatients carrying the G85E mutation compared with AF508 homozygotes Genotype Parameter G85Elany AF5081AF508 No of patients 13 30 Sex: female/male 6/7 12/18 Alive 11 28 Mean, SD (No studied) Current age (y) 8.4, 5.4 (11) 9.9, 4.3 (28) Age at diagnosis (y) 4.23,4.7 (13) 2.4, 2.3 (30)* Sweat sodium (mmol/l) 105.1, 17.0 (7) 94.3, 14.0 (23)* Sweat chloride (mmol/l) 107.2, 4.0 (5) 97.5, 14.0 (22) Schwachman score 87.5, 7.5 (10) 88.7, 9.4 (28) Chrispin-Norman chest x ray score 4.5, 3.1 (9) 4.7, 5.3 (27) FEVI (% predicted) 80.2, 8.2 (6) 87.1, 13 (24) FVC (% predicted) 85.3, 7.7 (6) 94.8, 14.6 (24) % Ideal body weight 101.4, 14.2 (10) 100.7, 11.2 (28) % Ideal body height 102.8, 4.7 (10) 97.8, 4.1 (28)** No positivelNo studied (%) Pancreatic insufficiency 4/13 (30.7) 30/30 (100)** Colonisation with P aeruginosa 10/13 (76.9) 24/30 (80) Chronic bronchial infection with P aeruginosa 5/13 (38.4) 10/30 (33.3) Meconium ileus 0/13 (0) 2/30 (6.7) Liver disease 1/13 (7.6) 2/30 (6.7) Dehydration and metabloic alkalosis 4/13 (30.7) 1/30 (3.4)* *p<0.05, **p<0.01. Login to comment
32 Direct sequencing allowed us to identify the G85E mutation. Login to comment
34 G85E was found in 13 samples. Login to comment
35 All chromosomes with mutation G85E were associated with the same microsatellite haplotype 16-24-13. Login to comment
36 Nine patients were compound heterozygotes for AF508/G85E, one pair of sibs were G85E/AI507, one was G85E/712-1 G>T, and one was a G85E homozygote. Login to comment
37 Data from G85E/AF508 and G85E/non-AF508 patients were pooled for comparison with those from AF508 homozygotes. Login to comment
38 The G85E/any mutation patients were older at diagnosis, had a higher percentage of ideal height, and a lower prevalence of PI (30% v 100%, p<0.01) compared with the AF508 homozygotes (table 1). Login to comment
42 Ages at death were 6 and 14 years in the G85E/any group, and 16 and 18 years in the AF508 homozygous group. Login to comment
44 The G85E homozygote was a 17 year old girl with pancreatic sufficiency (PS), who had recurrent coughing from the age of 4 and was diagnosed at the age of 13 years. Login to comment
51 The worldwide prevalence of G85E is 0.2%,4 but higher frequencies have been reported in the US (0.7%),24 Italy (1.7%),25 and Spain (1%) (T Casals, personal communication). Login to comment
53 The molecular mechanism of the CFTR dysfunction associated with G85E has recently been found to consist of a trafficking defect (class II mutation)6 with rapid degradation of the mutant protein before transit through the Golgi complex.26 There have been only three reports on the clinical characteristics associated with this mutation. Login to comment
54 Chalkley and Harris27 described an 11 year old boy, homozygous for G85E, with PS. Login to comment
55 Kerem et af8 reported five G85E homozygotes and eight compound Table 2 Characteristics of 13 CFpatients with mutation G85E* Patient Agelage at Pancreatic FEVI (% No Sex diagnosis (y) status Schwachman CBI pred) Other clinicalfeatures 1 F 17.6/13.3 PS 95 No 75 Mild pulmonary disease 2 F 14 (dead)/0.7 PSt - Yes 28 Dehydration. Login to comment
62 Older sib of patient 8 * Patient 1 was G85E/G85E, patients 8 and 13 G85E/AI507, patient 9 G85E/712-1G>T, and the rest G85E/AF508. Login to comment
65 Friedman et af4 reported that six out of their nine patients with G85E had PS. Login to comment
66 The data presented here suggest that G85E is predominantly associated with PS. Login to comment
67 This inconsistent association of G85E with PS, and the clinical discordance in our two pairs of sibs, suggest that genetic factors other than CF genotype, as well as environmental factors, are involved in the severity of the disease. Login to comment
68 These factors seem more evident when the CF mutations are mild, as has been shown for mutation R334W7 or for the IVS8-6(5T) mutation, which is associated with male infertility, mild CF, or even a normal phenotype.8 In summary G85E occurred with PS in 70% of the cases. Login to comment
69 Compared with AF508 homozygotes, patients with G85E were diagnosed later, were taller, and had a higher prevalence of dehydration, but no other difference was observed either in the severity of the pulmonary disease or in the prevalence of other CF related complications.This mutation also shows intrafamilial variability in its clinical presentation. Login to comment
70 Further experience over a longer time span is needed to clarify the comparative severity of the phenotype associated with G85E. Login to comment
134 Relatively high prevalence ofthe CFTR mutations G85E and 1154insTC. Login to comment
138 26 Skach WR. CFTR mutants G85E and G91 R insert charged residues into the first transmembrane segment and disrupt intracellular trafficking but do not alter transmembrane topology. Login to comment
141 A cystic fibrosis patient who is homozygous for the G85E mutation has very mild disease. Login to comment
143 28 Kerem E, Nissim M, Argaman Z, et al. Extremely high variability of clinical presentation among CF patients carrying the missense mutation G85E. Login to comment
145 G85E: a pancreatic sufficiency/insufficiency homozygous for the missense mutation compound heterozygous and one Thirteen cystic fibrosis patients, 12 http://jmg.bmj.com/content/33/10/820 Updated information and services can be found at: These include: References http://jmg.bmj.com/content/33/10/820#related-urls Article cited in: service Email alerting the box at the top right corner of the online article. Login to comment
130 24 Friedman KJ, Blalock ML, Silverman LM. Relatively high prevalence ofthe CFTR mutations G85E and 1154insTC. Login to comment
137 A cystic fibrosis patient who is homozygous for the G85E mutation has very mild disease. Login to comment
140 Extremely high variability of clinical presentation among CF patients carrying the missense mutation G85E. Login to comment
142 G85E: a pancreatic sufficiency/insufficiency homozygous for the missense mutation compound heterozygous and one Thirteen cystic fibrosis patients, 12 R Cabanas, C Soler and X Estivill C Vazquez, G Anti&#f1;olo, T Casals, J Dapena, J Elorz, J L Seculi, J Sirvent, doi: 10.1136/jmg.33.10.820 1996 33: 820-822 J Med Genet http://jmg.bmj.com/content/33/10/820 Updated information and services can be found at: These include: service Email alerting box at the top right corner of the online article. Login to comment

[hide] Survey of cystic fibrosis transmembrane conductanc... Dig Dis Sci. 1996 Mar;41(3):540-2. McGill JM, Williams DM, Hunt CM
Survey of cystic fibrosis transmembrane conductance regulator genotypes in primary sclerosing cholangitis.
Dig Dis Sci. 1996 Mar;41(3):540-2., [PMID:8617131]

Abstract [show]
A variety of cholestatic liver diseases appear to primarily affect the biliary epithelium, including cystic fibrosis (CF). CF results from a defect in the chloride channel protein, cystic fibrosis transmembrane conductance regulator (CFTR). Although the majority of CF patients have a genomic deletion in deltaF508, other mutations of CFTR may result in less severe clinical presentations and outcomes. Recently, CFTR has been shown to be involved in secretin-stimulated choleresis in intrahepatic bile duct epithelial cells. Cholestasis in cystic fibrosis appears to result from defective chloride transport across the biliary epithelium and is the only cholestatic disease of bile ducts for which a cellular defect has been identified. Primary sclerosing cholangitis (PSC) is a cholestatic disease with histological and cholangiographic features similar to CF. The purpose of this pilot study was to explore whether there is an increased prevalence of CFTR mutations. Two patients exhibited mutations in one allele, yielding a carrier rate of 10.6%, not statistically different from the general U.S. population carrier rate of 4%.

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33 In total, 32 mutations were evaluated, which represent 90% of the most common mutations (t4): AF508 G542X G551D W1282X 3905insT NI303K 3849+ 10kbC--~T R553X 621+ IG--*T 1717- IG--,A lt)78delT 2789+5G---~A 3849+4A--~G 711+ IG---oT R1162X 1898+IG----~A R117H 3659delC G85E 2184delA A1507 R347P Y1092X R560T A455E R334W Y122X S549R(T---~G) Q493X V520F $549N R347H Patient Selection. Login to comment

[hide] Mutation characterization of CFTR gene in 206 Nort... Hum Mutat. 1996;8(4):340-7. Hughes DJ, Hill AJ, Macek M Jr, Redmond AO, Nevin NC, Graham CA
Mutation characterization of CFTR gene in 206 Northern Irish CF families: thirty mutations, including two novel, account for approximately 94% of CF chromosomes.
Hum Mutat. 1996;8(4):340-7., [PMID:8956039]

Abstract [show]
A variety of mutation detection techniques, including restriction endonuclease digestion, allele specific oligonucleotides, and automated fluorescent sequencing, were used in the identification of 15 CFTR mutations representing 86.7% of CF chromosomes in 206 Northern Irish cystic fibrosis (CF) families. A systematic analysis of the 27 exons and intron/exon boundaries of the CFTR gene was performed using denaturing gradient gel electrophoresis (DGGE) in an attempt to characterise the 55 unknown CF mutations in 51 patients. Twenty different mutations were detected by DGGE on 30 chromosomes accounting for a further 7.3% of CF alleles. Fifteen of these mutations had not previously been found in Northern Ireland, and two are novel, M1I(G > T) and V562L. In total, 30 CFTR mutations account for 93.9% of the 412 Northern Irish CF chromosomes tested. The three major CF mutations in Northern Ireland are delta F508, G551D, and R117H with respective frequencies of 68.0%, 5.1%, and 4.1%. The efficacy of the DGGE technique was proven by the detection of 77 out of 77 control variants from all the CFTR exons. DGGE is a highly efficient and sensitive method for mutation screening especially in large genes where the mutation spectrum is known to be heterogeneous.

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73 G85E, L88S R117H, 621+1G>T, 435insA. Login to comment

[hide] Fluorescent multiplex microsatellites used to defi... Hum Mutat. 1996;8(3):229-35. Hughes D, Wallace A, Taylor J, Tassabehji M, McMahon R, Hill A, Nevin N, Graham C
Fluorescent multiplex microsatellites used to define haplotypes associated with 75 CFTR mutations from the UK on 437 CF chromosomes.
Hum Mutat. 1996;8(3):229-35., [PMID:8889582]

Abstract [show]
The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene contains three highly informative microsatellites: IVS8CA, IVS17bTA, and IVS17bCA. Their analysis improves prenatal/ carrier diagnosis and generates haplotypes from CF chromosomes that are strongly associated with specific mutations. Microsatellite haplotypes were defined for 75 CFTR mutations carried on 437 CF chromosomes (220 for delta F508, 217 for other mutations) from Northern Ireland and three English regions: the North-West, East Anglia, and the South. Fluorescently labelled microsatellites were amplified in a triplex PCR reaction and typed using an ABI 373A fluorescent fragment analyser. These mutations cover all the common and most of the rare CF defects found in the UK, and their corresponding haplotypes and geographic region are tabulated here. Ancient mutations, delta F508, G542X, N1303K, were associated with several related haplotypes due to slippage during replication, whereas other common mutations were associated with the one respective haplotype (e.g., G551D and R560T with 16-7-17, R117H with 16-30-13, 621 + 1G > T with 21-31-13, 3659delC with 16-35-13). This simple, fast, and automated method for fluorescent typing of these haplotypes will help to direct mutation screening for uncharacterised CF chromosomes.

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No. Sentence Comment
58 As the repeat number increasesbeyond 50, it probably becomes more disposed to the addition or deletion of dinucleotide repeats during replication as all other mutations where several alleles were tested gave the same respective 17bTA repeat number, i.e., G551D (17AT repeats = 7), R560T (7), G85E (24), R117H (30), 621+1G>T (31), 3659delC (354, 1898+lG>A (45). Login to comment
74 CF 8CA-17bTA-17bCA Mutation chromosomes % Normal Laboratoryb Reference' HaplotVpe 1)15-29-13 557delT Nl Graham et al.. 1992 21 16-07-17 MU (G>T) 3) 16-24-13 4) 16-25-13 5) 16-29-13 6) 16-30-13 7) 16-30-14 8) 16-31-13 9) 16-31-14 10) 16-32-13 12) 16-33-13 13) 16-34-13 14) 16-35-13 11)16-32-17 15)1645-13 16) 1646-13 17) 1646-14 19) 17-07-17 18)16-53-13 20)17-29-14 21) 17-31-13 22) 17-32-13 23) 17-35-13 24) 17-51-11 25) 17-55-13 27) 17-58-13 28) 21-31-13 29) 22-31-13 31)23-22-17 26) 17-56-13 30) 22-33-13 32) 23-29-13 33)23-31-13 34)23-32-13 35)23-33-13 36)23-34-13 37) 23-36-13 38)24-22-17 39) 24-31-13 182delT P67L R75X L206W 1154insTC 146linsAGAT Q493x V520F 1717-1G>A G551D R560T V562L R709X S1196X L1254X R1283M G85E 2184insA 711+lG>T 3495delA 4279insA SlOR L88S R117C R117H G178R 1717-1G>A Y563N W1098R G1123R 3850- 1G>A E6OX %%deIT 1138insG R34P 2183AA>G 2184delA R1158X 1078delT R1162X 3849G>A Q141W R347P Y917C G2iX 711+3A>G 441delA 3130de115 3659delC 1898+1G>A R709X 2711delT R1158X E92K 3849+lOkbC>T 2118delAACT 4048insCC 296+1 2 T S Q22OX R297Q A1507 2789+5G>A 3120+1G>A W128W 1811+lG>C AF508 E831X R116W AF508 W846X1 3120G>A R785X R553X R553X R553X 621+1G>T G542X G542X Y1182X N1303K AF508 G54W 3041delG 1525-1G>A N1303K G542X G542X G542X 394delTT R709X N1303K 1 1 1 2 1 1 4 2 3 4 2 26 8 1 1 1 1 1 8 1 1 1 1 1 1 1 19 1 2 1 1 1 1 7 1 1 2 1 1 2 1 1 1 1 1 1 1 1 2 1 1 7 4 1 2 1 1 2 1 1 4 Asian 1 2 1Asian 5 4 i Afro-Caribbean 5 1 42 (19%) 1 1 57 (26%) 1 2 1 1 1 2 12 2 11.4 0.4 4.9 16.3 1.1 3.8 1.9 10.6 2.3 1.5 2.3 1.5 2.7 4.5 0.4 0.8 0.8 0.4 0.8 0.4 1 2 1 7 1 1 1Asian 1 1.5 0.8 0.8 NI G NI, M M NI NI. Login to comment
83 G 23-31-13 2 NI,G 24-31-13 1 C R117H 16-30-13 19 16.3 NI.G, C 1717-1G>A 16-07-17 2 M, G 16-30-13 2 M, G R553X 17-55-13 1 M 17-56-13 1 C 17-58-13 1 0.4 NI 1898+1G>A 16-45-13 4 1.5 M E60X 0.2-0.5 16-31-13 7 3.8 NI.M G85E 16-24-13 8 M. G, C 1154insTC 16-07-17 4 NI, M, G A1507 17-07-17 5 4.5 NI R560T 16-07-17 8 N1 3659delC 16-35-13 7 2.3 NI. Login to comment

[hide] Haplotype analysis of 94 cystic fibrosis mutations... Hum Mutat. 1996;8(2):149-59. Morral N, Dork T, Llevadot R, Dziadek V, Mercier B, Ferec C, Costes B, Girodon E, Zielenski J, Tsui LC, Tummler B, Estivill X
Haplotype analysis of 94 cystic fibrosis mutations with seven polymorphic CFTR DNA markers.
Hum Mutat. 1996;8(2):149-59., [PMID:8844213]

Abstract [show]
We have analyzed 416 normal and 467 chromosomes carrying 94 different cystic fibrosis (CF) mutations with polymorphic genetic markers J44, IVS6aGATT, IVS8CA, T854, IVS17BTA, IVS17BCA, and TUB20. The number of mutations found with each haplotype is proportional to its frequency among normal chromosomes, suggesting that there is no preferential haplotype in which mutations arise and thus excluding possible selection for specific haplotypes. While many common mutations in the worldwide CF population showed absence of haplotype variation, indicating their recent origins, some mutations were associated with more than one haplotype. The most common CF mutations, delta F508, G542X, and N1303K, showed the highest number of slippage events at microsatellites, suggesting that they are the most ancient CF mutations. Recurrence was probably the case for 9 CF mutations (R117H, H199Y, R347YH, R347P, L558S, 2184insA, 3272-26A-->G, R1162X, and 3849 + 10kbC-->T). This analysis of 94 CF mutations should facilitate mutation screening and provides useful data for studies on population genetics of CF.

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105 CFTR Haplotypes for Diallelic and Multiallelic DNA Markers for 94 CF Mutations" J44-GATT- 8CA-17BTA- No. of T854-TUB20 17BCA Mutation chromosomes % Normal Laboratory Reference 2-7-1-2 17-47-13 (55.4%) 17-46-13 17-45-13 17-34-13 17-32-13 17-31-14 17-31-13 17-29-14 17-28-13 16-48-13 16-46-14 16-46-13 16-45-13 16-44-13 16-35-13 16-33-13 16-32-13 16-31-14 16-31-13 16-30-13 16-29-13 16-26-13 16-25-13 16-24-13 14-31-13 1-7-2-1 17-7-17 (16.8%) R334W R334W 3860ins31 G1244E R1162X R1162X R1162X G91R MllOlK R347P R334W R117C E92K 3849+lOkbC+T 3293delA 1811+1.6kb A-tG 1811+1.6kb A-tG 2184insA P205S 3659delC G673X 11005R I336K W58S R347P W846X 405+1-A G178R 3905insT R1162X R347H 3100insA E60X 1078delT 4005+1-A K710X 1677delTA H199Y 3601-2AjG 3850-3T+G 3272-26A-tG 3850-1-A 1812-1-A R117H L1059X S492F Y1092X Y569H 3732delA C866Y 711+1G+T 711+1-T G85E 1949del84 2789+5-A H1085R W1282X R1066C 2043delG V456F 2 1 1 1 2 1 6 2 2 1 2 1 1 2 1 1 4 1 1 1 3 2 1 1 1 1 1 1 2 7 1 1 1 1 2 1 1 3 19 3 3 1 1 2 1 1 5 1 1 1 1 3 6 3 5 1 13 2 1 1 - 0.48 0.48 - - - 0.24 - - - 2.65 2.40 1.93 2.65 1.68 2.65 0.72 13.94 13.46 1.93 - 0.72 0.24 3.37 - b b fP fP fP t b,fb.fP h fb t h t h h fP fP b.h b h h b h h h h h fb fb,fP.t fP fP fP9t fP b t fPh b h fb b.fb,h fb*fP b,fP h h t h fb fb,fp,h.t fP fP fb t b.fP,t b,fb,h,t b f b h h fb b,fb.fP,h fP h h Gasparini et al. (1991b) Chilldn et al. (1993a) Devoto et al. (1991) Gasparini et al. (1991b) Dork et al. (1993a) Guillermit et al. (1993) Zielenski et al. (1993) Dean et al. (1990) Dork et al. (1994a) Nunes et al. (1993) Highsmith et al. (1994) Ghanem et al. (1994) Chilldn et al. (1995) Dork et al. (1994a) Dork et al. (1993a) Chilldn et al. (1993b) Kerem et al. (1990) Dork et al. (1994a) Dork et al. (1994a) Cuppenset al. (1993) Fanen et al. (1992) Maggio et al. (personal communication) Audrezet et al. (1993) Vidaud et al. (1990) Dork et al. (1993b) Zielenski et al. (1991a) Chilldn et al. (1994b) Malik et al. (personal communication) Cremonesi et at. Login to comment
134 Several mutations with a relative frequency of between 0.1 and 0.7% (CFGAC, 1994) are each associated with a single haplotype (1078delT, G85E, 621+lG+T), suggesting that they have a recent origin. Login to comment

[hide] Cystic fibrosis mutation detection by hybridizatio... Hum Mutat. 1996;7(3):244-55. Cronin MT, Fucini RV, Kim SM, Masino RS, Wespi RM, Miyada CG
Cystic fibrosis mutation detection by hybridization to light-generated DNA probe arrays.
Hum Mutat. 1996;7(3):244-55., [PMID:8829658]

Abstract [show]
We have combined photochemistry and photolithography with solid-phase DNA synthesis chemistry to form a new technology that makes high density oligonucleotide probe array synthesis possible. Hybridization to these two-dimensional arrays containing hundreds or thousands of oligonucleotide probes provides a powerful DNA sequence analysis tool. Two types of light-generated DNA probe arrays have been used to test for a variety of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. One array, made up of 428 probes, was designed to scan through the length of CFTR exon 11 and identify differences from the wild type reference sequence. The second type of array contained 1480 probes chosen to detect known deletions, insertions, or base substitution mutations. The validity of the probe arrays was established by hybridizing them with fluorescently labeled control oligonucleotide targets. Characterized mutant CFTR genomic DNA samples were then used to further test probe array hybridization specificity. Finally, ten unknown patient samples were genotyped using the CFTR probe array assay. The genotype assignments were identical to those obtained by PCR product restriction fragment analysis. Our results show that light-generated DNA probe arrays are highly effective in analyzing complex mutation and polymorphism patterns in a relatively large gene such as CFTR.

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238 Cystic Fibrosis Mutation-Specific DNA Probe Array" Mutation Exon and column Tested Subarrayhow G85E R117H I148T 621 -+ l(G+T) 711 + 1(G+T) R334W R347H R347P 1078 delT A455E G480C Q493X A1507 F508C AF508 V520F G542X S549R(T-+ G) G551D Q552X R553X A559T R560T 1898 + l(G-,A) 2184 del A 2789 + 5(G+ A) R1066C L1077P Y1092X R1162X 3659 del C 1717-1(& A) 3272 - 26(A+ G) 3 4 4 in 4 in 5 7 7 7 7 9 10 10 10 10 10 10 in 10 11 11 11 11 11 11 11 in 12 13 in 14b in 17a 17b 17b 17b 19 19 * * * * * * * * * * * * * * * * * * * * * * * * * * * * 3849 + lOkb C-, T in 19 9,3 W1282X 20 994 3905insT 20 10.1 * N1303K 21 10,2 * * * "Row and column locations for each of the mutation specific,40 probe sets included in the specialized probe array design. Login to comment

[hide] Methods for screening in cystic fibrosis. Methods Mol Med. 1996;5:99-119. Schwarz M, Malone G
Methods for screening in cystic fibrosis.
Methods Mol Med. 1996;5:99-119., [PMID:21374513]

Abstract [show]
Cystic fibrosis (CF) is the most common lethal autosomal recessive disorder in Whites, with an incidence of approx 1 m 2500 live births and a carrier frequency of approx 1 in 25. Since the discovery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene m 1989 (1-3), molecular genetics laboratories throughout the world have endeavored to identify the mutations present in their population of CF-bearing chromosomes. Since the entire CFTR gene and its intron-exon boundaries have been sequenced, mutation analysis in CF has become relatively simple, although time consuming. Generally, a number of different methods are applied to mutation analysis, but all involve an imtial step of amplification of part of the gene by polymerase chain reaction (PCR) (4), or a derivative of it, such as amplification refractory mutation system (ARMS) (5).

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10 Molecular D/agnoss of GenetIc Diseases Edlted by R Elles Humana Press Inc , Totowa, NJ 99 Table1 RelativeFrequenciesoftheCommonCFMutationsintheUnitedKingdom(S),NorthAmerica, andNorthernandSouthernEurope(7)" NorthNorthernSouthern UKAmericaEuropeEurope MutationExonNo.%No.%No.%No%References AF508 G551D G542X 621+l(G>T) 1717-l(G>A) sR117H =R553x 1898+l(G>A) N1303K R560T AI507 G85E 1154insTC V520F W1282X E60X 3659delC 1078delT 10738775.32690066.114,86670.28400755.033 113023.082061.973561.68370.518 111651.682342.244392.082593.569 intron4910.931541.48970.46370.51IO intron10560.57440.421600.76650.899 4450.46610.586202930.04II 11450.46960.921650.7844068 intron12450.4620.02410.191001412 21450.461301.252090.991792.4613 11410.42240.2340019009 10300.31200.19570.2750.079,14 3210.211601530014140.19II 7190.19n/an/an/an/an/an/a15 10170.17n/an/an/an/an/an/a16 20170.172452.351200.57430.5917 3160.16n/an/an/an/an/an/aMaloneb 19140.14140.133901810.019 790.0910.015302520.318 S549N1180.0850.051800920038 Q493X1070.07n/an/adan/adan/a9 R347P760.06260.25550.26240.3311 3849+10kb(C>T)intron1950.05570.55230.1180.1119 A455E930.03270.26350.17009 %/a,Datanotavadable. Login to comment
42 Screening in Cystic Fibrosis 107 Table 5 CF Mutations Detectable by Restriction Endonuclease (RE) Digestion of PCR Products Mutation PCR primers0 RE RE digestion product sizes, bpbJ Normal Mutant G85E 621+ 1 (G`V 1154insTC R334W R347P G551D R553X R560T S549N 3849+ IOkb CC ` T) W1282X 3i5 and 313 4i5 and 4i3 Hinff MseI 105 + 204 33,35,71, 118, 181 7i5 and 7i3 MspI, RsaI 50,68,74 + 21V 715 and 7i3 MspI 192 + 218 7i5 and 7i3 CfoI 151+ 259 1li5 and 1113 Mb01 425 1115 and lli3 HzncII 186 + 239 lli5 and lli3 Mae11 425 lli5 and lli3 DdeI 13, 174 + 238 i19F and i19R HphI 88 + 349 2Oi5 and 2Oi3 Mnfl 185 + 288 309 33,35,54,71, 118, 127 50,68,76 + 21gc 410 410 182+243 425 215 + 210 13 + 412 88,127 + 222 473 'See Table 2 bThe expected digestion product sizes for both normal and mutant sequences are shown CTheseproducts may be d1stmgmshedby PAGE 1.2.3. Login to comment

[hide] Correlation of sweat chloride concentration with c... J Pediatr. 1995 Nov;127(5):705-10. Wilschanski M, Zielenski J, Markiewicz D, Tsui LC, Corey M, Levison H, Durie PR
Correlation of sweat chloride concentration with classes of the cystic fibrosis transmembrane conductance regulator gene mutations.
J Pediatr. 1995 Nov;127(5):705-10., [PMID:7472820]

Abstract [show]
OBJECTIVE: To compare differences in epithelial chloride conductance according to class of mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. METHODS: We evaluated the relationship between the functional classes of CFTR mutations and chloride conductance using the first diagnostic sweat chloride concentration in a large cystic fibrosis (CF) population. RESULTS: There was no difference in sweat chloride value value between classes of CFTR mutations that produce no protein (class I), fail to reach the apical membrane because of defective processing (class II), or produce protein that fails to respond to cyclic adenosine monophosphate (class III). Those mutations that produce a cyclic adenosine monophosphate-responsive channel with reduced conductance (class IV) were associated with a significantly lower, intermediate sweat chloride value. However, patients with the mutations that cause reduced synthesis or partially defective processing of normal CFTR (class V) had sweat chloride concentrations similar to those in classes I to III. CONCLUSION: Studies of differences in chloride conductance between functional classes of CFTR mutations provide insight into phenotypic expression of the disease.

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43 Defined mutations (each mutation cited in references 8, 23, and 24; numerals in parentheses indicate number of patients): Nonsense mutations-----class I: Frameshift mutations---class I: Splice site mutations-class I: Missense mutations---class HI: Missense mutations---class IV: Partially defective processing---class V: Alternative spficing-----classV: R1162X (3), Y1092X (3), G542X (21), Q552X (2), Q493X (2), w1282x (2), E1104X (1), R553X (6), E585X (l), (all PI) 3659delC (5), 2184delA (4), 4010de14 (1), 556delA (1), 3002delG (1) 3905insT (1), 4016insT (3), 1154insTC (l), 441delA (1), 2184insA (2), 1078delT (1), 4326delTC (3) (all PI) I717-1G--~A (4), 621+lG--*T (10), 711+IG--~T (3), 875+1G-+C (2), 3120+IG-~A (1) (18 PI, 2 PS) G551D (25), N1303K (7), R560T (8), I148T (1), G85E (3), A559T (1), L1077P (2), T1234V (1), (47 PI, 1 PS) R117H (10), R347H (3), R347P (1), D614G (1), S1251N (2), (all PS) P574H (2), A455E (2), (all PS) 3272-26A-+G (4), 3849+10KbC---~T (2), 3120G-+A (1), (all PS) analysis, we further grouped the patients according to the molecular consequences conferred by the CFTR alleles. Login to comment
58 Only one patient in class III (AF508/G85E) had a PS phenotype. Login to comment
82 In one patient homozygous for AF508 and one heterozygous for AF508/G85E, PS was diagnosed at the time of analysis. Login to comment
84 However, the G85E mutation (classified as class III in this study) has been associated with both PS and PI phenotypes.16 Complete sequencing of the coding region of the G85E alleles in the PI and PS patients did not reveal any second site mutations that might modify the molecular consequence. Login to comment

[hide] Screening Young syndrome patients for CFTR mutatio... Am J Respir Crit Care Med. 1995 Oct;152(4 Pt 1):1353-7. Friedman KJ, Teichtahl H, De Kretser DM, Temple-Smith P, Southwick GJ, Silverman LM, Highsmith WE Jr, Boucher RC, Knowles MR
Screening Young syndrome patients for CFTR mutations.
Am J Respir Crit Care Med. 1995 Oct;152(4 Pt 1):1353-7., [PMID:7551394]

Abstract [show]
Young syndrome is characterized by obstructive azoospermia associated with chronic sinobronchial disease of an infectious nature, but normal sweat-gland and pancreatic function as well as normal nasal potential differences. Congenital bilateral absence of the vas deferens (CBAVD) in some patients arises from mutations within the cystic fibrosis (CF) transmembrane regulator (CFTR) gene. Because of some similarities between Young syndrome, CF, and CBAVD, we evaluated 13 patients with Young syndrome, including screening for more than 30 different mutations within the CFTR gene. The mean age of the patients was 43 yr (range, 32 to 50 yr), and all were of northern European extraction. The sweat chloride concentration was normal in all patients (mean = 29 mEq/L; range, 8 to 43 mEq/L). Most had intermittent bronchial and sinus infections, but none was chronically colonized with Staphylococcus aureus or Pseudomonas aeruginosa. The FEV1 was normal or only mildly reduced in most patients (mean = 74%; range, 48 to 100% predicted). Of 26 Young syndrome chromosomes, we identified one with the recognized CF mutation delta F508. The incidence of CFTR mutations (1 in 26) did not differ significantly from the expected carrier frequency in this population. In summary, it is unlikely that the typical Young syndrome patient has a clinical disease associated with CFTR mutation on both alleles.

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78 Of the 13 Young syndrome patients, we identified one (Patient 5) who was het- CBAVD Dl152H D1270N G576A* R75Q* P67L Rl17H 3849 + 10 KB C > T G551S Rl17H Pancreatic Sufficient, Moderate Pulmonary Symptoms, Normal Sweat Chloride Concentrations Pancreatic Sufficient, Moderate Pulmonary Symptoms R347P 2789 + 5 G > A R334W G85E R347H R347L Rl17H G91R A455E S945L Y563N Q1291H R297Q R352Q L1065P 3850-3 T > G F1286S 3849 + 10 KB C > T TABLE 1 CFTR MUTATION SCREENING PANEL Severe M508 G551D R553X N1303K W1282X G542X 1717-1 G > A ~1507 R560T 3659deiC 621 + 1 G > T S549N TABLE 2 CLINICAL FEATURES OF YOUNG SYNDROME PATIENTS Patient Age Sweat CI- FEV, Paranasal Sputum No. Login to comment

[hide] Search for mutations in pancreatic sufficient cyst... Hum Genet. 1995 Sep;96(3):312-8. Brancolini V, Cremonesi L, Belloni E, Pappalardo E, Bordoni R, Seia M, Russo S, Padoan R, Giunta A, Ferrari M
Search for mutations in pancreatic sufficient cystic fibrosis Italian patients: detection of 90% of molecular defects and identification of three novel mutations.
Hum Genet. 1995 Sep;96(3):312-8., [PMID:7544319]

Abstract [show]
A cohort of 31 cystic fibrosis patients showing pancreatic sufficiency and bearing an unidentified mutation on at least one chromosome was analyzed through denaturing gradient gel electrophoresis of the whole coding region of the cystic fibrosis transmembrane conductance regulator gene, including intron-exon boundaries. Three new and 19 previously described mutations were detected. The combination of these with known mutations detected by other methods, allowed the characterization of mutations on 56/62 (90.3%) chromosomes. Among those identified, 17 can be considered responsible for pancreatic sufficiency, since they were found in patients carrying a severe mutation on the other chromosome. Among these presumed mild mutations, eight were detected more than once, R352Q being the most frequent in this sample (4.83%). Intragenic microsatellite analysis revealed that the six chromosomes still bearing unidentified mutations are associated with five different haplotypes. This may indicate that these chromosomes bear different mutations, rarely occurring among cystic fibrosis patients, further underlying the molecular heterogeneity of the genetic defects present in patients having pancreatic sufficiency.

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42 The remaining 19 included R352Q (Cremonesi et al. 1992) (three chromosomes), G85E (Zielenski et al. 1991a), Dl152H (High- Fig. 1 A-C Direct sequencing of PCR products from three cystic fibrosis patients (CF) carrying the W57G (A), E193K (B) and D579G (C) mutations, in parallel with control samples (C) displaying normal sequences (N/N) smith et al., personal communication to the CF Genetic Analysis Consortium), R1066H (Ferec et al. 1992), T338I (Saba et al. 1993), 711 +5G--+A (Gasparini et al., personal communication to the CF Genetic Analysis Consortium), M1V (Cheadle et al. 1993), R334W (Gasparini et al. 1991) (two chromosomes each), 4382delA (Claustres et al. 1993), R1158X (Ronchetto et al. 1992), F1052V (Mercier et al. 1993), G1349D (Beaudet et al. 1991), 1898+3A-+G (Cremonesi et al. 1992), $549N (Cutting et al. 1990), 711+ 3A-->G (Petreska et al. 1994), R347P (Dean et al. 1990), 2789+5G--+A (Highsmith et al. 1990), R1066C (Fanen et al. 1992) and S1251N (K~ilin et al. 1992) (one chromosome each). Login to comment
70 (UN yet unidentified mutation) Patient Genotype after Genotype at the end number preliminary screening of the analysis UN/UN M1V/4382delA 1717-1G---~A/UN 1717-1G---~A/R1066H AF508/UN AF508/D579G UN/UN M1V/UN AF508/UN AF508/UN UN/UN T338I/R1158X UN/UN G85E/71 I+5G---~A UN/UN D1152H/UN AF508/UN AF508/UN AF508/UN AF508/3849+ 10kbC---~T UN/UN 711+3A---~G/UN AF508/UN AF508/F1052V UN/UN R352Q/W57G UN/UN 1898+3A----~G/UN AF508/UN AF508/711+5G--~A G542X/UN G542X/DI 152H AF508/UN AF508/E193K 1717-1G---~A/UN 1717-1G---~A/2789+5A---)G AF508/UN AF508/G1349D AF508/UN AF508/G85E AF508/UN AF508/R347P AF508/UN AF508/R352Q AF508/UN AF508/R352Q AF508/UN AF508/S549N G542X/UN G542X/R1066H AF508/UN AF508/T338I AF508/UN AF508/R334W AF508/UN AF508/R334W AF508/UN AF508/S1251N AF508/UN AF508/R1066C AF508/UN AF508/D579G results) while the remaining three haplotypes had been found in association with other rare mutations, which were excluded by DGGE analysis in these patients (Table 3). Login to comment
85 In total, among the mutations detected in our PS patients, 17 (D579G, E193K, F1052V, 711+5G---~A, G1349D, G85E, R347R R352Q, $549N, 2789+5A---~G, D1152H, R1066H, R334W, T338I, 3849+10kbC---~T, S1251N, R1066C) have been detected in compound heterozygosity with a mutation already classified as severe (AF508, 1717-1G--~A, G542X) and thus can be considered as presumably mild. Login to comment
86 Of these mutations, seven (G85E, EI93K, 711+5G--qA, R347P, R334W, R352Q, T338|) are located in the first transmembrane (I TM) domain, five (2789+ 5A---~G, RI066H, F1052V, D1152H, R1066C) in the second transmembrane (II TM) domain, four in the nucleo- R334W R347P R352Q T338I E193K 711+.E G85E 1 2 3 4 D579G G->A I S 549N 5 6a 6b 7 8 9 10 11 12 13 3849+11 !11 ! Login to comment
91 Conversely, other presumably mild mutations such as R352Q (three chromosomes), and G85E, Dl152H, 711+5G--~A, R1066H, T338I, R334W, D579G (two chromosomes each), are more frequently detected in the PS cohort, accounting in total for 27.4% of chromosomes. Login to comment
93 Screening for only eight presumed mild mutations (R352Q, R1066H, G85E, Dl152H, 711+5G---~A, T338I, R334W and D579G) in addition to the predominant four severe mutations (AF508, G542X, 1717-1G-+A and N1303K), would have allowed the identification of 64.5% of the molecular defects in our patients having PS. Login to comment

[hide] Double mutant alleles: are they rare? Hum Mol Genet. 1995 Jul;4(7):1169-71. Savov A, Angelicheva D, Balassopoulou A, Jordanova A, Noussia-Arvanitakis S, Kalaydjieva L
Double mutant alleles: are they rare?
Hum Mol Genet. 1995 Jul;4(7):1169-71., [PMID:8528204]

Abstract [show]
The presence of two different mutations carried by the same CF allele has been demonstrated in four out of 44 Bulgarian CF patients during a systematic search of the entire coding sequence of the CFTR gene. Two of the double mutant alleles include one nonsense and one missense mutation and although the nonsense mutation can be considered to be the main defect, the amino acid substitutions are good candidates for disease-causing mutations as well. One double mutant carries two missense mutations whose contribution to the CF phenotype is difficult to evaluate. The findings suggest that double mutant alleles may be more common than expected and could account for some of the problems in phenotype-genotype correlations. Such alleles may have important implications for molecular diagnosis and genetic counselling.

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28 (A) SSCP analysis of the 309 bp PCR product of exon 3 of the CFTR gene (primers 3i-5 and 3i-3); 12% 37.5:1 acrylamide:bisacrylamide gel run at 4°C, 1600 V for 24 h. Lanes 1, 2, 8, 9, 10, normal controls; lane 3, G85E/N heterozygote; lanes 4, 5, 7, L88X/N heterozygotes; lane 6, L88X/ L88X homozygote. Login to comment

[hide] CFTR haplotype analysis reveals genetic heterogene... Am J Hum Genet. 1995 Jun;56(6):1359-66. Rave-Harel N, Madgar I, Goshen R, Nissim-Rafinia M, Ziadni A, Rahat A, Chiba O, Kalman YM, Brautbar C, Levinson D, et al.
CFTR haplotype analysis reveals genetic heterogeneity in the etiology of congenital bilateral aplasia of the vas deferens.
Am J Hum Genet. 1995 Jun;56(6):1359-66., [PMID:7539210]

Abstract [show]
Congenital bilateral aplasia of the vas deferens (CBAVD) was suggested to be a mild form of cystic fibrosis (CF). Mutation analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in males with CBAVD revealed that in some males CBAVD is caused by two defective CFTR alleles. The genetic basis of CBAVD in the other males and its association with CF remained unclear. We undertook this study to test the hypothesis of commonality of CBAVD and CF by haplotype analysis, in the CFTR locus, of males suffering from CBAVD and of their families. According to the hypothesis of commonality of CBAVD and CF, two brothers with CBAVD are expected to carry the same two CFTR alleles, while their fertile brothers are expected to carry at least one different allele. Eleven families were studied, of which two families, with unidentified CFTR mutations, did not support this hypothesis. In these families two brothers with CBAVD inherited different CFTR alleles. Their fertile brothers inherited the same CFTR alleles as their brothers with CBAVD. These results provide evidence for genetic heterogeneity in CBAVD. Though in some families CBAVD is associated with two CFTR mutations, we suggest that in others it is caused by other mechanisms, such as mutations at other loci or homozygosity or heterozygosity for partially penetrant CFTR mutations.

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39 1994a); G85E (Zielenski et al. Login to comment
58 Mutation Analysis Fourteen CF-R mutations that were elsewhere identified among the Israeli CF patient population (Kerem et al. 1994) were analyzed: W1282X, AF508, N1303K, G542X, 3849+10Kb C--T, S549R, S549I, W1089X, 4010delTATT, G85E, 1717-1G--A, D1152H, 405+1G--A, and Q359K1T360K. Login to comment
59 Mutation Analysis Fourteen CF-R mutations that were elsewhere identified among the Israeli CF patient population (Kerem et al. 1994) were analyzed: W1282X, AF508, N1303K, G542X, 3849+10Kb C--T, S549R, S549I, W1089X, 4010delTATT, G85E, 1717-1G--A, D1152H, 405+1G--A, and Q359K1T360K. Login to comment

[hide] Structural analysis of CFTR gene in congenital bil... Clin Chem. 1995 Jun;41(6 Pt 1):833-5. Jezequel P, Dorval I, Fergelot P, Chauvel B, Le Treut A, Le Gall JY, Le Lannou D, Blayau M
Structural analysis of CFTR gene in congenital bilateral absence of vas deferens.
Clin Chem. 1995 Jun;41(6 Pt 1):833-5., [PMID:7539342]

Abstract [show]
Congenital bilateral absence of the vas deferens (CBAVD) is found in most males with cystic fibrosis (CF), but this malformation can be observed without any pulmonary or digestive features. We have analyzed 13 exons of the CF gene in a cohort of 25 CBAVD patients. Among the 50 chromosomes studied, 24 mutations were identified: delta F508 (14 cases), R117H (7 cases), R1070W (2 cases), 621 + 1 G --> T (1 case), and A1067V (1 case). Except for delta F508, the most frequent mutations (R117H, R1070W) were not observed in the CF group (109 patients) studied in our laboratory. We discuss the significance of these results.

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46 SF508/ SF508 SF508 / N1303K AF508/ G551D SF508 / 3272-26G--*A SF508 / 1078 delT F508/Y1092X SF508 / Ai507 F5O8 / G542X SF508 / 621+1G-T F508 / 3898 insC SF508 / 574 delA AF508 / G85E SF508 / W1282X N1303K/F311L G551D/F311L R553X I? Login to comment
47 N1303K/? Login to comment

[hide] Analysis of the complete coding region of the CFTR... Hum Genet. 1995 Apr;95(4):397-402. Bonizzato A, Bisceglia L, Marigo C, Nicolis E, Bombieri C, Castellani C, Borgo G, Zelante L, Mastella G, Cabrini G, et al.
Analysis of the complete coding region of the CFTR gene in a cohort of CF patients from north-eastern Italy: identification of 90% of the mutations.
Hum Genet. 1995 Apr;95(4):397-402., [PMID:7535742]

Abstract [show]
A complete coding-region analysis on 225 cystic fibrosis (CF) chromosomes from a cohort that includes all the affected subjects born in two North-Eastern Italian regions over eight years was performed. In a previous study, we identified mutations on 166/225 (73.8%) CF chromosomes after screening for 62 mutations. To characterise the remaining 59 CF chromosomes, we carried out automated direct DNA sequencing (exons 9 and 13), RNA single-strand conformation polymorphism (exons 1-8 and 10-12) and denaturing gradient gel electrophoresis (exons 14a-24) of the 27 exons and flanking regions of the CF transmembrane conductance regulator gene. We identified 22 mutations, four of which are novel, viz. 711 + 5G-->A, R709X, 3132delTG and 2790-2A-->G, and we characterised 90.2% (203/225) of the CF chromosomes. Taking advantage of the homogeneity of the sample, an evaluation of the most important clinical parameters, assessed at the age of 12 years, is presented. We confirm some previously reported genotype-phenotype correlations and we report a new nonsense mutation (R709X) associated with a pancreatic sufficient phenotype.

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35 Table 1 CF mutations identified in this cohort study (225 chromosomes from Veneto and Trentino Alto-Adige) n Number of CF chromosomes, Cum fi cumulative fraction, wnovel mutation identified during this study " Cystic Fibrosis Genetic Analysis Consortium, personal comunication Table 2 DNA sequence variations identified in this cohort study (w Novel sequence variation identified during this study a Cystic Fibrosis Genetic Analysis Consortium, personal comunication Mutation Exon n % Cure fr References AF508 l0 107 47.56 47.56 Kerem et al. 1989 R1162X 19 22 9.78 57.33 Gasparini et al. 1991 2183AA----~G 13 21 9.33 66.67 Bozon et al. 1994 N1303K 21 9 4.00 70.67 Osborne et al. t991 G542X 11 6 2.67 73.33 Kerem et al. 1990 711+5G--~A intron 5 6 2.67 76.00 w 1717 1G--~A intron 10 5 2.22 78.22 Kerem et al. 1990 G85E 3 3 1.33 79.56 Zielenski et al. 1991~' R553X 11 3 1.33 80.89 Cutting et al. 1990 2789+5G--~A intron 14b 3 1.33 82.22 Highsmith* Q552X 11 3 1.33 83.56 Devoto et al. 1991 621+lG---~T intron 4 2 0.89 84.44 Zielenski et al. 1991b W1282X 20 2 0.89 85.33 Vidaud et al. 1990 3132delTG 17a 2 0.89 86.22 w 2790-2A---~G intron 14b 2 0.89 87.11 w 457TAT--)G 4 1 0.44 87.56 Ravnik-Glavac et al. 1993 R347P 7 1 0.44 88.00 Dean et al. 1990 G551D 11 .1 0.44 88.44 Cutting et al. 1990 1717-8G-+A intron 10 1 0.44 88.89 Savov et al. 1994 3849+ 10KbC--)T intron 19 1 0.44 89.33 Highsmith* R709X 13 1 0.44 89.78 w 1898+3A---~G intron 12 1 0.44 90.22 Cremonesi et al. 1992 Identified 203 90.22 Unidentified 22 9.78 Variatioh Exon References 1540 A orG Met or Val at 470 10 Kerem et al. 1990 1898+152 T or A intron 12 Chillon et al. 1991 2134 C or T Arg or Cys at 668 13 Fanen et al. 1992 2694 T or G No change Thr at 854 14a Zielenski et al. 199 lb 2752-22 A or G intron 14a w 3601-65 C or A intron 18 Dork et al. 199l 4029 A or G No change Thr at 1299 21 Fanen et al. 1992 4404 C or T No change Tyr at 1424 24 ShoshanP 711 +5G--+A This mutation was found in the splice donor site flanking the 3' end of exon 5. Login to comment

[hide] Increased incidence of cystic fibrosis gene mutati... Hum Mol Genet. 1995 Apr;4(4):635-9. Pignatti PF, Bombieri C, Marigo C, Benetazzo M, Luisetti M
Increased incidence of cystic fibrosis gene mutations in adults with disseminated bronchiectasis.
Hum Mol Genet. 1995 Apr;4(4):635-9., [PMID:7543317]

Abstract [show]
In order to identify a possible hereditary predisposition to the development of obstructive pulmonary disease of unknown origin, we have looked for the presence of Cystic Fibrosis Transmembrane Regulator (CFTR) gene mutations in unrelated patients with no signs of Cystic Fibrosis (CF). We screened for 70 common mutations, and also for rare mutations by denaturing gradient gel electrophoresis analysis. In this search, different CFTR gene mutations (R75Q, delta F508, R1066C, M1137V and 3667ins4) were found in five out of 16 adult Italian patients with disseminated bronchiectasis, a significant increase over the expected frequency of carriers. Moreover, three rare CFTR gene DNA polymorphisms (G576A, R668C, and 2736 A-->G), not deemed to be the cause of CF, were found in two patients, one of which was a compound heterozygote with R1066C. These results indicate that CFTR gene mutations, and perhaps also DNA polymorphisms, may be involved in the etiopathogenesis of at least some cases of bronchiectasis.

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25 RESULTS Common CF mutations All the study subjects were initially typed with respect to some CFTR mutations known to be present in CF patients in the North East Italian population: AF508, R1162X, 2183AA->G, NI303K, G542X, 711 + 5G->A, 1717-1 G^>A, 1717-8G->A, G85E, R553X, 2789 + 5 G->A, Q552X, 621 + 1 G->T, W1282X, 3132delTG, 2790-2A->G, 457 TAT->G, R347P, G551D, 1898 + 3A->G and 3849 + 10 kbC^T. Login to comment
31 List of CFTR gene mutations and DNA polymorphisms screened Mutations R75Q/X/L, G85E, 394deITT 457TAT->G, R117H 621 + 1G->T 711 + 5G->A L206W 875 + 40 A->G 936 del TA 1001 + 11C->T R334W, R347 P/H/L, 1154insTC A455E, V456F DF5O8 1717-IG->A, 1717-8G->A G542X, G551D, Q552X, R553X P574H 1898 + 3A->G 2183 AA->G, 2184delA, R709X D836Y, 2694 T/G 2752-22 A/G 2789 + 5 G->A, 2790-2 A-»G Q890X 3041-71 G/C 3132delTG 3271 + 18 C-»T, 3272-26 A->G H1054D, G1061R, R1066C/H, A1067T, H1085R, Y1092X, 3320 ins5 D1152H R1162X, 3667ins4, 3737delA, 11234V 3849 + 10 kb C-»T, 3850-1 G-»A SI25IN, S1255P, 3905insT, 3898insC, D127ON, W1282X, R1283M, 4002 A/G 4005 + 1 G-»A N1303 K/H, 4029 A/G D1377H Q1411 X 4404 C/T, 4521 G/A Location e 3 e 4 i 4 i 5 e 6a i 6a e 6b i 6b e 7 e 9 e 10 i 10 e 11 e 12 i 12 e 13 e 14a i 14a i 14b e 15 i 15 e 17a i 17a e 17b e 18 e 19 i 19 e 20 i 20 e2l e 22 e 23 e24 Listing is in order of location along the CFTR gene, e = exon; i = intron. Login to comment

[hide] Two CF patients, one homozygous for the 621 + 1G >... J Med Genet. 1995 Feb;32(2):158. Cheadle JP, Meredith AL, Millar-Jones L, Goodchild MC
Two CF patients, one homozygous for the 621 + 1G > T splice mutation, the other homozygous for the 1898 + 1G > A splice mutation.
J Med Genet. 1995 Feb;32(2):158., [PMID:7539080]

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5 To date, investigators have described homozygotes for G542X,2 R553X,3 G85E,4 S549N,5 Rl17H,6 2184delA,7 R1162X,8 and W128X.9 We report here two patients, one homozygous for 621 + 1G>T, the other homozygous for 1898 + 1G>A. Login to comment
36 A cystic fibrosis patient who is homozygous for the G85E mutation has very mild disease,JIMedGenet 199 1;28:875-7. Login to comment

[hide] Identification of seven rather infrequent and one ... Hum Mol Genet. 1994 Dec;3(12):2249-50. Teng H, Cuppens H, De Boeck C, Cassiman JJ
Identification of seven rather infrequent and one novel CFTR mutation in the Belgian population.
Hum Mol Genet. 1994 Dec;3(12):2249-50., [PMID:7881429]

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6 Seven of these were described previously: R117H (2), G551D (3), R553X (3), 394delTT (4), L206W (4), G85E (5) and D1152H (6). Login to comment
11 The G85E mutation was found in a mutant CFTR gene of Turkish ancestry. Login to comment

[hide] Association of pancreatic adenocarcinoma, mild lun... Clin Chem. 1994 Oct;40(10):1972-4. Tsongalis GJ, Faber G, Dalldorf FG, Friedman KJ, Silverman LM, Yankaskas JR
Association of pancreatic adenocarcinoma, mild lung disease, and delta F508 mutation in a cystic fibrosis patient.
Clin Chem. 1994 Oct;40(10):1972-4., [PMID:7522998]

Abstract [show]
A case of adenocarcinoma of the pancreas and mild lung disease in a 39-year-old man homozygous for the delta F508 cystic fibrosis mutation is presented. Cystic fibrosis is the most common lethal genetic disease in Caucasians, and is most commonly associated with severe obstructive lung disease. To our knowledge, this is only the fifth case of adenocarcinoma of the pancreas in a CF patient to be reported and the first case for which molecular data are available. The rare incidence of this type of malignancy in the general population suggests a possible association of CF with this malignant disease.

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44 CorrelatIon of phenotype and genotype of CFTR mutations Key phenotypic Lung disease SweatC1 Exocnne pancreas function Vasdeferens Associated CFTR mutations Pancreatic InsuffIcIent Pancreatic sufficient Normalsweat C1 Severe Less severe Relatively mild Elevated Elevated Normal Insufficient Sufficient Sufficient Absent Absent Absent SF508, G542X, R553X, G5510, Ni 303K, Wi 282X, RI 17H, and others 2789 + 5G>A, R117H, R334W, R347P, A455E, P574H, S945L, G85E, and others G551S, R117H, 3849 + 10kb C>T, and others Congenitalabsence of the vas deferens None Normal or elevated Sufficient Absent F508C, Ri 17H, Di D1152H, and others FIg. 2. Login to comment

[hide] Analysis of mutations and alternative splicing pat... Hum Mol Genet. 1994 Jul;3(7):1141-6. Hull J, Shackleton S, Harris A
Analysis of mutations and alternative splicing patterns in the CFTR gene using mRNA derived from nasal epithelial cells.
Hum Mol Genet. 1994 Jul;3(7):1141-6., [PMID:7526925]

Abstract [show]
Ten to fifteen percent of CF chromosomes carry mutations which are not detected by routine screening of the CFTR gene for known mutations. Many techniques have been used to screen the CFTR gene for these remaining mutations. Most of the methods use genomic DNA, and since the CFTR gene contains 27 exons, are necessarily labour intensive. We have screened the entire coding region of CFTR, by chemical cleavage of 7 overlapping segments of amplified cDNA. Using this method we have identified 4 sequence changes which had not been detected by screening genomic DNA, and successfully detected 10 out of 13 known mutations. In addition, we have identified 8 alternatively spliced forms of CFTR mRNA, 4 of which have not been described previously. These include transcripts lacking a) exon 3, b) exons 2 + 3, c) exons 9 + 12, and d) the final 357 bp of exon 15 as a result of use of the cryptic splice donor site CA2863/GTTCGT).

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33 Of the 13 known sequence changes, 9 (G85E (8), E92K (10), Q220X (11), AF508 (1), G542X (12), G551D (13), 3659delC (12), W1282X (14), 4271delC (11)) were readily identified by •To whom correspondence should be addressed A-6 \ B c ~~i r D t 1 F 1 2 3 4 5 Ga 6b 7 8 9 1 0 1 1 1 2 13 14i 14bl 516 17a 17bl S 19 202122 23 24 MSD 1 NBF 1 R domain MSD 2 NBF 2 Figure 1. Login to comment

[hide] Mutation analysis in 600 French cystic fibrosis pa... J Med Genet. 1994 Jul;31(7):541-4. Chevalier-Porst F, Bonardot AM, Gilly R, Chazalette JP, Mathieu M, Bozon D
Mutation analysis in 600 French cystic fibrosis patients.
J Med Genet. 1994 Jul;31(7):541-4., [PMID:7525963]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) gene of 600 unrelated cystic fibrosis (CF) patients living in France (excluding Brittany) was screened for 105 different mutations. This analysis resulted in the identification of 86% of the CF alleles and complete genotyping of 76% of the patients. The most frequent mutations in this population after delta F508 (69% of the CF chromosomes) are G542X (3.3%), N1303K (1.8%), W1282X (1.5%), 1717-1G-->A (1.3%), 2184delA + 2183 A-->G (0.9%), and R553X (0.8%).

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21 Among the 104 other CFTR mutations tested on the 373 non-AF508 CF chromosomes, none of the following 58 mutations were found: G91R, 435 insA, 444delA, D11OH, 556delA, 557delT, R297Q, 1154insTC, R347L, R352Q, Q359K/T360K, 1221delCT, G480C, Q493R, V520F, C524X, 1706dell7, S549R (A-C), S549N, S549I, G551S, 1784delG, Q552X, L558S, A559T, R560T, R560K, Y563N, P574H, 2307insA, 2522insC, 2556insAT, E827X, Q890X, Y913C, 2991de132 (Dork et al, personal communication), L967S, 3320ins5, 3359delCT, H1085R, R1158X, 3662delA, 3667del4, 3667ins4, 3732delA, 3737delA, W1204X, 3750delAG, I 1234V, Q1238X, 3850- 3T-+G, 3860ins31, S1255X, 3898insC, D1270N, R1283M, F1286S, 4005 + I G-A. Forty-six other mutations were found on at Distribution of CFTR mutations found in our sample ofpopulation (1200 CF chromosomes) Mutations tested No of CF chromosomes Haplotypes Method with the mutation XV2C-KM19 (% of total CF alleles) Exon 3: G85E 4 (033) 3C HinfI/ASO394delTT 2 2B PAGEExon 4: R117H 1 B ASOY122X 2 2C MseI/sequenceI148T 1 B ASO621+IG-J* 1 B MseIIASOExon 5: 711+1G--T 8(07) 8A ASOExon 7: AF311 1 C PAGE/sequencelO78delT 5 (0-42) 5C PAGE/ASOR334W 5 (0-42) 2A,2C,ID MspIlASOR347P 5 (042) 5A CfoI/NcoIR347H 1 Cfol/sequenceExon 9: A455E 1 B ASOExon 10: S492F I C DdeI/sequenceQ493X 1 D ASOl609deICA 1 C PAGE/Ddel/sequenceA1507 3 (025) 3D PAGE/ASOAF508 827 (69) 794B,30D,2C,IA PAGEl677delTA 1 A PAGE/sequenceExon I11: 1717-IG--.A 16(1-3) 14B Modified primers + AvaIIG542X 40 (3-3) 29B,5D,2A Modified primers + BstNiS549R(T--*G) 2 2B ASOG551D 3 (025) 3B HincII/Sau3AR553X 10(0-8) 6A,1B,2C,ID Hincll/sequenceExon 12: 1898+IG--A 1 C ASO1898+ IG-C 2 IC ASOExon 13: l9l8deIGC 1 A PAGE/sequence1949de184 I C PAGE/sequenceG628R(G-+A) 2 2A Sequence2118de14 I c PAGE/sequence2143de1T 1 B PAGE/modified primers2184de1A+2183A--*G 11 (0-9) lIB PAGE/ASO2184de1A 1 ASOK710X 3 (025) IC XmnI2372de18 1 B PAGE/sequenceExon 15: S945L 1 C TaqlExon 17b:L1065P I MnlIL1077P 1 A ASOY1092X 3 (025) 2C,IA Rsal/ASOExon 19: RI1162X 6 (0-5) 5C,IA DdeI/ASO3659delC 3 (025) 3C ASOExon 20: G1244E 2 2A MboIIS1251N 2 2C RsaI3905insT 4 (0-33) 4C PAGE/ASOW1282X 18 (105) 15B,1D MnlI/ASOR1283K 1 C Mnll/sequenceExon 21: N1303K 22 (1-8) 18B,lA,ID Modified primers+BstNI 47 mutations 1031 (85 9) least one CF chromosome (table): 21 of them are very rare as they were found on only one CF chromosome in our population. Login to comment

[hide] Sensitivity of single-strand conformation polymorp... Hum Mol Genet. 1994 May;3(5):801-7. Ravnik-Glavac M, Glavac D, Dean M
Sensitivity of single-strand conformation polymorphism and heteroduplex method for mutation detection in the cystic fibrosis gene.
Hum Mol Genet. 1994 May;3(5):801-7., [PMID:7521710]

Abstract [show]
The gene responsible for cystic fibrosis (CF) contains 27 coding exons and more than 300 independent mutations have been identified. An efficient and optimized strategy is required to identify additional mutations and/or to screen patient samples for the presence of known mutations. We have tested several different conditions for performing single-stranded conformation polymorphism (SSCP) analysis in order to determine the efficiency of the method and to identify the optimum conditions for mutation detection. Each exon and corresponding exon boundaries were amplified. A panel of 134 known CF mutations were used to test the efficiency of detection of mutations. The SSCP conditions were varied by altering the percentage and cross-linking of the acrylamide, employing MDE (an acrylamide substitute), and by adding sucrose and glycerol. The presence of heteroduplexes could be detected on most gels and in some cases contributed to the ability to distinguish certain mutations. Each analysis condition detected 75-98% of the mutations, and all of the mutations could be detected by at least one condition. Therefore, an optimized SSCP analysis can be used to efficiently screen for mutations in a large gene.

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120 Exon 1: S4X (24), 186-13C-G (F£rec et al., pers. comm.); Exon 2: G27X (Shacldeton and Harris, pers. comm.), Q30X (Chilldn aal., pers. comm.), R31L (Zielenski et al., pers. comm.), Q39X (25); Exon 3: 300delA (Malone et al., pers. comm.), W57G (Ferrari et al., pers. comm.), W57X (26), E60X (Malone et al., pers. comm.), R74W (Claustres et al., pers. comm.), R75Q (27), G85E (28), 394delTT (Claustres et al., pers. comm.), L88X (Maceketal., pers. comm.), L88S (Malone et al., pers. comm.), 405 + 1G-A (Dork and Tummler, pers. comm.); Exon 4: E92K (Chillon et al., pers. comm.), E92X (D6rk a al., pers. comm.), P99L (Schwartz and Holmberg, pers. comm.), 441delA (Zielenski et al., pers. comm.), 444delA (29), 457TAT-C- (F£rec et al., pers. comm., (21), Dl 10H (14), Rl 17C (D6rk et al., pers. comm.), Rl 17H (14), A120T (Chillon et al., pers. comm.), 541delC (30), 556delA (28), I148T (Rininsland et al., pers. comm.), Q151X (Shacldeton et al., pers. comm.), 621 + 1C-T (28), 622-2A-C (31); Exon5:G178R (28), 681delC (Zielenski a al., pers. comm.), 711 + 1G-T (28); Exon 6a: H199Y (Dork and Tummler, pers. comm.), H199Q (Dean etal., pers. comm.), L206W (Claustres et al., pers. comm.), Q220X (Shacldeton and Harris, pers. comm., Schwartz and Holmberg, pers. comm.), 852del22 (32); Exon 6b: 977insA (33); Exon7:F311L(34). Login to comment

[hide] Analysis of the CFTR gene confirms the high geneti... Hum Genet. 1994 Apr;93(4):447-51. Chillon M, Casals T, Gimenez J, Ramos MD, Palacio A, Morral N, Estivill X, Nunes V
Analysis of the CFTR gene confirms the high genetic heterogeneity of the Spanish population: 43 mutations account for only 78% of CF chromosomes.
Hum Genet. 1994 Apr;93(4):447-51., [PMID:7513293]

Abstract [show]
We have analysed 972 unrelated Spanish cystic fibrosis patients for 70 known mutations. Analysis was performed on exons 1, 2, 3, 4, 5, 6a, 6b, 7, 10, 11, 12, 13, 14a, 14b, 15, 16, 17b, 18, 19, 20 and 21 of the cystic fibrosis transmembrane regulator gene using single strand conformation polymorphism analysis and denaturing gradient gel electrophoresis. The major mutation delta F508 accounts for 50.6% of CF chromosomes, whereas another 42 mutations account for 27.6% of CF chromosomes, with 21.8% of Spanish CF chromosomes remaining uncharacterized. At present, we have identified 36 mutations that have frequency of less than 1% and that are spread over 15 different exons. This indicates that, in the Spanish population, with the exception of delta F508 (50.6%) and G542X (8%), the mutations are not concentrated in a few exons of the gene nor are there any predominating mutations. This high degree of genetic heterogeneity is mainly a result of the different ethnic groups that have populated Spain and of the maintenance of separated population sets (Basques, Arab-Andalusian, Mediterranean, Canarian and Gallician). The high proportion of CF chromosomes still unidentified (21.8%) together with association analysis with intragenic markers suggest that at least 100 different mutations causing CF are present in our population.

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40 Frequencies of CF mutations in the Spanish population Mutation Exon/intron N~chro- % mosomes Known (43) 760 78.18 AF508 Exon 10 492 50.61 G542X Exon 11 78 8.02 N1303K Exon 21 23 2.36 3601-111 G---~C Intron 18 19 1.95 R1162X Exon 19 18 1.85 711+1 G---~T Exon 5 12 1.23 R334W Exon 7 11 1.13 1609 del CA Exon 10 9 0.92 G85E Exon 3 8 0.82 2789+5 G---~A Intron 14b 7 0.72 2869 ins G Exon 15 7 0.72 R1066C Exon 17b 7 0.72 W1282X Exon 20 6 0.62 AI507 Exon 10 5 0.51 3272-26 A---~G Intron 17a 5 0.51 G551D Exon 11 4 0.41 1812-1 G---~A Intron 11 4 0.41 2184 de! Login to comment

[hide] A new missense mutation (G27E) in exon 2 of the CF... Hum Mol Genet. 1994 Feb;3(2):365-6. Bienvenu T, Cazeneuve C, Beldjord C, Dusser D, Kaplan JC, Hubert D
A new missense mutation (G27E) in exon 2 of the CFTR gene in a mildly affected cystic fibrosis patient.
Hum Mol Genet. 1994 Feb;3(2):365-6., [PMID:7516232]

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35 This observation is consistent with the mild clinical phenotype already observed in compound heterozygote patients with another missense mutation in exon 3 (G91R) (13) or in homozygous patient for the G85E mutation (14). Login to comment

[hide] Nasal epithelial ion transport and genetic analysi... Hum Mol Genet. 1993 Oct;2(10):1605-9. Osborne LR, Lynch M, Middleton PG, Alton EW, Geddes DM, Pryor JP, Hodson ME, Santis GK
Nasal epithelial ion transport and genetic analysis of infertile men with congenital bilateral absence of the vas deferens.
Hum Mol Genet. 1993 Oct;2(10):1605-9., [PMID:7505692]

Abstract [show]
It has been suggested that congenital bilateral absence of the vas deferens (CBAVD), an important cause of male infertility, is a variant of cystic fibrosis (CF). This study describes a defect in chloride conductance across the nasal epithelium of subjects with CBAVD which is dissimilar to that found in patients with CF. It also demonstrates normal sodium transport across the nasal epithelium in these men, in contrast to patients with CF who exhibit increased sodium absorption. The increased frequency of CFTR mutations in these men implicates the CFTR gene in the pathogenesis of this disorder. Genetic analysis of men with CBAVD who were heterozygous for a known CFTR mutation failed to identify a second mutation within any of the exons or introns of the CFTR gene. These results demonstrate that most men presenting with CBAVD are not compound heterozygotes for mutations within the CFTR gene and can be distinguished from individuals with atypical or asymptomatic CF on the basis of the bioelectric properties of their nasal epithelium. We postulate that mutations in the promoter region or at other regulatory sites of the CFTR gene may be responsible for the CBAVD phenotype in a proportion of cases.

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25 Nasal potential difference, sweat sodium concentration and CF genotype in subjects with CBAVD, CF patients and controls CF genotype Control cohort CBAVD cohort CF cohort N/N« AF5O8/R347H SM9N/R1070Q AF508/Other R553X/N*> Unknown/Unknowtf AF 5«/AF5O8 AFjQj/Other AF508/R347P AF50e/G542X AFjQg/RllTH AF5O8/G85E AF5Q8/R553X AFJO8/G551D AF5O8A^520F AFJO8/S549N AF^/DIjQ, N1303K/Other G542X/R117H AFJ08/R334W Other/Other Subjects 50 1 1 1 6 1 16 25 18 3 2 1 1 1 2 1 1 1 3 1 1 3 Mean (range) sweat Na+ concentration (mmol/1) 50 (27-78) 88 N/A 94 57 (47-70) 36 49 (32-76) 126(80-162) 99 (80-128) 108 (99-115) 128 (118-137) 95 107 130 122 (116-128) 90 80 118 96 (92-99) 84 123 108(83-130) Mean (range) nasal potential difference (-mV) 21 (8-30) 31 N/A 34 23 (13-29) -15 20 (-13-28) 45 (32-58) 41 (33-61) 50 (36-77) 43 (33-52) 51 42 39 60 (50-71) 32 37 41 38 (36-40) 32 34 44(31-57) N = non-CF chromosome Other = uncharacterised CF chromosome N/A not available • with CF carrier frequency of 1/20-1/25, it is likely that 2 or 3 of these individuals will be carriers. Login to comment

[hide] Direct sequencing of the complete CFTR gene: the m... Hum Mol Genet. 1993 Oct;2(10):1551-6. Cheadle JP, Goodchild MC, Meredith AL
Direct sequencing of the complete CFTR gene: the molecular characterisation of 99.5% of CF chromosomes in Wales.
Hum Mol Genet. 1993 Oct;2(10):1551-6., [PMID:7505689]

Abstract [show]
We have performed an extensive mutation analysis on 184 CF families in Wales. In our previous study, mutations on 329/369 CF chromosomes were identified after screening for delta F508 and sixteen other mutations. To identify the mutations on the remaining 40 uncharacterized CF chromosomes, we have carried out direct DNA sequencing over the complete coding region, intron splice sites, and part of the promoter region of the CFTR gene. During this study we have designed a set of internal sequencing primers which allow clear sequencing through the aforementioned regions. Sequence analysis revealed 15 further mutations (4 of which are novel), and 10 previously described polymorphisms. In total, we have identified 29 mutations, the distribution of which provides further insight into the functional domains of the CFTR protein. We have characterised 99.5% of the CF chromosomes (365/367, one sample degraded). In order to ascertain accurate frequency data for the Welsh population, CF families with at least 3 'Welsh' grandparents were strictly regarded as 'Welsh'. Of these 91 families, delta F508 accounts for 71.6%, 621 + 1G-->T 6.6% and 1898 + 1G-->A 5.5%. The implications for CF population screening in Wales are discussed.

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64 17b 18 19 20 21 22 23 24 M1V E60X O220X | R117H G85E 621*1G>T 977msA AF508 I AI607 1898*1G>A G642X 2184delA 1078delT I I 8549N 2184insA I I 1154insTC S549R G651D I R553X I R560T I 1717-1G>A W846X1 3272-26A>G L1077P R12B3U 3669delC 4 016in aT I N1303K 3849*10kbC>T Figure 2. Login to comment

[hide] Analysis of the 27 exons and flanking regions of t... Hum Mol Genet. 1993 Aug;2(8):1209-13. Claustres M, Laussel M, Desgeorges M, Giansily M, Culard JF, Razakatsara G, Demaille J
Analysis of the 27 exons and flanking regions of the cystic fibrosis gene: 40 different mutations account for 91.2% of the mutant alleles in southern France.
Hum Mol Genet. 1993 Aug;2(8):1209-13., [PMID:7691344]

Abstract [show]
In order to characterize the non-delta F508 mutations that account for 36% of cystic fibrosis (CF) chromosomes in Southern France in a sample of 137 patients, we have systematically screened the entire coding region and adjacent sequences of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by the single strand conformation polymorphism (SSCP) technique followed by direct sequencing of the mutant DNAs. We identified 13 novel mutations (9 reported in this paper) and 4 novel rare nucleotide sequence variations. Forty different mutations including delta F508, located in 15 exons, account for only 91.2% of mutants in a population originating from Southern France, in contrast with a recent report on the Celtic population of Brittany demonstrating that 90% of mutations can be detected with only three mutations. We present a very large spectrum of different CF mutations identified in a small geographical area.

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26 Mutations identified in a Southern french population mutation AF5O8 M1K 300delA P67L R74W G85E 394detTT 406-6 (T-C) Y122X I148T 621 + 1G-T 62/+2T-G L206W 1078deIT R334W R347H R347P AI507 1717-1G-A G542X R553X S549N G551D E585X 2184delA K710X R792X S945L Y1092X 3272-26A-G R1158X R1162X 3737delA 3659delC 11234V D1270N W1282X N13O3H N13O3K 4382delA Exon 10 1 3 3 3 3 3 intron 3 4 4 intron 4 intron 4 6a 7 7 7 7 10 intron 10 11 11 11 11 , 12 13 13 13 15 17b intron 17a 19 19 19 19 19 20 20 21 21 24 Amino acid change 3 bp deletion start-Lys at 1 frameshift Pro-Leu at67 Arg-Trp at 74 Gly-Glu at 85 frameshift splice mutation? Login to comment

[hide] A novel mutation in exon 3 of the CFTR gene. Hum Genet. 1993 Apr;91(3):233-5. Guillermit H, Jehanne M, Quere I, Audrezet MP, Mercier B, Ferec C
A novel mutation in exon 3 of the CFTR gene.
Hum Genet. 1993 Apr;91(3):233-5., [PMID:7682984]

Abstract [show]
We have screened the 27 exons of the cystic fibrosis transmembrane conductance regulator gene in 87 non-delta F508 chromosomes of Breton origin using the combined techniques of denaturing gradient gel electrophoresis and direct sequencing. By this process, we have detected a new missense mutation, G91R, which results in an arginine for glycine at codon 91. Three affected patients with a delta F508/G91R genotype are pancreatic sufficient. Such observations could facilitate a better understanding of the functional importance of different regions of the encoded product and of the pathogenesis of the disease.

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62 The missense mutation G85E (Zielenski et al. 1991b), which results in a glutamic acid for a glycine at position 85 of the first transmembrane domain produces very mild symptoms of the disease, despite the radical change in amino acid sequence (Chalkley and Harris 1991). Login to comment

[hide] Absence of cystic fibrosis mutations in a large As... J Med Genet. 1993 Feb;30(2):164-6. Curtis A, Richardson RJ, Boohene J, Jackson A, Nelson R, Bhattacharya SS
Absence of cystic fibrosis mutations in a large Asian population sample and occurrence of a homozygous S549N mutation in an inbred Pakistani family.
J Med Genet. 1993 Feb;30(2):164-6., [PMID:7680378]

Abstract [show]
The occurrence of cystic fibrosis is very rare in the Asian population. Carriers of the mutations most commonly found in Caucasians were not detected in a large Asian population sample of almost 900 chromosomes. However, an affected Pakistani child born to consanguineous parents was further investigated and shown to be homozygous for the mutation S549N (G-->A). Molecular and clinical data are presented which may improve our understanding of the phenotypic effects of the S549N mutation in the CFTR gene.

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19 Such homozygotes are likely to be extremely rare, but some useful cases have been identified, such as aG85E homozygotel' and an R553X" homozygote, in addition to the S549N case described here. Login to comment

[hide] Cystic fibrosis genotypes and views on screening a... Am J Hum Genet. 1992 Nov;51(5):943-50. Scriver CR, Fujiwara TM
Cystic fibrosis genotypes and views on screening are both heterogeneous and population related.
Am J Hum Genet. 1992 Nov;51(5):943-50., [PMID:1384327]

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78 Ten alleles at 0.27% each .................... Total ............................................ French Canadians from northeastern Quebec (Rozen et al. 1992 [181]): 48 30 12 4 3 97 60 22 4 0 4 92a 82 4 1 1 1 2 3 98a AFS08 ......... ..................... 58 621+1G-T .............................. 23 A455E .......... .................... 8 G85E ......... ..................... 1 711+1 G-T ............................... 1 G542X .......... .................... 1 Y1092X .............................. 1 N1303K ........... ................... 1 Total .......... .................... 93a a Because of rounding, individual values do not sum to the total. Login to comment

[hide] The spectrum of cystic fibrosis mutations. Trends Genet. 1992 Nov;8(11):392-8. Tsui LC
The spectrum of cystic fibrosis mutations.
Trends Genet. 1992 Nov;8(11):392-8., [PMID:1279852]

Abstract [show]
Although the major mutation causing cystic fibrosis accounts for almost 70% of mutant chromosomes screened, almost 300 sequence alterations have been identified in the gene during the past two and a half years. At least 230 of these mutations are probably associated with disease. This rapid accumulation of data is in part due to the highly coordinated effort by members of the Cystic Fibrosis Genetic Analysis Consortium. The information is not only essential to genetic diagnosis, but also will aid in understanding the structure and function of the protein, and possibly in correlating genotype with phenotype.

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64 Frequent cystic fibrosis mutations Name Relative freqeenc~ Mutation Con~,~'~luence Ref. Z~508 67.2 G542X 3.4 G551D 2.4 W1282X 2.1 3905insT 2.1 N1303K 1.8 3849+10kbC-+T 1.4 1717-1G-+A 1078delT 2789+5G--+A Deletion of 3 bp between nt 1652 and t655 in exon 10 G-+T at nt 1756 in exon 11 G-+A at nt 1784 in exon 1I G-+A at nt 3978 in exon 20 Insertion of T after nt 3905 in exon 20 C-+G at nt 4041 in exon 21 C-->T in a 6.2 kb EcoRI fragment 10 kb from 5' junction of intron 19 3849+4A-+G 1.0 7tt÷IG--+T 0.9 Rl162X 0.9 1898+lG-+A 0.9 Rll7H 0.8 3659delc 0.8 G85E 0.7 2184delA 0.7 AI5W 0.5 R347P 0.5 R~ 0.4 1,3 C-+T at nt 1"789in exon 11 1.3 G-+T at nt 1 from 5' junction of intron 4 1.1 G--+A at nt 1 from 3' junction of intron 10 1.1 Deletion of T at nt 1078 in exon 7 1.1 G-cA at 5 nt from 5' end of intron 14b A-->G at 4 nt from 5' end of intron 19 G-+T at nt 1 from 5' junction of intron 5 C-+T at nt 3616 in exon 19 G-+A at nt 1 from 5' junction of intron 12 G--)A at nt 482 in exon Deletion of C at nt 3659 in exon 19 G-+A at nt 386 in exon 3 A-->G at nt 2183 and deletion of A at nt 2184 in exon 13 Deletion of 3 bp between nt 1648 and 1653 in exon 10 G-+C at nt 1172 in exon 7 G-~C at nt 1811 in exon 11 A455E 0.4 R334W 0.4 Y122X 0.3 S549R(T-+G) 0.3 Q493X 0.3 V520F 0.2 S549N 0.2 C-+A at nt 1496 in exon 9 C-+T at nt 1132 in exon 7 T-cA at nt ~i98 in exon 4 T--+G at nt 1779 in exon 11 C-+T at nt 1609 in exon 10 G-+T at nt 1690 in exon 10 G-->A at nt I778 in exon !1 Deletion of Phe at codon 508 Gly-+Stop at codon 542 12 Gly-~Asp at codon 551 10 l"rp-->Stop at codon t282 35 Frameshift -~ Asn-+Lys at codon 1303 36 Aberrant splicing -~ Arg~Stop ~ codon 553 Splice mutation 10 37 Splice mutation 12 Frameshift 38 Splice mutation _c Splice mutation? Login to comment

[hide] Milestones in cystic fibrosis. Br Med Bull. 1992 Oct;48(4):717-37. Super M
Milestones in cystic fibrosis.
Br Med Bull. 1992 Oct;48(4):717-37., [PMID:1281032]

Abstract [show]
The study of cystic fibrosis (CF) provides a fascinating insight into developments in medicine in the 20th century. Milestones include the first clear clinical descriptions in the 1930s, discovery of a sweat electrolyte abnormality, establishing the autosomal recessive mode of inheritance and improvements in treatment. Microdissection experiments on sweat glands allowed the main defect to be delineated as one of chloride transport. Location of the gene to chromosome 7 made prenatal diagnosis feasible and carrier detection in siblings. The CF gene--its product being the cystic fibrosis transmembrane conductance regulator (CFTR), and its major mutation Delta F508 was discovered in 1989. World-wide collaboration has resulted in discovery of more than 150 further mutations. Incorporation of CFTR into non-chloride transporting insect cells by conferring chloride transport, proved it a chloride channel. CFTR incorporated into adenovirus results in correction of the chloride transport defect in airway cells, bringing gene therapy closer.

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160 Thus a protein conformational change in CFTR resulting in a signif- FIBROSIS Table 4 CF Mutations encountered in United Kingdom Mutation Delta F508 G551D R553 G542X R56OT N1303K DI507 R117H 621+1G-T G85E W1282X E60X R75Q V520F 1717-1 G-A CF chromosomes screened 1 Mutations encountered 1062 199 (non Delta F508) 199 199 199 199 199 199 199 199 199 30 15 199 199 CF chromosomes with mutation in North-West England 863 37 8 11 6 6 4 5 10 4 2 2 1 3 3 Percentage 81.2 3.48 0 75 1.03 0.58 0 58 038 0.47 0.98 0.38 0 19 ? Login to comment

[hide] Mutation analysis of 184 cystic fibrosis families ... J Med Genet. 1992 Sep;29(9):642-6. Cheadle J, Myring J, al-Jader L, Meredith L
Mutation analysis of 184 cystic fibrosis families in Wales.
J Med Genet. 1992 Sep;29(9):642-6., [PMID:1357180]

Abstract [show]
We describe a molecular analysis of 184 cystic fibrosis (CF) families in Wales. To determine accurate frequency data for the CF mutations in the Welsh population, families with at least three Welsh grandparents were strictly regarded as Welsh. Of these 74 families, we have identified approximately 90% of mutations causing CF, with delta F508 accounting for 71.8% and 621 + 1G greater than T 6.7%. We observed a significant difference between the Welsh and Scottish frequencies of 621 + 1G greater than T. To allow the rapid and efficient screening for the more common mutations we modified a multiplex used by Watson et al enabling the detection of delta F508, G551D, and R553X simultaneously with 621 + 1G greater than T. In parallel to this system we ran the Cellmark Diagnostics ARMS multiplex kit, which detects delta F508, 621 + 1G greater than T, G551D, and G542X. RFLP analysis of the 184 families shows that the delta F508 chromosomes are almost exclusively found on the B haplotype (XV2c 1, KM19 2); the other CF mutations have more heterogeneous backgrounds. Strong haplotype correlations exist between the markers XV2c, KM19, D9, and G2 and the other CF mutations. Haplotype data suggest that there are at least seven mutations that remain to be identified in these families.

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61 Welsh Mixed Undefined Total Mutation No % No % No % No % AF508 107/149 71-8 92/126 73 0 69/94 73 4 268/369 72-6 621 + 1G>T 10/42* 6-7 5/34* 4-0 4/25* 4-3 19/101* 51 G551D 2/42* 1-3 6/34* 4-8 3/25* 3-2 11/101* 3 0 G542X 4/42* 2-7 4/34* 3-2 1/25* 1.1 9/101* 2-4 G85E 0/41* 0-0 2/34* 1 6 3/24* 3*4 5/99* 1-4 R553X 2/42* 1-3 2/34* 16 0/25* 00 4/101* 1-1 R1283M 3/42* 2.0 0/34* 0.0 0/25* 0.0 3/101* 0-8 N1303K 1/42* 0 7 1/34* 0-8 0/24* 0.0 2/100* 0-6 AI507 2/149 1-3 0/126 0.0 0/94 0.0 2/369 0-5 R117H 1/42* 0 7 1/34* 0-8 0/25* 0.0 2/101* 0-5 1717- 1G>A 2/42* 1-3 0/34* 0 0 0/25* 0 0 2/101* 0-5 R560T 0/42* 00 0/34* 00 1/25* 1 1 1/101* 03 1154InsTC 0/40* 0 0 1/33* 0 9 0/24* 0.0 1/97* 0-3 V520F 0/42* 0 0 0/34* 0 0 0/25* 0.0 0/101* 0 0 W1282X 0/42* 0 0 0/34* 0.0 0/25* 0.0 0/101* 0 0 R347P 0/42* 0 0 0/34* 0 0 0/24* 0.0 0/100* 0 0 Q493X 0/42* 0 0 0/34* 0 0 0/24* 0 0 0/100* 00 Total (%) 89-8 90 7 86-5 891 * Non-AF508 chromosomes. Login to comment
80 G85E has been categorised as a severe allele by Zielenski et al.6 However, more recently, Chalkley and Harris24 have reported that G85E may be associated with milder disease (that is, a mild allele). Login to comment
81 In either case, the fact that one of our five AF508/G85E affected subjects is pancreatic sufficient and the other four are insufficient is in conflict with the hypothesis proposed by Kerem et al.3 Table 3 Distribution of XV2c and KM1 9 haplotypes in CF and normal chromosomes. Login to comment
97 We wish to thank Professor P S Harper, Dr D Shaw, and Dr A Clarke for their useful comments on this manuscript, Dr L Sandkuijl for his help with the statistical analyses, Dr M 0 621 + 1G> T Total G551D Total G542X Total G85E Total R553X Total R1283M Total N1303K Total AI507 Total RI 17H Total 1717-1 Total R560T Total 1154Ins TC Total 0 1 1 o o o 0 o 0 0 2 0 o o 0 1 0 0 I 1 1 1 I I I Al-Jader, Meredith Table S Distribution of haplotypes in the uncharacterised CF chromosomes. Login to comment
155 A cystic fibrosis patient who is homozygous for the G85E mutation has very mild disease. Login to comment

[hide] The gene defect in cystic fibrosis and clinical ap... J R Soc Med. 1992;85 Suppl 19:6-8. Super M
The gene defect in cystic fibrosis and clinical applications of the knowledge.
J R Soc Med. 1992;85 Suppl 19:6-8., [PMID:1375961]

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55 V520F 63* 3 0.90% This supports a crucial role for the nucleotide binding G85E 40* 2 0.48% folds in determining severity-R117H is in exon 3, Total 91.99% part ofthe intramural anchoringprotein presumably not playing a major role in ion transport. Login to comment

[hide] Analysis of 31 CFTR mutations in 55 families from ... Early Hum Dev. 2001 Nov;65 Suppl:S161-4. Gomez-Llorente MA, Suarez A, Gomez-Llorente C, Munoz A, Arauzo M, Antunez A, Navarro M, Gil A, Gomez-Capilla JA
Analysis of 31 CFTR mutations in 55 families from the South of Spain.
Early Hum Dev. 2001 Nov;65 Suppl:S161-4., [PMID:11755047]

Abstract [show]
We carried out a molecular analysis of 350 chromosomes from 55 families originating from the South of Spain (Andalucia) who were diagnosed with cystic fibrosis (CF). We used polymerase chain reaction, followed by an oligonucleotide ligation assay (OLA) and sequence-coded separation using capillary electrophoresis. A frequency of 43.5% for DeltaF508 was found, making it the most common CF mutation in our sample. Seven more mutations (G542X, R334W, R1162X, 2789+5G-->A, R117H, DeltaI507 and W1282X) were detected and accounted for 24.7% of the total. The remaining mutations (31.8%) were undetectable with the methodology used in this study.

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27 The patients and their families were referred to us from six Table 1 Listing of the CFTR mutations which are interrogated in the CF assay used in this study Mutation Location Mutation Location Exon/Intron Exon/Intron DF508 E.10 W1282X E.20 F508C E.10 3905insT E.20 DI507 E.10 N1303K E.21 Q493X E.10 G85E E.3 V520F E.10 621 + 1G ! Login to comment

[hide] Effect of genotype on phenotype and mortality in c... Lancet. 2003 May 17;361(9370):1671-6. McKone EF, Emerson SS, Edwards KL, Aitken ML
Effect of genotype on phenotype and mortality in cystic fibrosis: a retrospective cohort study.
Lancet. 2003 May 17;361(9370):1671-6., [PMID:12767731]

Abstract [show]
BACKGROUND: Over 1000 mutations of the cystic fibrosis transmembrane conductance regulator gene (CFTR) that cause cystic fibrosis have been identified. We examined the effect of CFTR genotype on mortality and disease phenotype. METHODS: Using the US Cystic Fibrosis Foundation National Registry, we did a retrospective cohort study to compare standardised mortality rates for the 11 most common genotypes heterozygous for DeltaF508 with those homozygous for DeltaF508. Of the 28455 patients enrolled in the registry at the time of our analysis, 17853 (63%) were genotyped. We also compared the clinical phenotype, including lung function, age at diagnosis, and nutritional measures, of 22 DeltaF508 heterozygous genotypes. Mortality rates and clinical phenotype were also compared between genotypes classified into six classes on the basis of their functional effect on CFTR production. FINDINGS: Between 1991 and 1999, genetic and clinical data were available for 17853 patients with cystic fibrosis, which was 63% of the total cohort. There were 1547 deaths during the 9 years of follow-up. In the analysis of the 11 most common genotypes, DeltaF508/R117H, DeltaF508/DeltaI507, DeltaF508/3849+10kbC-->T, and DeltaF508/2789+5G-->A had a significantly lower mortality rate (4.7, 8.0, 11.9, and 4.4, respectively) than the genotype homozygous for DeltaF508 (21.8, p=0.0060). DeltaF508/R117H, DeltaF508/DeltaI507, DeltaF508/ 3849+10 kbC-->T, DeltaF508/2789+5G-->A, and DeltaF508/A455E have a milder clinical phenotype. Outcomes for all functional classes were compared with that of class II (containing DeltaF508 homozygotes) and classes IV and V had a significantly lower mortality rate and milder clinical phenotype. INTERPRETATION: Patients with cystic fibrosis have distinct genetic subgroups that are associated with mild clinical manifestations and low mortality. These differences in phenotype are also related to the functional classification of CFTR genotype.

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47 ARTICLES 1672 THE LANCET ߦ Vol 361 ߦ May 17, 2003 ߦ www.thelancet.com Panel 1: Frequencies of CFTR mutations* CFTR Allele CFTR Allele mutation frequency (%) mutation frequency èc;F508 69&#b7;4% 2789+5G࢐A 0&#b7;3% Unknown 15&#b7;7% R1162X 0&#b7;3% G542X 2&#b7;3% G85E 0&#b7;3% G551D 2&#b7;2% R560T 0&#b7;2% èc;I507 1&#b7;6% R334W 0&#b7;2% W1282X 1&#b7;4% 3659èc;C 0&#b7;2% N1303K 1&#b7;2% A455E 0&#b7;1% R553X 0&#b7;9% 711+1G࢐T 0&#b7;1% 621+1G࢐T 0&#b7;8% 1898+1G࢐A 0&#b7;1% R117H 0&#b7;7% 2184èc;A 0&#b7;1% 3849+10 kbC࢐T 0&#b7;7% S549N 0&#b7;1% 1717-IG࢐A 0&#b7;5% 1078èc;T 0&#b7;03% R347P 0&#b7;3% *n=17 853. Login to comment
48 Panel 2: Functional classification of CFTR alleles Class Functional effect of Allele mutation I Defective protein G542X, R553X, W1282X, production R1162X, 621-1G࢐T, 1717-1G࢐A, 1078èc;T, 3659èc;C II Defective protein èc;F508, èc;I507, N1303K, processing S549N III Defective protein G551D, R560T regulation IV Defective protein R117H, R334W, G85E, conductance R347P V Reduced amounts of 3849+10KbC࢐T, functioning CFTR protein 2789+5G࢐A, A455E Unknown 711+1G࢐T, 2184DA, 1898+1G࢐A Total cohort Genotyped cohort (n=28 455) (n=17 853) Person-years at risk 152 011 96 870 Sex (% male) 53% 52% Race (% white) 96% 96% Age (years) 11&#b7;9 (11&#b7;1) 10&#b7;9 (11&#b7;2) Age at diagnosis (years) 3&#b7;5 (7&#b7;1) 3&#b7;6 (7&#b7;5) Sweat test (mmol/L) 101 (19) 100 (20) FEV1 (L) 1&#b7;72 (0&#b7;91) 1&#b7;80 (0&#b7;92) FEV1 (% predicted) 69 (29) 72 (28) FVC (L) 2&#b7;41 (1&#b7;18) 2&#b7;50 (1&#b7;21) FVC (% predicted) 81% (28) 84% (24) Height (cm) 121% (41) 117% (41) Weight (kg) 30&#b7;0 (21&#b7;3) 28&#b7;6 (21&#b7;8) Pancreatic insufficiency (%) 90% 87% P aeruginosa colonisation (%) 49% 46% Number of deaths (%) 3548 (12%) 1547 (9%) Data are mean (SD) unless otherwise stated. Login to comment
67 Table 2: Standardised and crude mortality rates (including organ transplantation) by genotype Genotype No of Age at Sweat FEV1 FVC Height Weight Pancreatic P&#b7; aeruginosa Subjects Diagnosis Chloride (% predicted)* (% predicted)* (cms)* (kg)* Insufficiency Colonization (yrs) (mmol) (%)ߤ (%)ߤ èc;F508/èc;F508 6 213 2&#b7;5 &#b1; 0&#b7;1 104 &#b1; 0&#b7;2 77 &#b1; 0&#b7;3 89 &#b1; 0&#b7;3 141 &#b1; 0&#b7;2 37&#b7;0 &#b1; 0&#b7;1 92 (91-92) 60 (59-61) èc;F508/G551D 411 3&#b7;7 &#b1; 0&#b7;3ߥ 108 &#b1; 0&#b7;9ߥ 76 &#b1; 1&#b7;2 89 &#b1; 1&#b7;2 142 &#b1; 0&#b7;7&#a7; 38&#b7;2 &#b1; 0&#b7;6&#a7; 92 (89-94) 59 (54-64) èc;F508/G542X 389 1&#b7;9 &#b1; 0&#b7;2 104 &#b1; 0&#b7;8 79 &#b1; 1&#b7;2 91 &#b1; 1&#b7;2 141 &#b1; 0&#b7;7 37&#b7;3 &#b1; 0&#b7;5 93 (89-95) 57 (52-62) èc;F508/N1303K 213 2&#b7;1 &#b1; 0&#b7;3 106 &#b1; 1&#b7;2 80 &#b1; 1&#b7;8 91 &#b1; 1&#b7;7 141 &#b1; 1&#b7;0 37&#b7;1 &#b1; 0&#b7;6 92 (87-95) 61 (55--68) èc;F508/W1282X 205 1&#b7;6 &#b1; 0&#b7;2 103 &#b1; 1&#b7;2 80 &#b1; 1&#b7;7 92 &#b1; 1&#b7;6 141 &#b1; 0&#b7;9 37&#b7;4 &#b1; 0&#b7;7 94 (90-97) 59 (52-65) èc;F508/R553X 164 2&#b7;5 &#b1; 0&#b7;4 106 &#b1; 1&#b7;4 76 &#b1; 1&#b7;8 89 &#b1; 1&#b7;6 139 &#b1; 0&#b7;9 35&#b7;4 &#b1; 0&#b7;7&#a7; 90 (85-94) 60 (53-67) èc;F508/621-1G 162 2&#b7;5 &#b1; 0&#b7;4 107 &#b1; 1&#b7;3 78 &#b1; 1&#b7;8 89 &#b1; 1&#b7;5 143 &#b1; 1&#b7;0&#a7; 38&#b7;8 &#b1; 0&#b7;8&#a7; 87 (80-91)&#a7; 57 (49-64) èc;F508/èc;I507 149 8&#b7;5 &#b1; 1&#b7;1ߥ 95 &#b1; 1&#b7;9ߥ 86 &#b1; 2&#b7;1ߥ 93 &#b1; 1&#b7;8&#a7; 137 &#b1; 1&#b7;4&#a7; 37&#b7;4 &#b1; 1&#b7;25 84 (78-89)ߥ 39 (31-48)ߥ èc;F508/R117H 123 13&#b7;7 &#b1; 1&#b7;2ߥ 80 &#b1; 1&#b7;9ߥ 91 &#b1; 2&#b7;1ߥ 97 &#b1; 1&#b7;7ߥ 143 &#b1; 1&#b7;8 42&#b7;9 &#b1; 1&#b7;7ߥ 65 (55-73)ߥ 22 (16-29)ߥ èc;F508/3849+10 kB 114 11&#b7;3 &#b1; 0&#b7;9ߥ 72 &#b1; 2&#b7;5ߥ 77 &#b1; 2&#b7;1 87 &#b1; 1&#b7;9 144 &#b1; 1&#b7;4&#a7; 41&#b7;2 &#b1; 1&#b7;2ߥ 66 (57-74)ߥ 69 (59-77) èc;F508/2789+5G 63 13&#b7;4 &#b1; 1&#b7;6ߥ 102 &#b1; 2&#b7;1 88 &#b1; 2&#b7;8ߥ 97 &#b1; 2&#b7;3ߥ 140 &#b1; 2&#b7;5 41&#b7;8 &#b1; 2&#b7;2&#a7; 71 (59-81)ߥ 32 (22-44)ߥ èc;F508/1717-1G 74 1&#b7;3 &#b1; 0&#b7;3 103 &#b1; 2&#b7;0 75 &#b1; 2&#b7;7 86 &#b1; 2&#b7;4 139 &#b1; 1&#b7;5 35&#b7;7 &#b1; 0&#b7;9 96 (88-99) 59 (48-69) èc;F508/R560T 46 1&#b7;7 &#b1; 0&#b7;5 104 &#b1; 2&#b7;0 84 &#b1; 3&#b7;3ߥ 96&#b1; 2&#b7;8&#a7; 142 &#b1; 1&#b7;9 38&#b7;4 &#b1; 1&#b7;4 91 (79-97) 63 (48-75) èc;F508/R347P 44 5&#b7;9 &#b1; 1&#b7;1&#a7; 105 &#b1; 2&#b7;6 76 &#b1; 3&#b7;0 90 &#b1; 2&#b7;9 142 &#b1; 2&#b7;4 38&#b7;7 &#b1; 1&#b7;8 67 (52-79)ߥ 53 (38-68) èc;F508/G85E 43 9&#b7;2 &#b1; 1&#b7;8ߥ 99 &#b1; 2&#b7;3&#a7; 76 &#b1; 2&#b7;5 90 &#b1; 2&#b7;5 142 &#b1; 2&#b7;9 38&#b7;3 &#b1; 2&#b7;2 88 (75-95) 52 (35-68) èc;F508/3659DC 40 1&#b7;1 &#b1; 0&#b7;4 105 &#b1; 2&#b7;1 76 &#b1; 3&#b7;9 88 &#b1; 4&#b7;1 139 &#b1; 1&#b7;9 36&#b7;6 &#b1; 1&#b7;2 92 (77-97) 55 (39-69) èc;F508/A455E 29 14&#b7;3 &#b1; 2&#b7;0ߥ 89 &#b1; 3&#b7;1ߥ 98 &#b1; 4&#b7;0ߥ 104 &#b1; 3&#b7;4ߥ 138 &#b1; 3&#b7;4 42&#b7;1 &#b1; 2&#b7;5&#a7; 60 (41--76)ߥ 17 (8-32)ߥ èc;F508/R334W 28 13&#b7;2 &#b1; 3&#b7;0ߥ 104 &#b1; 3&#b7;2 86 &#b1; 3&#b7;4&#a7; 94 &#b1; 3&#b7;3 138 &#b1; 3&#b7;2 42&#b7;3 &#b1; 3&#b7;5 67 (46-82)ߥ 51 (32--70) èc;F508/R1162X 26 1&#b7;9 &#b1; 1&#b7;1 101 &#b1; 2&#b7;3 77 &#b1; 4&#b7;2 92 &#b1; 4&#b7;6 138 &#b1; 1&#b7;8 36&#b7;5 &#b1; 1&#b7;4 92 (75-98) 65 (47-80) èc;F508/1898+1G 20 1&#b7;2 &#b1; 0&#b7;3 99 &#b1; 2&#b7;8 83 &#b1; 4&#b7;1 94 &#b1; 4&#b7;4 138 &#b1; 3&#b7;3 35&#b7;1 &#b1; 2&#b7;1 85 (61--95) 63 (39-82) èc;F508/2184DA 20 2&#b7;3 &#b1; 0&#b7;9 106 &#b1; 5&#b7;3 82 &#b1; 4&#b7;3 92 &#b1; 4&#b7;4 141 &#b1; 3&#b7;0 36&#b7;5 &#b1; 1&#b7;5 94 (69-99) 60 (38-79) èc;F508/711+1G 17 1&#b7;3 &#b1; 0&#b7;5 108 &#b1; 4&#b7;6 83 &#b1; 4&#b7;2 94 &#b1; 4&#b7;4 137 &#b1; 3&#b7;4 36&#b7;7 &#b1; 2&#b7;9 100 73 (50-88) èc;F508/S549N 11 6&#b7;4 &#b1; 1&#b7;9&#a7; 109 &#b1; 5&#b7;7 67 &#b1; 6&#b7;1 77 &#b1; 7&#b7;2 140 &#b1; 3&#b7;2 36&#b7;7 &#b1; 2&#b7;6 92 (62-99) 71 (40--90) èc;F508/Other 2 262 5&#b7;8 &#b1; 0&#b7;2ߥ 99 &#b1; 0&#b7;4ߥ 80 &#b1; 0&#b7;5ߥ 91 &#b1; 0&#b7;5ߥ 141 &#b1; 0&#b7;3 38&#b7;1 &#b1; 0&#b7;3ߥ 86 (84-87)ߥ 50 (48-52)ߥ Other/Other 1 551 7&#b7;5 &#b1; 0&#b7;3ߥ 93 &#b1; 0&#b7;6ߥ 82 &#b1; 0&#b7;6ߥ 90 &#b1; 0&#b7;6&#a7; 141 &#b1; 0&#b7;4 38&#b7;3 &#b1; 0&#b7;3ߥ 81 (80-84)ߥ 40 (38-43)ߥ Data are mean (SE) unless otherwise indicated. Login to comment

[hide] Cystic fibrosis. Am J Clin Pathol. 2003 Dec;120 Suppl:S3-13. Lewis MJ, Lewis EH 3rd, Amos JA, Tsongalis GJ
Cystic fibrosis.
Am J Clin Pathol. 2003 Dec;120 Suppl:S3-13., [PMID:15298139]

Abstract [show]
On a daily basis, pathologists examine the fundamental basis of human diseases using morphologic, immunologic, and molecular techniques. Cystic fibrosis (CF), as a clinically heterogeneous disease, exemplifies the complex challenges of genetic diseases for the pathologist who attempts to explain the mechanisms of disease and provide rationale for clinical management. This review includes an overview of CF and a discussion of pathophysiologic features and practical components of clinical and anatomic pathology, and concludes with a review of molecular diagnostics.

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95 ēa; ēa;Table 3ēa; ēa; Recommended Mutation Panel for Cystic Fibrosis Carrier Screening ࢞F508 ࢞I507 G542X G551D W1282X N1303K R553X 621+1G>T R117H 1717-1G>A A455E R560T R1162X G85E R334W R347P 711+1G>T 1898+1G>A 2184delA 1078delT 3849+10kbC>T 2789+5G>A 3659delC I148T 3120+1G>A I506V* I507V* F508C* 5T/7T/9T* * Reflex tests. Login to comment

[hide] Diagnosis of cystic fibrosis in adults with diffus... J Cyst Fibros. 2004 Mar;3(1):15-22. Hubert D, Fajac I, Bienvenu T, Desmazes-Dufeu N, Ellaffi M, Dall'ava-Santucci J, Dusser D
Diagnosis of cystic fibrosis in adults with diffuse bronchiectasis.
J Cyst Fibros. 2004 Mar;3(1):15-22., [PMID:15463882]

Abstract [show]
We assessed the contribution of the sweat test, genotyping and nasal potential difference (NPD) in the diagnosis of cystic fibrosis (CF) in adults with diffuse bronchiectasis (DB). Among 601 adults referred for DB from 1992 to 2001, 46 were diagnosed with CF. The sweat test was positive in 37 patients and normal or intermediate in nine patients. Two CF mutations were identified in 18 patients (39%) by screening for 31 mutations and in 36 patients (78%) after complete genetic analysis. NPD was suggestive of CF in 71% of the patients. The combination of the sweat test and genetic analysis led to the diagnosis of CF in 45 patients. In the nine patients with normal or intermediate sweat test, the diagnosis was confirmed by screening for 31 mutations in five, by complete genetic screening in three, and by NPD in the remaining patient. Searching for CF should start with sweat test. If the sweat test is normal or intermediate, screening for 31 mutations may help to diagnose CF. A complete genetic analysis is indicated when only one mutation is detected and/or when other clinical features, such as obstructive azoospermia or pancreatic insufficiency, are suggestive of CF. NPD measurement is indicated in controversial cases.

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47 We used an oligonucleotide ligation assay using a commercially available kit (Cystic Fibrosis Assay, Applied Biosystems, Foster City, CA, USA) to seek 31 mutations in the CFTR gene (F508del, I507del, Q943X, V520F, 1717y1GࡊA, G542X, G551D, R553X, R560T, S549R, S549 N, 3849q10kbCࡊT, 3849q4AࡊG, R1162X, 3659delC, W1282X, 3905insT, 621q1GࡊT, R117H, Y122X, 711q1GࡊT, 1078delT, R347P, R347H, R334 W, A455E, N1303K, G85E, 1898q1GࡊA, 2183AAࡊG, 2789q5GࡊA) which allowed to detect 82% of the CF alleles in France. Login to comment
128 The consensus opinion is Table 3 Sensitivity of the sweat test and genotyping for the diagnosis of cystic fibrosis Positive diagnosis of CF Number of patients (percentage) Sweat test (sweat chloride)60 mmol l ) y1 37 (80%) Molecular analysis: -screening for 31 mutations 18 (39%) -complete screening 36 (78%) Combination of sweat test and molecular analysis: -sweat testqscreening for 31 mutations 42 (91%) -sweat testqcomplete screening 45 (98%) Table 4 Efficacy of the CFTR genetic analysis according to the screening method used Identified mutations Two mutations At least one mutation No mutations Screening for F508del Number of patients (%) 3 (7%) 31 (67%) 15 (33%) Screening for nine mutations* Number of patients (%) 6 (13%) 36 (78%) 10 (22%) Screening for 31 mutations** Number of patients (%) 18 (39%) 43 (93%) 3 (7%) Complete screening Number of patients (%) 36 (78%) 46 (100%) 0 Nine mutations: F508del, I507del, G542X, G551D, R553X, 621q1GࡊT, G85E, R117H, N1303K. Login to comment
129 * 31 mutations: F508del, I507del, Q493X, V520F, 1717y1GࡊA, G542X, G551D, R553X, R560T, S549R, S549 N, 3849q10kbCࡊT, 3849q ** 4AࡊG, R1162X, 3659delC, W1282X, 3905insT, 621q1GࡊT, R117H, Y122X, 711q1GࡊT, 1078delT, R347P, R347H, R334 W, A455E, N1303K, G85E, 1898q1GࡊA, 2183AAࡊG, 2789q5GࡊA. that the laboratory criteria for the diagnosis of CF should be expanded to include identification of CFTR mutations and abnormal bioelectrical properties of the nasal epithelium, in addition to the sweat test w7x. Login to comment

[hide] A 96-well formatted method for exon and exon/intro... Anal Biochem. 2006 Jun 15;353(2):226-35. Epub 2006 Apr 5. Lucarelli M, Narzi L, Piergentili R, Ferraguti G, Grandoni F, Quattrucci S, Strom R
A 96-well formatted method for exon and exon/intron boundary full sequencing of the CFTR gene.
Anal Biochem. 2006 Jun 15;353(2):226-35. Epub 2006 Apr 5., [PMID:16635477]

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Full genotypic characterization of subjects affected by cystic fibrosis (CF) is essential for the definition of the genotype-phenotype correlation as well as for the enhancement of the diagnostic and prognostic value of the genetic investigation. High-sensitivity diagnostic methods, capable of full scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, are needed to enhance the significance of these genetic assays. A method for extensive sequencing of the CFTR gene was optimized. This method was applied to subjects clinically positive for CF and to controls from the general population of central Italy as well as to a single subject heterozygous for a mild mutation and with an uncertain diagnosis. Some points that are crucial for the optimization of the method emerged: a 96-well format, primer project and purification, and amplicon purification. The optimized method displayed a high degree of diagnostic sensitivity; we identified a subset of 13 CFTR mutations that greatly enhanced the diagnostic sensitivity of common methods of mutational analysis. A novel G1244R disease causing mutation, leading to a CF phenotype with pancreatic sufficiency but early onset of pulmonary involvement, was detected in the subject with an uncertain diagnosis. Some discrepancies between our results and previously published CFTR sequence were found.

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26 None of these subjects showed any clinical manifestations of CF, nor were any positive for CFTR mutations when analyzed by means of the PCR/OLA/SCS method (Celera Diagnostics) [21], which searches for the most common worldwide 31 CFTR mutations (G85E, R117H, Y122X, 621+1G->T, 711+1G->T, 1078delT, R347P, R347H, R334W, A455E, DF508, DI507, Q493X, V520F, 1717-1G->A, G542X, G551D, R553X, R560T, S549R(T->G), S549N, 1898+1G->A, 2183AA->G, 2789+5G->A, R1162X, 3659delC, 3849+10kbC->T, 3849+4A->G, W1282X, 3905insT, N1303K), including the 12 most common in Italy [1,22]. Login to comment

[hide] The genetic background of osteoporosis in cystic f... J Cyst Fibros. 2006 Dec;5(4):229-35. Epub 2006 May 18. Castellani C, Malerba G, Sangalli A, Delmarco A, Petrelli E, Rossini M, Assael BM, Mottes M
The genetic background of osteoporosis in cystic fibrosis: association analysis with polymorphic markers in four candidate genes.
J Cyst Fibros. 2006 Dec;5(4):229-35. Epub 2006 May 18., [PMID:16713399]

Abstract [show]
BACKGROUND: Reduced Bone Mass Density (BMD) is frequent in Cystic Fibrosis (CF). Potentially, other genes than the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene may contribute to the bone phenotype variability in CF patients. METHODS: Four candidate genes likely associated with BMD variability were studied: the vitamin D receptor (VDR) gene, the estrogen receptor alpha (ESR1), the calcitonin receptor (CALCR) and the type I alpha 1 collagen (COL1A1) gene. A complete bone and CF evaluation was obtained for 82 subjects (39 m, 43 f): 15 had normal BMD (group 1), 46 were osteopenic (group 2), and 21 were osteoporotic (group 3). RESULTS: No statistical difference was found among the three groups for age, sex, pancreatic status, and vertebral fractures, nor for any of the biochemical markers. Weight, Body Mass Index (BMI), and FEV1, scored significantly worse in the two groups with the lowest T score. The CFTR mutations R1162X and F508del were more frequent in patients with lower BMD (p=0.044 and p=0.071). There was no significant difference in the distribution of the five marker genotypes among the 3 groups defined according to the unadjusted or adjusted (BMI and FEV1) BMD T score. No significant correlation was found between the VDR, CALCR, or COL1A1 gene polymorphisms and reduced BMD values. The individual ESR1 PvuII-XbaI haplotype C-A is associated to elevated u-calcium levels whereas the haplotype T-A is associated to lower values (p=0.00251). CONCLUSIONS: There was no evidence that the genes under study, with the possible exception of ESR1 gene variants, may modulate bone phenotype in CF.

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80 assay which allows the simultaneous analysis of the commonest CFTR mutations in North-eastern Italy (F508del, I507del, R117H, R1162X, 2183AA>G, N1303K, 3849+10KbC>T, G542X, 1717-1G>A, R553X, Q552X, G85E, 711+5G>A, W1282X, 3132delTG and 2789+5G>A) [25]. Login to comment

[hide] CFTR gene analysis in Latin American CF patients: ... J Cyst Fibros. 2007 May;6(3):194-208. Epub 2006 Sep 11. Perez MM, Luna MC, Pivetta OH, Keyeux G
CFTR gene analysis in Latin American CF patients: heterogeneous origin and distribution of mutations across the continent.
J Cyst Fibros. 2007 May;6(3):194-208. Epub 2006 Sep 11., [PMID:16963320]

Abstract [show]
BACKGROUND: Cystic Fibrosis (CF) is the most prevalent Mendelian disorder in European populations. Despite the fact that many Latin American countries have a predominant population of European-descent, CF has remained an unknown entity until recently. Argentina and Brazil have detected the first patients around three decades ago, but in most countries this disease has remained poorly documented. Recently, other countries started publishing their results. METHODS: We present a compilation and statistical analysis of the data obtained in 10 countries (Argentina, Brazil, Chile, Colombia, Costa Rica, Cuba, Ecuador, Mexico, Uruguay and Venezuela), with a total of 4354 unrelated CF chromosomes studied. RESULTS: The results show a wide distribution of 89 different mutations, with a maximum coverage of 62.8% of CF chromosomes/alleles in the patient's sample. Most of these mutations are frequent in Spain, Italy, and Portugal, consistent with the origin of the European settlers. A few African mutations are also present in those countries which were part of the slave trade. New mutations were also found, possibly originating in America. CONCLUSION: The profile of mutations in the CFTR gene, which reflects the heterogeneity of its inhabitants, shows the complexity of the molecular diagnosis of CF mutations in most of the Latin American countries.

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42 Some have concentrated in the search of specific mutations that are Table 1 Mutations found in the Latin American CF patients Exon 1 p.L6VÌe; Exon 3 p.W57X, p.R75X, p.G85E Exon 4 p.R117H Exon 6a p.H199Y, p.V201M, p.L206W, p.Q220X, p.V232D, c.846delTÌe; Exon 6b p.Y275XÌe;, c.935delA Exon 7 p.R334W, p.R347P, p.Y362XÌe;, c.1078delT, c.1215delG Exon 8 c.1323_1324insAÌe; Exon 9 c.1460_1461delATÌe;, c.1353_1354insTÌe;,# Exon 10 p.I506T, p.I507del, p.F508del Exon 11 p.G542X, p.S549N, p.S549R, p.G551D, p.G551S, p.R553X, p.L558S, p.A559T, c.1782delA Exon 12 p.S589I Exon 13 p.H609RÌe;, p.P750L, p.V754M, c.1924_1930del, c.2055_2063del, c.2183AA NG;c.2184delA, c.2184delA, c.2185_2186insC, c.2347delG, c.2566_2567insTÌe;, c.2594_2595delGTÌe; Exon 14a p.R851L, c.2686_2687insTÌe; Exon 15 c.2869_2870insG Exon 16 c.3120+1GNA Exon 17a p.I1027T, c.3171delC, c.3199_3204del Exon 17b p.G1061R, p.R1066C, p.W1069X#, p.W1089X, p.Y1092X, p.W1098CÌe; Exon 19 p.R1162X, p.W1204X, p.Q1238X, c.3617_3618delGAÌe;#, c.3659delC Exon 20 p.W1282X, p.R1283M Exon 21 p.N1303K, c.4016_4017insT Exon 22 c.4160_4161insGGGGÌe; 5' flanking c.-834GNT Intron 2 c.297-1GNAÌe;, c.297-2ANG Intron 3 c.406-1GNA Intron 4 c.621+1GNT Intron 5 c.711+1GNT Intron 8 c.IVS8-5T Intron 10 c.1716GNA, c.1717-1GNA Intron 11 c.1811+1.6KbANG, c.1812-1GNA Intron 12 c.1898+1GNA, c.1898+3ANG Intron 14 c.2789+2_2789+3insA, c.2789+5GNA Intron 17a c.3272-26ANG Intron 17b c.3500-2ANGÌe; Intron 19 c.3849+1GNA, c.3849+10KbCNT Intron 20 c.4005+1GNA, c.4005-1GNA# Mutations are listed according to their position in the gene. Login to comment
46 of chromosomes analysed p.F508del p.G542X p.W1282X p.N1303K p.R1162X p.L6VÌe; p.W57X p.R75X p.G85E p.R117H p.H199Y p.V201M p.L206W p.Q220X p.V232D p.Y275XÌe; p.R334W p.R347P p.Y362XÌe; p.I506T Argentina 98 61 440 258 18 12 12 2 1 1 3 1 5 1 310 181 20 7 5 5 7 0 5 0 222 135 15 7 5 1 26 14 2 1 1 150 88 6 6 1 2 3 Subtotal and frequency (%) 1246 100 737 59.15 61 4.90 27 2.17 28 2.25 9 0.72 1 0.08 1 0.08 13 1.04 1 0.08 13 1.04 1 0.08 Brazil 468 221 26 11 74 38 2 1 320 155 28 3 8 8 4 1 2 1 1 8 122 62 120 38 10 3 148 38 4 0 0 48 15 154 75 5 1 0 2 0 386 154 24 6 10 17 9 0 10 1 18 4 0 0 2 0 0 0 0 Subtotal and frequency (%) 1858 100 800 43.06 99 5.33 11 0.59 34 1.83 25 1.35 13 0.70 1 0.05 2 0.11 1 0.05 1 0.05 20 1.07 1 0.05 Chile 72 21 36 11 3 0 44 22 4 3 1 1 100 45 7 5 0 2 0 2 0 Subtotal and frequency (%) 252 100 99 41.28 14 5.55 8 3.17 3 1.19 3 1.19 Colombia 184 77 7 2 1 2 1 34 13 2 1 1 Subtotal and frequency (%) 218 100 90 41.28 9 4.13 3 1.38 2 0.92 2 0.92 1 0.46 Costa Rica Frequency (%) 48 100 11 22.91 12 25.00 0 0 0 0 0 Cuba Frequency (%) 144 100 49 34.03 Ecuador 32 11 1 50 16 2 2 20 5 0 0 0 Subtotal and frequency (%) 102 100 32 31.37 2 1.96 1 0.98 2 1.96 Mexico 194 79 12 4 3 1 1 1 2 80 36 4 1 Subtotal and frequency (%) 274 100 115 41.97 16 5.84 5 1.82 3 1.09 1 0.36 1 0.36 1 0.36 2 0.73 Uruguay Frequency (%) 76 100 43 56.58 6 7.89 2 2.63 3 3.95 3 3.95 2 2.63 Venezuela 54 16 2 82 41 Subtotal and frequency (%) 136 100 57 41.91 2 1.47 Total 4354 2033 221 49 72 42 1 1 3 32 1 1 1 2 1 1 1 39 1 1 2 Frequency (%) 100 46.69 5.08 1.13 1.65 0.96 0.02 0.02 0.07 0.73 0.02 0.02 0.02 0.05 0.02 0.02 0.02 0.90 0.02 0.02 0.05 The five most frequent mutations are shown on the left-hand side, followed by the rest of the mutations in 5'-3' and exon-intron order. Login to comment
66 The p.G85E mutation was found in several countries such as Argentina [[12,18,19], Oller Ramirez, personal communication], Brazil [15,22], Ecuador (Cassiman, 2004, personal communication), Mexico [13] and Uruguay [41] with frequencies ranging from 0.36% in Mexico up to 3.95% in Uruguay. Login to comment
69 As shown in Table 4, 19 of them have overall frequencies of 0.1% to 1% in Latin America and could be considered as rare, but some of them have local elevated frequencies, like c.1811+1.6KbANG in Colombia (from 4.2% to 10.5% in the different geographical regions studied) [14, 46], p.G85E in Ecuador (8.9%) (Ruiz et al., personal communication), Uruguay (3.95%) [41], Brazil (2.33%) [15] and Argentina (2.26%) [18], p.R334W in Brazil (2.6%) [15, 22] and p.I507del (2.58%) and p.S549N (2.5%) in Mexico [13] (Table 2). Login to comment
90 Table 4 Mutations with frequencies between 0.1% and 1% Mutation Frequency Country Number of chromosomes % p.R334W 39 0.90 Argentina, Brazil, Chile, Colombia, Uruguay p.G85E 32 0.73 Argentina, Brazil, Ecuador, Mexico, Uruguay p.R553X 22 0.51 Argentina, Brazil, Chile, Mexico, Uruguay c.1811+1.6KbANG 18 0.41 Argentina, Colombia c.3849+10KbCNT 18 0.41 Argentina, Mexico c.1717-1GNA 14 0.32 Argentina, Brazil, Uruguay c.3120+1GNA 12 0.28 Argentina, Brazil Colombia p.I507del 10 0.23 Brazil, Mexico, Uruguay c.2789+5GNA 9 0.21 Argentina, Brazil, Colombia, Uruguay c.621+1GNT 7 0.16 Argentina, Brazil, Mexico p.S549N 6 0.14 Mexico p.S549R 6 0.14 Argentina, Brazil, Colombia p.G551D 6 0.14 Argentina, Mexico p.R1066C 6 0.14 Argentina, Colombia, Mexico c.2183ANG;c.2184delA 6 0.14 Argentina, Mexico p.Y1092X 5 0.11 Colombia, Mexico c.1812-1GNA 5 0.11 Brazil c.IVS8-5T 5 0.11 Argentina c.3659delC 4 0.09 Argentina Unfortunately, at present not all Latin-American countries have started molecular studies in their patients with a probable Cystic Fibrosis diagnosis. Login to comment
111 As discussed, another way to disclose similarities or differences in the distribution of mutations in the CF patients from Latin Table 6 Screening panel of CFTR mutations Country Total number of mutations Minimum panel Detection power Uruguay 12 6 mutations: p.F508del, p.G542X, p.R1162X, p.N1303K (p.R334W, p.G85E) 78% Argentina 52 7 mutations: p.F508del, p.G542X, p.R1162X, p.W1282X, p.N1303K (p.R334W, p.G85E) 71% M&#e9;xico 35 8 mutations: p.F508del, p.G542X, p.N1303K (p.R75X, p.I507del, p.S549N,c.406-1GNA, c.3849+10kbGNA) 58% Colombia 19 7 mutations: p.F508del, p.G542X, p.R1162X, p.W1282X, p.N1303K (p.S549R, c.1811+1.6kbANG) 56% Brazil 41 6 mutations: p.F508del, p.G542X, p.R1162X, p.W1282X, p.N1303K (p.R334W) 53% The total number of mutations found in each country is indicated in the second column from left. Login to comment
134 This panel, besides the common mutations shown in Table 3, should include the p.R334W and p.G85E mutations. Login to comment

[hide] [The French nationwide cystic fibrosis newborn scr... Arch Pediatr. 2008 Jun;15 Suppl 1:S1-6. doi: 10.1016/S0929-693X(08)73940-X. Munck A, Roussey M
[The French nationwide cystic fibrosis newborn screening program: strategy and results].
Arch Pediatr. 2008 Jun;15 Suppl 1:S1-6. doi: 10.1016/S0929-693X(08)73940-X., [PMID:18822253]

Abstract [show]
In 2002 France implemented a nationwide newborn screening program for cystic fibrosis (CF). The strategy combined immunoreactive trypsinogen and, in case of a value over the cut-off level, DNA analysis in dried blood samples at day 3. Data were centralized and periodically analyzed thus maintaining the percentage of samples requiring mutation analysis (0.6%), limiting the number of false-positive cases (0.1%) without increasing the number of false-negative cases (3.2%). 3.527.353 infants were screened between 2002 and 2006. The overall cystic fibrosis incidence was 1/ 4136 with a wide range of regional variations. Dilemma case presentation occurred for 14 % of the patients; an European working group is actively working on this topic, attempting to establish a consensus on the adequate procedures. Cystic fibrosis newborn screening is feasible all over a nation but needs a strong organization from maternity wards to CF care centers.

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50 L`organigramme du DNN (fig. 1) pr&#e9;voit une valeur seuil de TIR &#e0; J3 d&#e9;termin&#e9;e sur les donn&#e9;es des r&#e9;gions fran&#e7;aises ayant d&#e9;but&#e9; ce d&#e9;pistage il y a plus de 10 ans afin de s&#e9;lec- Mutations recherch&#e9;es par le Kit Elucigen dans le cadre du d&#e9;pistage n&#e9;onatal de la mucoviscidose (Kit CF30) : F508del ; I 507del ; 1078delT, 1717-1 G>A ; 2183AA>G ; 3659delC ; 3849+10kbC>T ; 621+1G>T ; A455E ; E60X ; G542X ; G551D ; N1303K ; R1162X ; R117H ; R334W ; R347P ; R553X ; S1251N ;W1282X ; 1811+1.6kbA>G ; 2789+5G>A ; 3120+1G>A ; 3272-26A>G ; 394delT ; 711+1G>T ; G85E ; Y1092X ; Y122X ;W846X. Login to comment

[hide] [Mild cystic fibrosis: genetics - extending follow... Arch Pediatr. 2009 Apr;16(4):387-90. doi: 10.1016/j.arcped.2008.12.008. Epub 2009 Jan 31. Gaillard D, Clavel C, Bessaci-Kabouya K, Abely M
[Mild cystic fibrosis: genetics - extending follow-up is necessary].
Arch Pediatr. 2009 Apr;16(4):387-90. doi: 10.1016/j.arcped.2008.12.008. Epub 2009 Jan 31., [PMID:19181498]

Abstract [show]
The diagnosis of mild cystic fibrosis is first suspected on mild lung disease or absence of pancreatic insufficiency and is assessed by biological analysis. The sweat test is not always conclusive. The nasal potential difference and molecular analysis of CFTR gene allow confirming diagnosis. A regular follow-up in cystic fibrosis clinical centre is essential all life long. The genotype, especially during neonatal period, cannot be used to predict individually the course of the disease. Genetic counselling must be recommended to the parents in order to propose an analysis of CFTR gene to give the appropriate genetic counselling and to consider with them which family members could be concerned, especially in the event of parental project. The research of heterozygote status in related for prenatal diagnosis is not recommended for all mutations.

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43 Parmi les variations de s&#e9;quence identifi&#e9;es en p&#e9;riode n&#e9;onatale par le kit &#c9;lucig&#e8;ne1 CF30, plusieurs sont reconnues comme ayant un effet mod&#e9;r&#e9;, comme R117H, R334W, R2789 + 5G > A, 3272-26A > G, 3849 + 10kbC > T [7,9,10], voire variable, notamment G85E, A455E [3] ; les 23 autres mutations sont reconnues comme s&#e9;v&#e8;res. Login to comment

[hide] Frequency of 8 CFTR gene mutations in cystic fibro... Braz J Med Biol Res. 2010 Feb;43(2):134-8. Epub 2010 Jan 15. Perone C, Medeiros GS, del Castillo DM, de Aguiar MJ, Januario JN
Frequency of 8 CFTR gene mutations in cystic fibrosis patients in Minas Gerais, Brazil, diagnosed by neonatal screening.
Braz J Med Biol Res. 2010 Feb;43(2):134-8. Epub 2010 Jan 15., [PMID:20098842]

Abstract [show]
The nature and frequency of cystic fibrosis mutations in Brazil is not uniform due to the highly varied ethnic composition of the population. The average frequency of the F508del mutation has been reported to be 48.6%. Other common mutations in Brazil are G542X, R1162X, and N1303K. The aim of this study was to analyze the frequency of 8 mutations (F508del, G542X, R1162X, N1303K, W1282X, G85E, 3120+1G>A, and 711+1G>T) in a sample of 111 newborn patients with cystic fibrosis diagnosed by the Cystic Fibrosis Neonatal Screening Program of Minas Gerais State. The mutations were tested by allele-specific oligonucleotide PCR with specially designed primers. An allele frequency of 48.2% was observed for the F508del mutation, and allele frequencies of 5.41, 4.50, 4.05, and 3.60% were found for the R1162X, G542X, 3120+1G>A, and G85E mutations, respectively. The genotypes obtained were in Hardy-Weinberg equilibrium. These data demonstrate that the 8-mutation panel studied here has extensive coverage (68%) for the cystic fibrosis mutations in Minas Gerais. These data improve our knowledge of cystic fibrosis in Brazil, particularly in this region. In addition, this investigation contributed to the establishment of a sensitive and population-specific mutation panel, which can be helpful for molecular diagnosis of cystic fibrosis.

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3 The aim of this study was to analyze the frequency of 8 mutations (F508del, G542X, R1162X, N1303K, W1282X, G85E, 3120+1G>A, and 711+1G>T) in a sample of 111 newborn patients with cystic fibrosis diagnosed by the Cystic Fibrosis Neonatal Screening Program of Minas Gerais State. Login to comment
5 An allele frequency of 48.2% was observed for the F508del mutation, and allele frequencies of 5.41, 4.50, 4.05, and 3.60% were found for the R1162X, G542X, 3120+1G>A, and G85E mutations, respectively. Login to comment
29 The objective of the present investigation was to determine the frequency of 8 CFTR mutations (G85E, 711+1G>T, F508del, G542X, 3120+1G>A, R1162X, W1282X, and N1303K) in 111 sweat test-positive newborns screened by the CFNS program in the State of Minas Gerais, Brazil. Login to comment
43 The mutations are: G85E, 711+1G>T, F508del, G542X, 3120+1G>A, R1162X, W1282X, and N1303K. Login to comment
47 Mutation Primer sequence (5` ࢐ 3`) Amplicon size (bp) Annealing temperature (&#b0;C) G85E-S GGA GAT TTA TGT TCT ATG G 245 52 G85E-M GGA GAT TTA TGT TCT ATG A G85E-R GTA AAT TGC CAC CCG TGT TCC AGG 711+1G>T-S CCA ACA ACC TGA ACA AAT TTG ATG AAG 340 64 711+1G>T-M CCA ACA ACC TGA ACA AAT TTG ATG AAT 711+1G>T-R TTG CTC AGG TAT CAT ATC TGG CC F508del-S ACC ATT AAA GAA AAT ATC ATC TT 262 54 F508del-M ACC ATT AAA GAA AAT ATC ATT GG F508del-R TGC AAG CTT CTT AAA GCA TA G542X-S GCA GAG AAA GAC AAT ATA GTT CTT G 213/217 58 G542X-M GTT TGC AGA GAA AGA CAA TAT AGT TCT TTT G542X-R CCA CTA GCC ATA AAA CCC CAG G 3120+1G>A-S CTT ACC ATA TTT GAC TTC ATC CAG G 191 62 3120+1G>A-M CTT ACC ATA TTT GAC TTC ATC CAG A 3120+1G>A-R TTA CTA AAC TTA TGT CTA TTT TGA AGG C R1162X-S TTA TTT CAG ATG CGA TCT GTG AGC C 117 63 R1162X-M TTA TTT CAG ATG CGA TCT GTG AGC TT R1162X-R AAT CAT AAC TTT CGA GAG TTG GCC W1282X-S GGG ATT CAA TAA CTT TGC AAC AGT GG 203 67 W1282X-M GGG ATT CAA TAA CTT TGC AAC AGT GA W1282X-R TCT GCC TAT GAG AAA ACT GCA CTG GAG N1303K-S TTT TTT CTG GAA CAT TTA GAA AAA AC 137 58 N1303K-M TTT TTT CTG GAA CAT TTA GAA AAA AG N1303K-R GCC ATT TGT GTT GGT ATG AGT TAC CCC The -S suffix indicates a wild allele specific primer, the -M suffix a mutant allele primer, and the -R suffix the primer used in both wild and mutant allele amplification. Login to comment
84 Mutation N Frequency (%) Cumulative frequency (%) G85E 8 3.60 3.60 711+1G>T 2 0.90 4.50 F508del 107 48.20 52.70 G542X 10 4.50 57.20 3120+1G>A 9 4.05 61.25 R1162X 12 5.41 66.66 W1282X 1 0.45 67.11 N1303K 2 0.90 68.01 Unknown alleles 71 31.99 Total 222 100.00 100.00 N = number of observed alleles. Login to comment

[hide] [Cystic fibrosis in a woman aged seventy]. Ned Tijdschr Geneeskd. 2010;154:A1342. Ras JE, van Velzen E, van Berkhout FT, van den Brand JJ
[Cystic fibrosis in a woman aged seventy].
Ned Tijdschr Geneeskd. 2010;154:A1342., [PMID:20619026]

Abstract [show]
A seventy-year-old woman was admitted to hospital with a Staphylococcus aureus respiratory tract infection. She had a history of extensive bronchiectasis and allergic bronchopulmonary aspergillosis (ABPA). Cystic fibrosis (CF) was suspected and cystic fibrosis transmembrane conductance regulator (CFTR) gene analysis showed F508del and R117H-7T mutations. In these mutations there is residual activity in the chloride channel in the cell membrane coded by the CFTR gene. This results in a much milder disease pattern varying from no disease at all to isolated organ disease. This type of disease is known as non-classical cystic fibrosis. In our patient the diagnosis of cystic fibrosis was made exceptionally late in life.

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63 TABEL 1 Classificatie van mutaties in het 'cystic fibrosis transmembrane conductance regulator`(CFTR)-gen op chromosoom 7 klasse mechanisme enkele bekende mutaties I geen synthese van het CFTR-eiwit G542X R553X W1282X R1162X 621-1G࢐T 1717-1G࢐A 1078࢞T 3659࢞C II defect in eiwitrijping met voortijdig afbraak ࢞F508 ࢞I507 N1303K S549N III verstoorde regulatie van de CFTR-functie G551D R56OT IV verstoorde conductie van chloride of verstoorde kanaalopening R117H R334W G85E R347P V minder synthese van het CFTR-eiwit 3849+10KbC࢐T 2789+5G࢐A A455E TABEL 2 Diagnostiek van cystische fibrose test testuitslag klassieke CF* niet-klassieke CFߤ zweettest chlorideconcentratie > 60 mmol/l chlorideconcentratie ࣘ 60 mmol/l neuspotentiaalmeting afwijkend niet-afwijkend CFTR-mutatie-analyse 2 mutaties 2 mutaties CF = cystische fibrose; CFTR = 'cystic fibrosis transporter regulator`-gen. Login to comment

[hide] Newborn screening for cystic fibrosis in Alberta: ... Paediatr Child Health. 2010 Nov;15(9):590-4. Lilley M, Christian S, Hume S, Scott P, Montgomery M, Semple L, Zuberbuhler P, Tabak J, Bamforth F, Somerville MJ
Newborn screening for cystic fibrosis in Alberta: Two years of experience.
Paediatr Child Health. 2010 Nov;15(9):590-4., [PMID:22043142]

Abstract [show]
On April 1, 2007, Alberta became the first province in Canada to introduce cystic fibrosis (CF) to its newborn screening program. The Alberta protocol involves a two-tier algorithm involving an immunoreactive trypsinogen measurement followed by molecular analysis using a CF panel for 39 mutations. Positive screens are followed up with sweat chloride testing and an assessment by a CF specialist. Of the 99,408 newborns screened in Alberta during the first two years of the program, 221 had a positive CF newborn screen. The program subsequently identified and initiated treatment in 31 newborns with CF. A relatively high frequency of the R117H mutation and the M1101K mutation was noted. The M1101K mutation is common in the Hutterite population. The presence of the R117H mutation has created both counselling and management dilemmas. The ability to offer CF transmembrane regulator full sequencing may help resolve diagnostic dilemmas. Counselling and management challenges are created when mutations are mild or of unknown clinical significance.

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46 These include the following mutations: delF508, I507del, G542X, G85E, R117H, 621+1G࢐T, 711+1G࢐T, G551D, R334W, R347P, A455E, 1717-1G࢐A, R560T, R553X, N1303K, 1898+1G࢐A, 2184delA, 2789+5G࢐A, 3120+1G࢐A, R1162X, 3659delC, 3849+10kbC࢐T, W1282X, 1078delT, 394delTT, Y122X, R347H, V520F, A559T, S549N, S549R, 1898+5G࢐T, 2183AA࢐G, 2307insA, Y1092X, M1101K, S1255X, 3876delA and 3905insT. Login to comment

[hide] Development of CFTR Structure. Front Pharmacol. 2012 Sep 6;3:162. doi: 10.3389/fphar.2012.00162. eCollection 2012. Patrick AE, Thomas PJ
Development of CFTR Structure.
Front Pharmacol. 2012 Sep 6;3:162. doi: 10.3389/fphar.2012.00162. eCollection 2012., [PMID:22973227]

Abstract [show]
Cystic fibrosis is a lethal genetic disease caused by lack of functional cystic fibrosis transmembrane conductance regulator (CFTR) proteins at the apical surface of secretory epithelia. CFTR is a multidomain protein, containing five domains, and its functional structure is attained in a hierarchical folding process. Most CF-causing mutations in CFTR, including the most common mutation, a deletion of phenylalanine at position 508 (DeltaF508), are unable to properly fold into this functional native three dimensional structure. Currently, no high-resolution structural information about full length CFTR exists. However, insight has been gained through examining homologous ABC transporter structures, molecular modeling, and high-resolution structures of individual, isolated CFTR domains. Taken together, these studies indicate that the prevalent DeltaF508 mutation disrupts two essential steps during the development of the native structure: folding of the first nucleotide binding domain (NBD1) and its later association with the fourth intracellular loop (ICL4) in the second transmembrane domain (TMD2). Therapeutics to rescue DeltaF508 and other mutants in CFTR can be targeted to correct defects that occur during the complex folding process. This article reviews the structural relationships between CFTR and ABC transporters and current knowledge about how CFTR attains its structure-with a focus on how this process is altered by CF-causing mutations in a manner targetable by therapeutics.

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160 An example of mutants similarly located within CFTR with different local mechanisms of misfolding are the G85E and G91R mutations. Login to comment
163 Recently, G85E was found to dramatically alter the conformation/integration profile of TM1 (Patrick et al., 2011). Login to comment
166 Interestingly, the corrector compound four rescues G91R but not G85E-CFTR (Grove et al., 2009), suggesting the differences in the mutant molecular pathologies may be relevant for their ability to benefit from specific treatments to rescue defective CFTR. Login to comment

[hide] Mechanisms of CFTR Folding at the Endoplasmic Reti... Front Pharmacol. 2012 Dec 13;3:201. doi: 10.3389/fphar.2012.00201. eCollection 2012. Kim SJ, Skach WR
Mechanisms of CFTR Folding at the Endoplasmic Reticulum.
Front Pharmacol. 2012 Dec 13;3:201. doi: 10.3389/fphar.2012.00201. eCollection 2012., [PMID:23248597]

Abstract [show]
In the past decade much has been learned about how Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) folds and misfolds as the etiologic cause of cystic fibrosis (CF). CFTR folding is complex and hierarchical, takes place in multiple cellular compartments and physical environments, and involves several large networks of folding machineries. Insertion of transmembrane (TM) segments into the endoplasmic reticulum (ER) membrane and tertiary folding of cytosolic domains begin cotranslationally as the nascent polypeptide emerges from the ribosome, whereas posttranslational folding establishes critical domain-domain contacts needed to form a physiologically stable structure. Within the membrane, N- and C-terminal TM helices are sorted into bundles that project from the cytosol to form docking sites for nucleotide binding domains, NBD1 and NBD2, which in turn form a sandwich dimer for ATP binding. While tertiary folding is required for domain assembly, proper domain assembly also reciprocally affects folding of individual domains analogous to a jig-saw puzzle wherein the structure of each interlocking piece influences its neighbors. Superimposed on this process is an elaborate proteostatic network of cellular chaperones and folding machineries that facilitate the timing and coordination of specific folding steps in and across the ER membrane. While the details of this process require further refinement, we finally have a useful framework to understand key folding defect(s) caused by DeltaF508 that provides a molecular target(s) for the next generation of CFTR small molecule correctors aimed at the specific defect present in the majority of CF patients.

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66 An important implication of this topogenesis mechanism is highlighted by two CF-causing mutations, G85E and G91R, each of which introduces an additional ionizable residue into TM1. Login to comment
68 Despite achieving correct topology, however, G85E and G91R still disrupt CFTR folding and trafficking (Xiong et al., 1997; Patrick et al., 2011). Login to comment

[hide] PGD for cystic fibrosis patients and couples at ri... Reprod Biomed Online. 2013 May;26(5):420-30. doi: 10.1016/j.rbmo.2013.01.006. Epub 2013 Jan 29. Rechitsky S, Verlinsky O, Kuliev A
PGD for cystic fibrosis patients and couples at risk of an additional genetic disorder combined with 24-chromosome aneuploidy testing.
Reprod Biomed Online. 2013 May;26(5):420-30. doi: 10.1016/j.rbmo.2013.01.006. Epub 2013 Jan 29., [PMID:23523379]

Abstract [show]
Preimplantation genetic diagnosis (PGD) for inherited disorders is presently applied for more than 300 different conditions. The most frequent PGD indication is cystic fibrosis (CF), the largest series of which is reviewed here, totalling 404 PGD cycles. This involved testing for 52 different CFTR mutations with almost half of the cases (195/404 cycles) performed for DeltaF508 mutation, one-quarter (103/404 cycles) for six other frequent mutations and only a few for the remaining 45 CFTR mutations. There were 44 PGD cycles performed for 25 CF-affected homozygous or double-heterozygous CF patients (18 male and seven female partners), which involved testing simultaneously for three mutations, resulting in birth of 13 healthy CF-free children and no misdiagnosis. PGD was also performed for six couples at a combined risk of producing offspring with CF and another genetic disorder. Concomitant testing for CFTR and other mutations resulted in birth of six healthy children, free of both CF and another genetic disorder in all but one cycle. A total of 96 PGD cycles for CF were performed with simultaneous aneuploidy testing, including microarray-based 24-chromosome analysis, as a comprehensive PGD for two or more conditions in the same biopsy material.

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41 Mutation Region Legacy name cDNA name Protein name # of Patient Number of cycles Number of transfers Number of embryos transferred Pregnancy Birth 125G/C c.-8G>C NA Promoter 1 2 2 2 1 (1) 0 E60X c.178G>T p.Glu60X Exon 3 1 1 1 1 0 0 G85E c.254G>A p.Gly85Glu Exon 3 1 1 1 2 1 1 R75Q c.224G>A p.Arg75Gln Exon 3 1 1 1 1 1 1 R75X c.223C>T p.Arg75X Exon 3 1 1 1 2 1 2 A120T c.358G>A p.Ala120Thr Exon 4 1 1 1 1 0 0 R117C c.349C>T p.Arg117Cys Exon 4 2 6 3 5 1 1 R117H c.350G>A p.Arg117His Exon 4 14 22 19 38 8 6 621+1G-T c.489 &#b1; 1G>T - Intron 4 4 7 4 6 2 1 852del22 c.720_741 p.Gly241GlufsX13 Exon 6 1 1 0 0 0 0 L206W c.617T>G p.Leu206Trp Exon 6 1 2 1 2 0 0 A349V c.1046C>T p.Ala349Val Exon 8 1 2 2 4 2 4 1078delT c.948delT p.Phe316LeufsX12 Exon 8 1 1 1 1 1 0 1154ins-TC c.1022_1023insTC p.Phe342HisfsX28 Exon 8 1 2 1 2 0 0 Q359K/T360K c. Login to comment

[hide] Elevated levels of miR-145 correlate with SMAD3 do... J Cyst Fibros. 2013 Dec;12(6):797-802. doi: 10.1016/j.jcf.2013.03.007. Epub 2013 Apr 28. Megiorni F, Cialfi S, Cimino G, De Biase RV, Dominici C, Quattrucci S, Pizzuti A
Elevated levels of miR-145 correlate with SMAD3 down-regulation in cystic fibrosis patients.
J Cyst Fibros. 2013 Dec;12(6):797-802. doi: 10.1016/j.jcf.2013.03.007. Epub 2013 Apr 28., [PMID:23632450]

Abstract [show]
MicroRNAs (miRNAs) have recently emerged as important gene regulators in Cystic Fibrosis (CF), a common monogenic disease characterized by severe infection and inflammation, especially in the airway compartments. In the current study, we show that both miR-145 and miR-494 are significantly up-regulated in nasal epithelial tissues from CF patients compared with healthy controls (p<0.001 and p<0.01, respectively) by Quantitative Real-Time PCR. Only miR-494 levels showed a trend of correlation with reduced CFTR mRNA expression and positive sweat test values, supporting the negative regulatory role of this miRNA on CFTR synthesis. Using computational prediction algorithms and luciferase reporter assays, SMAD family member 3 (SMAD3), a key element of the TGF-beta1 inflammatory pathway, was identified as a target of miR-145. Indeed, miR-145 synthetic mimics suppressed by approximately 40% the expression of a reporter construct containing the SMAD3 3'-UTR. Moreover, we observed an inverse correlation between SMAD3 mRNA expression and miR-145 in CF nasal tissues (r=-0.68, p=0.0018, Pearson's correlation). Taken together, these results confirm the pivotal role of miRNAs in the CF physio-pathogenesis and suggest that miRNA deregulation play a role in the airway disease severity by modulating CFTR levels as well as the expression of important molecules involved in the inflammatory response. miR-494 and miR-145 may, therefore, be potential biomarker and therapeutic target to specific CF clinical manifestations.

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33 CF cases were F508del/F508del homozygotes (11/18) or carried at least one F508del variant: F508del/W1282X (3/18), F508del/N1303K (1/18), F508del/G85E (1/18), F508del/S549R(A N C) (1/18); one individual was homozygote for CFTR mutations different from F508del (R553X/N1303K). Login to comment

[hide] The cystic fibrosis of exocrine pancreas. Cold Spring Harb Perspect Med. 2013 May 1;3(5):a009746. doi: 10.1101/cshperspect.a009746. Wilschanski M, Novak I
The cystic fibrosis of exocrine pancreas.
Cold Spring Harb Perspect Med. 2013 May 1;3(5):a009746. doi: 10.1101/cshperspect.a009746., [PMID:23637307]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) protein is highly expressed in the pancreatic duct epithelia and permits anions and water to enter the ductal lumen. This results in an increased volume of alkaline fluid allowing the highly concentrated proteins secreted by the acinar cells to remain in a soluble state. This work will expound on the pathophysiology and pathology caused by the malfunctioning CFTR protein with special reference to ion transport and acid-base abnormalities both in humans and animal models. We will also discuss the relationship between cystic fibrosis (CF) and pancreatitis, and outline present and potential therapeutic approaches in CF treatment relevant to the pancreas.

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41 A few missense mutations (e.g., G85E) conferavariable pancreatic phenotype. Login to comment

[hide] Clinical and genetic features in patients with cys... Iran J Pediatr. 2013 Apr;23(2):212-5. Farjadian S, Moghtaderi M, Kashef S, Alyasin S, Najib K, Saki F
Clinical and genetic features in patients with cystic fibrosis in southwestern iran.
Iran J Pediatr. 2013 Apr;23(2):212-5., [PMID:23724185]

Abstract [show]
OBJECTIVE: Cystic fibrosis (CF) is a common autosomal recessive genetic disease caused by a mutation in the CF transmembrane conductance regulatory (CFTR) gene. This study attempted to identify the most common CFTR mutations and any correlations between certain mutations and the clinical presentation of the disease in CF patients in southwestern Iran. METHODS: Twenty nine common CFTR gene mutations were examined in 45 CF patients. FINDINGS: Chronic cough, intestinal obstruction, dehydration, heat exhaustion and steatorrhea were the most common early clinical symptoms among our patients. The most common mutation was DeltaF508, with an allele frequency of 21%. The homozygous DeltaF508 mutation was observed in eight patients (18%), and three patients (7%) were DeltaF508 carriers. The 2183AA > G mutation was observed in four patients, one of whom was also a DeltaF508 carrier. The R1162X mutation was detected in two patients. The G542X, R334W and N1303K mutations were detected each in one patient, the first of whom was also a DeltaF508 carrier. CONCLUSION: Out of 45 patients, 27 (60%) had none of the CFTR gene mutations we tested for. The most frequent mutations in southwestern Iranian patients with CF should be identified by sequencing the entire CFTR gene in order to optimize the design of a diagnostic kit for common regional mutations.

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26 Genomic DNA was extracted from 200 &#b5;L of whole blood with the QiaAmp DNA Mini Kit (Qiagen, Valencia, CA, USA) and 29 common CFTR gene mutations (D1152H, 1717-1G>A, G542X, W1282X, N1303K, ࢞F508, 3849+10kbC>T, 394delTT, 621+1G>T, S1251N, G551D, R117H, R1162X, R334W, A455E, 2183AA>G, 3659delC, 1078delT, ࢞I507, R347P, R553X, E60X, 3120+1G>A, 2789+5G>A, 1898+1G>A, 711+1G>T, G85E, 2184delA and R560T) were analyzed with the ELUCIGENE CF29 v. 2 kit using four multiplex PCR. Login to comment

[hide] A comprehensive assay for CFTR mutational analysis... Clin Chem. 2013 Oct;59(10):1481-8. doi: 10.1373/clinchem.2013.206466. Epub 2013 Jun 17. Abou Tayoun AN, Tunkey CD, Pugh TJ, Ross T, Shah M, Lee CC, Harkins TT, Wells WA, Tafe LJ, Amos CI, Tsongalis GJ
A comprehensive assay for CFTR mutational analysis using next-generation sequencing.
Clin Chem. 2013 Oct;59(10):1481-8. doi: 10.1373/clinchem.2013.206466. Epub 2013 Jun 17., [PMID:23775370]

Abstract [show]
BACKGROUND: Cystic fibrosis is a life-threatening genetic disorder that has been associated with mutations in the CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] gene. Hundreds of CFTR mutations have been detected to date. Current CFTR genotyping assays target a subset of these mutations, particularly a mutation panel recommended by the American College of Medical Genetics for carrier screening of the general population. Fast sequencing of the entire coding sequence in a scalable manner could expand the detection of CFTR mutations and facilitate management of costs and turnaround times in the clinical laboratory. METHODS: We describe a proof-of-concept CFTR assay that uses PCR target enrichment and next-generation sequencing on the Ion Torrent Personal Genome Machine (PGM) platform. RESULTS: The scalability of the assay was demonstrated, with an average mean depth of coverage ranging from 500x to 3500x, depending on the number of multiplexed patient samples and the Ion Torrent chip used. In a blinded study of 79 previously genotyped patient DNA samples and cell lines, our assay detected most of the mutations, including single-nucleotide variants, small insertions and deletions, and large copy-number variants. The reproducibility was 100% for detecting mutations in independent runs. Our assay demonstrated high specificity, with only 2 false-positive calls (at 2184delA) found in 2 samples caused by a sequencing error in a homopolymer stretch of sequence. The detection rate for variants of unknown significance was very low in the targeted region. CONCLUSIONS: With continued optimization and system refinements, PGM sequencing promises to be a powerful, rapid, and scalable means of clinical diagnostic sequencing.

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53 of cases af9;/af9; c.1521_1523delCTT; c.1521_1523delCTT èc;F508; èc;F508 CF Yes 97 Dartmouth 1 af9;/af9; c.1521_1523delCTT; c.350Gb0e;A èc;F508; R117H CF Yes 53; 50 Dartmouth 1 af9;/af9; c.350Gb0e;A; c.1477Cb0e;T R117H; Q493*b CF Yes 52; 49 Dartmouth 1 af9;/af9; c.1521_1523delCTT; c.1000Cb0e;T èc;F508; R334W CF Yes 49; 54 Dartmouth 1 af9;/af9; c.1521_1523delCTT; c.489af9;1Gb0e;T èc;F508; 621af9;1Gb0e;T CF Yes 48; 47 Dartmouth 1 af9;/af9; c.1521_1523delCTT; c.1364Cb0e;A èc;F508; A455E CF Yes 51; 46 Dartmouth 1 af9;/af9; c.489af9;1Gb0e;T; c.2988af9;1Gb0e;A 621af9;1Gb0e;T; 3120af9;1Gb0e;A CF Yes 48; 49 Coriell 1 af9;/af9; c.1521_1523delCTT; c.1657Cb0e;T èc;F508; R553* CF Yes 44; 49c Coriell 2 af9;/af9; c.1521_1523delCTT; c.3528delC èc;F508; 3659delC CF Yes 46; 46 Coriell 1 af9;/af9; c.489af9;1Gb0e;T; c.579af9;1Gb0e;T 621af9;1Gb0e;T; 711af9;1Gb0e;T CF Yes 50; 51 Coriell 1 af9;/af9; c.489af9;1Gb0e;T; c.254Gb0e;A 621af9;1Gb0e;T; G85E CF Yes 50; 45 Coriell 1 af9;/af9; c.1521_1523delCTT; c.1679Gb0e;C èc;F508; R560T CF Yes 44; 52 Coriell 1 af9;/af9; c.489af9;1Gb0e;T; c.1364Cb0e;A 621af9;1Gb0e;T; A455E CF Yes 50; 49 Coriell 1 af9;/af9; c.3909Cb0e;G; c.4046Gb0e;A N1303K; G1349D CF Yes 47; 52 Coriell 1 af9;/af9; c.2657af9;5Gb0e;A; c.2657af9;5Gb0e;A 2789af9;5Gb0e;A; 2789af9;5Gb0e;A CF Yes 100 Coriell 1 af9;/af9; c.1040Gb0e;C; c.1652Gb0e;A R347P; G551D CF Yes 51; 49 Coriell 1 af9;/af9; c.1000Cb0e;T; c.3368-2Ab0e;T R334W; 3500-2Ab0e;G CF Yes 53; 45 Coriell 1 af9;/af9; c.254Gb0e;A; c.3454Gb0e;C G85E; D1152H CF Yes 44; 47 Coriell 1 af9;/af9; c.1521_1523delCTT; c.350Gb0e;A èc;F508; R117H CF Yes 49; 50 Coriell 1 af9;/af9; c.1521_1523delCTT; c.54-5940_273af9;10250del21kb èc;F508; CFTRdel2,3 CF Yes 47; N/Ad Coriell 1 af9;/af9; c.1521_1523delCTT; c.1766af9;1Gb0e;A èc;F508; 1898af9;1Gb0e;A CF Yes 47; 50 Coriell 1 af9;/af9; c.1521_1523delCTT; c.2051_2052delAAinsG èc;F508; K684Sfs CF Yes 47; 50 Coriell 1 af9;/af9; c.1521_1523delCTT; c.2052del èc;F508; K684Nfs*38 CF Yes 51; 55 Coriell 1 af9;/afa; c.1521_1523delCTT èc;F508 Carrier Yes 50c Dartmouth 16 af9;/afa; c.1652Gb0e;A G551D Carrier Yes 50c Dartmouth 5 af9;/afa; c.1519_1521delATC èc;I507 Carrier Yes 46 Dartmouth 1 af9;/afa; c.3454Gb0e;C D1152H Carrier Yes 50 Dartmouth 1 af9;/afa; c.1657Cb0e;T R553* Carrier Yes 51 Dartmouth 1 af9;/afa; c.178Gb0e;T E60* Carrier Yes 51 Dartmouth 1 af9;/afa; c.3846Gb0e;A W1282* Carrier Yes 45c Dartmouth 3 af9;/afa; c.1000Cb0e;T R334W Carrier Yes 51 Dartmouth 1 af9;/afa; c.1624Gb0e;T G542* Carrier Yes 47c Dartmouth 4 af9;/afa; c.3484Cb0e;T R1162* Carrier Yes 43 Dartmouth 1 af9;/afa; c.1766af9;1Gb0e;A 1898af9;1Gb0e;A Carrier Yes 57 Dartmouth 1 af9;/afa; c.3773_3774insT 3905insT (L1258Ffs*7) Carrier Yes 37 Dartmouth 1 af9;/afa; c.350Gb0e;A R117H Carrier Yes 50c Dartmouth 3 af9;/afa; c.1645Ab0e;C S549R Ab0e;C Carrier No N/A Dartmouth 1 af9;/afa; c.1040Gb0e;A R347H Carrier Yes 47 Dartmouth 1 af9;/afa; c.3909Cb0e;G N1303K Carrier Yes 46 Dartmouth 1 af9;/afa; c.3718-2477Cb0e;T 3849af9;10kbCb0e;T Carrier Yes 51 Coriell 1 af9;/afa; c.2988af9;1Gb0e;A 3120af9;1Gb0e;A Carrier Yes 49 Coriell 1 af9;/afa; c.489af9;1Gb0e;T 621af9;1Gb0e;T Carrier Yes 50 Coriell 1 af9;/afa; c.1585-1Gb0e;A 1717-1Gb0e;A Carrier Yes 51 Coriell 1 afa;/afa;e N/Af N/A Normal N/A N/A Dartmouth 9 a af9;/af9;, 2 pathogenic mutations; af9;/afa;, carrier of a single pathogenic mutation; afa;/afa;, absence of any pathogenic mutations. Login to comment
115 Mutation cDNA position Mutation class Sensitivity (TPa ) Specificity (TN) Accuracyb G85E c.254Gb0e;A Missense Detected (2/2) 100% (77/77) 100% R117H c.350Gb0e;A Missense Detected (6/6) 100% (73/73) 100% 621af9;1Gb0e;T c.489af9;1Gb0e;T Splice site Detected (6/6) 100% (73/73) 100% 711af9;1Gb0e;T c.579af9;1Gb0e;T Splice site Detected (1/1) 100% (78/78) 100% R334W c.1000Cb0e;T Missense Detected (3/3) 100% (76/76) 100% R347P c.1040Gb0e;C Missense Detected (1/1) 100% (78/78) 100% A455E c.1364Cb0e;A Missense Detected (2/2) 100% (77/77) 100% èc;I507 c.1519_1521delATC In-frame deletion Detected (1/1) 100% (78/78) 100% èc;F508 c.1521_1523delCTT In-frame deletion Detected (30/30) 100% (49/49) 100% G542* c.1624Gb0e;T Nonsense Detected (4/4) 100% (75/75) 100% G551D c.1652Gb0e;A Missense Detected (6/6) 100% (73/73) 100% R553* c.1657Cb0e;T Nonsense Detected (3/3) 100% (76/76) 100% R560T c.1679Gb0e;C Missense Detected (1/1) 100% (78/78) 100% 1898af9;1Gb0e;A c.1766af9;1Gb0e;A Splice site Detected (2/2) 100% (77/77) 100% 2789af9;5Gb0e;A c.2657af9;5Gb0e;A Splice site Detected (1/1) 100% (78/78) 100% 3120af9;1Gb0e;A c.2988af9;1Gb0e;A Splice site Detected (2/2) 100% (77/77) 100% R1162* c.3484Cb0e;T Nonsense Detected (1/1) 100% (78/78) 100% 3659delC c.3528del Frameshift deletion Detected (1/1) 100% (78/78) 100% 3849af9;10kbCb0e;T c.3718-2477Cb0e;T Splice site Detected (1/1) 100% (78/78) 100% W1282* c.3846Gb0e;A Nonsense Detected (3/3) 100% (75/75) 100% N1303K c.3909Cb0e;G Missense Detected (1/1) 100% (78/78) 100% 2184delA c.2052del Frameshift deletion Detected (1/1) 97% (76/78) 97% 1717-1Gb0e;A c.1585-1Gb0e;A Splice site Detected (1/1) 100% (78/78) 100% a TP, true-positive rate; TN, true-negative rate. Login to comment

[hide] Novel CFTR variants identified during the first 3 ... J Mol Diagn. 2013 Sep;15(5):710-22. doi: 10.1016/j.jmoldx.2013.05.006. Epub 2013 Jun 28. Prach L, Koepke R, Kharrazi M, Keiles S, Salinas DB, Reyes MC, Pian M, Opsimos H, Otsuka KN, Hardy KA, Milla CE, Zirbes JM, Chipps B, O'Bra S, Saeed MM, Sudhakar R, Lehto S, Nielson D, Shay GF, Seastrand M, Jhawar S, Nickerson B, Landon C, Thompson A, Nussbaum E, Chin T, Wojtczak H
Novel CFTR variants identified during the first 3 years of cystic fibrosis newborn screening in California.
J Mol Diagn. 2013 Sep;15(5):710-22. doi: 10.1016/j.jmoldx.2013.05.006. Epub 2013 Jun 28., [PMID:23810505]

Abstract [show]
California uses a unique method to screen newborns for cystic fibrosis (CF) that includes gene scanning and DNA sequencing after only one California-40 cystic fibrosis transmembrane conductance regulator (CFTR) panel mutation has been identified in hypertrypsinogenemic specimens. Newborns found by sequencing to have one or more additional mutations or variants (including novel variants) in the CFTR gene are systematically followed, allowing for prospective assessment of the pathogenic potential of these variants. During the first 3 years of screening, 55 novel variants were identified. Six of these novel variants were discovered in five screen-negative participants and three were identified in multiple unrelated participants. Ten novel variants (c.2554_2555insT, p.F1107L, c.-152G>C, p.L323P, p.L32M, c.2883_2886dupGTCA, c.2349_2350insT, p.K114del, c.-602A>T, and c.2822delT) were associated with a CF phenotype (42% of participants were diagnosed at 4 to 25 months of age), whereas 26 were associated with CFTR-related metabolic syndrome to date. Associations with the remaining novel variants were confounded by the presence of other diseases or other mutations in cis or by inadequate follow-up. These findings have implications for how CF newborn screening and follow-up is conducted and will help guide which genotypes should, and which should not, be considered screen positive for CF in California and elsewhere.

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26 Newborns were screened using the California method, which includes i) analysis of serum immunoreactive trypsinogen (IRT) levels using the AutoDELFIA neonatal IRT L kit (PerkinElmer, Waltham, MA) in all newborn blood spot specimens, ii) CFTR mutation panel [29-40 mutations (the mutations on the California panel were selected for the most part according to allelic frequencies found in a comprehensively genotyped group of California CF cases to achieve a 95% race/ethnicity-specific rate of CF case detection in black, white, and Hispanic individuals in California and include c.1585-1G>A, c.1680-1G>A, c.1973-1985del13insAGAAA, c.2175_2176insA, c.164 &#fe; 2T>A (removed on August 12, 2008), c.2988 &#fe; 1G>A, c.3717 &#fe; 12191C>T, c.3744delA, c.274-1G>A, c.489 &#fe; 1G>T, c.579 &#fe; 1G>T, p.A559T, p.F311del, p.F508del, p.I507del, p.G542X, p.G551D, p.G85E, p.H199Y, p.N1303K, p.R1066C, p.R1162X, p.R334W, p.R553X, p.S549N, p.W1089X, p.W1204X (c.3611G>A), p.W1282X, c.1153_1154insAT [added October 4, 2007], c.1923_1931del9insA, c.3140-26A>G, c.531delT, c.803delA, c.54-5940_273 &#fe; 10250del21kb, p.P205S, p.Q98R, p.R75X, p.S492F [added December 12, 2007], c.3659delC, p.G330X, p.W1204X [c.3612G>A] [added August 12, 2008] [Signature CF 2.0 ASR; Asuragen Inc., Austin, TX])] testing of specimens with IRT 62 ng/mL (highest 1.5%), iii) CFTR gene scanning and sequence analysis (Ambry Test: CF; Ambry Genetics, Aliso Viejo, CA) for specimens found to have only one mutation after CFTR mutation panel testing, and iv) referral to 1 of 15 pediatric CF care centers (CFCs) for sweat chloride (SC) testing and follow-up of all newborns with either two CFTR mutations detected during panel testing or one CFTR mutation detected during panel testing and one (or more) additional CFTR mutation and/or variant detected during sequencing. Login to comment
184 Parental testing for participants 45, 46, and 59, all diagnosed as CFTR carriers, showed the following mutation pairs to be in cis: p.G85E with c.744-15T>C (novel), p.N1303K with c.2490 &#fe; 14G>A (novel), and c.164 &#fe; 4T>A (novel) with p.G1173S (novel), respectively. Login to comment

[hide] Genetic testing of sperm donors for cystic fibrosi... Eur J Obstet Gynecol Reprod Biol. 2013 Sep;170(1):183-7. doi: 10.1016/j.ejogrb.2013.06.022. Epub 2013 Jul 15. Landaburu I, Gonzalvo MC, Clavero A, Ramirez JP, Yoldi A, Mozas J, Zamora S, Martinez L, Castilla JA
Genetic testing of sperm donors for cystic fibrosis and spinal muscular atrophy: evaluation of clinical utility.
Eur J Obstet Gynecol Reprod Biol. 2013 Sep;170(1):183-7. doi: 10.1016/j.ejogrb.2013.06.022. Epub 2013 Jul 15., [PMID:23866907]

Abstract [show]
OBJECTIVE: To evaluate the clinical utility of genetic testing for cystic fibrosis (CF) and spinal muscular atrophy (SMA) in sperm donors. STUDY DESIGN: We studied the results of the genetic tests for CF and SMA applied to 372 sperm donor candidates. The CF carrier screening test analysed 32 mutations on the CFTR gene. Regarding SMA, the carrier test studied possible deletions of SMN1/2 by Multiplex Ligation-dependent Probe Amplification (MLPA) methodology. RESULTS: The carrier frequency obtained was greater for SMA than for CF. After adjusting the results obtained for the sensitivity of the tests, and taking into account the prevalence of female carriers in our population, the probability of transmission of the disease to the child from a donor with a negative genetic test was about five times lower in the case of SMA than in CF, although this difference was not statistically significant. The number of donors needed to screen (NNS) to avoid the occurrence of a child being affected by CF and SMA in our population was similar in both cases (1591 vs. 1536). CONCLUSIONS: This study demonstrates the need to include SMA among the diseases for which genetic screening is performed in the process of sperm donor selection. We believe that testing donors for SMA is as important and as useful as doing so for CF.

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51 The panel of mutations studied was: S549N, S549R, R553X, G551D, V520F, I507del, F508del, 3876delA, 1717-1G->A, G542X, R560T, 3120+1G->A, A455E, R117H, 394delTT, 2183AA- >G, 2184delA, 2789+5G->A, 1898+1G->A, 621+1G->T, 711+1G- >T, G85E, R347P, R347H, W1282X, R334W, 1078delT, 3849+10kbC->T, R1162X, N1303K, 3659delC, 3905insT. Login to comment

[hide] Effect of ivacaftor on CFTR forms with missense mu... J Cyst Fibros. 2014 Jan;13(1):29-36. doi: 10.1016/j.jcf.2013.06.008. Epub 2013 Jul 23. Van Goor F, Yu H, Burton B, Hoffman BJ
Effect of ivacaftor on CFTR forms with missense mutations associated with defects in protein processing or function.
J Cyst Fibros. 2014 Jan;13(1):29-36. doi: 10.1016/j.jcf.2013.06.008. Epub 2013 Jul 23., [PMID:23891399]

Abstract [show]
BACKGROUND: Ivacaftor (KALYDECO, VX-770) is a CFTR potentiator that increased CFTR channel activity and improved lung function in patients age 6 years and older with CF who have the G551D-CFTR gating mutation. The aim of this in vitro study was to evaluate the effect of ivacaftor on mutant CFTR protein forms with defects in protein processing and/or channel function. METHODS: The effect of ivacaftor on CFTR function was tested in electrophysiological studies using a panel of Fischer rat thyroid (FRT) cells expressing 54 missense CFTR mutations that cause defects in the amount or function of CFTR at the cell surface. RESULTS: Ivacaftor potentiated multiple mutant CFTR protein forms that produce functional CFTR at the cell surface. These included mutant CFTR forms with mild defects in CFTR processing or mild defects in CFTR channel conductance. CONCLUSIONS: These in vitro data indicated that ivacaftor is a broad acting CFTR potentiator and could be used to help stratify patients with CF who have different CFTR genotypes for studies investigating the potential clinical benefit of ivacaftor.

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No. Sentence Comment
44 None M1V A46D E56K P67L R74W G85E E92K D110E D110H R117C R117H E193K L206W R334W I336K T338I S341P R347H R347P R352Q A455E L467P S492F F508del V520F A559T R560S R560T A561E Y569D D579G R668C L927P S945L S977F L997F F1052V H1054D K1060T L1065P R1066C R1066H R1066M A1067T R1070Q R1070W F1074L L1077P H1085R M1101K D1152H S1235R D1270N N1303K 0 100 200 300 400 500 600 * * * CFTR Mutation mRNA (% Normal CFTR) Fig. 1. Login to comment
64 Mutant CFTR form CFTR processing Mature/total % Normal CFTR Normal 0.89 &#b1; 0.01 100.0 &#b1; 18.5 G85E -0.05 &#b1; 0.04 -1.0 &#b1; 0.9 R560S 0.00 &#b1; 0.00 0.0 &#b1; 0.0 R1066C 0.02 &#b1; 0.01 0.0 &#b1; 0.0 S492F 0.00 &#b1; 0.00 0.1 &#b1; 0.1 R560T 0.01 &#b1; 0.01 0.2 &#b1; 0.1 V520F 0.05 &#b1; 0.03 0.3 &#b1; 0.2 M1101K 0.05 &#b1; 0.03 0.3 &#b1; 0.1 A561E 0.08 &#b1; 0.04 0.5 &#b1; 0.2 R1066M 0.02 &#b1; 0.02 0.5 &#b1; 0.4 N1303K 0.02 &#b1; 0.02 0.5 &#b1; 0.3 A559T 0.16 &#b1; 0.09 0.6 &#b1; 0.2 M1V 0.06 &#b1; 0.06 0.7 &#b1; 0.6 Y569D 0.11 &#b1; 0.04 0.6 &#b1; 0.2 R1066H 0.08 &#b1; 0.02a 0.7 &#b1; 0.2a L1065P 0.05 &#b1; 0.05 1.0 &#b1; 0.8 L467P 0.10 &#b1; 0.07 1.2 &#b1; 0.8 L1077P 0.08 &#b1; 0.04 1.5 &#b1; 0.6 A46D 0.21 &#b1; 0.08 1.9 &#b1; 0.5a E92K 0.06 &#b1; 0.05 1.9 &#b1; 1.3 H1054D 0.09 &#b1; 0.04 1.9 &#b1; 0.8 F508del 0.09 &#b1; 0.02a 2.3 &#b1; 0.5a H1085R 0.06 &#b1; 0.01a 3.0 &#b1; 0.7a I336K 0.42 &#b1; 0.05a 6.5 &#b1; 0.7a L206W 0.35 &#b1; 0.10a 6.8 &#b1; 1.7a F1074L 0.52 &#b1; 0.03a 10.9 &#b1; 0.6a A455E 0.26 &#b1; 0.10a 11.5 &#b1; 2.5a E56K 0.29 &#b1; 0.04a 12.2 &#b1; 1.5a R347P 0.48 &#b1; 0.04a 14.6 &#b1; 1.8a R1070W 0.61 &#b1; 0.04a 16.3 &#b1; 0.6a P67L 0.36 &#b1; 0.04a 28.4 &#b1; 6.8a R1070Q 0.90 &#b1; 0.01a 29.5 &#b1; 1.4a S977F 0.97 &#b1; 0.01a 37.3 &#b1; 2.4a A1067T 0.78 &#b1; 0.03a 38.6 &#b1; 6.1a D579G 0.72 &#b1; 0.02a 39.3 &#b1; 3.1a D1270N 1.00 &#b1; 0.00a,c 40.7 &#b1; 1.2a S945L 0.65 &#b1; 0.04a 42.4 &#b1; 8.9a L927P 0.89 &#b1; 0.01a,b 43.5 &#b1; 2.5a,b R117C 0.87 &#b1; 0.02a,b 49.1 &#b1; 2.9a,b T338I 0.93 &#b1; 0.03a,b 54.2 &#b1; 3.7a,b L997F 0.90 &#b1; 0.04a,b 59.8 &#b1; 10.4a,b D110H 0.97 &#b1; 0.01a,b 60.6 &#b1; 1.5a,b S341P 0.79 &#b1; 0.02a 65.0 &#b1; 4.9a,b R668C 0.94 &#b1; 0.03a,b 68.5 &#b1; 1.9a,b R74W 0.78 &#b1; 0.01a 69.0 &#b1; 2.7a,b D110E 0.92 &#b1; 0.05a,b 87.5 &#b1; 9.5a,b R334W 0.91 &#b1; 0.05a,b 97.6 &#b1; 10.0a,b K1060T 0.87 &#b1; 0.02a,b 109.9 &#b1; 28.0a,b R347H 0.96 &#b1; 0.02a,c 120.7 &#b1; 2.8a,b S1235R 0.96 &#b1; 0.00a,c 139.0 &#b1; 9.0a,b E193K 0.84 &#b1; 0.02a,b 143.0 &#b1; 17.1a,b R117H 0.86 &#b1; 0.01a,b 164.5 &#b1; 34.2a,b R352Q 0.98 &#b1; 0.01a,b 179.9 &#b1; 8.0a,c F1052V 0.90 &#b1; 0.01a,b 189.9 &#b1; 33.1a,b D1152H 0.96 &#b1; 0.02a,c 312.0 &#b1; 45.5a,b Notes to Table 1: Quantification of steady-state CFTR maturation expressed as the mean (&#b1;SEM; n = 5-9) ratio of mature CFTR to total CFTR (immature plus mature) or level of mature mutant CFTR relative to mature normal-CFTR (% normal CFTR) in FRT cells individually expressing CFTR mutations. Login to comment
74 Because the level of CFTR mRNA was similar across the panel of cell lines tested, the range in baseline activity and ivacaftor response likely reflects the severity of the functional defect and/or the 0 50 100 150 200 S341P R347P L467P S492F A559T A561E Y569D L1065P R1066C R1066M L1077P M1101K N1303K R560S L927P R560T H1085R V520F E92K M1V F508del H1054D I336K A46D G85E R334W T338I R1066H R352Q R117C L206W R347H S977F S945L A455E F1074L E56K P67L R1070W D110H D579G D110E R1070Q L997F A1067T E193K R117H R74W K1060T R668C D1270N D1152H S1235R F1052V Baseline With ivacaftor * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Chloride transport (% Normal) Mutant CFTR form 0 100 200 300 400 S341P R347P L467P S492F A559T A561E Y569D L1065P R1066C R1066M L1077P M1101K N1303K R560S L927P R560T H1085R V520F E92K M1V F508del H1054D I336K A46D G85E R334W T338I R1066H R352Q R117C L206W R347H S977F S945L A455E F1074L P67L E56K R1070W D110H D579G D110E R1070Q L997F A1067T E193K R117H R74W K1060T R668C D1270N D1152H S1235R F1052V * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Mature CFTR (% Normal) Mutant CFTR form A B Fig. 2. Login to comment
82 Mutation Patientsa Chloride transport (bc;A/cm2 ) Chloride transport (% normal) EC50 Baseline With ivacaftor Baseline With ivacaftor Fold increase over baselineb Normal 204.5 &#b1; 33.3 301.3 &#b1; 33.8c 100.0 &#b1; 16.3 147.3 &#b1; 16.5c 1.5 266 &#b1; 42 G551D 1282 1.5 &#b1; 0.7 113.2 &#b1; 13.0c 1.0 &#b1; 0.5 55.3 &#b1; 6.3c 55.3 312 &#b1; 73 F1052V 12 177.3 &#b1; 13.7 410.2 &#b1; 11.3c 86.7 &#b1; 6.7 200.7 &#b1; 5.6c 2.3 177 &#b1; 14 S1235R ND 160.6 &#b1; 25.7 352.1 &#b1; 43.4c 78.5 &#b1; 12.6 172.2 &#b1; 21.2c 2.2 282 &#b1; 104 D1152H 185 117.3 &#b1; 23.0 282.7 &#b1; 46.9c 57.4 &#b1; 11.2 138.2 &#b1; 22.9c 2.4 178 &#b1; 67 D1270N 32 109.5 &#b1; 20.5 209.5 &#b1; 27.4c 53.6 &#b1; 10.0 102.4 &#b1; 13.4c 1.9 254 &#b1; 56 R668C 45 99.0 &#b1; 9.4 217.6 &#b1; 11.7c 48.4 &#b1; 4.6 106.4 &#b1; 5.7c 2.2 517 &#b1; 105 K1060T ND 89.0 &#b1; 9.8 236.4 &#b1; 20.3c 43.5 &#b1; 4.8 115.6 &#b1; 9.9c 2.7 131 &#b1; 73 R74W 25 86.8 &#b1; 26.9 199.1 &#b1; 16.8c 42.5 &#b1; 13.2 97.3 &#b1; 8.2c 2.3 162 &#b1; 17 R117H 739 67.2 &#b1; 13.3 274.1 &#b1; 32.2c 32.9 &#b1; 6.5 134.0 &#b1; 15.7c 4.1 151 &#b1; 14 E193K ND 62.2 &#b1; 9.8 379.1 &#b1; 1.1c 30.4 &#b1; 4.8 185.4 &#b1; 1.0c 6.1 240 &#b1; 20 A1067T ND 55.9 &#b1; 3.2 164.0 &#b1; 9.7c 27.3 &#b1; 1.6 80.2 &#b1; 4.7c 2.9 317 &#b1; 214 L997F 27 43.7 &#b1; 3.2 145.5 &#b1; 4.0c 21.4 &#b1; 1.6 71.2 &#b1; 2.0c 3.3 162 &#b1; 12 R1070Q 15 42.0 &#b1; 0.8 67.3 &#b1; 2.9c 20.6 &#b1; 0.4 32.9 &#b1; 1.4c 1.6 164 &#b1; 20 D110E ND 23.3 &#b1; 4.7 96.4 &#b1; 15.6c 11.4 &#b1; 2.3 47.1 &#b1; 7.6c 4.1 213 &#b1; 51 D579G 21 21.5 &#b1; 4.1 192.0 &#b1; 18.5c 10.5 &#b1; 2.0 93.9 &#b1; 9.0c 8.9 239 &#b1; 48 D110H 30 18.5 &#b1; 2.2 116.7 &#b1; 11.3c 9.1 &#b1; 1.1 57.1 &#b1; 5.5c 6.2 249 &#b1; 59 R1070W 13 16.6 &#b1; 2.6 102.1 &#b1; 3.1c 8.1 &#b1; 1.3 49.9 &#b1; 1.5c 6.2 158 &#b1; 48 P67L 53 16.0 &#b1; 6.7 88.7 &#b1; 15.7c 7.8 &#b1; 3.3 43.4 &#b1; 7.7c 5.6 195 &#b1; 40 E56K ND 15.8 &#b1; 3.1 63.6 &#b1; 4.4c 7.7 &#b1; 1.5 31.1 &#b1; 2.2c 4.0 123 &#b1; 33 F1074L ND 14.0 &#b1; 3.4 43.5 &#b1; 5.4c 6.9 &#b1; 1.6 21.3 &#b1; 2.6c 3.1 141 &#b1; 19 A455E 120 12.9 &#b1; 2.6 36.4 &#b1; 2.5c 6.3 &#b1; 1.2 17.8 &#b1; 1.2c 2.8 170 &#b1; 44 S945L 63 12.3 &#b1; 3.9 154.9 &#b1; 47.6c 6.0 &#b1; 1.9 75.8 &#b1; 23.3c 12.6 181 &#b1; 36 S977F 9 11.3 &#b1; 6.2 42.5 &#b1; 19.1c 5.5 &#b1; 3.0 20.8 &#b1; 9.3c 3.8 283 &#b1; 36 R347H 65 10.9 &#b1; 3.3 106.3 &#b1; 7.6c 5.3 &#b1; 1.6 52.0 &#b1; 3.7c 9.8 280 &#b1; 35 L206W 81 10.3 &#b1; 1.7 36.4 &#b1; 2.8c 5.0 &#b1; 0.8 17.8 &#b1; 1.4c 3.6 101 &#b1; 13 R117C 61 5.8 &#b1; 1.5 33.7 &#b1; 7.8c 2.9 &#b1; 0.7 16.5 &#b1; 3.8c 5.7 380 &#b1; 136 R352Q 46 5.5 &#b1; 1.0 84.5 &#b1; 7.8c 2.7 &#b1; 0.5 41.3 &#b1; 3.8c 15.2 287 &#b1; 75 R1066H 29 3.0 &#b1; 0.3 8.0 &#b1; 0.8c 1.5 &#b1; 0.1 3.9 &#b1; 0.4c 2.6 390 &#b1; 179 T338I 54 2.9 &#b1; 0.8 16.1 &#b1; 2.4c 1.4 &#b1; 0.4 7.9 &#b1; 1.2c 5.6 334 &#b1; 38 R334W 150 2.6 &#b1; 0.5 10.0 &#b1; 1.4c 1.3 &#b1; 0.2 4.9 &#b1; 0.7c 3.8 259 &#b1; 103 G85E 262 1.6 &#b1; 1.0 1.5 &#b1; 1.2 0.8 &#b1; 0.5 0.7 &#b1; 0.6 NS NS A46D ND 2.0 &#b1; 0.6 1.1 &#b1; 1.1 1.0 &#b1; 0.3 0.5 &#b1; 0.6 NS NS I336K 29 1.8 &#b1; 0.2 7.4 &#b1; 0.1c 0.9 &#b1; 0.1 3.6 &#b1; 0.1c 4 735 &#b1; 204 H1054D ND 1.7 &#b1; 0.3 8.7 &#b1; 0.3c 0.8 &#b1; 0.1 4.2 &#b1; 0.1c 5.3 187 &#b1; 20 F508del 29,018 0.8 &#b1; 0.6 12.1 &#b1; 1.7c 0.4 &#b1; 0.3 5.9 &#b1; 0.8c 14.8 129 &#b1; 38 M1V 9 0.7 &#b1; 1.4 6.5 &#b1; 1.9c 0.4 &#b1; 0.7 3.2 &#b1; 0.9c 8.0 183 &#b1; 85 E92K 14 0.6 &#b1; 0.2 4.3 &#b1; 0.8c 0.3 &#b1; 0.1 2.1 &#b1; 0.4c 7.0 198 &#b1; 46 V520F 58 0.4 &#b1; 0.2 0.5 &#b1; 0.2 0.2 &#b1; 0.1 0.2 &#b1; 0.1 NS NS H1085R ND 0.3 &#b1; 0.2 2.1 &#b1; 0.4 0.2 &#b1; 0.1 1.0 &#b1; 0.2 NS NS R560T 180 0.3 &#b1; 0.3 0.5 &#b1; 0.5 0.1 &#b1; 0.1 0.2 &#b1; 0.2 NS NS L927P 15 0.2 &#b1; 0.1 10.7 &#b1; 1.7c 0.1 &#b1; 0.1 5.2 &#b1; 0.8c 52.0 313 &#b1; 66 R560S ND 0.0 &#b1; 0.1 -0.2 &#b1; 0.2 0.0 &#b1; 0.0 -0.1 &#b1; 0.1 NS NS N1303K 1161 0.0 &#b1; 0.0 1.7 &#b1; 0.3 0.0 &#b1; 0.0 0.8 &#b1; 0.2 NS NS M1101K 79 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS L1077P 42 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS R1066M ND 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS R1066C 100 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS L1065P 25 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS Y569D 9 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS A561E ND 0.0 &#b1; 0.1 0.0 &#b1; 0.1 0.0 &#b1; 0.0 0.0 &#b1; 0.1 NS NS A559T 43 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS S492F 16 0.0 &#b1; 0.0 1.7 &#b1; 1.2 0.0 &#b1; 0.0 0.8 &#b1; 0.6 NS NS L467P 16 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS R347P 214 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 0.0 &#b1; 0.0 NS NS S341P 9 0.0 &#b1; 0.0 0.2 &#b1; 0.2 0.0 &#b1; 0.0 0.1 &#b1; 0.1 NS NS a Number of individuals with the individual mutation in the CFTR-2 database (www.CFTR2.org). Login to comment

[hide] Cystic fibrosis carrier screening in a North Ameri... Genet Med. 2014 Jul;16(7):539-46. doi: 10.1038/gim.2013.188. Epub 2013 Dec 19. Zvereff VV, Faruki H, Edwards M, Friedman KJ
Cystic fibrosis carrier screening in a North American population.
Genet Med. 2014 Jul;16(7):539-46. doi: 10.1038/gim.2013.188. Epub 2013 Dec 19., [PMID:24357848]

Abstract [show]
PURPOSE: The aim of this study was to compare the mutation frequency distribution for a 32-mutation panel and a 69-mutation panel used for cystic fibrosis carrier screening. Further aims of the study were to examine the race-specific detection rates provided by both panels and to assess the performance of extended panels in large-scale, population-based cystic fibrosis carrier screening. Although genetic screening for the most common CFTR mutations allows detection of nearly 90% of cystic fibrosis carriers, the large number of other mutations, and their distribution within different ethnic groups, limits the utility of general population screening. METHODS: Patients referred for cystic fibrosis screening from January 2005 through December 2010 were tested using either a 32-mutation panel (n = 1,601,308 individuals) or a 69-mutation panel (n = 109,830). RESULTS: The carrier frequencies observed for the 69-mutation panel study population (1/36) and Caucasian (1/27) and African-American individuals (1/79) agree well with published cystic fibrosis carrier frequencies; however, a higher carrier frequency was observed for Hispanic-American individuals (1/48) using the 69-mutation panel as compared with the 32-mutation panel (1/69). The 69-mutation panel detected ~20% more mutations than the 32-mutation panel for both African-American and Hispanic-American individuals. CONCLUSION: Expanded panels using race-specific variants can improve cystic fibrosis carrier detection rates within specific populations. However, it is important that the pathogenicity and the relative frequency of these variants are confirmed.

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63 This threshold could not be reached Table 1ߒ CFTR allele frequency identified by the CF32 mutation panel Varianta Number of detected alleles Mutation (%) Legacy nomenclature HGVS nomenclature F508delb p.F508del 31,142 68.69 R117Hb p.R117H 5,198 11.46 G542Xb p.G542X 1,162 2.56 G551Db p.G551D 989 2.18 W1282Xb p.W1282X 824 1.82 3120ߙ+ߙ1G>Ab c.2988ߙ+ߙ1G>A 706 1.56 N1303Kb p.N1303K 648 1.43 R553Xb p.R553X 487 1.07 3849ߙ+ߙ10kbC>Tb c.3717ߙ+ߙ12191C>T 436 0.96 621ߙ+ߙ1G>Tb c.489ߙ+ߙ1G>T 410 0.90 1717-1G>Ab c.1585-1G>A 388 0.86 2789ߙ+ߙ5G>Ab c.2657ߙ+ߙ5G>A 382 0.84 I507delb p.I507del 258 0.57 R334Wb p.R334W 257 0.57 R1162Xb p.R1162X 211 0.47 G85Eb p.G85E 199 0.44 1898ߙ+ߙ1G>Ab c.1766ߙ+ߙ1G>A 170 0.37 R347Hc p.R347H 160 0.35 3659delCb c.3528delC 155 0.34 3876delAc c.3744delA 153 0.34 R560Tb p.R560T 132 0.29 S549Nc p.S549N 125 0.28 3905insTc c.3773dupT 121 0.27 R347Pb p.R347P 117 0.26 2184delAb c.2052delA 107 0.24 A455Eb p.A455E 106 0.23 711ߙ+ߙ1G>Tb c.579ߙ+ߙ1G>T 65 0.14 394delTTc c.262_263delTT 56 0.12 V520Fc p.V520F 54 0.12 1078delTc c.948delT 52 0.11 2183AA>Ga,c c.2051_2052delAAinsG 37 0.08 S549Rc p.S549R 31 0.07 Total 45,338 100 a 2183AA>G variant was added to the panel in 2010. b Variants from ACMG/ACOG CF screening panel. c Classified as a CF-causing mutation by the CFTR2 Database. ACMG, American College of Medical Genetics and Genomics; ACOG, American College of Obstetricians and Gynecologists; CF, cystic fibrosis; HGVS, Human Genome Variation Society. Table 2ߒ Continued on next page Table 2ߒ CFTR allele frequency identified by the CF69 mutation panel Varianta Allele frequency Mutation (%) Legacy nomenclature HGVS nomenclature F508delb p.F508del 1,868 60.49 R117Hb p.R117H 274 8.87 D1152Hc p.D1152H 125 4.05 G542Xb p.G542X 98 3.17 L206Wd p.L206W 73 2.36 3120ߙ+ߙ1G>Ab c.2988ߙ+ߙ1G>A 65 2.10 G551Db p.G551D 47 1.52 N1303Kb p.N1303K 42 1.36 W1282Xb p.W1282X 38 1.23 3849ߙ+ߙ10kbC>Tb c.3717ߙ+ߙ12191C>T 28 0.91 3876delAd c.3744delA 28 0.91 F311dele p.F312del 24 0.78 I507delb p.I507del 24 0.78 R553Xb p.R553X 24 0.78 R117Cd p.R117C 22 0.71 621ߙ+ߙ1G>Tb c.489ߙ+ߙ1G>T 21 0.68 1717-1G>Ab c.1585-1G>A 18 0.58 S549Nd p.S549N 18 0.58 R334Wb p.R334W 17 0.55 2789ߙ+ߙ5G>Ab c.2657ߙ+ߙ5G>A 16 0.52 G85Eb p.G85E 14 0.45 3199del6e c.3067_3072delATAGTG 12 0.39 R1066Cd p.R1066C 11 0.36 1898ߙ+ߙ1G>Ab c.1766ߙ+ߙ1G>A 10 0.32 R347Hd p.R347H 10 0.32 R1162 Xb p.R1162X 9 0.29 W1089Xd p.W1089X 9 0.29 2184delAb c.2052delA 8 0.26 2307insAd c.2175dupA 8 0.26 1078delTd c.948delT 7 0.23 R75Xd p.R75X 7 0.23 3120G>Ad c.2988 G>A 6 0.19 3659delCb c.3528delC 6 0.19 Q493Xd p.Q493X 6 0.19 R1158Xd p.R1158X 6 0.19 R560Tb p.R560T 6 0.19 1812-1G>Ad c.1680-1G>A 5 0.16 2055del9>Ad c.1923_1931del9insA 5 0.16 406-1G>Ad c.274-1G>A 5 0.16 A559Td p.A559T 5 0.16 R347Pb p.R347P 5 0.16 S1255Xd p.S1255X 5 0.16 1677delTAd c.1545_1546delTA 4 0.13 711ߙ+ߙ1G>Tb c.579ߙ+ߙ1G>T 4 0.13 E60Xd p.E60X 4 0.13 R352Qd p.R352Q 4 0.13 Y1092Xd p.Y1092X 4 0.13 2183AA>Gd c.2051_2052delAAinsG 3 0.10 3791delCd c.3659delC 3 0.10 3905insTd c.3773dupT 3 0.10 by 10 variants: the 2143delT, A455E, S549R, Y122X, and M1101K mutations, typically observed in Caucasians; 935delA, 2869insG, and Q890X in Hispanics; and 405+3A>C and G480C in the African-American population. Login to comment

[hide] The relative frequency of CFTR mutation classes in... J Cyst Fibros. 2014 Jul;13(4):403-9. doi: 10.1016/j.jcf.2013.12.003. Epub 2014 Jan 16. De Boeck K, Zolin A, Cuppens H, Olesen HV, Viviani L
The relative frequency of CFTR mutation classes in European patients with cystic fibrosis.
J Cyst Fibros. 2014 Jul;13(4):403-9. doi: 10.1016/j.jcf.2013.12.003. Epub 2014 Jan 16., [PMID:24440181]

Abstract [show]
More than 1900 different mutations in the CFTR gene have been reported. These are grouped into classes according to their effect on the synthesis and/or function of the CFTR protein. CFTR repair therapies that are mutation or mutation class specific are under development. To progress efficiently in the clinical phase of drug development, knowledge of the relative frequency of CFTR mutation classes in different populations is useful. Therefore, we describe the mutation class spectrum in 25,394 subjects with CF from 23 European countries. In 18/23 countries, 80% or more of the patients had at least one class II mutation, explained by F508del being by far the most frequent mutation. Overall 16.4% of European patients had at least one class I mutation but this varied from 3 countries with more than 30% to 4 countries with less than 10% of subjects. Overall only respectively 3.9, 3.3 and 3.0% of European subjects had at least one mutation of classes III, IV and V with again great variability: 14% of Irish patients had at least one class III mutation, 7% of Portuguese patients had at least one class IV mutation, and in 6 countries more than 5% of patients had at least one class V mutation.

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49 We modified the classification proposed by McKone et al. [21] based on new insights: G85E was reassigned to class II [22], and recently recognized gating mutations with proof of CFTR potentiator responsiveness were added to class III [23]. Login to comment
56 Class Type of defect List of mutations attributed to this class Class I Defective protein production Nonsense mutations Large deletions and insertions 1078delT; 1717-1G࢐A; 3659delC; 621+1G࢐T Class II Defective protein processing G85E, F508del, I507del, R560T, N1303K Class III Defective protein regulation ('gating`) G178R, S549N, S549R, G551D, G551S, G970R, G1244E, S1251N, S1255P, G1349D Class IV Defective protein conductance R117H, R334W, R347P Class V Reduced amount of functioning protein 2789+5G࢐A, 3849+10KbC࢐T, A455E Unclassified All other mutations, including those unknown. Login to comment

[hide] Impact of heterozygote CFTR mutations in COPD pati... Respir Res. 2014 Feb 11;15:18. doi: 10.1186/1465-9921-15-18. Raju SV, Tate JH, Peacock SK, Fang P, Oster RA, Dransfield MT, Rowe SM
Impact of heterozygote CFTR mutations in COPD patients with chronic bronchitis.
Respir Res. 2014 Feb 11;15:18. doi: 10.1186/1465-9921-15-18., [PMID:24517344]

Abstract [show]
BACKGROUND: Cigarette smoking causes Chronic Obstructive Pulmonary Disease (COPD), the 3rd leading cause of death in the U.S. CFTR ion transport dysfunction has been implicated in COPD pathogenesis, and is associated with chronic bronchitis. However, susceptibility to smoke induced lung injury is variable and the underlying genetic contributors remain unclear. We hypothesized that presence of CFTR mutation heterozygosity may alter susceptibility to cigarette smoke induced CFTR dysfunction. Consequently, COPD patients with chronic bronchitis may have a higher rate of CFTR mutations compared to the general population. METHODS: Primary human bronchial epithelial cells derived from F508del CFTR heterozygotes and mice with (CFTR+/-) and without (CFTR+/+) CFTR heterozygosity were exposed to whole cigarette smoke (WCS); CFTR-dependent ion transport was assessed by Ussing chamber electrophysiology and nasal potential difference measurements, respectively. Caucasians with COPD and chronic bronchitis, age 40 to 80 with FEV1/FVC < 0.70 and FEV1 < 60% predicted, were selected for genetic analysis from participants in the NIH COPD Clinical Research Network's Azithromycin for Prevention of Exacerbations of COPD in comparison to 32,900 Caucasian women who underwent prenatal genetic testing. Genetic analysis involved an allele-specific genotyping of 89 CFTR mutations. RESULTS: Exposure to WCS caused a pronounced reduction in CFTR activity in both CFTR (+/+) cells and F508del CFTR (+/-) cells; however, neither the degree of decrement (44.7% wild-type vs. 53.5% F508del heterozygous, P = NS) nor the residual CFTR activity were altered by CFTR heterozygosity. Similarly, WCS caused a marked reduction in CFTR activity measured by NPD in both wild type and CFTR heterozygous mice, but the severity of decrement (91.1% wild type vs. 47.7% CF heterozygous, P = NS) and the residual activity were not significantly affected by CFTR genetic status. Five of 127 (3.9%) COPD patients with chronic bronchitis were heterozygous for CFTR mutations which was not significantly different from controls (4.5%) (P = NS). CONCLUSIONS: The magnitude of WCS induced reductions in CFTR activity was not affected by the presence of CFTR mutation heterozygosity. CFTR mutations do not increase the risk of COPD with chronic bronchitis. CFTR dysfunction due to smoking is primarily an acquired phenomenon and is not affected by the presence of congenital CFTR mutations.

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81 As expected based on genotype-phenotype correlations in the disease [33], HBE cells derived from a F508del CFTR heterozygote had slightly lower CFTR activity at baseline than wild type monolayers as measured by Table 1 List of CFTR mutations analyzed F508del R117H 1717-1G > A R117C G85E R334W 1898 + 1G > A Y122X A455E R347P 2184delA G178R I507del R553X 2789 + 5G > A G314E G542X R560T 3120 + 1G > A G330X G551D W1282X 3659delC R347H N1303K 621 + 1G > T K710X 406-1G > A R1162X 711 + 1G > T E60X G480C R1066C W1089X V520F A559T S1196X Q1238X S1251N S1255X 663delT 935delA 1161delC 1288insTA 2184insA 2307insA 2711delT 2869insG R709X R764X R1158X 574delA Q493X 1898 + 5G > T 3905insT I506T 3849 + 10kbC > T 712-1G > T Q98R Q552X S549N 1078delT H199Y 444delA S549R (T > G) 2143delT P205S 2043delG 1811 + 1.6kbA > G 3272-26A > G L206W 3791delC Y1092X (C > G) 3199del6 F508C 2108delA Y1092X (C > A) D1152H V520I 3667del4 394delTT 3876delA M1101K 1677delTA W1098X (TGA) 1812-1G > A 4016insT 1609delCA 3171delC response to forskolin stimulation (49.3 &#b1; 11.5 bc;A/cm2 in CFTR (+/+) vs. 40.5 &#b1; 5.3 bc;A/cm2 in CFTR (+/-), although this was not statistically significant (Figure 1A,B). Login to comment

[hide] An image analysis method to quantify CFTR subcellu... Mol Cell Probes. 2014 Aug;28(4):175-80. doi: 10.1016/j.mcp.2014.02.004. Epub 2014 Feb 21. Pizzo L, Fariello MI, Lepanto P, Aguilar PS, Kierbel A
An image analysis method to quantify CFTR subcellular localization.
Mol Cell Probes. 2014 Aug;28(4):175-80. doi: 10.1016/j.mcp.2014.02.004. Epub 2014 Feb 21., [PMID:24561544]

Abstract [show]
Aberrant protein subcellular localization caused by mutation is a prominent feature of many human diseases. In Cystic Fibrosis (CF), a recessive lethal disorder that results from dysfunction of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the most common mutation is a deletion of phenylalanine-508 (pF508del). Such mutation produces a misfolded protein that fails to reach the cell surface. To date, over 1900 mutations have been identified in CFTR gene, but only a minority has been analyzed at the protein level. To establish if a particular CFTR variant alters its subcellular distribution, it is necessary to quantitatively determine protein localization in the appropriate cellular context. To date, most quantitative studies on CFTR localization have been based on immunoprecipitation and western blot. In this work, we developed and validated a confocal microscopy-image analysis method to quantitatively examine CFTR at the apical membrane of epithelial cells. Polarized MDCK cells transiently transfected with EGFP-CFTR constructs and stained for an apical marker were used. EGFP-CFTR fluorescence intensity in a region defined by the apical marker was normalized to EGFP-CFTR whole cell fluorescence intensity, rendering "apical CFTR ratio". We obtained an apical CFTR ratio of 0.67 +/- 0.05 for wtCFTR and 0.11 +/- 0.02 for pF508del. In addition, this image analysis method was able to discriminate intermediate phenotypes: partial rescue of the pF508del by incubation at 27 degrees C rendered an apical CFTR ratio value of 0.23 +/- 0.01. We concluded the method has a good sensitivity and accurately detects milder phenotypes. Improving axial resolution through deconvolution further increased the sensitivity of the system as rendered an apical CFTR ratio of 0.76 +/- 0.03 for wild type and 0.05 +/- 0.02 for pF508del. The presented procedure is faster and simpler when compared with other available methods and it is therefore suitable as a screening method to identify mutations that completely or mildly affect CFTR processing. Moreover, it could be extended to other studies on the biology underlying protein subcellular localization in health and disease.

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210 Structural cues involved in endoplasmic reticulum degradation of G85E and G91R mutant cystic fibrosis transmembrane conductance regulator. Login to comment

[hide] CFTR mutations spectrum and the efficiency of mole... PLoS One. 2014 Feb 26;9(2):e89094. doi: 10.1371/journal.pone.0089094. eCollection 2014. Zietkiewicz E, Rutkiewicz E, Pogorzelski A, Klimek B, Voelkel K, Witt M
CFTR mutations spectrum and the efficiency of molecular diagnostics in Polish cystic fibrosis patients.
PLoS One. 2014 Feb 26;9(2):e89094. doi: 10.1371/journal.pone.0089094. eCollection 2014., [PMID:24586523]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane regulator gene (CFTR). In light of the strong allelic heterogeneity and regional specificity of the mutation spectrum, the strategy of molecular diagnostics and counseling in CF requires genetic tests to reflect the frequency profile characteristic for a given population. The goal of the study was to provide an updated comprehensive estimation of the distribution of CFTR mutations in Polish CF patients and to assess the effectiveness of INNOLiPA_CFTR tests in Polish population. The analyzed cohort consisted of 738 patients with the clinically confirmed CF diagnosis, prescreened for molecular defects using INNOLiPA_CFTR panels from Innogenetics. A combined efficiency of INNOLiPA CFTR_19 and CFTR_17_TnUpdate tests was 75.5%; both mutations were detected in 68.2%, and one mutation in 14.8% of the affected individuals. The group composed of all the patients with only one or with no mutation detected (109 and 126 individuals, respectively) was analyzed further using a mutation screening approach, i.e. SSCP/HD (single strand conformational polymorphism/heteroduplex) analysis of PCR products followed by sequencing of the coding sequence. As a result, 53 more mutations were found in 97 patients. The overall efficiency of the CF allele detection was 82.5% (7.0% increase compared to INNOLiPA tests alone). The distribution of the most frequent mutations in Poland was assessed. Most of the mutations repetitively found in Polish patients had been previously described in other European populations. The most frequent mutated allele, F508del, represented 54.5% of Polish CF chromosomes. Another eight mutations had frequencies over 1%, 24 had frequencies between 1 and 0.1%; c.2052-2053insA and c.3468+2_3468+3insT were the most frequent non-INNOLiPA mutations. Mutation distribution described herein is also relevant to the Polish diaspora. Our study also demonstrates that the reported efficiency of mutation detection strongly depends on the diagnostic experience of referring health centers.

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71 Exon / intron (legacy) Exon / intron (Ensembl) Protein change SVM value cDNA (HGVS nomenclature) gDNA (cDNA +132 bp) Number of PL CF chromosomes Reference a Mutations in trans Pathogenic mutations 1 1 L15Ffs10X c.43delC 175delC 1 CFMDB 1717-1G.A 2 2 G27V 21.92 c.80G.T 212G.T 1 Novel F508del 2 2 S18RfsX16 c.54-5940_273 +10250del21kb exon2,3del21kb 66 IL19 various CF mutations i2 i2 IVS2_Donor c.164+1G.A 296+1G.A 3 CFMDB various CF mutations 3 3 G85E 22.61 c.254G.A 386G.A 1 IL17 unknown 3 3 E60X c.178G.T 310G.T 0 IL17 x 3 3 L88IfsX22 c.262_263delTT 394delTT 0 IL17 x 4 4 E92K 21.92 c.274G.A 406G.A 2 CFMDB c.164+1G.A; c.2051- 2AA.G 4 4 L101X c.302T.G 434T.G 1 CFMDB c.3717+12191C.T 4 4 K114IfsX5 c.341_353del13bp 473del13bp 1 Novel F508del 4 4 R117H 20.35 c.350G.A 482G.A 5 IL17 F508del; 2x unknown 4 4 R117C 22.07 c.349C.T 481C.T 2 CFMDB S1206X;1x unknown 4 4 L137_L138insT c.412_413insACT L138ins 1 CFMDB F508del 4 4 R153I 22.61 c.458G.T 590G.T 2 Novel F508del; c.3527delC i4 i4 IVS4_Donor c.489+1G.T 621+1G.T 5 IL17 F508del; c.489+1G.T 5 5 L165X c.494T.A 626T.A 1 Novel F508del i5 i5 IVS5_Donor c.579+1G.T 711+1G.T 0 IL19 x i5 i5 IVS5_Donor c.579+3A.G 711+3A.G 2 CFMDB 2,3del21kb; c.2052-3insA i5 i5 IVS5_Donor c.579+5G.A 711+5G.A 0 IL17 x 7 8 F311L 20.90 c.933C.G 965C.G 2 CFMDB 2x F508 7 8 G314R 20.58 c.940G.A 1072G.A 4 CFMDB various CF mutations 7 8 F316LfsX12 c.948delT 1078delT 1 IL17 unkown 7 8 R334W 22.41 c.1000C.T 1132C.T 6 IL17 various CF mutations 7 8 I336K 22.07 c.1007T.A 1139T.A 2 CFMDB 2,3de21kb; F508del 7 8 R347P 22.27 c.1040G.C 1172G.C 11 IL17 various CF mutations i7 i8 IVS8_Donor c.1116+2T.A 1248+2T.A 1 Novel Q1412X 9 10 A455E 22.61 c.1364C.A 1496C.A 0 IL17 x i9 i10 IVS10_Donor c.1392+1G.A 1524+1G.A 1 CFMDB c.3816-7delGT 10 11 S466X c.1397C.G 1529C.G 1 CFMDB G542X 10 11 I507del c.1519_1521delATC 1651delATC 2 IL19 F508del 10 11 F508del c.1521_1523delCTT 1654delCTT 805 IL19 various CF mutations i10 i11 IVS11_Acceptor c.1585-1G.A 1717-1G.A 27 IL19 various CF mutations 11 12 G542X c.1624G.T 1756G.T 25 IL19 various CF mutations 11 12 G551D 21.24 c.1624G.T 1756G.T 5 IL19 various CF mutations 11 12 Q552X c.1654C.T 1786C.T 0 IL19 x 11 12 R553X c.1657C.T 1789C.T 14 IL19 various CF mutations 11 12 R560T 21.92 c.1679G.C 1811G.C 0 IL19 x i12 i13 IVS13_Donor c.1766+1G.A 1898+1G.A 6 IL19 various CF mutations i12 i13 IVS13_Donor c.1766+1G.C 1898+1G.C 1 CFMDB F508del 13 14 H620P 21.73 c.1859A.C 1991A.C 1 CFMDB F508del 13 14 R668C//G576A 21.61//1.73 c.2002C.T//c.1727G.C 2134C.T// 1859G.C 5 b CFMDB// rs1800098 c.1585-1G.A; 4 unknown 13 14 L671X c.2012delT 2143delT 27 IL17 various CF mutations 13 14 K684SfsX38 c.2051_2052delAAinsG 2183AA.G 10 IL17 various CF mutations 13 14 K684NfsX38 c.2052delA 2184delA 0 IL17 x 13 14 Q685TfsX4 c.2052_2053insA 2184insA 15 CFMDB various CF mutationsc , 1 unknown Table 2. Cont. Exon / intron (legacy) Exon / intron (Ensembl) Protein change SVM value cDNA (HGVS nomenclature) gDNA (cDNA +132 bp) Number of PL CF chromosomes Reference a Mutations in trans 13 14 L732X c.2195T.G 2327T.G 1 CFMDB F508del 14A 15 R851X c.2551C.T 2683C.T 3 CFMDB various CF mutations 14A 15 I864SfsX28 c.2589_2599del11bp 2721del11bp 2 CFMDB F508del; 2,3del21kb i14B i16 IVS16_Donor c.2657+2_2657+3insA 2789+2insA 1 CFMDB F508del i14B i16 IVS16_Donor c.2657+5G.A 2789+5G.A 0 IL17 unkown 15 17 Y919C 21.02 c.2756A.G 2888A.G 1 CFMDB unknown 15 17 H939HfsX27 c.2817_2820delTACTC 2949delTACTC 1 Novel unkown i15 i17 IVS17_Donor c.2908+3A.C 3040+3A.C 1 Novel F508del i16 i18 IVS18_Donor c.2988+1G.A 3120+1G.A 0 IL19 x 17A 19 I1023_V1024del c.3067_3072delATAGTG 3199del6 0 IL19 x i17A i19 IVS19 c.3140-26A.G 3272-26A.G 9 IL19 various CF mutations 17B 20 L1065R 21.90 c.3194T.G 3326T.G 1 CFMDB F508del 17B 20 Y1092X c.3276C.A 3408C.A 1 CFMDB R334W i18 i21 IVS21_Donor c.3468+2_3468+3insT 3600+2insT 11 CFMDB various CF mutationsd , 1 unknown 18 21 E1126EfsX7 c.3376_3379delGAAG 3508delGAAG 1 Novel F508del 19 22 R1158X c.3472C.T 3604C.T 2 CFMDB F508del; R553X 19 22 R1162X c.3484C.T 3616C.T 1 IL17 F508del 19 22 L1177SfsX15 c.3528delC 3659delC 4 IL17 various CF mutations 19 22 S1206X c.3617C.A 3749C.A 1 CFMDB R117C i19 i22 IVS22 c.3717+12191C.T 3849+10kbC.T 58 IL17 various CF mutations 20 23 G1244R 22.62 c.3730G.C 3862G.C 1 CFMDB F508del 20 23 S1251N 22.28 c.3752G.A 3884G.A 0 IL19 x 20 23 L1258FfsX7 c.3773_3774insT 3905insT 0 IL19 x 20 23 V1272VfsX28 c.3816_3817delGT 3944delGT 1 CFMDB c.1392+1G.A 20 23 W1282X c.3846G.A 3978G.A 9 IL19 various CF mutations 21 24 N1303K 22.62 c.3909C.G 4041C.G 18 IL19 various CF mutations 22 25 V1327X c.3979delG 4111delG 1 Novel F508del 22 25 S1347PfsX13 c.4035_4038dupCCTA c.4167dupCCTA 1 CFMDB 2,3del21kb 23 26 Q1382X c.4144C.T 4276C.T 1 CFMDB F508del 23 26 Q1412X c.4234C.T 4366C.T 2 CFMDB F508del; c.1116+2T.A i23 i26 IVS26_Donor c.4242+1G.T 4374+1G.T 1 CFMDB F508del Sequence changes of uncertain pathogenic effect, tentatively counted as mutations 6A 6 E217G 0.30 c.650A.G 782A.G 1 CFMDB; rs1219109046 unknown 7 8 R352Q 20.01 c.1055G.A 1187G.A 1 CFMDB; rs121908753 F508del 7 8 Q359R 0.33 c.1076A.G 1208A.G 1 CFMDB F508del i8 i9 IVS9 c.1210-12T5_1210- 34_35 (TG)12 1332-12Tn_- 34TGm 6 CFMDB F508del; 3x unknown i8 i9 IVS9 c.1210-12T5_1210- 34_35 (TG)13 1332-12Tn_- 34TGm 2 CFMDB 2143delT; 1x unknown i8 i9 IVS9 c.1210-12T8 1332-12Tn 1 Novel unknown 10 11 I506V 20.21 c.1516A.G 1648A.G 1 CFMDB; rs1800091 unknown 12 13 V562L 0.79 c.1684G.C 1816G.C 1 CFMDB; rs1800097 unknown 13 14 G723V 0.44 c.2168G.T 2300G.T 1 CFMDB; rs200531709 unknown 15 17 D924N 0.03 c.2770G.A 2902G.A 1 CFMDB; rs201759207 unknown patient with F508del on another allele) was not supported by the SVM value (+0.35); the patient was PS and had ambiguous chloride values (45, 64 and 83 mmol/L). 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103 IL17 (INNOLiPA_CFTR17_TnUpdate): 621+1G.T; 3849+10kbC.T; 2183AA.G; 394delTT; 2789+5G.A; R1162X; 3659delC; R117H; R334W; R347P; G85E; 1078delT; A455E; 2143delT; E60X; 2184delA; 711+5G.A; polymorphism 5T/7T/9T. Login to comment

[hide] Genetics of cystic fibrosis: CFTR mutation classif... Int J Biochem Cell Biol. 2014 Jul;52:94-102. doi: 10.1016/j.biocel.2014.02.023. Epub 2014 Mar 12. Fanen P, Wohlhuter-Haddad A, Hinzpeter A
Genetics of cystic fibrosis: CFTR mutation classifications toward genotype-based CF therapies.
Int J Biochem Cell Biol. 2014 Jul;52:94-102. doi: 10.1016/j.biocel.2014.02.023. Epub 2014 Mar 12., [PMID:24631642]

Abstract [show]
Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an epithelial anion channel. Since the identification of the disease in 1938 and up until 2012, CF patients have been treated exclusively with medications aimed at bettering their respiratory, digestive, inflammatory and infectious symptoms. The identification of the CFTR gene in 1989 gave hopes of rapidly finding a cure for the disease, for which over 1950 mutations have been identified. Since 2012, recent approaches have enabled the identification of small molecules targeting either the CFTR protein directly or its key processing steps, giving rise to novel promising therapeutic tools. This review presents the current CFTR mutation classifications according to their clinical consequences and to their effect on the structure and function of the CFTR channel. How these classifications are essential in the establishment of mutation-targeted therapeutic strategies is then discussed. The future of CFTR-targeted treatment lies in combinatory therapies that will enable CF patients to receive a customized treatment.

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74 23 ACMG recommended panel of classic CF-causing mutations G85E R117H R334W R347P A455E I507del F508del G542X G551D R553X R560T R1162X W1282X N1303K 621 + 1G > T 711 + 1G > T 1717 - 1G > A 1898 + 1G > A 2184delA 2789 + 5G > A 3120 + 1G > A 3659delC 3849 + 10kbC > T Additional or alternative mutations present at significant frequencies in an ethnic population served by a newborn screening program may be assessed. Login to comment

[hide] Biosynthesis of cystic fibrosis transmembrane cond... Int J Biochem Cell Biol. 2014 Jul;52:26-38. doi: 10.1016/j.biocel.2014.03.020. Epub 2014 Mar 28. Pranke IM, Sermet-Gaudelus I
Biosynthesis of cystic fibrosis transmembrane conductance regulator.
Int J Biochem Cell Biol. 2014 Jul;52:26-38. doi: 10.1016/j.biocel.2014.03.020. Epub 2014 Mar 28., [PMID:24685677]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride (Cl(-)) channel. Mutations of its gene lead to the disease of cystis fibrosis (CF) among which the most common is the deletion of phenylalanine at position 508 (Phe508del). CFTR is a multi-domain glycoprotein whose biosynthesis, maturation and functioning as an anion channel involve multi-level post-translational modifications of CFTR molecules and complex folding processes to reach its native, tertiary conformation. Only 20-40% of the nascent chains achieve folded conformation, while the remaining molecules are targeted for degradation by endoplasmic reticulum, lysosomes, or autophagy. A large number of mutations causing CF impair processing of CFTR. Growing knowledge of CFTR biosynthesis has enabled understanding the cellular basis of CF and has brought to light various potential targets for novel, promising therapies.

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1536 It was reported that Derlin1- and p97-containing complexes mediate the retro-translocation of both misfolded WT and mutant CFTR (Phe508del and Gly85Glu). Login to comment

[hide] New pharmacological approaches for cystic fibrosis... Pharmacol Ther. 2015 Jan;145:19-34. doi: 10.1016/j.pharmthera.2014.06.005. Epub 2014 Jun 14. Bell SC, De Boeck K, Amaral MD
New pharmacological approaches for cystic fibrosis: promises, progress, pitfalls.
Pharmacol Ther. 2015 Jan;145:19-34. doi: 10.1016/j.pharmthera.2014.06.005. Epub 2014 Jun 14., [PMID:24932877]

Abstract [show]
With the discovery of the CFTR gene in 1989, the search for therapies to improve the basic defects of cystic fibrosis (CF) commenced. Pharmacological manipulation provides the opportunity to enhance CF transmembrane conductance regulator (CFTR) protein synthesis and/or function. CFTR modulators include potentiators to improve channel gating (class III mutations), correctors to improve abnormal CFTR protein folding and trafficking (class II mutations) and stop codon mutation read-through drugs relevant for patients with premature stop codons (most class I mutations). After several successful clinical trials the potentiator, ivacaftor, is now licenced for use in adults and children (>six years), with CF bearing the class III G551D mutation and FDA licence was recently expanded to include 8 additional class III mutations. Alternative approaches for class I and class II mutations are currently being studied. Combination drug treatment with correctors and potentiators appears to be required to restore CFTR function of F508del, the most common CFTR mutation. Alternative therapies such as gene therapy and pharmacological modulation of other ion channels may be advantageous because they are mutation-class independent, however progress is less well advanced. Clinical trials for CFTR modulators have been enthusiastically embraced by patients with CF and health care providers. Whilst novel trial end-points are being evaluated allowing CFTR modulators to be efficiently tested, many challenges related to the complexity of CFTR and the biology of the epithelium still need to be overcome.

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544 Mutation Alternative name Allele frequency (% of total known) in ECFSPR 2010 Allele frequency (% of total known mutations) in 2010 ECFSPR F508del 64.5 Most frequent mutation worldwide Southeast to Northwest increasing prevalence in Europe IL 25.5 to DK 82.6 Mutations with an overall EU prevalence above 1% G542X Mediterranean mutation 2.5 GR 6.7, ES 6.0 N1303K Ancient Phoenician mutation 1.9 IT 4.2 W1282X Jewish Ashkenazi mutation 1.2 IL 22.4 G551D Celtic mutation 1.1 IE 7.3 1717-1GNA Italian mutation 1.0 IT 3.7 Mutations with an overall EU prevalence below 0.5% G85E PT 3.5 A455E Dutch mutation NL 3.5 CFTR dele 2,3 Slavic mutation CZ 5.2, BY 6.7 394delTT Nordic mutation SE 7.9, DK 2.0 3905insT Swiss mutation CH 2.4 R1162X Italian mutation IT 7.8 A561E Portuguese mutation PT 3.2 Abbreviations ECFSPR - European Cystic Fibrosis Society Patient Registry. Login to comment
547 Class Type of defect List of mutations attributed to this class Class I Defective protein production Nonsense mutations: G542X, R1162X, RW1282X Deletions and insertions: CFTRdele2,3; 1078delT; 1717-1G ࢐ A; 3659delC; 621+1G N T Class II Defective protein processing G85E, F508del, I507del, R560T, A561E, R1066C, N1303K Class III Defective protein regulation (gating) G178R, S549N, S549R, G551D, G551S, G970R, G1244E, S1251N, S1255P, G1349D Class IV Defective protein conductance R334W, R347P, R117H Class V Reduced amount of functioning protein 2789+5G ࢐ A, 3272-26ANG, 3849+10KbC ࢐ T, A455E Class VI Reduced cell surface stability Rescued F508del, c.120del23 Unclassified All other mutations, including those unknown a F508del-CFTR pocket (at NBD1:ICL4 interface) (Farinha et al., 2013). Login to comment

[hide] Mechanisms of CFTR functional variants that impair... PLoS Genet. 2014 Jul 17;10(7):e1004376. doi: 10.1371/journal.pgen.1004376. eCollection 2014 Jul. LaRusch J, Jung J, General IJ, Lewis MD, Park HW, Brand RE, Gelrud A, Anderson MA, Banks PA, Conwell D, Lawrence C, Romagnuolo J, Baillie J, Alkaade S, Cote G, Gardner TB, Amann ST, Slivka A, Sandhu B, Aloe A, Kienholz ML, Yadav D, Barmada MM, Bahar I, Lee MG, Whitcomb DC
Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis.
PLoS Genet. 2014 Jul 17;10(7):e1004376. doi: 10.1371/journal.pgen.1004376. eCollection 2014 Jul., [PMID:25033378]

Abstract [show]
CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTRBD) affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens) but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a) screened 984 well-phenotyped pancreatitis cases for candidate CFTRBD mutations from among 81 previously described CFTR variants; (b) conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c) computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d) tested the newly defined CFTRBD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N) not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002). Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTRBD increased risk for rhinosinusitis (OR 2.3, p<0.005) and male infertility (OR 395, p<<0.0001). WNK1-SPAK pathway-activated increases in CFTR bicarbonate permeability are altered by CFTRBD variants through multiple mechanisms. CFTRBD variants are associated with clinically significant disorders of the pancreas, sinuses, and male reproductive system.

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269 67 SNPs (125GtoC, 1716G.A, 1717-1G.A, 1898+1G.A, 2183AA.G, 2184delA, 2789+5G.A, 3120+1G.A, 3659delC, 3849+10kbC.T, 621+ 1G.T, 711+5G.A, A455E, D110H, D1152H, D1270N, D443Y, D579G, F1052V, F1074L, F508C, F508del, G1069R, G1244E, G1349D, G178R, G542X, G551D, G551S, I1131L/V, I148T, I336K/T, I507del, I807M, IVS8T5, K1180T, L1065P, L967S, L997F, M1V, M470V, M952I, M952T, N1303K, P67L, Q1463Q, R1070Q, R1162X, R117C, R117H, R170H, R258G, R297Q, R31C, R352Q, R553X, R668C, R74W, R75Q, S1235R, S1255P, S485R, S977F, T338I, T854T, V201M, W1282X) were multiplexed into 6 wells; 14 SNPs (S492F, S945L, R74Q, R560T, R1162L, G85E, I1027T, R334W, R347P, G576A, 711+1G.T, 1001+11C.T, P1290P, 3199del6) were ascertained separately via TaqMan Gene Expression Assays, with repeat confirmation of all positive results. Login to comment

[hide] Comprehensive CFTR gene analysis of the French cys... Genet Med. 2015 Feb;17(2):108-16. doi: 10.1038/gim.2014.113. Epub 2014 Aug 14. Audrezet MP, Munck A, Scotet V, Claustres M, Roussey M, Delmas D, Ferec C, Desgeorges M
Comprehensive CFTR gene analysis of the French cystic fibrosis screened newborn cohort: implications for diagnosis, genetic counseling, and mutation-specific therapy.
Genet Med. 2015 Feb;17(2):108-16. doi: 10.1038/gim.2014.113. Epub 2014 Aug 14., [PMID:25122143]

Abstract [show]
PURPOSE: Newborn screening (NBS) for cystic fibrosis (CF) was implemented throughout France in 2002. It involves a four-tiered procedure: immunoreactive trypsin (IRT)/DNA/IRT/sweat test [corrected] was implemented throughout France in 2002. The aim of this study was to assess the performance of molecular CFTR gene analysis from the French NBS cohort, to evaluate CF incidence, mutation detection rate, and allelic heterogeneity. METHODS: During the 8-year period, 5,947,148 newborns were screened for cystic fibrosis. The data were collected by the Association Francaise pour le Depistage et la Prevention des Handicaps de l'Enfant. The mutations identified were classified into four groups based on their potential for causing disease, and a diagnostic algorithm was proposed. RESULTS: Combining the genetic and sweat test results, 1,160 neonates were diagnosed as having cystic fibrosis. The corresponding incidence, including both the meconium ileus (MI) and false-negative cases, was calculated at 1 in 4,726 live births. The CF30 kit, completed with a comprehensive CFTR gene analysis, provides an excellent detection rate of 99.77% for the mutated alleles, enabling the identification of a complete genotype in 99.55% of affected neonates. With more than 200 different mutations characterized, we confirmed the French allelic heterogeneity. CONCLUSION: The very good sensitivity, specificity, and positive predictive value obtained suggest that the four-tiered IRT/DNA/IRT/sweat test procedure may provide an effective strategy for newborn screening for cystic fibrosis.

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53 Because only a limited number of functional studies have assessed the pathogenicity of variants, mutations have been classified in previous studies according to their disease-causing potential.16,22,23 Based on the recommendations and data from these studies (UMD-CFTR-France),24 variants were classified into four groups: A, CF-causing; B, associated with CFTR-RDs; C, no clinical consequences; and D, unknown or Table 1ߒ Allelic frequencies of CF30-kit mutations, identified in neonates with CF, and correspondence between traditional mutation nomenclature and that on the Human Genome Variation Society website Frequency (F) % Mutation Legacy mutation nomenclature Number of alleles/2,320 % of alleles/2,320 Cumulative % ࣙ5 p.Phe508del F508del 1,560 67.24 67.24 p.Gly542* G542X 113 3.19 10.51 p.Asn1303Lys N1303K 81 1.98 c.1585-1G>A 1717-1G>A 48 1.47 1.00ࣙFࣙ4.99 c.2657ߙ+ߙ5G>A 2789ߙ+ߙ5G>A 37 1.42 p.Arg553* R553X 36 1.29 p.Gly551Asp G551D 31 1.16 p.Tyr122* Y122X 26 0.97 6.86 c.2988ߙ+ߙ1G>A 3120ߙ+ߙ1G>A 22 0.82 c.579ߙ+ߙ1G>T 711ߙ+ߙ1G>T 18 0.67 p.Ile507del I507del 17 0.63 c.3140-26A>G 3272-26A>G 16 0.59 0.40ࣙFࣙ0.99 p.Arg347Pro R347P 15 0.56 p.Arg1162* R1162X 15 0.56 p.Trp1282* W1282X 14 0.52 p.Tyr1092* Y1092X 13 0.48 c.2051_2052delinsG 2183AA>G 12 0.45 c.3528delC 3659delC 11 0.41 c.1680-886A>G 1811ߙ+ߙ1.6kbA>G 9 0.39 p.Gly85Glu G85E 8 0.34 3.06 p.Ser1251Asn S1251N 7 0.30 p.Arg334Trp R334W 7 0.30 p.Arg117His R117H 7 0.30 0.1ࣙFࣙ0.39 p.Trp846* W846X 6 0.26 c.489ߙ+ߙ1G>T 621ߙ+ߙ1G>T 6 0.26 c.948delT 1078delT 5 0.22 p.Ala455Glu A455E 5 0.22 p.Glu60* E60X 4 0.17 c.262_263delTT 394delTT 4 0.17 c.3718-2477C>T 3849ߙ+ߙ10kbC>T 3 0.13 Total 2,034 87.67 87.67 Mutations are clustered into four groups of frequency intervals (>5%, 1-4.99%, 0.99-0.4%, and <0.4%). Login to comment

[hide] Full-open and closed CFTR channels, with lateral t... Cell Mol Life Sci. 2015 Apr;72(7):1377-403. doi: 10.1007/s00018-014-1749-2. Epub 2014 Oct 7. Mornon JP, Hoffmann B, Jonic S, Lehn P, Callebaut I
Full-open and closed CFTR channels, with lateral tunnels from the cytoplasm and an alternative position of the F508 region, as revealed by molecular dynamics.
Cell Mol Life Sci. 2015 Apr;72(7):1377-403. doi: 10.1007/s00018-014-1749-2. Epub 2014 Oct 7., [PMID:25287046]

Abstract [show]
In absence of experimental 3D structures, several homology models, based on ABC exporter 3D structures, have provided significant insights into the molecular mechanisms underlying the function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a chloride channel whose defects are associated with cystic fibrosis (CF). Until now, these models, however, did not furnished much insights into the continuous way that ions could follow from the cytosol to the extracellular milieu in the open form of the channel. Here, we have built a refined model of CFTR, based on the outward-facing Sav1866 experimental 3D structure and integrating the evolutionary and structural information available today. Molecular dynamics simulations revealed significant conformational changes, resulting in a full-open channel, accessible from the cytosol through lateral tunnels displayed in the long intracellular loops (ICLs). At the same time, the region of nucleotide-binding domain 1 in contact with one of the ICLs and carrying amino acid F508, the deletion of which is the most common CF-causing mutation, was found to adopt an alternative but stable position. Then, in a second step, this first stable full-open conformation evolved toward another stable state, in which only a limited displacement of the upper part of the transmembrane helices leads to a closure of the channel, in a conformation very close to that adopted by the Atm1 ABC exporter, in an inward-facing conformation. These models, supported by experimental data, provide significant new insights into the CFTR structure-function relationships and into the possible impact of CF-causing mutations.

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346 First, almost all CF-causing mutations involving residues located in the MSD transmembrane segments are encountered in MSD1 and generally concern positions lining the pore (G85E, E92K, D110H, P205S, R334W, I336K, T338I, S341P, R347H/R347P, and R352Q) (Fig. 7a). Login to comment

[hide] Analysis of cystic fibrosis gene mutations in chil... J Med Case Rep. 2014 Oct 10;8:339. doi: 10.1186/1752-1947-8-339. Dell'Edera D, Benedetto M, Gadaleta G, Carone D, Salvatore D, Angione A, Gallo M, Milo M, Pisaturo ML, Di Pierro G, Mazzone E, Epifania AA
Analysis of cystic fibrosis gene mutations in children with cystic fibrosis and in 964 infertile couples within the region of Basilicata, Italy: a research study.
J Med Case Rep. 2014 Oct 10;8:339. doi: 10.1186/1752-1947-8-339., [PMID:25304080]

Abstract [show]
INTRODUCTION: Cystic fibrosis is the most common autosomal recessive genetic disease in the Caucasian population. Extending knowledge about the molecular pathology on the one hand allows better delineation of the mutations in the CFTR gene and the other to dramatically increase the predictive power of molecular testing. METHODS: This study reports the results of a molecular screening of cystic fibrosis using DNA samples of patients enrolled from January 2009 to December 2013. Patients were referred to our laboratory for cystic fibrosis screening for infertile couples. In addition, we identified the gene mutations present in 76 patients affected by cystic fibrosis in the pediatric population of Basilicata. RESULTS: In the 964 infertile couples examined, 132 subjects (69 women and 63 men) resulted heterozygous for one of the CFTR mutations, with a recurrence of carriers of 6.85%. The recurrence of carriers in infertile couples is significantly higher from the hypothetical value of the general population (4%). CONCLUSIONS: This study shows that in the Basilicata region of Italy the CFTR phenotype is caused by a small number of mutations. Our aim is to develop a kit able to detect not less than 96% of CTFR gene mutations so that the relative risk for screened couples is superimposable with respect to the general population.

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59 As mentioned before, molecular screening Table 2 Comparison between the results obtained in this study and those obtained in a previous study Castaldo et al. [14] Mutations observed in the present study F508del 55.8% (29) 48.62% (141) N1303K 3.8% (2) 9.31% (27) G542X 3.8% (2) 8.96% (26) W1282X 3.8% (2) 1.03% (3) 2183AA>G 5.8% (3) 2.76% (8) R1162X 0 0 1717-1G>A 1.9% (1) 0 T338I 0 0 R347P 0 0.69% (2) 711+5G>A 0 0 852del22 5.8% (3) 1.03% (3) 4382delA 0 0.69% (2) 1259insA 0 0.34% (1) 4016insT 0 0.34% (1) R553X 0 0.34% (1) R1158X 0 0 L1077P 0 1.03% (3) I502T 0 0 3849+10kbC>T 1.9% (1) 0.34% (1) D579G 0 0.69% (2) G1244E 3.8% (2) 0 G1349D 0 0.34% (1) 2789+5G>A 0 1.03% (3) 711+1G>T 0 0 L1065P 0 0 2522insC 0 0 E585X 0 0 G85E 0 0 G178R 0 0 D1152H 0 3.10% (9) I148T-3195del6 0 0 I148T (alone) 0 4.48% (13) R334W 0 0 DI507 0 0.69% (2) I1005R 0 0 3272-26A>G 0 0 2711delT 0 0 L558S 1.9% (1) 0.34% (1) W1063X 0 0 D110H 0 0 S549R (A>C) 1.9% (1) 0.69% (2) 2184insA 0 0 3131del22 0 0 Table 2 Comparison between the results obtained in this study and those obtained in a previous study (Continued) R709N 0 0 A349V 0 0 4015insA 0 0 Y849X 1.9% (1) 0.34% (1) G551D 0 1.03% (3) 621+3A>G 0 0.34% (1) E831X 0 0 I507del 0 0.69% (2) IVS8 TG12/t5 0 1.03% (3) H139R (A->G) 0 0.34% (1) 1248+1G>A 0 0.34% (1) R74W;V201M;D1270N 0 0.69% (2) S1455X 0 0.34% (1) dele 2,3 (21kb) 0 0.34% (1) 991del5 0 0.34% (1) UNKNOWN 7 %(4) 4.83% (14) F508C 0 0.69% (2) TOTAL 52 290 of CF is highly recommended in the USA by the National Institutes of Health Consensus Development Conference Statement on genetic testing for cystic fibrosis [17]. Login to comment
79 The test has a sensitivity and a specificity of more than Table 3 List of 60 mutations in the cystic fibrosis transmembrane regulator gene (specificity 100%) F508del I507del F508C 621+1G>T D110H E585X G1349D I502T 1706del17 1677delTA R117H H139R 1898+1G>A 4015delA G542X 1717-1G>A Q552X 852del22 G178R 1898+3A>G G551D S549R(A>C) 2183AA>G T338I 991del5 1898+5G>T N1303K 4016insT 3849+10kb C>T R347P R334W 2184insA G85E 711+5G>A 711+1G>T 1259insA R347H 2522insC 2789+5G>A W1282X G1244E R1066H R352Q 3120+1G>A I148T 3199del6 S912X R1158X 1717-8G>A R1066C R1162X 4382delA D1152H L1077P D579G 3272-26A>G L1065P R553X PoliT: 5T, 7T, 9T 1874insT 3659delC 99%. Login to comment

[hide] Improving newborn screening for cystic fibrosis us... Genet Med. 2015 Feb 12. doi: 10.1038/gim.2014.209. Baker MW, Atkins AE, Cordovado SK, Hendrix M, Earley MC, Farrell PM
Improving newborn screening for cystic fibrosis using next-generation sequencing technology: a technical feasibility study.
Genet Med. 2015 Feb 12. doi: 10.1038/gim.2014.209., [PMID:25674778]

Abstract [show]
Purpose:Many regions have implemented newborn screening (NBS) for cystic fibrosis (CF) using a limited panel of cystic fibrosis transmembrane regulator (CFTR) mutations after immunoreactive trypsinogen (IRT) analysis. We sought to assess the feasibility of further improving the screening using next-generation sequencing (NGS) technology.Methods:An NGS assay was used to detect 162 CFTR mutations/variants characterized by the CFTR2 project. We used 67 dried blood spots (DBSs) containing 48 distinct CFTR mutations to validate the assay. NGS assay was retrospectively performed on 165 CF screen-positive samples with one CFTR mutation.Results:The NGS assay was successfully performed using DNA isolated from DBSs, and it correctly detected all CFTR mutations in the validation. Among 165 screen-positive infants with one CFTR mutation, no additional disease-causing mutation was identified in 151 samples consistent with normal sweat tests. Five infants had a CF-causing mutation that was not included in this panel, and nine with two CF-causing mutations were identified.Conclusion:The NGS assay was 100% concordant with traditional methods. Retrospective analysis results indicate an IRT/NGS screening algorithm would enable high sensitivity, better specificity and positive predictive value (PPV). This study lays the foundation for prospective studies and for introducing NGS in NBS laboratories.Genet Med advance online publication 12 February 2015Genetics in Medicine (2015); doi:10.1038/gim.2014.209.

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15 Correspondence: Mei W. Baker (mwbaker@wisc.edu) Improving newborn screening for cystic fibrosis using next-generation sequencing technology: a technical feasibility study Mei W. Baker, MD1,2 , Anne E. Atkins, MPH2 , Suzanne K. Cordovado, PhD3 , Miyono Hendrix, MS3 , Marie C. Earley, PhD3 and Philip M. Farrell, MD, PhD1,4 Table 1ߒ CF-causing or varying consequences mutations in the MiSeqDx IUO Cystic Fibrosis System c.1521_1523delCTT (F508del) c.2875delG (3007delG) c.54-5940_273ߙ+ߙ10250del21kb (CFTRdele2,3) c.3909C>G (N1303K) c.3752G>A (S1251N) Mutations that cause CF when combined with another CF-causing mutation c.1624G>T (G542X) c.2988ߙ+ߙ1G>A (3120ߙ+ߙ1G->A) c.3964-78_4242ߙ+ߙ577del (CFTRdele22,23) c.613C>T (P205S) c.1021T>C (S341P) c.948delT (1078delT) c.2988G>A (3120G->A) c.328G>C (D110H) c.200C>T (P67L) c.1397C>A (S466X(C>A)) c.1022_1023insTC (1154insTC) c.2989-1G>A (3121-1G->A) c.3310G>T (E1104X) c.3937C>T (Q1313X) c.1397C>G (S466X(C>G)) c.1081delT (1213delT) c.3140-26A>G (3272-26A->G) c.1753G>T (E585X) c.658C>T (Q220X) c.1466C>A (S489X) c.1116ߙ+ߙ1G>A (1248ߙ+ߙ1G->A) c.3528delC (3659delC) c.178G>T (E60X) c.115C>T (Q39X) c.1475C>T (S492F) c.1127_1128insA (1259insA) c.3659delC (3791delC) c.2464G>T (E822X) c.1477C>T (Q493X) c.1646G>A (S549N) c.1209ߙ+ߙ1G>A (1341ߙ+ߙ1G->A) c.3717ߙ+ߙ12191C>T (3849ߙ+ߙ10kbC->T) c.2491G>T (E831X) c.1573C>T (Q525X) c.1645A>C (S549R) c.1329_1330insAGAT (1461ins4) c.3744delA (3876delA) c.274G>A (E92K) c.1654C>T (Q552X) c.1647T>G (S549R) c.1393-1G>A (1525-1G->A) c.3773_3774insT (3905insT) c.274G>T (E92X) c.2668C>T (Q890X) c.2834C>T (S945L) c.1418delG (1548delG) c.262_263delTT (394delTT) c.3731G>A (G1244E) c.292C>T (Q98X) c.1013C>T (T338I) c.1545_1546delTA (1677delTA) c.3873ߙ+ߙ1G>A (4005ߙ+ߙ1G->A) c.532G>A (G178R) c.3196C>T (R1066C) c.1558G>T (V520F) c.1585-1G>A (1717-1G->A) c.3884_3885insT (4016insT) c.988G>T (G330X) c.3197G>A (R1066H) c.3266G>A (W1089X) c.1585-8G>A (1717-8G->A) c.273ߙ+ߙ1G>A (405ߙ+ߙ1G->A) c.1652G>A (G551D) c.3472C>T (R1158X) c.3611G>A (W1204X) c.1679ߙ+ߙ1.6kbA>G (1811ߙ+ߙ1.6kbA->G) c.274-1G>A (406-1G->A) c.254G>A (G85E) c.3484C>T (R1162X) c.3612G>A (W1204X) c.1680-1G>A (1812-1G->A) c.4077_4080delTGTTinsAA (4209TGTT->AA) c.2908G>C (G970R) c.349C>T (R117C) c.3846G>A (W1282X) c.1766ߙ+ߙ1G>A (1898ߙ+ߙ1G->A) c.4251delA (4382delA) c.595C>T (H199Y) c.1000C>T (R334W) c.1202G>A (W401X) c.1766ߙ+ߙ3A>G (1898ߙ+ߙ 3A->G) c.325_327delTATinsG (457TAT->G) c.1007T>A (I336K) c.1040G>A (R347H) c.1203G>A (W401X) c.2012delT (2143delT) c.442delA (574delA) c.1519_1521delATC (I507del) c.1040G>C (R347P) c.2537G>A (W846X) c.2051_2052delAAinsG (2183AA->G) c.489ߙ+ߙ1G>T (621ߙ+ߙ 1G->T) c.2128A>T (K710X) c.1055G>A (R352Q) c.3276C>A (Y1092X (C>A)) c.2052delA (2184delA) c.531delT (663delT) c.3194T>C (L1065P) c.1657C>T (R553X) c.3276C>G (Y1092X (C>G)) c.2052_2053insA (2184insA) c.579ߙ+ߙ1G>T (711ߙ+ߙ 1G->T) c.3230T>C (L1077P) c.1679G>A (R560K) c.366T>A (Y122X) c.2175_2176insA (2307insA) c.579ߙ+ߙ3A>G (711ߙ+ߙ 3A->G) c.617T>G (L206W) c.1679G>C (R560T) - c.2215delG (2347delG) c.579ߙ+ߙ5G>A (711ߙ+ߙ 5G->A) c.1400T>C (L467P) c.2125C>T (R709X) - c.2453delT (2585delT) c.580-1G>T (712-1G->T) c.2195T>G (L732X) c.223C>T (R75X) - c.2490ߙ+ߙ1G>A (2622ߙ+ߙ1G->A) c.720_741delAGGGAG AATGATGATGAAGTAC (852del22) c.2780T>C (L927P) c.2290C>T (R764X) - c.2583delT (2711delT) c.1364C>A (A455E) c.3302T>A (M1101K) c.2551C>T (R851X) - c.2657ߙ+ߙ5G>A (2789ߙ+ߙ5G->A) c.1675G>A (A559T) c.1A>G (M1V) c.3587C>G (S1196X) - Mutations/variants that were validated in this study are in bold. CF, cystic fibrosis. Table 1ߒ Continued on next page reduce carrier detection and potentially improve the positive predictive value (PPV), the NBS goals of equity and the highest possible sensitivity become more difficult to achieve. Login to comment

[hide] Mutation analysis of PRSS1, SPINK1 and CFTR gene i... Turk J Gastroenterol. 2015 Mar;26(2):176-80. doi: 10.5152/tjg.2015.4287. Sisman G, Tugcu M, Ayla K, Sebati O, Senturk H
Mutation analysis of PRSS1, SPINK1 and CFTR gene in patients with alcoholic and idiopathic chronic pancreatitis: A single center study.
Turk J Gastroenterol. 2015 Mar;26(2):176-80. doi: 10.5152/tjg.2015.4287., [PMID:25835118]

Abstract [show]
BACKGROUND/AIMS: A relation between some genetic mutations and chronic pancreatitis (CP) has been reported. However, the relation of genetic mutation to alcoholic CP (ACP) and idiopathic CP (ICP) still remains controversial. In this study, we investigated the prevalence of protease serine 1 (PRSS1), serine protease inhibitor, Kazal type 1 (SPINK1) SPINK1 and cystic fibrosis transmembrane conductance regulator (CFTR) mutations in ACP and ICP patients in Turkey. MATERIALS AND METHODS: Forty-one patients with ACP and 38 patients with ICP were enrolled, and 35 healthy individuals served as controls. The PRSS1 and SPINK1 mutations were investigated by the polymerase chain reaction (PCR)-restriction fragment-length polymorphism (RFLP) technique. The CFTR mutation was examined with PCR direct sequencing. RESULTS: The mean ages of the ACP, ICP and healthy control groups were 53.2, 40.4 and 46.3 years, respectively. A CFTR F508 mutation was detected as a heterozygote in one (2.4%) patient with ACP. In the ICP and control populations, PRSS1, SPINK1 and CFTR mutations were not detected. CONCLUSION: This study shows that PRSS1, SPINK1 and CFTR mutations do not play a role in ACP and ICP patients.

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45 DNA samples were multiplied by multiplex PCR with a CF 22Mut and CF 14Mut+Tn strip assay kit which has 36 common mutations of the CFTR gene (DF508, DI507, F508C, I502T, 1706del17, 1677del TA, G542X, 1717-1G>A, R553X, Q552X, G551D, S549R(A>C), N1303K, 4016insT, R1162X, R1158X, W1282X, G1244E, 2789+5G>A, 2183AA>G, 711+5G>A, 711+1G>T, G85E, 3849+10kbC>T, 621+1G>T, R117H, D1152H, L1065P, R1066H, L1077P, 4382delA, 1259insA, 852del22, R347P, T338I, S912X and Allele5T-7T-9T). Login to comment

[hide] Clinical diagnostic Next-Generation sequencing: th... Scand J Clin Lab Invest. 2015 Sep;75(5):374-81. doi: 10.3109/00365513.2015.1031689. Epub 2015 Apr 15. Loukas YL, Thodi G, Molou E, Georgiou V, Dotsikas Y, Schulpis KH
Clinical diagnostic Next-Generation sequencing: the case of CFTR carrier screening.
Scand J Clin Lab Invest. 2015 Sep;75(5):374-81. doi: 10.3109/00365513.2015.1031689. Epub 2015 Apr 15., [PMID:25874479]

Abstract [show]
A 23-mutation panel for CFTR carrier screening is recommended to women of reproductive age by the American College of Obstetricians and Gynecologists. In the present study the optimized efficiency regarding the carrier rate of Next-Generation sequencing (NGS) technology is compared to the one of limited mutation detection panels. A total of 824 consequent cases were subjected to the commercial Cystic Fibrosis Genotyping Assay. Some 188 negative samples randomly selected from the initial group of probands were further subjected to an extended mutation panel characterized by 92% detection rate, as well as to massive parallel sequencing. Twenty-two probands subjected to the commercial assay proved to carry one mutation included in the ACOG panel (carrier rate 0.0267). The latter panels revealed the presence of mutations not included in the ACOG panel in four probands, resulting to an increase of carrier rate of 0.0106 in the case of in-house panel and an increase of rate of 0.0213 if NGS was used. The above data seem to support the implementation of NGS in the routine CFTR carrier screening.

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84 The other six individuals carried one of the below mutations p.Gly85Glu, p.Gly542Ter, c.489af9;1Gb0e;T, p.Arg334Trp. Login to comment

[hide] A Genotypic-Oriented View of CFTR Genetics Highlig... Mol Med. 2015 Apr 21;21:257-75. doi: 10.2119/molmed.2014.00229. Lucarelli M, Bruno SM, Pierandrei S, Ferraguti G, Stamato A, Narzi F, Amato A, Cimino G, Bertasi S, Quattrucci S, Strom R
A Genotypic-Oriented View of CFTR Genetics Highlights Specific Mutational Patterns Underlying Clinical Macrocategories of Cystic Fibrosis.
Mol Med. 2015 Apr 21;21:257-75. doi: 10.2119/molmed.2014.00229., [PMID:25910067]

Abstract [show]
Cystic fibrosis (CF) is a monogenic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The genotype-phenotype relationship in this disease is still unclear, and diagnostic, prognostic and therapeutic challenges persist. We enrolled 610 patients with different forms of CF and studied them from a clinical, biochemical, microbiological and genetic point of view. Overall, there were 125 different mutated alleles (11 with novel mutations and 10 with complex mutations) and 225 genotypes. A strong correlation between mutational patterns at the genotypic level and phenotypic macrocategories emerged. This specificity appears to largely depend on rare and individual mutations, as well as on the varying prevalence of common alleles in different clinical macrocategories. However, 19 genotypes appeared to underlie different clinical forms of the disease. The dissection of the pathway from the CFTR mutated genotype to the clinical phenotype allowed to identify at least two components of the variability usually found in the genotype-phenotype relationship. One component seems to depend on the genetic variation of CFTR, the other component on the cumulative effect of variations in other genes and cellular pathways independent from CFTR. The experimental dissection of the overall biological CFTR pathway appears to be a powerful approach for a better comprehension of the genotype-phenotype relationship. However, a change from an allele-oriented to a genotypic-oriented view of CFTR genetics is mandatory, as well as a better assessment of sources of variability within the CFTR pathway.

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296 These patients had the following mutations on the other allele: F508del (p.Phe508del) (1 CF-PI), G85E (p.Gly85Glu) (1 CF-PS), R334W (p.Arg334Trp) (2 CF-PS siblings) and W1282X (p.Trp1282*) (2 CF-PS). Login to comment
300 These patients had the following mutations on the other allele: F508del (p.Phe508del) (1 CF-PS, 4 CFTR-RD and 1 CBAVD, including 2 siblings), G85E (p.Gly85Glu) (1 CF-PS), W1282X (p.Trp1282*) (2 CFTR-RD siblings), L320V (p.Leu320Val) (1 CFTR-RD), S549R(A>C) (p.Ser549Arg) (1 CFTR-RD), 711+5G>A (c.579+5G>A) (1 CBAVD) and unknown (1 CBAVD). Login to comment
366 [227_228insT;1210-14TG[12];1210-12T[5]] uncertain: CF-PI and/or CF-PS and/or CFTR-RD 359insT nd; T5 varying clinical consequence G85E c.254G>A CF-PI,CF-PS CF-causing p.Gly85Glu D110H c.328G>C CF-PS CF-causing p.Asp110His R117C c.349C>T CF-PS CF-causing p.Arg117Cys R117H c.350G>A CFTR-RD varying clinical consequence p.Arg117His [R117L;L997F] c. Login to comment

[hide] Translating the genetics of cystic fibrosis to per... Transl Res. 2015 Apr 15. pii: S1931-5244(15)00131-0. doi: 10.1016/j.trsl.2015.04.008. Corvol H, Thompson KE, Tabary O, le Rouzic P, Guillot L
Translating the genetics of cystic fibrosis to personalized medicine.
Transl Res. 2015 Apr 15. pii: S1931-5244(15)00131-0. doi: 10.1016/j.trsl.2015.04.008., [PMID:25940043]

Abstract [show]
Cystic fibrosis (CF) is the most common life-threatening recessive genetic disease in the Caucasian population. This multiorgan disease is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, a chloride channel recognized as regulating several apical ion channels. The gene mutations result either in the lack of the protein at the apical surface or in an improperly functioning protein. Morbidity and mortality because of the mutation of CFTR are mainly attributable to lung disease resulting from chronic infection and inflammation. Since its discovery as the causative gene in 1989, much progress has been achieved not only in clinical genetics but also in basic science studies. Recently, combinations of these efforts have been successfully translated into development and availability for patients of new therapies targeting specific CFTR mutations to correct the CFTR at the protein level. Current technologies such as next gene sequencing and novel genomic editing tools may offer new strategies to identify new CFTR variants and modifier genes, and to correct CFTR to pursue personalized medicine, which is already developed in some patient subsets. Personalized medicine or P4 medicine ("personalized," "predictive," "preventive," and "participatory") is currently booming for CF. The various current and future challenges of personalized medicine as they apply to the issues faced in CF are discussed in this review.

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64 A p.Gly85Glu (G85E) 0.44% c.1364C . Login to comment

[hide] Prevalence of meconium ileus marks the severity of... Genet Med. 2015 Jun 18. doi: 10.1038/gim.2015.79. Dupuis A, Keenan K, Ooi CY, Dorfman R, Sontag MK, Naehrlich L, Castellani C, Strug LJ, Rommens JM, Gonska T
Prevalence of meconium ileus marks the severity of mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene.
Genet Med. 2015 Jun 18. doi: 10.1038/gim.2015.79., [PMID:26087176]

Abstract [show]
RATIONALE: Meconium ileus (MI) is a perinatal complication in cystic fibrosis (CF), which is only minimally influenced by environmental factors. We derived and examined MI prevalence (MIP) scores to assess CFTR phenotype-phenotype correlation for severe mutations. METHOD: MIP scores were established using a Canadian CF population (n = 2,492) as estimates of the proportion of patients with MI among all patients carrying the same CFTR mutation, focusing on patients with p.F508del as the second allele. Comparisons were made to the registries from the US CF Foundation (n = 43,432), Italy (Veneto/Trentino/Alto Adige regions) (n = 1,788), and Germany (n = 3,596). RESULTS: The prevalence of MI varied among the different registries (13-21%). MI was predominantly prevalent in patients with pancreatic insufficiency carrying "severe" CFTR mutations. In this severe spectrum MIP scores further distinguished between mutation types, for example, G542X (0.31) with a high, F508del (0.22) with a moderate, and G551D (0.08) with a low MIP score. Higher MIP scores were associated with more severe clinical phenotypes, such as a lower forced expiratory volume in 1 second (P = 0.01) and body mass index z score (P = 0.04). CONCLUSIONS: MIP scores can be used to rank CFTR mutations according to their clinical severity and provide a means to expand delineation of CF phenotypes.Genet Med advance online publication 18 June 2015Genetics in Medicine (2015); doi:10.1038/gim.2015.79.

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63 Canadian studies for CF modfier genes 2,492 3,153 43,432 3,596 1,788 2,230 23,397 16,023 3 716 3,438 860 15% (19%) 1,902 2,576 PIP and MIP derivation FEV1 and zBMI modeling MIP calculation following correction of MI variable 23,301 2,413 510 21% (25%) 20% (23%) 13% (15%) Total F508del/others MI prevalence uncorrected (estimated) Missing or incomplete genotype Available for analysis Canadian CF patient registry, born after 1980 US CF patient registry German CF patient registry CF patient registry, North Italy Table 1ߒ Meconium ileus prevalence scores for the most common cystic fibrosis-causing variants p. F508del/other variants Class PIP Canada, (n) MIP, (n) Canada United States Germany Italy HGVS Legacy name c.262_263delTT 394delTT I 0.38 (50) c.3472C>T R1158X I 0.37 (35) c.1558G>T V520F 0.35 (43) c.3484C>T R1162X I 0.34 (135) 0.17 (14) 0.22 (45) c.2012delT 2143delT I 0.33 (13) c.3276C>A or G Y1092X I 0.92 (13) 0.09 (12) 0.33 (55) c.3846G>A W1282X I 1.00 (13) 0.29 (13) 0.32 (442) 0.17 (20) c.1477C>T Q493X I 1.00 (11) 0.19 (11) 0.32 (102) c.3528delC 3659delC I 0.31 (139) c.579ߙ+ߙ1G>T 711ߙ+ߙ1G>T 0.97 (39) 0.30 (38) 0.31 (54) c.178G>T E60X I 0.30 (66) c.1657C>T R553X I 1.00 (16) 0.28 (16) 0.30 (415) 0.24 (107) c.1585-1G>A 1717-1G>A I 1.00 (12) 0.23 (12) 0.29 (367) 0.22 (38) 0.16 (22) c.1766ߙ+ߙ1G>A 1898ߙ+ߙ1G>A 0.29 (139) c.1624G>T G542X I 0.99 (73) 0.31 (72) 0.29 (976) 0.21 (79) 0.22 (33) c.1521_1523delCTT F508del II 0.99 (1292) 0.22 (1260) 0.27 (15391) 0.21 (1910) 0.20 (230) c.1679G>C R560T II 0.27 (123) c.3744delA 3876delA 0.27 (22) c.2128A>T K710X I 0.26 (12) c.1519_1521delATC I507del II 1.00 (20) 0.21 (19) 0.25 (162) c.3909C>G N1303K II 0.98 (40) 0.13 (39) 0.25 (534) 0.23 (80) 0.14 (62) c.489ߙ+ߙ1G>T 621ߙ+ߙ1G>T I 1.00 (90) 0.24 (88) 0.25 (369) 0.21 (11) c.3266G>A W1089X I 0.25 (17) c.1675G>A A559T 0.24 (21) c.988G>T G330X 0.24 (10) c.3773_3774insT 3905insT 0.23 (78) c.2988ߙ+ߙ1G>A 3120ߙ+ߙ1G>A 0.22 (121) c.443T>C I148T;3199del6 1.00 (15) 0.22 (15) c.2052delA 2184delA I 0.21 (89) 0.22 (10) c.2051_2052delAAinsG 2183AA>G 0.20 (73) 0.20 (42) c.948delT 1078delT 0.19 (20) c.1652G>A G551D III 0.96 (54) 0.08 (53) 0.15 (979) 0.09 (84) c.254G>A G85E 0.50 (24) 0.06 (24) 0.14 (137) 0.00 (10) c.3196C>T R1066C 0.14 (42) c.1466C>A S489X 1.00 (14) 0.14 (14) c.3808G>A D1270N 0.13 (19) c.1055G>A R352Q 0.12 (18) c.579ߙ+ߙ5G>A 711ߙ+ߙ5G>A 0.12 (30) c.2175_2176insA 2307insA 0.11 (24) c.349C>T R117C 0.10 (37) c.1040G>C R347P IV 0.18 (11) 0.19 (11) 0.10 (130) 0.02 (56) c.350G>A R117H IV 0.05 (21) 0.00 (21) 0.07 (666) 0.02 (19) c.2657ߙ+ߙ5G>A 2789ߙ+ߙ5G>A V 0.25 (20) 0.00 (20) 0.06 (271) 0.01 (21) c.1040G>A R347H 0.06 (55) c.2988G>A 3120G->A 0.06 (36) c.328G>C D1152H IV 0.06 (124) c.3717ߙ+ߙ12191C>T 3849ߙ+ߙ10kbC>T V 0.07 (14) 0.00 (14) 0.05 (299) 0.01 (42) 0.00 (15) c.1364C>A A455E V 0.16 (45) 0.01 (41) 0.05 (109) c.1000C>T R334W IV 0.18 (11) 0.00 (10) 0.05 (92) c.617T>G L206W 0.06 (18) 0.05 (17) 0.04 (52) c.3302T>A M1101K 0.04 (17) c.200C>T P67L V 0.07 (14) 0.00 (14) Meconium ileus prevalence (MIP) and pancreas insufficiency prevalence (PIP) scores are presented. Login to comment
109 While non-CFTR modifier genes as well as environmental factors largely influence the development and progression of lung disease and nutritional decline,33-36 we demonstrate that the severity of the underlying CFTR genotype Table 2ߒ Meconium ileus prevalence scores and CFTR function CFTR mutation MIP score CFTR function (%wt) High MIP score ߓ V520F 0.38 0.2 ߓ N1303K 0.25 0.5 ߓ F508del 0.27 0.4 ߓ R560T 0.27 0.1 ߓ A559T 0.24 0 ߓ G551D 0.15 1 ߓ G85E 0.14 0.8 ߓ R1066C 0.13 0 Low MIP score ߓ R347P 0.1 0 ߓ R117C 0.1 2.9 ߓ R117H 0.07 33 ߓ R347H 0.06 5 ߓ R334W 0.05 1.3 ߓ A455E 0.05 6 ߓ L206W 0.04 5 ߓ M1101K 0.04 0 ߓ P67L 0.0 8 The table compares meconium ileus prevalence (MIP) scores and measured cystic fibrosis transmembrane conductance regulator (CFTR) function in Fisher rat thyroid determined by VanGoor et al.24 for the major and missense cystic fibrosis-causing variants for which patient group size was ࣙ10 in at least the US group. Login to comment

[hide] Distribution of Cystic Fibrosis Transmembrane Cond... PLoS One. 2015 Jul 24;10(7):e0133890. doi: 10.1371/journal.pone.0133890. eCollection 2015. Siryani I, Jama M, Rumman N, Marzouqa H, Kannan M, Lyon E, Hindiyeh M
Distribution of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Mutations in a Cohort of Patients Residing in Palestine.
PLoS One. 2015 Jul 24;10(7):e0133890. doi: 10.1371/journal.pone.0133890. eCollection 2015., [PMID:26208274]

Abstract [show]
Cystic fibrosis (CF) is an autosomal recessive inherited life-threatening disorder that causes severe damage to the lungs and the digestive system. In Palestine, mutations in the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) that contributes to the clinical presentation of CF are ill defined. A cohort of thirty three clinically diagnosed CF patients from twenty one different Palestinian families residing in the central and southern part of Palestine were incorporated in this study. Sweat chloride testing was performed using the Sweat Chek Conductivity Analyzer (ELITECH Group, France) to confirm the clinical diagnosis of CF. In addition, nucleic acid from the patients' blood samples was extracted and the CFTR mutation profiles were assessed by direct sequencing of the CFTR 27 exons and the intron-exon boundaries. For patient's DNA samples where no homozygous or two heterozygous CFTR mutations were identified by exon sequencing, DNA samples were tested for deletions or duplications using SALSA MLPA probemix P091-D1 CFTR assay. Sweat chloride testing confirmed the clinical diagnosis of CF in those patients. All patients had NaCl conductivity >60 mmol/l. In addition, nine different CFTR mutations were identified in all 21 different families evaluated. These mutations were c.1393-1G>A, F508del, W1282X, G85E, c.313delA, N1303K, deletion exons 17a-17b-18, deletion exons 17a-17b and Q1100P. c.1393-1G>A was shown to be the most frequent occurring mutation among tested families. We have profiled the underling mutations in the CFTR gene of a cohort of 21 different families affected by CF. Unlike other studies from the Arab countries where F508del was reported to be the most common mutation, in southern/central Palestine, the c.1393-1G>A appeared to be the most common. Further studies are needed per sample size and geographic distribution to account for other possible CFTR genetic alterations and their frequencies. Genotype/phenotype assessments are also recommended and finally carrier frequency should be ascertained.

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9 These mutations were c.1393-1G>A, F508del, W1282X, G85E, c.313delA, N1303K, deletion exons 17a-17b-18, deletion exons 17a-17b and Q1100P. Login to comment
34 Few studies from different countries reported the mutations present in the Palestinian patients they treated in those countries of which F508del, N1303K, W1282X, 3120+1Kbdel8.6Kb and G85E were the most common [9, 10]. Login to comment
46 Code Family Members Age (Years) District Result /Mutations Sweat Conductivity Equivalent NaCl (mmol/L) Pancreaticassessment Sputum Culture Results BMI 001 Pal-1 Daughter-1 5 Hebron c.1393-1G>A 125 PI P. aeruginosa 15.2 002 Pal-1 Daughter- 2 11 Hebron c.1393-1G>A 110 PI P. aeruginosa 16.1 027 Pal-1/ Cos Daughter-1 7 Hebron c.1393-1G>A 125 PI P. aeruginosa 14.0 005 Pal-2 Daughter-1 5 Hebron F508del 108 PI P. aeruginosa 14.3 011 Pal-3 Son-1 25 Bethlehem Deletion exons 17a-17b 137 PI P. aeruginosa 18.4 013 Pal-4 Son-1 16 Hebron W1282X 104 PI P. aeruginosa 18.6 014 Pal-4 Daughter-1 5 Hebron W1282X 119 PI P. aeruginosa 16.1 015 Pal-4 Daughter- 2 8 Hebron W1282X 92 PI P. aeruginosa 13.4 021 Pal-5 Son-1 14 Hebron c.1393-1G>A 135 PI NA 16.9 030 Pal-6 Daughter-1 2 Hebron Het (c.1393-1G>A) Het (W1282X) 103 PI P. aeruginosa 15.0 040 Pal-6/ Cos Son-1 11 Hebron c.1393-1G>A 108 PI P. aeruginosa 15.7 035 Pal-7 Son-1 6 Hebron F508del 130 PI Negative 14.0 036 Pa1-7 Daughter-1 10 Hebron F508del 132 PI Negative 15.7 038 Pal-8 Daughter-1 8 Hebron Het (F508del) Deletion Exons 17a-17b-18 110 PI P. aeruginosa 17.8 050 Pal-9 Daughter-1 14 Hebron N1303K 132 PI P. aeruginosa 12.6 058 Pal-10 Son-1 10 Hebron Het (F508del) Deletion Exons 17a-17b-18 111 PI P. aeruginosa 12.7 070 Pal-11 Daughter-1 4 Hebron W1282X 101 PI P. aeruginosa and MRSA 15.5 117 Pal-11/ Cos Daughter 0.5 Hebron W1282X 120 PI P. aeruginosa 13.3 072 Pa1-12 Daughter-1 5 Hebron G85E 102 PI P. aeruginosa 14.2 073 Pal-12 Daughter- 2 7 Hebron G85E 115 PI P. aeruginosa 15.3 074 Pal-12/ Cos Daughter-1 11 Hebron G85E 129 PI P. aeruginosa and MRSA 16.2 079 Pal-13 Son-1 4 Hebron 444DelA 116 PI MRSA 16.2 080 Pal-13 Son-2 7 Hebron 444DelA 101 PI Negative 15.2 081 Pal-13 Son-3 1 Hebron 444DelA 90 PI Negative 9.7 091 Pal-14 Son-1 7 Hebron c.1393-1G>A 117 PI Negative 15.2 093 Pal-15 Son-1 1 Hebron F508del 117 PI P. aeruginosa 15.2 099 Pal-16 Daughter-1 1 Hebron W1282X 124 PI P. aeruginosa 14.9 102 Pal-17 Son-1 30 Hebron Het (G85E)Het (Q1100P) 130 PI P. aeruginosa 20.1 (Continued) distantly related, rather than possessing a true founder mutation. Login to comment
76 The CFTR mutations detected were c.1393-1G>A, F508del, W1282X, G85E, c.313delA, N1303K, deletion exons 17a-17b-18, deletion exons 17a-17b and Q1100P. Login to comment
101 CFTR Mutations Exon/Intron # of Families Frequency (%) c.1393-1G>A / c.1393-1G>A Intron 9 6 28.58 F508del / F508delA Exon 10 4 19.05 W1282X / W1282X Exon 20 3 14.29 F508del / Deletion exons 17a-17b-18 Exon 10 / Exons 17a-18 2 9.52 G85E / G85E Exon 3 1 4.76 c.313delA / c.313delA Exon 4 1 4.76 Deletion exons 17a-17b-18 / Deletion exons 17a-17b-18 Exons 17a-18 1 4.76 N1303K / N1303K Exon 21 1 4.76 G85E / Q1100P Exon 3 / Exon 17b 1 4.76 Deletion exons 17a-17b / Deletion exons 17a-17b Exons 17a-17b 1 4.76 doi:10.1371/journal.pone.0133890.t002 mutation was detected in six families out of the twenty one involved in our study in both homozygous (28.6%), and heterozygous (4.8%) forms. Login to comment
110 These mutations were G85E, N1303K, Q1100P, c.313delA, deletion Exons 17a-17b and 3120+1Kbdel8.6Kb (deletion in exons 17a, 17b, 18) [23]. Login to comment

[hide] The impact of a national population carrier screen... J Cyst Fibros. 2015 Sep 16. pii: S1569-1993(15)00203-9. doi: 10.1016/j.jcf.2015.08.007. Stafler P, Mei-Zahav M, Wilschanski M, Mussaffi H, Efrati O, Lavie M, Shoseyov D, Cohen-Cymberknoh M, Gur M, Bentur L, Livnat G, Aviram M, Alkrinawi S, Picard E, Prais D, Steuer G, Inbar O, Kerem E, Blau H
The impact of a national population carrier screening program on cystic fibrosis birth rate and age at diagnosis: Implications for newborn screening.
J Cyst Fibros. 2015 Sep 16. pii: S1569-1993(15)00203-9. doi: 10.1016/j.jcf.2015.08.007., [PMID:26386752]

Abstract [show]
BACKGROUND: Population carrier screening (PCS) has been available in Israel since 1999 and universally subsidized since 2008. We sought to evaluate its impact. METHODS: A retrospective review of governmental databanks, the national CF registry and CF centers. RESULTS: CF rate per 100,000 live births has decreased from 14.5 in 1990 to 6 in 2011. From 2004-2011 there were 95 CF births: 22 utilized PCS; 68 (72%) had 2 known CFTR mutations; 37% were pancreatic sufficient. At diagnosis, age was 6 (0-98) months; 53/95 had respiratory symptoms, 41/95 failure to thrive and 19/95 pseudomonas. Thirty-four (36%) were Arabs and 19 (20%) orthodox Jews, compared to 20% and 8% respectively, in the general population. CONCLUSIONS: PCS markedly reduced CF birth rates with a shift towards milder mutations, but was often avoided for cultural reasons. As children regularly have significant disease at diagnosis, we suggest a balanced approach, utilizing both PCS and newborn screening.

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55 Mutation DF508 G542X W1282X N1303K 3849 + 10kbC- N T D1152H 405 + 1G࢐A G85E S549R W1089X 1717 + 1G࢐A I1234Va Y1092Xb 3121-1G N Ab 3120 + 1kbdel8.6 kbc 2183AA N Gc 4010delTATTc The first 14 mutations served as the panel used for Jewish population carrier screening program during the study period. Login to comment

[hide] Identification and frequencies of cystic fibrosis ... Clin Biochem. 2015 Oct 21. pii: S0009-9120(15)00473-7. doi: 10.1016/j.clinbiochem.2015.10.007. Pepermans X, Mellado S, Chialina S, Wagener M, Gallardo L, Lande H, Bordino W, Baran D, Bours V, Leal T
Identification and frequencies of cystic fibrosis mutations in central Argentina.
Clin Biochem. 2015 Oct 21. pii: S0009-9120(15)00473-7. doi: 10.1016/j.clinbiochem.2015.10.007., [PMID:26500004]

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99 rs name HGVS p. name HGVS c. name Legacy name n (%) Screening panel CFTR1 database CFTR2 database rs199826652 p.Phe508del c.1521_1523delCTT F508del 94 (56.6) Yes Yes CF-causing rs113993959 p.Gly542* c.1624G N T G542X 7 (4.2) Yes Yes CF-causing No p.Asn1303Lys c.3909C N G N1303K 5 (3) Yes Yes CF-causing rs74767530 p.Arg1162* c.3484C N T R1162X 4 (2.4) Yes Yes CF-causing rs75961395 p.Gly85Glu c.254G N A G85E 3 (1.8) Yes Yes CF-causing rs78756941 NA c.489 + 1G N T 621 + 1G N T 3 (1.8) Yes Yes CF-causing rs76713772 NA c.1585-1G N A 1717-1G N A 3 (1.8) Yes Yes CF-causing No p.Lys684Serfs*38 c.2051_2052delAAinsG 2183AA N G 3 (1.8) Yes Yes CF-causing rs397508173 p.Ser4* c.11C N A S4X 2 (1.2) No Yes No rs121909011 p.Arg334Trp c.1000C N T R334W 2 (1.2) Yes Yes CF-causing rs77010898 p.Trp1282* c.3846G N A W1282X 2 (1.2) Yes Yes CF-causing rs397508141 p.Leu34_Gln39del c.100_117delTTGTCAGACATATACCAA 232del18 1 (0.6) No Yes No No p.Leu49Pro c.146 T N C L49P &#a7; 1 (0.6) No No No rs77834169 p.Arg117Cys c.349C N T R117C 1 (0.6) Yes Yes CF-causing No p.Arg117Pro c.350G N C R117P 1 (0.6) No Yes No rs80282562 p.Gly178Arg c.532G N A G178R 1 (0.6) Yes Yes CF-causing rs121908803 p.Pro205Ser c.613C N T P205S 1 (0.6) No Yes CF-causing rs121908752 p.Leu206Trp c.617 T N G L206W 1 (0.6) Yes Yes CF-causing No p.Arg347Pro c.1040G N C R347P 1 (0.6) Yes Yes CF-causing rs397508155 p.Tyr362* c.1086 T N A Y362X 1 (0.6) No Yes No rs74597325 p.Arg553* c.1657C N T R553X 1 (0.6) Yes Yes CF-causing rs1800098 + rs1800100 p.[Gly576Ala(;)Arg668Cys] c. Login to comment
113 Genotypes associated with nasal polyps were p.Phe508del/p.Phe508del, p.Phe508del/p.Gly85Glu and p.Gly542*/p.Asn1303Lys. Login to comment
126 Genotype N Frequency (%) Total N Total frequency (%) Category I: p.Phe508del/p.Phe508del p.Phe508del/p.Phe508del 30 36.1 30 36.1 Category II: p.Phe508del/Other p.Phe508del/p.Gly542* 5 6 p.Phe508del/p.Asn1303Lys 3 3.6 p.Phe508del/p.Gly85Glu 2 2.4 p.Phe508del/c.1585-1G N A 2 2.4 p.Phe508del/c.2051_2052delAAinsG 2 2.4 p.Phe508del/p.Trp1282* 2 2.4 p.Phe508del/p.Arg117Pro 1 1.2 p.Phe508del/p.Pro205Ser 1 1.2 p.Phe508del/p.Leu206Trp 1 1.2 p.Phe508del/p.Arg553* 1 1.2 p.Phe508del/p.Ser589Ile 1 1.2 p.Phe508del/p.Ser737Phe 1 1.2 p.Phe508del/p.Arg1162* 1 1.2 p.Phe508del/c.1766 + 1G N A 1 1.2 p.Phe508del/p.Leu34_Gln39del 1 1.2 p.Phe508del/p.Leu812Phefs*11 1 1.2 p.Phe508del/c.3140-26A N G 1 1.2 p.Phe508del/c.3873 + 1G N A 1 1.2 p.Phe508del/p.Ser1297Phefs*5 1 1.2 p.Phe508del/c.4242_4242 + 1delGGinsTT 1 1.2 p.Phe508del/c.489 + 1G N T 1 1.2 31 37.5 Category III: Other/other p.Gly542*/p.Asn1303Lys 1 1.2 p.Asn1303Lys/p.Gly85Glu 1 1.2 c.489 + 1G N T/p.Lys684Serfs*38 1 1.2 c.489 + 1G N T/p.Gly542* 1 1.2 p.Arg1162*/p.Ser4* 1 1.2 p.Arg1162*/p.Tyr362* 1 1.2 p.Arg334Trp/c.1585-1G N A 1 1.2 p.Arg334Trp/p.Ser821Argfs*4 1 1.2 p.Arg347Pro/p.Ser4* 1 1.2 c.2657 + 5G N A/p.Tyr852Leufs*44 # 1 1.2 p.Arg1162*/p.Leu49Pro # 1 1.2 11 13.2 Category IV: A single mutation p.Phe508del/WT 3 3.6 c.2988 + 1G N A/WT 1 1.2 p.Arg117Cys/WT 1 1.2 p.Gly178Arg/WT 1 1.2 p.[Gly576Ala(;)Arg668Cys]/TG11-5T 1 1.2 7 8.4 Category V: Wild type 4 4.8 #: new mutation submitted to CFTR1 database [1]; other = other mutation than p.Phe508del. Login to comment

[hide] Newborn Screening for Cystic Fibrosis in Californi... Pediatrics. 2015 Dec;136(6):1062-72. doi: 10.1542/peds.2015-0811. Epub 2015 Nov 16. Kharrazi M, Yang J, Bishop T, Lessing S, Young S, Graham S, Pearl M, Chow H, Ho T, Currier R, Gaffney L, Feuchtbaum L
Newborn Screening for Cystic Fibrosis in California.
Pediatrics. 2015 Dec;136(6):1062-72. doi: 10.1542/peds.2015-0811. Epub 2015 Nov 16., [PMID:26574590]

Abstract [show]
OBJECTIVES: This article describes the methods used and the program performance results for the first 5 years of newborn screening for cystic fibrosis (CF) in California. METHODS: From July 16, 2007, to June 30, 2012, a total of 2 573 293 newborns were screened for CF by using a 3-step model: (1) measuring immunoreactive trypsinogen in all dried blood spot specimens; (2) testing 28 to 40 selected cystic fibrosis transmembrane conductance regulator (CFTR) mutations in specimens with immunoreactive trypsinogen values >/=62 ng/mL (top 1.6%); and (3) performing DNA sequencing on specimens found to have only 1 mutation in step 2. Infants with >/=2 mutations/variants were referred to CF care centers for diagnostic evaluation and follow-up. Infants with 1 mutation were considered carriers and their parents offered telephone genetic counseling. RESULTS: Overall, 345 CF cases, 533 CFTR-related metabolic syndrome cases, and 1617 carriers were detected; 28 cases of CF were missed. Of the 345 CF cases, 20 (5.8%) infants were initially assessed as having CFTR-related metabolic syndrome, and their CF diagnosis occurred after age 6 months (median follow-up: 4.5 years). Program sensitivity was 92%, and the positive predictive value was 34%. CF prevalence was 1 in 6899 births. A total of 303 CFTR mutations were identified, including 78 novel variants. The median age at referral to a CF care center was 34 days (18 and 37 days for step 2 and 3 screening test-positive infants, respectively). CONCLUSIONS: The 3-step model had high detection and low false-positive levels in this diverse population.

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77 July 16, 2007 c.164+2T.A (296+2T.A) 28 c.254G.A (G85E) c.274-1G.A (406-1G.A) c.489+1G.T (621+1G.T) c.579+1G.T (711+1G.T) c.595C.T (H199Y) c.933_935delCTT (F311del) c.1000C.T (R334W) c.1519_1521delATC (I507del) c.1521_1523delCTT (F508del) c.1585-1G.A (1717-1G.A) c.1624G.T (G5423) c.1646G.A (S549N) c.1652G.A (G551D) c.1657C.T (R5533) c.1675G.A (A559T) c.1680-1G.A (1812-1G.A) c.1973-1985del13insAGAAA (2105-2117del13insAGAAA) c.2175_2176insA (2307insA) c.2988+1G.A (3120+1G.A) c.3196C.T (R1066C) c.3266G.A (W10893) c.3485G.T (R11623) c.3611G.A (W12043 [3743G.A]) c.3717+12191C.T (3849+10kbC.T) c.3744delA (3876delA) c.3846G.A (W12823) c.3909C.G (N1303K) October 4, 2007 c.1153_1154insAT (1288insTA) 29 December 12, 2007 c.54-5940_273+10250del21kb (CFTRdele2,3(21kb)) 38 c.531delT (663delT) c.613C.T (P205S) c.803delA (935delA) c.1475C.T (S492F) c.1923_1931del9insA (2055del9.A) c.223C.T (R753) c.293A.G (Q98R) c.3140-26A.G (3272-26A.G) August 12, 2008 c.988G.T (G3303) 40 c.3612G.A (W12043 [3744G.A]) c.3659delC (3791delC) c.164+2T.A (296+2T.A), removed cDNA, complementary DNA. Login to comment

[hide] [Male infertility caused by bilateral agenesis of ... Rev Med Interne. 1997;18(2):114-8. Durieu I, Bey-Omar F, Rollet J, Boggio D, Bellon G, Morel Y, Vital Durand D
[Male infertility caused by bilateral agenesis of the vas deferens: a new clinical form of cystic fibrosis?].
Rev Med Interne. 1997;18(2):114-8., [PMID:9092029]

Abstract [show]
Congenital bilateral absence of vas deferens causes male excretory infertility and represents 1 to 2% of male infertility. Because of a genotypic similarity with cystic fibrosis, the possible in vitro fertilization with epididymal sperm requires careful genetic counselling. We studied genotype, sweat chloride concentration, respiratory function tests, sinus abnormalities, pancreatic and hepatic functions in 22 subjects with congenital bilateral absence of vas deferens. Among them, four were compound heterozygotus, all of them with the R117H mutation. Ten had a positive sweat test, one of them also being compound heterozygotus. Congenital bilateral absence of vas deferens and double mutation or positive sweat test led to high probable cystic fibrosis diagnosis in 13 subjects. Six subjects were heterozygotus for one cystic fibrosis mutation, criterium which is not sufficient for cystic fibrosis diagnosis; five of them had sinus abnormalities, present in 11 of the 22 subjects. Only three patients had no mutation nor sweat chloride abnormalities. This work confirms the high frequency of cystic fibrosis mutations in males with congenital bilateral absence of vas deferens, with a higher frequency of positive sweat test than in other publications, and a high frequency of sinus abnormalities. This monosymptomatic phenotype of cystic fibrosis suggests new hypotheses for a relationship between genotype and phenotype.

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46 Vingt-deux mutations du gene CFTR ont Cte recher- chtes : les cinq plus frequentes (AF508, G542X, N1303K, 1717-G--A, G85E) et les 17 suivantes : R117H, 556delA, R334W, R347H, R347P, S549N, S5491, S549R, G551D, R553X,R560T,G1244E3,S1255X,W1282X,R1283K,3898 ins C, D1270N. Login to comment