ABCMdb

ABCC7 p.Phe1052Val

Admin's notes:	Class III (gating defect) Veit et al.
ClinVar:	c.3154T>G , p.Phe1052Val D , Likely pathogenic
CF databases:	c.3154T>G , p.Phe1052Val ? , Varying clinical consequence ; CFTR1:
Predicted by SNAP2:	A: D (91%), C: D (91%), D: D (95%), E: D (95%), G: D (95%), H: D (95%), I: D (85%), K: D (95%), L: D (85%), M: D (91%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: D (95%), T: D (95%), V: D (53%), W: D (95%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: N, Y: N,

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

Publications
[hide] 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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381 When R1066 was mutated into R1066C, the protein was not correctly processed, not fully glycosylated, reflecting a defect in protein biosynthesis and little discernible effect on function; in contrast, mutant F1052V was processed normally but had dramatically altered function [181,182]. Login to comment

[hide] Complete mutational screening of the cystic fibros... Hum Reprod. 1999 Dec;14(12):3035-40. Pallares-Ruiz N, Carles S, Des Georges M, Guittard C, Arnal F, Humeau C, Claustres M
Complete mutational screening of the cystic fibrosis transmembrane conductance regulator gene: cystic fibrosis mutations are not involved in healthy men with reduced sperm quality.
Hum Reprod. 1999 Dec;14(12):3035-40., [PMID:10601093]

Abstract [show]
Based on the analysis of the most frequent mutations responsible for cystic fibrosis (CF), a higher than expected frequency of CF mutations was recently reported in men with infertility due to reduced sperm quality. To further document whether this condition is associated with severe or mild abnormalities of cystic fibrosis transmembrane conductance regulator (CFTR) functions, we carried out a complete scanning of CFTR sequences using a strategy that detects almost all 850 mutations and 150 polymorphisms reported to date in the CFTR gene. We have investigated a cohort of 56 patients with severe oligoasthenoteratozoospermia (OAT) and 50 controls from southern France for CFTR gene mutations and variations. The frequencies of CF-causing mutations and CFTR variations identified in this OAT sample did not differ significantly from the frequencies found in the normal population. However, we observed a 1.7-fold increase in the proportion of homozygotes for a specific CFTR haplotype (TG11-T7-G1540) in the OAT group (P = 0.025). Our results do not confirm a link between CF mutations and reduced sperm quality. Further studies are needed to substantiate the hypothesis that a combination of variants affecting expression and function of the CFTR protein is associated with male infertility.

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24 The main objective of this F1052V (Mercier et al., 1993) and M952I (Girodon et al., study was to compare the complete CFTR genotypes of 56 1996) have been previously reported in CF and CBAVD men with OAT and 50 controls from the same population patients to the CF Genetics Analysis Consortium (CFGAC, background, in order to evaluate a putative involvement of http://www.genet.sickkids.on.ca/CFTR). Login to comment
56 Four OAT men had a missense mutation on one chromosome, 1540 (M470V) in exon 10 was significantly different between Table I. Characterization of CFTR genotypes in 56 patients with oligoasthenoteratozoospermia (OAT) and in 50 controls Mutations IVS8(T)n 1540A/G Other variations IVS8(TG)n OAT 1 I1230T 7/7 A/G 1655T/G 12/12 1 D1152H 7/7 G/G - 11/11 1 F1052V 7/7 A/A 875ϩ40A/G 10/10 1 M952I 7/7 G/G 4404C/T 11/11 1 - 7/7 G/G 4404C/T 11/11 2 - 7/7 A/G 875ϩ40A/G 10/11 2 - 7/7 A/G 125G/C 11/12 1 - 7/7 A/A 125G/C 12/12 1 - 7/9 A/A 1716G/A, 3041-71G/C ϩ 4002A/Ga 11/10 1 - 7/7 G/G 356G/A, 405ϩ46G/T, 4374ϩ13A/G 11/11 1 - 7/7 A/A 875ϩ40A/G, 3499ϩ37G/A 10/10 1 - 7/9 A/A 1859G/C ϩ 2134C/Ta 10/10 1 - 7/7 A/G 4002A/G 12/12 1 - 7/9 A/G 4002A/G 11/10 1 - 7/7 G/G 2377C/T 11/11 1 - 7/7 A/A 875ϩ40A/G, 1716G/A 10/10 1 - 7/7 G/G 3417A/T 11/11 1 - 7/7 A/G 3417A/T 11/12 24 - 7/7 G/G - 11/11 2 - 7/9 A/G - 10/12 3 - 7/9 A/G - 11/10 2 - 7/9 A/A - 10/10 1 - 9/9 A/A - 10/10 2 - 7/7 A/G - 10/11 1 - 7/7 A/A - 10/10 1 - 7/7 G/G - 8/11 Controls 1 ∆F508 7/9 A/G - 10/12 1 ∆F508 7/9 A/A 875ϩ40A/G 10/11 1 V562L 7/7 A/A 223C/T 10/10 1 G622D 7/9 A/G 3041-71G/C ϩ 4002A/Ga 10/11 1 - 7/7 A/A 3419T/G 10/11 1 - 7/7 G/G 4002A/G 11/11 3 - 7/7 A/G 125G/C 11/11 1 - 7/7 G/G 125G/C 11/11 1 - 7/7 A/A 125G/C 10/11 1 - 7/7 A/A 125G/C 11/12 2 - 7/5 A/G 875ϩ40A/G 11/11 1 - 7/7 A/G 875ϩ40A/G 10/10 1 - 7/7 A/A 875ϩ40A/G, 125G/C 10/11 1 - 7/7 G/G 356G/A 11/11 1 - 7/7 A/G 356G/A 10/10 1 - 9/9 A/A 3041-71G/C ϩ 4002A/Ga 10/10 1 - 7/7 G/G 406-6T/C 11/11 1 - 7/7 A/G 3417A/T 10/11 1 - 7/7 G/G 4404C/T 11/11 1 - 7/7 A/G 1859G/Cϩ2134C/Ta 10/11 11 - 7/7 G/G - 11/11 6 - 7/7 A/G - 10/10 2 - 7/7 A/G - 11/11 1 - 7/7 A/G - 10/11 5 - 7/9 A/G - 10/12 2 - 7/5 G/G - 10/11 aDouble mutant alleles, In bold: mutations or variations previously undescribed. 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
88 In three further cases where more than one sporadic mutation was observed in the same individual (1341+28 C/T and F1052V and S1235R; ∆F508 and I1027T; 1716G/A and N1303K), data from the literature were not available, and segregation analysis was not possible; thus, their phase could not be established. Login to comment
96 Moreover, 1525-61 A/G (i 9) and 3601-65 C/A (i 18) were detected by SSCA performed in the Spanish sample only (14/82 and 12/80, respectively); these mutations were not identifiable by DGGE as used in the present work The totals are: a378; b362; c380; d356 genes eCertainly a CF-causing mutations fThe most common allele at this site is (TTGA)7 gThe most common allele at this site is T7 hThe frequency shown is that of the M allele Mutation Position North-Central Southern Spain Total East Italy Italy France 82 genes 100 genes 100 genes 100 genes 382 genes % 125 G/C 5`UTR 1 2 7 3 13 3.4 R31C 2 1 1 1 0 3 0.8 K68E 3 1 0 0 0 1 0.3 R75Q 3 1 1 2 0 4 1.0 G85Ee 3 0 1 0 0 1 0.3 406-6 T/C i 3 0 0 1 0 1 0.3 I148T 4 1 0 0 0 1 0.3 621+3 A/G i 4 0 1 0 0 1 0.3 R170H 5 1 0 0 0 1 0.3 875+40 A/G i 6a 11 5 5 2 23 6.0 (TTGA)6 f i 6a 17 11 7 13 48 12.6 1341+28 C/T i 8 1 0 0 0 1 0.3 IVS8-6g T5 i 8 8 2 4 3/78 17a 4.5 IVS8-6g T9 i 8 10 7 10 11/78 38a 10.0 M470Vh 10 42 30 39 27 138 36.1 I506V 10 1 0 0 0 1 0.3 ∆F508e 10 1 0 2 0 3 0.8 1716 G/A 10 2 1 0 5 8 2.1 V562L 12 0 0 1 0 1 0.3 G576A 12 1 0/80 1 0 2b 0.6 G622D 13 0 0/80 1 0 1b 0.3 R668C 13 1 0/80 1 0 2b 0.6 2082 C/T 13 1 0/80 0 0 1b 0.3 2377 C/T 13 0 0/80 0 1 1b 0.3 2694 T/G i 14a 33 23 33 14/80 103c 27.1 2752-15 C/G i 14b 0 3 0 0 3 0.8 3041-71 G/C i 15 0 1 2 0 3 0.8 L997F 17a 0 2 0 0 2 0.5 I1027T 17a 1 0 0 0 1 0.3 F1052V 17b 1 0 0 0 1 0.3 L1096R 17b 0 0 1 0 1 0.3 3417 A/T 17b 1 0 1 0 2 0.5 I1131V 18 0 1 0 0 1 0.3 R1162L 19 0 1 0 0 1 0.3 3690 A/G 19 0 0 0 1/80 1c 0.3 S1235R 19 1 0 0 0 1 0.3 4002 A/G 20 2 3 3 3/80 11c 2.9 4005+28insA i 20 0 1 0 0 0.3 4029 A/G 21 1 0 0 0 1 0.3 N1303Ke 21 1 0 0 0 1 0.3 4404 C/T 24 1 0 1 0 2 0.5 4521 G/A 24 21 16 14/80 15/76 66d 18.5 Total 165 113 137 98 513 encountered in the present survey are possible. Login to comment

[hide] Many deltaF508 heterozygote neonates with transien... J Med Genet. 2000 Jul;37(7):543-7. Boyne J, Evans S, Pollitt RJ, Taylor CJ, Dalton A
Many deltaF508 heterozygote neonates with transient hypertrypsinaemia have a second, mild CFTR mutation.
J Med Genet. 2000 Jul;37(7):543-7., [PMID:10970190]

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538 These have been reported in patients with presenting phenotypes ranging from "cystic fibrosis" to oligospermia, but there have been too few cases Table 2 Compound heterozygotes detected Domain and mutation type Genotype Exon 1st IRT 2nd IRT Transmembrane, missense F508/P67L 3 129 34* F508/R117H 4 110 21* F508/R117H 4 84 34 F508/R117H 4 95 39 F508/R117H 4 104 40 F508/R117H 4 146 41 F508/R117H 4 104 48* F508/R117H 4 120 53 F508/R117H 4 111 54 F508/R117H 4 175 72* F508/R117L 4 129 70 F508/L967S 15 122 15 F508/F1052V 17b 189 29 F508/R1066H 17b 94 18 Transmembrane, nonsense F508/R75X 3 86 26 F508/R75X 3 171 27 F508/R851X 14a 112 76 Regulatory, missense F508/F693L 13 109 29 Alternate splice site F508/3849+10KB C→T i19 99 26* F508/3849+10KB C→T i19 112 36* None of these samples had the IVS8-5T variant sequence. Login to comment

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

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

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8 The cystic brosis transmembrane regulator (CFTR) gene mutations identi ed were delF508, D1152H, R1066C, R334W, G542X, N1303K, F1052V, A120T, 3849 ‡ 10kbC ® T, 2789 ‡ 5G ® A, 5T-12TG and the novel mutation D110E. Login to comment
70 Year of birth Patient Sex Age at diagnosis Genotype Sweat test (chloride mEq l¡1 ) 1990 1 BA F 8 mo DF508/2789 ‡ 5G ® A 74, 79 2 LG M 4 y ¡/¡ 84, 83 1991 3 BV F 6 y ¡/¡ a 61, 85, 70 4 CA F 8 y R1066C/D1152H 58, 59 5 CA F 8 y DF508/5T-TG12 65, 67 6 PS M 5 y N1303K/-a 41, 43, 55, 63, 85, 89 1992 7 AE F 1 y R334W/-a 57, 42, 78, 82 8 DA M 4 mo ¡/¡ 85, 101, 143, 9 FA M 1 y ¡/¡ a 70, 75, 98, 114 1993 10 CA F 7 y DF508/5T-TG12 45, 50 1995 11 BM M 3 y DF508/DF508 117, 123 1997 12 DG M 6 mo G542X/D110E 59, 88, 80, 70 13 DE F 2 y D1152H/3849 ‡ 10kbC ® T 31, 35 14 TL M 2 y ¡/¡ a 115, 136 1998 15 CM M 5 mo F1052V/A120T 20, 25 F: female; M: male. Login to comment
80 The CFTR alterations identi ed were D1152H, R1066C, R334W, G542X, N1303K, F1052V, A120T, 3849 ‡ 10kbC ® T, 2789 ‡ 5G ® A, 5T-12TG and the new mutation D110E (19). Login to comment

[hide] 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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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

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

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

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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] Multiplex sequence variation detection throughout ... Mol Hum Reprod. 2002 Sep;8(9):880-6. Vrettou C, Tzetis M, Traeger-Synodinos J, Palmer G, Kanavakis E
Multiplex sequence variation detection throughout the CFTR gene appropriate for preimplantation genetic diagnosis in populations with heterogeneity of cystic fibrosis mutations.
Mol Hum Reprod. 2002 Sep;8(9):880-6., [PMID:12200467]

Abstract [show]
Cystic fibrosis (CF) is one of the most important genetic diseases requiring prevention programmes. Preimplantation genetic diagnosis (PGD) represents an alternative to prenatal diagnosis, and is especially appropriate for couples with an unsuccessful reproductive history. For clinical application, protocols must be optimized to minimize PCR failure, allelic drop-out (ADO) and contamination, while simultaneously detecting a wide spectrum of CF genotypes. We have developed a flexible multiplex PCR protocol allowing analysis of sequence variations in any combination amongst seven CFTR gene exons (4, 10, 11, 13 in two parts, 14b, 17b and 21) by nested PCR and denaturing gradient gel electrophoresis analysis, along with analysis of a fluorescently labelled intragenic microsatellite (IVS8CA). The experiments were carried out on 390 single lymphocytes from three CF patients, one heterozygote and one non-CF individual. PCR efficiency of the exons ranged from 90 to 100%, and ADO from 0 to 3.8%. IVS8CA was co-amplified with a PCR efficiency of 92.4 and 10.8% ADO. The present method overcomes the need for separate assays for each CFTR gene mutation. Additionally, it facilitates analysis of any informative linked polymorphic sequence variation (within the seven exons) along with analysis of a microsatellite, which is useful (when informative) for minimizing misdiagnosis and/or indirect diagnosis. This method proved robust and flexible for diagnosing diverse CF genotype combinations in single cells.

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24 cells PCR ADO/total polymorphism (length bp) amplified product (%) cells (%) Patient 1 F508del 25 (196) 10 50 47 (94.0) 0/47 (0) 621 ϩ 1G→T 23 (192) 4 48 (96.0) 1/48 (2.1) Patient 2 N1303K 25 (196) 21 85 80 (94.1) 3/80 (3.8) 2789 ϩ 5G→A 18 (182) 14b 85 (100) 2/85 (2.4) Patient 3 E822X 17 (180) 13 part b 80 72 (90.0) 1/72 (1.4) F1052V 18 (182) 17b 75 (93.8) 2/75 (2.6) Heterozygotea 1719-9T→C 17 (180) 11 75 75 (100.0) 0/75 (0) R668C 13 part a Normal allele 18 (182) 74 (98.7) 1/74 (1.4) Microsatellite 290 268 (92.4) 29/268 (10.8) IVS8CA aIndividual heterozygote for D565G mutation in exon 12 (not included in assay) had two polymorphisms in cis to D565G (1719-9T→C in exon 11 and R668C in exon 13 part a), which were also in cis with 17 CA repeats in IVS8. Login to comment

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

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

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

[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] 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] Contribution of the CFTR gene, the pancreatic secr... Clin Genet. 2007 May;71(5):451-7. Tzetis M, Kaliakatsos M, Fotoulaki M, Papatheodorou A, Doudounakis S, Tsezou A, Makrythanasis P, Kanavakis E, Nousia-Arvanitakis S
Contribution of the CFTR gene, the pancreatic secretory trypsin inhibitor gene (SPINK1) and the cationic trypsinogen gene (PRSS1) to the etiology of recurrent pancreatitis.
Clin Genet. 2007 May;71(5):451-7., [PMID:17489851]

Abstract [show]
Acute recurrent/chronic pancreatitis (CP) is a complex multigenic disease. This is a case-control study consisting of 25 Greek patients with CP and a control population of 236 healthy Greek subjects. The whole coding area and neighboring intronic regions of the three genes were screened. Seventeen of 25 patients (68%) had mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene: nine compound heterozygotes with either mild or severe mutations and eight heterozygotes. Four patients (16%) carried CFTR-modulating haplotypes V470-TG11-T5 and V470-TG12-T7. All were negative for PRSS1 gene mutations, while variants c.486C/T and c.738C/T were found in nine patients each, three homozygotes for the minor alleles. Two carried SPINK1 gene mutation p.N34S, one being transheterozygote with CFTR mutation p.F1052V. The promoter variant -253T>C was found in four individuals (one homozygous for the minor allele), all four being transheterozygotes with mutations in the CFTR gene as well. Finally two carried c.272C/T in the 3' untranslated region, one being a p.N34S carrier as well. In total, 80% (20/25) of patients had a molecular defect in one or both of the CFTR and SPINK1 genes, suggesting that mutations/variants in the CFTR plus or minus mutations in the SPINK1, but not the PRSS1 gene, may confer a high risk for recurrent pancreatitis.

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10 Two carried SPINK1 gene mutation p.N34S, one being transheterozygote with CFTR mutation p.F1052V. Login to comment
62 Molecular findings for the CFTR gene Eight patients were carriers (16%): three with p.R1070Q and five with p.F508del, p.G576A, p.F1052V, CFTRdel2,3 (21 kb), and c.2752215G/C, each, representing a heterozygote frequency 2.1-fold higher than that found in the 211 general population controls (7.6%, p , 0.0001). Login to comment
72 One patient was additionally transheterozygote for CFTR mutation p.F1052V. Login to comment
90 Mutations and variants in the CFTR gene CFTR mutation/variant Patients with pancreatitis, n ¼ 25 (%) Controlsa , n ¼ 211 (%) Classic patients with CF, n ¼ 426 (%) p vs controls p vs patients with CF p.F508del 5 (10) 2 (0.47) 465 (54.6) ,0.0001 ,0.0001 p.R334W 4 (8) - 7 (8.2) 0.00011 0.0019 c.444delA 1 (2) - 1 (0.1) c.278915G.A 2 (4) - 11 (1.3) 0.011 CFTRdel2,3 (21 kb) 1 (2) - 2 (0.2) c.E822X 2 (4) - 12 (1.5) 0.011 p.R347H 1 (2) - - 0.055 p.R1070Q 3 (6) 1 (0.24) 7 (0.8) 0.004 0.013 p.G576A 1 (2) - 1 (0.1) p.F1052V 1 (2) 4 (0.95) 1 (0.1) p.I148T 1 (2) - 1 (0.1) c.3272226A.G 1 (2) - 7 (0.82) p.R75Q 2 (4) 4 (0.95) 1 (0.1) 0.0086 c.2752215G/C 1 (2) 4 (1) 5 (0.6) TG11T7 26 (52) 286 (67.7) ND TG11T5 2 (4) 5 (1.18) ND TG10T7 8 (16) 79 (18.7) ND TG10T9 8 (16) 14 (3.3) ND 0.0005 TG12T7 2 (4) 8 (1.9) ND M470 6 (12) 48 (11.4) ND V470 8 (16) 166 (39.3) ND 0.008 CF, cystic fibrosis; ND, not determined. Login to comment
97 We detected two heterozygotes (4%) for p.N34S mutation, cosegregating with IVS1237T.C one was additionally transheterozygote with CFTR mutation p.F1052V. Login to comment

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

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

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90 In some additional examples, a number of mutations found in the fourth intracellular loop (H1054D, G1061R, L1065P, R1066C/H/L, Q1071P, L1077P, H1085R, W1098R, M1101K/ R) also affect the biosynthetic processing of CFTR (although function was not tested) (73); some intracellular loop 4 mutants (F1052V, K1060T, A1067T, G1069R, R1070Q/W) can process CFTR to the complex-glycosylated ("Band C") form but have altered channel activity compared to wild type. 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] Phenotypic characterisation of patients with inter... Thorax. 2009 Aug;64(8):683-91. Epub 2009 Mar 23. Goubau C, Wilschanski M, Skalicka V, Lebecque P, Southern KW, Sermet I, Munck A, Derichs N, Middleton PG, Hjelte L, Padoan R, Vasar M, De Boeck K
Phenotypic characterisation of patients with intermediate sweat chloride values: towards validation of the European diagnostic algorithm for cystic fibrosis.
Thorax. 2009 Aug;64(8):683-91. Epub 2009 Mar 23., [PMID:19318346]

Abstract [show]
BACKGROUND: In patients with symptoms suggestive of cystic fibrosis (CF) and intermediate sweat chloride values (30-60 mmol/l), extensive CFTR gene mutation analysis and nasal potential difference (NPD) measurement are used as additional diagnostic tests and a positive result in either test provides evidence of CFTR dysfunction. To define the phenotype of such patients and confirm the validity of grouping them, patients with intermediate sweat chloride values in whom either additional CF diagnostic test was abnormal were compared with subjects in whom this was not the case and patients with classic CF. METHODS: The phenotypic features of four groups were compared: 59 patients with CFTR dysfunction, 46 with an intermediate sweat chloride concentration but no evidence of CFTR dysfunction (CF unlikely), 103 patients with CF and pancreatic sufficiency (CF-PS) and 62 with CF and pancreatic insufficiency (CF-PI). RESULTS: The CFTR dysfunction group had more lower respiratory tract infections (p = 0.01), more isolation of CF pathogens (p<0.001) and clubbing (p = 0.001) than the CF unlikely group, but less frequent respiratory tract infections with CF pathogens than the CF-PS group (p = 0.05). Patients in the CF-PS group had a milder phenotype than those with PI. Many features showed stepwise changes through the patient groups. CONCLUSION: Patients with intermediate sweat chloride values and two CFTR mutations or an abnormal NPD measurement have a CF-like phenotype compatible with CFTR dysfunction and, as a group, differ phenotypically from patients with intermediate sweat chloride values in whom further CF diagnostic tests are normal as well as from CF-PS and CF-PI patients.

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60 Table 2 CFTR mutations in the patient subgroups CF-PS CFTR dysfunction CF unlikely Genotype Subjects (n) Genotype Subjects (n) Genotype Subjects (n) F508del*/Not found 12 F508del*/3849+10 kb(C.T){ 11 Not found/Not found 39 Not found/Not found 10 F508del*/R117H{ 7 F508del*/Not found 4 F508del*/3849+10 kb(C.T){ 7 F508del*/Not found 7 IVS8-5T{/Not found 1 F508del*/R347P{ 5 Not found/Not found 5 S1235E/E528E 1 F508del*/R117H{ 4 F508del*/D1152H{ 4 No mutation analysis 1 F508del*/2789+5G.A{ 4 F508del*/IVS8-5T{ 4 Total 46 F508del*/S945L* 3 F508del*/S945L* 2 2789+5G.A{/Not found 3 W1282X*/IVS8-5T{ 2 F508del*/3272-26 A.G{ 2 F508del*/R1070W{ 1 F508del*/A455E{ 2 F508del*/L159S 1 F508del*/711+5G.A 2 F508del*/T1246I 1 F508del*/2789+5G.A 2 F508del*/L165S 1 G542X*/R334W{ 2 W1282X*/D1152H{ 1 F508del*/R334W{ 2 R1162X*/D1152H{ 1 R347P{/Not found 2 R347Hu/D1152H{ 1 F508del*/2116delCTAA 1 R553X*/R117H{ 1 F508del*/IVS8-5T{ 1 3659delC*/R117H{ 1 F508del*/D1152H{ 1 3849+10kb(C.T){/G551R 1 F508del*/711+3A.G 1 R1162X*/3849+10 kb(C.T){ 1 F508del*/L206W{ 1 2789+5G.A{/Not found 1 F508del*/I336K{ 1 G542X*/T854A 1 F508del*/G970D 1 R553X*/Q1463H 1 F508del*/L159S 1 S1235R/R668C 1 F508del*/R751L 1 2789+5G.A{/S977F 1 F508del*/E656X 1 No mutation analysis 1 F508del*/4015delA 1 Total 59 F508del*/Y913S 1 F508del*/L165S 1 F508del*/2143delT 1 G551D*/I336K{ 1 G551D*/3272-26A.G{ 1 G551D*/711+3A.G 1 R553X*/4005+2T.C 1 R553X*/E92K{ 1 G542X*/L206W{ 1 W1282X*/I336K 1 R1162X*/3849+10 kb(C.T){ 1 R1162X*/2789+5G.A{ 1 574delA*/3141del9 1 9890X/I105N 1 R334W{/R1070Q{ 1 3272-26A.G{/4218insT 1 3272-26A.G{/L165S 1 711+3A.G/G1244E 1 R352Q/1812-1G.A 1 F1052V/IVS8-5T{ 1 R74W/D1270N 1 1898-3G.A/1898-3G.A 1 1717-1G.A*/R334W{ 1 3659delC*/Not found 1 394delTT/Not found 1 R1162X*/Not found 1 R553X*/Not found 1 R117H{/Not found 1 G85E*/Not found 1 3849+10k(C.T){/Not found 1 Total 103 *Mutation class I, II or III. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Cancer. 2010 Jan 1;116(1):203-9. McWilliams RR, Petersen GM, Rabe KG, Holtegaard LM, Lynch PJ, Bishop MD, Highsmith WE Jr
Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations and risk for pancreatic adenocarcinoma.
Cancer. 2010 Jan 1;116(1):203-9., 2010-01-01 [PMID:19885835]

Abstract [show]
BACKGROUND: Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are common in white persons and are associated with pancreatic disease. The purpose of this case-control study was to determine whether CFTR mutations confer a higher risk of pancreatic cancer. METHODS: In a case-control study, the authors compared the rates of 39 common cystic fibrosis-associated CFTR mutations between 949 white patients with pancreatic adenocarcinoma and 13,340 white controls from a clinical laboratory database for prenatal testing for CFTR mutations. The main outcome measure was the CFTR mutation frequency in patients and controls. RESULTS: Overall, 50 (5.3%) of 949 patients with pancreatic cancer carried a common CFTR mutation versus 510 (3.8%) of 13,340 controls (odds ratio [OR], 1.40; 95% confidence interval [CI], 1.04-1.89; P = .027). Among patients who were younger when their disease was diagnosed (<60 years), the carrier frequency was higher than in controls (OR, 1.82; 95% CI, 1.14-2.94; P = .011). In patient-only analyses, the presence of a mutation was associated with younger age (median 62 vs 67 years; P = .034). In subgroups, the difference was seen only among ever-smokers (60 vs 65 years, P = .028). Subsequent sequencing analysis of the CFTR gene detected 8 (16%) compound heterozygotes among the 50 patients initially detected to have 1 mutation. CONCLUSIONS: Carrying a disease-associated mutation in CFTR is associated with a modest increase in risk for pancreatic cancer. Those affected appear to be diagnosed at a younger age, especially among smokers. Clinical evidence of antecedent pancreatitis was uncommon among both carriers and noncarriers of CFTR mutations.

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95 S42F has been described in 1 Italian CF patient.25 E528E is a synonymous polymorphism involving the last base pair in exon 10 (1716 G > A), which has been reported to affect splicing, but has not been linked to severe pulmonary disease.26 S912L is thought to be a neutral variant (serine for a leucine), unless in cis position to another mutation.27 The F1052V missense mutation is a variant with a modest effect, with normal or near-normal sweat chloride tests in combination with other mild variants.28,29 N1088S is a novel mutation that substitutes asparagine for a serine amino acid, both positively charged, and its functionality is unclear. Login to comment
103 Compound Heterozygotes Among Pancreatic Cancer Cases CFTR Mutations Sex Age at Diagnosis, y Ever/Never Smoker Family History of Pancreatic Cancer Pancreatitis ‡3 Years Before Cancer Diagnosis df508/S42F M 70 Nonsmoker No No R117H/E528E (splice site) M 75 Smoker Yes No df508/S912L W 56 Smoker No No df508/N1088S W 73 Smoker No No df508/M1191I M 79 Smoker No No df508/S1235R M 73 Smoker No No df508/F1052V M 49 Smoker No No df508/5T M 60 Smoker No No Man indicates man; W, woman. 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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130 Recently, a study of 335,204 patients screened for their CF carrier status revealed 4 individuals with Table 3 Frequency of less common CFTR mutations in the general population. Mutation Frequency Reference S1235R 1/77 [22,23] L997F 1/77 [24] I148T 1/129 [19] F1052V 1/200 [25] 621+3A→G 1/335 [26] 3601-111 G→C 1/690 [27] Table 4 New CFTR mutations found in the general population following 2nd level analysis. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Reprod Biomed Online. 2009 Nov;19(5):685-94. Gallati S, Hess S, Galie-Wunder D, Berger-Menz E, Bohlen D
Cystic fibrosis transmembrane conductance regulator mutations in azoospermic and oligospermic men and their partners.
Reprod Biomed Online. 2009 Nov;19(5):685-94., [PMID:20021716]

Abstract [show]
The objective of this study was to investigate the contribution of cystic fibrosis transmembrane conductance regulator (CFTR) to human infertility and to define screening and counselling procedures for couples asking for assisted reproduction treatment. Extended CFTR mutation screening was performed in 310 infertile men (25 with congenital absence of the vas deferens (CAVD), 116 with non-CAVD azoospermia, 169 with severe oligospermia), 70 female partners and 96 healthy controls. CFTR mutations were detected in the majority (68%) of CAVD patients and in significant proportions in azoospermic (31%) and oligospermic (22%) men. Carrier frequency among partners of infertile men was 16/70, exceeding that of controls (6/96) significantly (P = 0.0005). Thus, in 23% of infertile couples both partners were carriers, increasing the risk for their offspring to inherit two mutations to 25% or 50%. This study emphasizes the necessity to offer extended CFTR mutation screening and counselling not only to patients with CAVD but also to azoospermic and oligozoospermic men and their partners before undergoing assisted reproduction techniques. The identification of rare and/or mild mutations will not be a reason to abstain from parenthood, but will allow adequate treatment in children at risk for atypical or mild cystic fibrosis as soon as they develop any symptoms.

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99 Couple no. Infertile male CFTR mutation Female partner CFTR mutation Offspring genotype Risk for genotype (%) 01 F508del/wt azoospermia F508del/wt F508del/ F508del 25 F508del/wt 50 wt/wt 25 02 F508del/T5 CAVD F508del/wt F508del/ F508del 25 F508del/T5 25 F508del/wt 25 T5/wt 25 03 F508del/S13Ya azoospermia T5/wt F508del/T5 25 S13Y/T5 25 F508del/wt 25 S13Y/wt 25 04 I148T/wt oligospermia F508del/wt F508del/ I148T 25 I148T/wt 25 F508del/wt 25 wt/wt 25 05 1717À1G>A/wt oligospermia T5/wt 1717À1G>A/ T5 25 1717À1G>A/ wt 25 T5/wt 25 wt/wt 25 06 T5/wt oligospermia 3905insT/wt 3905insT/T5 25 3905insT/wt 25 T5/wt 25 wt/wt 25 07 T5/wt azoospermia D1152H/wt D1152H/T5 25 D1152H/wt 25 T5/wt 25 wt/wt 25 08 T5/F1052V oligospermia S1235R/wt F1052V/ S1235R 25 S1235R/T5 25 F1052V/wt 25 T5/wt 25 09 S1235R/wt oligospermia T5/wt S1235R/T5 25 S1235R/wt 25 T5/wt 25 wt/wt 25 10, 11 T5/wt oligospermia S1235R/wt S1235R/T5 25 S1235R/wt 25 T5/wt 25 wt/wt 25 12 V754M/wt oligospermia T5/wt V754M/T5 25 V754M/wt 25 T5/wt 25 wt/wt 25 13 T5/wt oligospermia Q1352H/wt Q1352H/T5 25 Q1352H/wt 25 T5/wt 25 wt/wt 25 (continued on next page)(continued) female partner is a carrier. Login to comment

[hide] The etiology of acute recurrent pancreatitis in ch... Pancreas. 2011 May;40(4):517-21. Lucidi V, Alghisi F, Dall'Oglio L, D'Apice MR, Monti L, De Angelis P, Gambardella S, Angioni A, Novelli G
The etiology of acute recurrent pancreatitis in children: a challenge for pediatricians.
Pancreas. 2011 May;40(4):517-21., [PMID:21499205]

Abstract [show]
OBJECTIVES: To assess specific etiologies of acute recurrent pancreatitis at a single Italian pediatric cystic fibrosis (CF) center. METHODS: We studied, retrospectively, 78 young patients (39 female subjects; mean age at diagnosis, 8.8 +/- 5.1 years) affected by acute recurrent episodes of pancreatitis, remained etiologically undiagnosed at first-level assessment. All patients were submitted to endoscopic retrograde cholangiopancreatography to exclude biliopancreatic malformations and tested for CF by a sweat chloride test. Most patients also were studied for the research of CFTR, PRSS1, and SPINK1 gene mutations. RESULTS: A high percentage of family history for chronic pancreatitis was observed (20.5%). The sweat test identified 8 subjects (10.3%) with classic CF (2 patients) or at risk for CF (6 patients). Genetic analysis showed mutations in CFTR, SPINK1, and PRSS1 genes in 39.6%, 7.1%, and 4.5% of patients, respectively. A biliopancreatic malformation was diagnosed in 15 patients (19.2%). We also observed biliary lithiasis (5 patients [6.5%]), congenital pancreatic polycystosis (2 patients), a case of dyslipidemia, and 1 patient with a posttransplantation, drug-induced pancreatitis. CONCLUSIONS: Recurrent pancreatitis in children has several etiologies. Genetic testing confirms the high frequency of CFTR mutations. This suggests that it is of some value to identify patients with late-onset CF and CFTR-related disorders.

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46 Genetic Findings Observed in Our Study Population and Related Clinical Features CFTR PRSS1 SPINK1 Clinical CharacteristicsMutations IVS8 F508del/UN 9T/9T S181G/- NEG No respiratory symptoms 3849+10KbC9T/UN 7T/7T NEG NEG No respiratory symptoms UN/UN 7T/7T NEG N34S/- UN/UN 5T/7T NEG NEG No respiratory symptoms 1899-136T/C/UN 5T/7T NEG NEG No respiratory symptoms F508del/UN 5T/9T NEG NEG No respiratory symptoms D1152H/D1152H NEG NEG No respiratory symptoms R75Q/UN 5T/7T NEG NEG No respiratory symptoms L997F/UN 7T/9T NEG NEG No respiratory symptoms UN/UN 7T/7T NEG N34S/- W1282X/I148T 7T/9T NEG NEG No respiratory symptoms NEG N34S/- R75Q/F1052V NEG NEG No respiratory symptoms F508del/D1152H NEG NEG Bronchiectasis-CF 406-6T/C/E528E 7T/7T NEG NEG No respiratory symptoms F508del/UN 7T/9T Mild respiratory symptomsYCF L967S/L997F NEG NEG No respiratory symptoms E528E/UN 5T/7T Crohn disease, food allergy 1716 G/A/UN 7T/7T NEG NEG No respiratory symptoms 1898+1G9A/UN 7T/7T No respiratory symptoms R31C/UN No respiratory symptoms R75Q/UN 7T/7T NEG NEG No respiratory symptoms N29T;V212I; D217Y NEG F508del/UN 7T/9T NEG NEG Pancreas divisum S1235R/UN 7T/9T NEG NEG Duodenal stenosis Entries in bold font undelines the detection of mutations or polymorphisms in the studied genes. Login to comment

[hide] Cystic fibrosis mutation screening in CBAVD patien... Mol Hum Reprod. 1998 Apr;4(4):333-7. Kanavakis E, Tzetis M, Antoniadi T, Pistofidis G, Milligos S, Kattamis C
Cystic fibrosis mutation screening in CBAVD patients and men with obstructive azoospermia or severe oligozoospermia.
Mol Hum Reprod. 1998 Apr;4(4):333-7., [PMID:9620832]

Abstract [show]
Congenital bilateral absence of the vas deferens (CBAVD) found in otherwise healthy infertile males, is associated with a high incidence of mutated cystic fibrosis transmembrane conductance regulator (CFTR) alleles, and is considered a genital form of cystic fibrosis (CF). The CF gene may also be involved in the aetiology of male infertility in cases other than CBAVD. The present study was undertaken to test the involvement of CFTR gene mutations in 14 CBAVD males and additionally in cases of male infertility caused by obstructive azoospermia (n = 10) and severe oligozoospermia (n = 3). The entire coding region of the CFTR gene was analysed using denaturing gradient gel electrophoresis (DGGE). The three allele (5T, 7T, 9T) polymorphic tract of thymidines in intron 8 (IVS8-polyT) of which the 5T allele acts as a mild mutation, causing reduced levels of normal CFTR mRNA due to deletion of exon 9, was also analysed. Of the 14 CBAVD cases, four (28.6%) were found to have mutations in both copies of the CFTR gene, six (42.8%) had one CFTR mutation, and in the remaining four (28.6%) no CFTR mutations were found. Of the 10 cases with obstructive azoospermia, three (30%) had one CFTR mutation and in the remaining seven (70%) no mutations were found. None of the three severe oligozoospermia cases carried a CFTR mutation. The frequency of the IVS8(5T) allele was 14.3% (4/28) for the CBAVD cases and 5% (1/20) for the obstructive azoospermia cases, none of the severe oligozoospermia males carried the IVS8-5(5T) allele. The data indicate that while there is a strong association between male infertility caused by CBAVD and mutations in the CFTR gene, cases of obstructive azoospermia without CBAVD also seem to be associated with CFTR gene mutations.

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64 Cystic fibrosis transmembrane conductance regulator (CFTR), PolyT genotypes and clinical data of men with congenital bilateral absence of the vas deferens (CBAVD, n ϭ 14), obstructive azoospermia (ObsA, n ϭ 10) and oligozoospermia (n ϭ 3) Patients Sweat chloride CFTR IVS8-polyT Other clinical (mEq/l) mutations alleles features Two mutations detected MS1 (CBAVD) 107.7 ∆F508/M1I 5T/9T Recurrent bronchitis MS6 (CBAVD) 74.5 ∆F508/711ϩ3AϾG 9T/7T Chronic cough MS19 (CBAVD) 51 W496X/F1052V 9T/9T MS24 (CBAVD) Ͻ40 D565G/R668C 7T/7T One mutation detected MS5 (CBAVD) Ͻ40 3272-26AϾG/- 7T/7T MS12 (CBAVD) Ͻ40 ∆F508/- 9T/7T MS14 (CBAVD) Ͻ40 ∆F508/- 9T/5T MS15 (CBAVD) 57.7 L732X/- 7T/5T Dehydration/recurrent bronchitis MS16 (CBAVD) Ͻ40 711ϩ3AϾG/- 7T/5T MS20 (CBAVD) Ͻ40 4010delTAT/- 7T/7T MS18 (ObsA) 48 ∆F508/- 5T/9T MS11 (ObsA) Ͻ40 R75Q/- 7T/7T MS23 (ObsA) Ͻ40 2790-8CϾG/- 7T/7T No mutation detected MS7 (CBAVD) Ͻ40 -/- 7T/7T MS10 (CBAVD) Ͻ40 -/- 7T/7T MS21 (CBAVD) Ͻ40 -/- 7T/9T MS28 (CBAVD) Ͻ40 -/- 7T/7T MS2 (ObsA) 54.2 -/- 7T/7T MS8 (ObsA) Ͻ40 -/- 7T/7T MS17 (ObsA) 50 -/- 7T/7T MS22 (ObsA) Ͻ40 -/- 7T/9T MS25 (ObsA) Ͻ40 -/- 7T/7T MS26 (ObsA) Ͻ40 - / - 7T/7T MS27 (ObsA) Ͻ40 -/- 7T/7T MS3 (oligozoospermia) 50 -/- 7T/7T MS4 (oligozoospermia) Ͻ40 -/- 7T/7T MS13 (oligozoospermia) Ͻ40 -/- 7T/7T Table II. 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
71 Lane 1, F1052V (T3G at 3286)/wt; lane 2, R1066C (C3T at 3328)/wt; lane 3, R1066H (G3A at 3329)/wt; lane 4, wt. Login to comment

[hide] Structure and function of the CFTR chloride channe... Physiol Rev. 1999 Jan;79(1 Suppl):S23-45. Sheppard DN, Welsh MJ
Structure and function of the CFTR chloride channel.
Physiol Rev. 1999 Jan;79(1 Suppl):S23-45., [PMID:9922375]

Abstract [show]
Structure and Function of the CFTR Chloride Channel. Physiol. Rev. 79, Suppl.: S23-S45, 1999. - The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ABC transporter family that forms a novel Cl- channel. It is located predominantly in the apical membrane of epithelia where it mediates transepithelial salt and liquid movement. Dysfunction of CFTR causes the genetic disease cystic fibrosis. The CFTR is composed of five domains: two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. Here we review the structure and function of this unique channel, with a focus on how the various domains contribute to channel function. The MSDs form the channel pore, phosphorylation of the R domain determines channel activity, and ATP hydrolysis by the NBDs controls channel gating. Current knowledge of CFTR structure and function may help us understand better its mechanism of action, its role in electrolyte transport, its dysfunction in cystic fibrosis, and its relationship to other ABC transporters.

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177 Because DPCA1067T altered the relationship between open probability (Po) and ATP concentration (33), and the response of inhibition of CFTR was voltage dependent and enhanced when the external Cl0 concentration was reduced,R1066L and F1052V to pyrophosphate (PPi) was less than wild type (33). Login to comment

[hide] Mutations of the cystic fibrosis gene in patients ... Ann Rheum Dis. 2011 Apr;70(4):653-9. Epub 2010 Dec 3. Puechal X, Bienvenu T, Genin E, Berthelot JM, Sibilia J, Gaudin P, Marcelli C, Lasbleiz S, Michou L, Cornelis F, Kahan A, Dusser DJ
Mutations of the cystic fibrosis gene in patients with bronchiectasis associated with rheumatoid arthritis.
Ann Rheum Dis. 2011 Apr;70(4):653-9. Epub 2010 Dec 3., [PMID:21131649]

Abstract [show]
OBJECTIVES: In cystic fibrosis, mutations of the CFTR gene lead to diffuse bronchiectasis (DB). DB is also associated with other diseases including rheumatoid arthritis (RA) in which the role of genetic factors in the predisposition to DB remains unclear. METHODS: A family-based association study was carried out to determine whether the frequency of CFTR mutations was higher in patients with RA-associated DB and to determine whether a causal relationship could be established between the variant and the disease by evaluating its cosegregation with DB within families. Families of probands with RA-DB were included if one first-degree relative had RA and/or DB. The controls comprised healthy subjects requesting genetic counselling because their partner had cystic fibrosis. RESULTS: The frequency of CFTR mutations was higher in family members with RA-DB or DB only than in unaffected relatives (p<0.005 for each comparison) and in unrelated healthy controls (p<0.001 for each comparison) but not in family members with RA only. CFTR mutations were more frequent in family members with RA-DB than in those with RA only (OR 5.30, 95% CI 2.48 to 11.33; p<5x10(-5)). They cosegregated with RA-DB in the families (sib-TDT=10.82, p=0.005). CONCLUSIONS: RA-DB should be added to the list of phenotypes in which CFTR mutations are pathogenic. CFTR mutation is the first genetic defect linked to an extra-articular feature of RA to be described. CFTR mutations in patients with RA appear to be an important marker of the risk of associated DB, which has been linked to a less favourable prognosis.

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61 Table 1 CFTR genotypes of the family population Family (N=24) CFTR genotypes RA-DB (N=30) DB only (N=8) RA only (N=24) Unaffected individuals (N=76) 1 c.1584G>A (p.Glu528Glu ):- 1 -:- 1 2 p.Gly424Ser+p.Gly576Ala 1 p.Gly424Ser:- 1 p.Arg75Gln:- 1 1 3 p.Phe508del (c.1521_1523delCTT)+5T (c.1210-12[5]) 1 p.Phe508del:- 1 2 2 5T (c.1210-12[5]):- 1 -:- 1 4 5T (c.1210-12[5]):- 1 1 2 -:- 1 2 1 p.Phe1052Val:- 1 5 5T (c.1210-12[5]):- 1 6 p.Phe508del+p.Ser977Phe-5T (c.1210-12[5]) 1 1 1 p.Phe508del:- 1 p.Ser977Phe-5T (c.1210-12[5]):- 3 7 p.Arg75Gln:- 2 8 p.Asp1152His+c.262_263delTT 1 1 p.Asp1152His+c.-7G>C 1 c.262_263delTT:- 1 9 c.1584G>A (p.Glu528Glu ):- 1 p.Arg75Gln:- 1 -:- 1 5 10 10 c.-7G>C:- 1 -:- 1 8 11 p.Ser1235Arg:- 1 1 -:- 1 12 p.Ala923Ala:- 1 1 13 c.1584G>A (p.Glu528Glu ):- 1 1 1 p.Ala923Ala:- 1 14 c.1584G>A (p.Glu528Glu ):- 2 1 -:- 1 3 15 -:- 1 1 5 16 -:- 1 1 7 17 -:- 2 1 3 18 -:- 1 1 19 -:- 1 1 2 20 -:- 1 1 21 -:- 1 1 3 22 -:- 1 1 6 23 -:- 1 1 3 24 -:- 1 1 7 Total no. 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] Borderline sweat test: Utility and limits of genet... Clin Biochem. 2009 May;42(7-8):611-6. Epub 2009 Jan 24. Seia M, Costantino L, Paracchini V, Porcaro L, Capasso P, Coviello D, Corbetta C, Torresani E, Magazzu D, Consalvo V, Monti A, Costantini D, Colombo C
Borderline sweat test: Utility and limits of genetic analysis for the diagnosis of cystic fibrosis.
Clin Biochem. 2009 May;42(7-8):611-6. Epub 2009 Jan 24., [PMID:19318035]

Abstract [show]
OBJECTIVE: The sweat test remains the gold standard for the diagnosis of Cystic Fibrosis (CF) even despite the availability of molecular analysis of Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR). We investigated the relationship between CFTR mutation analysis and sweat chloride concentration in a cohort of subjects with borderline sweat test values, in order to identify misdiagnosis of CF. DESIGN AND METHODS: In the period between March 2006 and February 2008 we performed 773 sweat tests in individuals referred for suspect CF. Ninety-one subjects had chloride values in the border-line range. Clinicians required CFTR gene complete scanning on 66 of them. RESULTS: The mean value of sweat chloride in the DNA negative subjects was lower than in those with at least one CFTR mutation. Our data indicate that 39 mEq/l is the best sensitivity trade off for the sweat test with respect to genotype. CONCLUSIONS: To optimise diagnostic accuracy of reference intervals, it may be useful to modify from 30 to 39 mEq/l the threshold for sweat chloride electrolytes.

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59 In order to evaluate the relationship between the presence of CFTR mutation and sweat chloride concentration, we focused our attention on the 91 individuals (11.8%) in whom borderline sweat chloride values (31-59 mEq/l) were recorded (mean sweat electrolyte value was 40.0 mEq/l): 25 refused to be referred to the local Table 2 Demographic and clinical features of subjects with positive DNA analysis Patient Initials Gender Age at test years/ months Sweat chloride mEq/l Clinical indication DNA results IRT Right arm Left arm 1 CA M 49y5m 34 34 CBAVD G542X/5T-TG12 ND 2 SA M 45y2m 45 43 Pancreatitis F508del/R117H-7T ND 3 PD F 43y7m 33 38 Recurrent bronchitis F508del/5T-TG12 ND 4 CA M 36y1m 31 29 CBAVD R117H-7T/R117C-7T ND 5 SC M 36y1m 33 40 Pneumonia F508del/D1152H ND 6 MG M 25Y5m 41 45 CBAVD Q552X/D1152H NEG 7 SG M 18y5m 49 54 Pancreatitis 4016insT/dupl.prom.-3 ND 8 LS F 10y4m 41 38 Pancreatitis D1152H/L997F NEG 9 CM M 8y3m 30 31 Pneumonia F1052V/A120T NEG 10 PT M 7y3m 41 39 Positive screening F508del/Y1032C POS 11 ME F 7y1m 44 44 Positive screening 2789+5GNA/5T-TG12 POS 12 PM F 6y4m 35 36 Positive screening 2183AANG/5T-TG12 POS 13 BM F 6y3m 36 39 Positive screening F508del/5T-TG12 POS 14 CD M 5y8m 40 41 Chronic bronchitis 5T-TG12/5T-TG12 NEG 15 CG F 4y5m 33 37 Recurrent bronchitis R553X/L997F POS 16 CS F 3y8m 53 58 Family history G542X/D614G POS 17 VA M 4y2m 49 43 Pneumonia E831X/5T-TG12 ND 18 SC M 3y4m 39 39 Positive screening R352Q/G213E POS 19 CC F 2y3m 31 31 Positive screening F508del/5T-TG12 POS 20 CA F 2y5m 51 52 Recurrent bronchitis E831X/5T-TG12 ND 21 MR F 3y+7m 29 31 Family history G542X/5T-TG12 POS 22 CM F 2y3m 60 58 Pneumonia T338I/L997F POS 23 LM F 2y1m 50 52 Positive screening F508del/E1473X POS 24 CGE F 0y8m 46 47 Positive screening E92K/5T-TG13 POS 25 NF M 0y7m 32 30 Positive screening F508del/P5L POS 26 RG M 0y7m 45 40 Positive screening N1303K/P5L POS 27 PE M 47y4m 60 58 Nasal polyposis R1066H/UN ND 28 LS M 39y9m 39 38 Azoospermy N1303K/UN ND 29 TM M 38y4m 40 45 Azoospermy N1303K/UN ND 30 DF M 34y2m 52 58 Bronchiectasis 3849+10 kbCNT/UN ND 31 TV F 30y5m 35 34 Recurrent bronchitis L997F/UN ND 32 FA F 18y7m 53 49 Family history Del es.2/UN NEG 33 DG M 17y8m 43 47 Recurrent bronchitis 5T-TG12/UN NEG 34 LN F 13y7m 54 53 Nasal poliposis, malnutrition R74W-V855I/UN NEG 35 FKT M 15y4m 54 53 Chronic bronchitis R352Q/UN NEG 36 BM M 10y9m 48 51 Chronic bronchitis T1263I/UN NEG 37 SV F 11y1m 60 58 Chronic bronchitis R347H/UN NEG 38 CV F 10y10m 38 39 Recurrent bronchitis 5T-TG12/UN NEG 39 BF F 9y10m 37 38 Chronic bronchitis L997F/UN NEG 40 CA M 8y2m 33 32 Pneumonia F508del/UN NEG 41 RX F 8y7m 29 31 Chronic bronchitis V920L/UN NEG 42 MG F 4y3m 51 51 Positive screening F508del/UN POS Sweat chloride concentration and mutations/variants detected are also reported. Login to comment
57 In order to evaluate the relationship between the presence of CFTR mutation and sweat chloride concentration, we focused our attention on the 91 individuals (11.8%) in whom borderline sweat chloride values (31-59 mEq/l) were recorded (mean sweat electrolyte value was 40.0 mEq/l): 25 refused to be referred to the local Table 2 Demographic and clinical features of subjects with positive DNA analysis Patient Initials Gender Age at test years/ months Sweat chloride mEq/l Clinical indication DNA results IRT Right arm Left arm 1 CA M 49y5m 34 34 CBAVD G542X/5T-TG12 ND 2 SA M 45y2m 45 43 Pancreatitis F508del/R117H-7T ND 3 PD F 43y7m 33 38 Recurrent bronchitis F508del/5T-TG12 ND 4 CA M 36y1m 31 29 CBAVD R117H-7T/R117C-7T ND 5 SC M 36y1m 33 40 Pneumonia F508del/D1152H ND 6 MG M 25Y5m 41 45 CBAVD Q552X/D1152H NEG 7 SG M 18y5m 49 54 Pancreatitis 4016insT/dupl.prom.-3 ND 8 LS F 10y4m 41 38 Pancreatitis D1152H/L997F NEG 9 CM M 8y3m 30 31 Pneumonia F1052V/A120T NEG 10 PT M 7y3m 41 39 Positive screening F508del/Y1032C POS 11 ME F 7y1m 44 44 Positive screening 2789+5GNA/5T-TG12 POS 12 PM F 6y4m 35 36 Positive screening 2183AANG/5T-TG12 POS 13 BM F 6y3m 36 39 Positive screening F508del/5T-TG12 POS 14 CD M 5y8m 40 41 Chronic bronchitis 5T-TG12/5T-TG12 NEG 15 CG F 4y5m 33 37 Recurrent bronchitis R553X/L997F POS 16 CS F 3y8m 53 58 Family history G542X/D614G POS 17 VA M 4y2m 49 43 Pneumonia E831X/5T-TG12 ND 18 SC M 3y4m 39 39 Positive screening R352Q/G213E POS 19 CC F 2y3m 31 31 Positive screening F508del/5T-TG12 POS 20 CA F 2y5m 51 52 Recurrent bronchitis E831X/5T-TG12 ND 21 MR F 3y+7m 29 31 Family history G542X/5T-TG12 POS 22 CM F 2y3m 60 58 Pneumonia T338I/L997F POS 23 LM F 2y1m 50 52 Positive screening F508del/E1473X POS 24 CGE F 0y8m 46 47 Positive screening E92K/5T-TG13 POS 25 NF M 0y7m 32 30 Positive screening F508del/P5L POS 26 RG M 0y7m 45 40 Positive screening N1303K/P5L POS 27 PE M 47y4m 60 58 Nasal polyposis R1066H/UN ND 28 LS M 39y9m 39 38 Azoospermy N1303K/UN ND 29 TM M 38y4m 40 45 Azoospermy N1303K/UN ND 30 DF M 34y2m 52 58 Bronchiectasis 3849+10 kbCNT/UN ND 31 TV F 30y5m 35 34 Recurrent bronchitis L997F/UN ND 32 FA F 18y7m 53 49 Family history Del es.2/UN NEG 33 DG M 17y8m 43 47 Recurrent bronchitis 5T-TG12/UN NEG 34 LN F 13y7m 54 53 Nasal poliposis, malnutrition R74W-V855I/UN NEG 35 FKT M 15y4m 54 53 Chronic bronchitis R352Q/UN NEG 36 BM M 10y9m 48 51 Chronic bronchitis T1263I/UN NEG 37 SV F 11y1m 60 58 Chronic bronchitis R347H/UN NEG 38 CV F 10y10m 38 39 Recurrent bronchitis 5T-TG12/UN NEG 39 BF F 9y10m 37 38 Chronic bronchitis L997F/UN NEG 40 CA M 8y2m 33 32 Pneumonia F508del/UN NEG 41 RX F 8y7m 29 31 Chronic bronchitis V920L/UN NEG 42 MG F 4y3m 51 51 Positive screening F508del/UN POS Sweat chloride concentration and mutations/variants detected are also reported. Login to comment

[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
122 N1303K).8,9,11,34 -36 Mutants that have been shown previously to form plasma membrane Cl- channels with altered single-channel properties in heterologous cells (S108F, R117H, R334W, F1052V)10,34,35,37 were associated with residual cAMP-mediated Cl- secretion of ϳ12%-54% of control rectal epithelia. Login to comment

[hide] High frequency of the R75Q CFTR variation in patie... J Cyst Fibros. 2004 Aug;3(3):189-91. Divac A, Nikolic A, Mitic-Milikic M, Nagorni-Obradovic L, Petrovic-Stanojevic N, Dopudja-Pantic V, Nadaskic R, Savic A, Radojkovic D
High frequency of the R75Q CFTR variation in patients with chronic obstructive pulmonary disease.
J Cyst Fibros. 2004 Aug;3(3):189-91., [PMID:15463907]

Abstract [show]
We performed the complete screening of the CFTR gene in a group of 31 patients with COPD in order to investigate the impact of mutations and polymorphisms in the CFTR gene. The cumulative frequency of CFTR mutations (17.74%) was significantly higher than in our general population (P < 0.0001). The R75Q was significantly overrepresented in COPD patients (8.06%; P = 0.002). In all patients carrying the R75Q chronic bronchitis was a dominant symptom of COPD, and all were homozygous for the V470 allele. These findings suggest that R75Q mutation could be characteristic CFTR variant for COPD patients.

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39 Six different mutations (R75Q, F508del, G126D, L997F, F1052V, R74W) were identified on 11 (17.74%) of the 62 chromosomes, giving a significantly higher frequency than in our general population ( P < 0.0001, 95%CI: 2.60-36.21). Login to comment
59 Table 1 CFTR genotypes in COPD patients No. of cases CFTR gene mutation IVS8 Tn M470V genotype 1 R75Q/R75Q 7/7 V470/V470 1 L997F/R75Q 7/9 V470/V470 2 R75Q/- 7/7 V470/V470 1 F508del/- 7/9 M470/V470 1 F508del/- 5/9 M470/M470 1 G126D/- 7/9 M470/M470 1 F1052V/- 7/7 M470/V470 1 R74W/- 7/7 M470/M470 2 -/- 5/7 V470/V470 3 -/- 5/7 M470/V470 1 -/- 5/7 M470/M470 1 -/- 5/9 M470/V470 3 -/- 7/9 M470/V470 6 -/- 7/7 V470/V470 4 -/- 7/7 M470/V470 -/- 7/7 M470/M470 A. Divac et al. / Journal of Cystic Fibrosis 3 (2004) 189-191190 Acknowledgements This work was supported by grant 1417 from Ministry for Science, Technologies and Development of Serbia. Login to comment

[hide] Analysis of the CFTR gene in Turkish cystic fibros... Hum Genet. 1998 Feb;102(2):224-30. Onay T, Topaloglu O, Zielenski J, Gokgoz N, Kayserili H, Camcioglu Y, Cokugras H, Akcakaya N, Apak M, Tsui LC, Kirdar B
Analysis of the CFTR gene in Turkish cystic fibrosis patients: identification of three novel mutations (3172delAC, P1013L and M1028I).
Hum Genet. 1998 Feb;102(2):224-30., [PMID:9521595]

Abstract [show]
In order to determine the spectrum of cystic fibrosis (CF) mutations in the Turkish population, a complete coding region of the cystic fibrosis transmembrane conductance regulator (CFTR) gene including exon-intron boundaries, on 122 unrelated CF chromosomes from 73 Turkish CF families was analysed by denaturing gradient gel electrophoresis and multiplex heteroduplex analysis on MDE gel matrix. In addition to 15 previously reported mutations and 12 polymorphisms, three novel mutations, namely 3172delAC, P1013L and M1028I, were detected. DeltaF508 was found to be present on 18.8% of CF chromosomes. The second most common mutation was 1677delTA, with a frequency of 7.3%, followed by G542X and 2183AA-->G mutations, with frequencies of 4.9%. These four most common mutations in Turkish CF population account for approximately 36% of mutations. This study could only detect 52.5% of disease-causing mutations in this population; 47.5% of CF alleles remain to be identified, reflecting the high molecular heterogeneity of the Turkish population.

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52 This patient was found to be a compound heterozygote for 3172delAC/F1052V. Login to comment
67 Mutations 1677delTA, G542X and 2183AA→G have frequencies greater or equal to approximately 5%, whereas F1052V, 2043delG, D110H, N1303K, L571S and 296+9 A→T have frequencies of 2%. Login to comment
76 F1052V 17b Phe→Val at 1052 T→G at 3286 2 (1.64) Mercier et al. 1993 6. Login to comment
124 Other mutations, namely F1052V, 2043delG, D110H, L571S and 296+9 A→T, have been detected with frequencies of 1.6% in Turkish CF chromosomes. Login to comment

[hide] Effect of cystic fibrosis-associated mutations in ... J Biol Chem. 1996 Aug 30;271(35):21279-84. Cotten JF, Ostedgaard LS, Carson MR, Welsh MJ
Effect of cystic fibrosis-associated mutations in the fourth intracellular loop of cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 1996 Aug 30;271(35):21279-84., [PMID:8702904]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) contains multiple membrane spanning sequences that form a Cl- channel pore and cytosolic domains that control the opening and closing of the channel. The fourth intracellular loop (ICL4), which connects the tenth and eleventh transmembrane spans, has a primary sequence that is highly conserved across species, is the site of a preserved sequence motif in the ABC transporter family, and contains a relatively large number of missense mutations associated with cystic fibrosis (CF). To investigate the role of ICL4 in CFTR function and to learn how CF mutations in this region disrupt function, we studied several CF-associated ICL4 mutants. We found that most ICL4 mutants disrupted the biosynthetic processing of CFTR, although not as severely as the most common DeltaF508 mutation. The mutations had no discernible effect on the channel's pore properties; but some altered gating behavior, the response to increasing concentrations of ATP, and stimulation in response to pyrophosphate. These effects on activity were similar to those observed with mutations in the nucleotide-binding domains, suggesting that ICL4 might help couple activity of the nucleotide-binding domains to gating of the Cl- channel pore. The data also explain how these mutations cause a loss of CFTR function and suggest that some patients with mutations in ICL4 may have a milder clinical phenotype because they retain partial activity of CFTR at the cell membrane.

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81 For example, levels of mature F1052V were similar to wild-type, whereas L1065P, R1070Q, and H1085R were similar to ⌬F508 in that they produced little mature protein (Fig. 2, A and B). Login to comment
84 In Fig. 2 the processing of ⌬F508 and the milder CF-associated mutant, P574H (10), are provided for reference. Login to comment
85 Whole Cell Function of ICL4 Mutants-To evaluate the effect of ICL4 mutations on Cl- channel activity, we selected the mutants R1066C, R1066H, R1066L, A1067T, and F1052V for study. Login to comment
93 selected F1052V, which lies outside the cluster of mutations and the ABC transporter consensus sequence, because we thought it would likely have a functional defect since it is associated with CF yet is processed like wild-type protein. Login to comment
95 We found that cells expressing all of the ICL4 mutants (F1052V, R1066C, R1066H, R1066L, and A1067T) generated cAMP-stimulated Cl- selective currents that showed time-and voltage-independent behavior identical to that of wild-type CFTR (data not shown). Login to comment
103 Mutations did not alter single-channel conductance: wild-type, 8.9 Ϯ 0.3 pS; F1052V, 9.6 Ϯ 0.2 pS; R1066C, 8.9 Ϯ 0.4 pS; R1066H, 8.5 Ϯ 0.7; R1066C, 8.6 Ϯ 0.3; and A1067T, 9.2 Ϯ 0.2 pS. Login to comment
105 Table I shows that F1052V, R1066L, and A1067T did not alter the relative permeability or conductivity sequence for Cl- , Br- , or I- . Login to comment
110 The most noticeable differences compared to wild-type were that the R1066C channels had longer closed times between bursts of activity. Login to comment
111 In addition, the Arg-1066 mutants and F1052V appeared to have bursts of activity with altered durations. Login to comment
144 Fig. 7A shows that application of PPi to the cytosolic surface of an excised macropatch reversibly increased the activity of R1066L channels. Login to comment
145 However, Fig. 7B shows that the response of R1066L and F1052V to PPi was less than that of wild-type CFTR. Login to comment
152 n Px/PCL Gx/GCL Br- Cl- IBr- ClI- Wild-type 3 1.29 Ϯ 0.07 1.00 0.56 Ϯ 0.13 1.18 Ϯ 0.56 1.00 0.35 Ϯ 0.06 F1052V 2 1.41 1.00 0.50 0.98 1.00 0.53 R1066L 4 1.36 Ϯ 0.07 1.00 0.88 Ϯ 0.11 1.15 Ϯ 0.22 1.00 0.40 Ϯ 0.11 A1067T 4 1.29 Ϯ 0.15 1.00 0.66 Ϯ 0.04 1.00 Ϯ 0.10 1.00 0.43 Ϯ 0.06 FIG. 4. Login to comment
156 Data are mean Ϯ S.E. of (6/5) measurements for Po and burst duration, respectively: wild-type (19/18), F1052V (6/5) R1066C (3/3), R1066H (6/7), R1066L (12/5), and A1067T (9/3). Login to comment
181 For example, the mutant F1052V was processed normally but had dramatically altered function, whereas the R1066H mutation had a dramatic effect on processing but little discernible effect on function. Login to comment
200 Data are mean Ϯ S.E. of (n) measurements for: wild-type (9), F1052V (3), R1066L (4), A1067T (4), G551S (6), K464A (4), G1349D (5), K1250 M at 5 mM PPi (5), wild-type at 5 mM PPi (16). Login to comment
218 There are reports that several of the mutations (for example, F1052V, H1054D, and L1077P) are associated with a milder, pancreatic sufficient phenotype in which pancreatic function is not completely defective (17, 20, 46). Login to comment
80 For example, levels of mature F1052V were similar to wild-type, whereas L1065P, R1070Q, and H1085R were similar to DF508 in that they produced little mature protein (Fig. 2, A and B). Login to comment
92 selected F1052V, which lies outside the cluster of mutations and the ABC transporter consensus sequence, because we thought it would likely have a functional defect since it is associated with CF yet is processed like wild-type protein. Login to comment
94 We found that cells expressing all of the ICL4 mutants (F1052V, R1066C, R1066H, R1066L, and A1067T) generated cAMP-stimulated Cl2 selective currents that showed time-and voltage-independent behavior identical to that of wild-type CFTR (data not shown). Login to comment
102 Mutations did not alter single-channel conductance: wild-type, 8.9 6 0.3 pS; F1052V, 9.6 6 0.2 pS; R1066C, 8.9 6 0.4 pS; R1066H, 8.5 6 0.7; R1066C, 8.6 6 0.3; and A1067T, 9.2 6 0.2 pS. Login to comment
104 Table I shows that F1052V, R1066L, and A1067T did not alter the relative permeability or conductivity sequence for Cl2 , Br2 , or I2 . Login to comment
151 n Px/PCL Gx/GCL Br2 Cl2 I2 Br2 Cl2 I2 Wild-type 3 1.29 6 0.07 1.00 0.56 6 0.13 1.18 6 0.56 1.00 0.35 6 0.06 F1052V 2 1.41 1.00 0.50 0.98 1.00 0.53 R1066L 4 1.36 6 0.07 1.00 0.88 6 0.11 1.15 6 0.22 1.00 0.40 6 0.11 A1067T 4 1.29 6 0.15 1.00 0.66 6 0.04 1.00 6 0.10 1.00 0.43 6 0.06 FIG. 4. Login to comment
155 Data are mean 6 S.E. of (6/5) measurements for Po and burst duration, respectively: wild-type (19/18), F1052V (6/5) R1066C (3/3), R1066H (6/7), R1066L (12/5), and A1067T (9/3). Login to comment
180 For example, the mutant F1052V was processed normally but had dramatically altered function, whereas the R1066H mutation had a dramatic effect on processing but little discernible effect on function. Login to comment
199 Data are mean 6 S.E. of (n) measurements for: wild-type (9), F1052V (3), R1066L (4), A1067T (4), G551S (6), K464A (4), G1349D (5), K1250 M at 5 mM PPi (5), wild-type at 5 mM PPi (16). Login to comment
217 There are reports that several of the mutations (for example, F1052V, H1054D, and L1077P) are associated with a milder, pancreatic sufficient phenotype in which pancreatic function is not completely defective (17, 20, 46). Login to comment

[hide] Disease-associated mutations in the fourth cytopla... J Biol Chem. 1996 Jun 21;271(25):15139-45. Seibert FS, Linsdell P, Loo TW, Hanrahan JW, Clarke DM, Riordan JR
Disease-associated mutations in the fourth cytoplasmic loop of cystic fibrosis transmembrane conductance regulator compromise biosynthetic processing and chloride channel activity.
J Biol Chem. 1996 Jun 21;271(25):15139-45., [PMID:8662892]

Abstract [show]
A cluster of 18 point mutations in exon 17b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has been detected in patients with cystic fibrosis. These mutations cause single amino acid substitutions in the most C-terminal cytoplasmic loop (CL4, residues 1035-1102) of the CFTR chloride channel. Heterologous expression of the mutants showed that 12 produced only core-glycosylated CFTR, which was retained in the endoplasmic reticulum; the other six mutants matured and reached the cell surface. In some cases substitution of one member of pairs of adjacent residues resulted in misprocessing, whereas the other did not. Thus, the secondary structure of CL4 may contribute crucially to the proper folding of the entire CFTR molecule. Cyclic AMP-stimulated iodide efflux was not detected from cells expressing the misprocessed variants but was from the other six, indicating that their mutations cause relatively subtle channel defects. Consistent with this, these latter mutations generally are present in patients who are pancreatic-sufficient, while the processing mutants are mostly from patients who are pancreatic-insufficient. Single-channel patch-clamp analysis demonstrated that the processed mutants had the same ohmic conductance as wild-type CFTR, but a lower open probability, generally due to an increase in channel mean closed time and a reduction in mean open time. This suggests that mutations in CL4 do not affect pore properties of CFTR, but disrupt the mechanism of channel gating.

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64 The mature forms of the other six mutants (F1052V, K1060T, A1067T, G1069R, R1070W, R1070Q) were produced in relatively normal amounts (band C), although for A1067T and R1070W CFTR the ratio of the complex-glycosylated to core-glycosylated bands was significantly lower than for wild-type CFTR. Login to comment
81 This showed that the level of activity for cells expressing each CFTR mutant did approximately correspond to the amount of mature protein in the sample, i.e. in COS-1 cells F1052V, K1060T, and G1069R produced efflux levels similar to wild-type CFTR in accordance with a level of protein expression similar to wild type. Login to comment
88 Other disease-causing CFTR mutants, which are appropriately processed and trafficked to the plasma membrane, show defective ion conduction properties (e.g. R334W, R347H, and R347P; Sheppard et al., 1993; Tabcharani et al., 1993) or defective regulation of channel activity (e.g. G551S, G1244E, S1255P, and G1349D; Anderson and Welsh, 1992). Login to comment
94 As shown in Fig. 5, B and C, in some mutants this reduction in open probability was associated with a significant reduction in mean burst duration (F1052V, G1069R) or an increase in mean interburst duration (R1070W). Login to comment
127 Symbols are as follows: A: छ, WT; E, F1052V; Ç, K1060T; µ, A1067T; Ⅺ, vector only control. Login to comment
142 A, examples of wild-type, F1052V, K1060T, A1067T, G1069R, R1070Q, and R1070W CFTR single channel currents recorded from inside-out membrane patches at a membrane potential of -30 mV. Login to comment
71 The mature forms of the other six mutants (F1052V, K1060T, A1067T, G1069R, R1070W, R1070Q) were produced in relatively normal amounts (band C), although for A1067T and R1070W CFTR the ratio of the complex-glycosylated to core-glycosylated bands was significantly lower than for wild-type CFTR. Login to comment
101 As shown in Fig. 5, B and C, in some mutants this reduction in open probability was associated with a significant reduction in mean burst duration (F1052V, G1069R) or an increase in mean interburst duration (R1070W). Login to comment
133 Symbols are as follows: A: L, WT; E, F1052V; &#c7;, K1060T; &#b5;, A1067T; M, vector only control. Login to comment
148 A, examples of wild-type, F1052V, K1060T, A1067T, G1069R, R1070Q, and R1070W CFTR single channel currents recorded from inside-out membrane patches at a membrane potential of 230 mV. 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
87 R1066C R1066H F1052V 2789+5A->G D1152H 14a14b 15 1617a 17b 18 19 S1251N ItKbC->T G1349D m III! Login to comment

[hide] Is congenital bilateral absence of vas deferens a ... Am J Hum Genet. 1995 Jan;56(1):272-7. Mercier B, Verlingue C, Lissens W, Silber SJ, Novelli G, Bonduelle M, Audrezet MP, Ferec C
Is congenital bilateral absence of vas deferens a primary form of cystic fibrosis? Analyses of the CFTR gene in 67 patients.
Am J Hum Genet. 1995 Jan;56(1):272-7., [PMID:7529962]

Abstract [show]
Congenital bilateral absence of the vas deferens (CBAVD) is an important cause of sterility in men. Although the genetic basis of this condition is still unclear, it has been shown recently that some of these patients carry mutations in their cystic fibrosis transmembrane conductance regulator (CFTR) genes. To extend this observation, we have analyzed the entire coding sequence of the CFTR gene in a cohort of 67 men with CBAVD, who are otherwise healthy. We have identified four novel missense mutations (A800G, G149R, R258G, and E193K). We have shown that 42% of subjects were carriers of one CFTR allele and that 24% are compound heterozygous for CFTR alleles. Thus, we have been unable to identify 76% of these patients as carrying two CFTR mutations. Furthermore, we have described the segregation of CFTR haplotypes in the family of one CBAVD male; in this family are two male siblings, with identical CFTR loci but displaying different phenotypes, one of them being fertile and the other sterile. The data presented in this family, indicating a discordance between the CBAVD phenotype and a marked carrier (delta F508) chromosome, support the involvement of another gene(s), in the etiology of CBAVD.

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65 In addition, we identified the following missense mutations: four R668C, one A800G, one (G628R + S1235R, borne on the same chromosome), one (R74W + D1270N, borne on the same chromosome), six R117H, one F1052V, one R117C, one S1235R, one G149R, one R258G, two R347H, one R1066H, one R75L, and one E193K. Login to comment
77 of Patients Genotypea 1 AF508 + (G628R + S1235R) 1 AF508 + (R74W + D1270N) 2 AF508 + R668C 4 AF508 + R117H 1 AF508 + R258G 1 AF508 + R75L 1 E193K + N1303K 1 R347H + R1066H 1 R117C + W1282X 1 R553X + R668C 1 G149R + R668C 1 R117H+R117H 18 AF508/unidentified 4 W1282X/unidentified 1 G542X/unidentified 1 N1303K/unidentified 1 S1235R/unidentified 1 R347H/unidentified 1 A800G/unidentified 1 F1052V/unidentified 23 unidentified/unidentified a In parentheses are the two mutations located on the same haplotype. Login to comment

[hide] A cluster of cystic fibrosis mutations in exon 17b... J Med Genet. 1994 Sep;31(9):731-4. Mercier B, Lissens W, Novelli G, Kalaydjieva L, de Arce M, Kapranov N, Canki Klain N, Estivill X, Palacio A, Cashman S, et al.
A cluster of cystic fibrosis mutations in exon 17b of the CFTR gene: a site for rare mutations.
J Med Genet. 1994 Sep;31(9):731-4., [PMID:7529319]

Abstract [show]
Intensive screening has improved our understanding of the profile of mutations in the CFTR gene in which more than 400 mutations have been detected to date. In collaboration with several European laboratories we are involved in such analysis. We have identified 14 new mutations in exon 17b of CFTR, having analysed 780 CF chromosomes, and have compared the frequency of mutations in this exon with that of other regions of the CFTR gene. The results obtained indicate an accumulation of mutations, not only in regions encoding the two nucleotide binding folds, but also in those encoding transmembrane domains of the CFTR gene, in particular exon 17b.

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33 (1) HI085R, (2) 3320 ins 5, (3) R1066C, (4)R1066H, (5) A1067V, (6) 3272-16 GA, (7) F1052V, (8) R1070Q, (9) nornmal, (10) Y1092X, (11) G1069R,(12) nornial A cluster of cystic fibrosis mutations in exon I 7b of the CFTR gene: a site for rare mutations 14b, 17a, 23, 24) (table 3). Login to comment

[hide] High frequency of cystic fibrosis transmembrane re... Am J Hum Genet. 2000 Jun;66(6):2013-4. Gomez Lira M, Benetazzo MG, Marzari MG, Bombieri C, Belpinati F, Castellani C, Cavallini GC, Mastella G, Pignatti PF
High frequency of cystic fibrosis transmembrane regulator mutation L997F in patients with recurrent idiopathic pancreatitis and in newborns with hypertrypsinemia.
Am J Hum Genet. 2000 Jun;66(6):2013-4., [PMID:10801389]

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12 Among these rare mutations, L997F was identified in 4 (12.5%) of 32 patients with idiopathic pancreatitis (genotypes L997F/DF508, L997F/5T, and twice L997F/no mutation identified, respectively), and in 4 (8%) of 49 newborns with hypertrypsinemia (genotypes L997F/G542X, L997F/R553X, L997F/DF508, and L997F-F1052V phase unknown, respectively). Login to comment

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

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

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42 [1075C>A; 1079C>A] p.[Gln359Lys; Thr360Lys] Exon 8 1 1 1 4 1 1 R297Q c.890G>A p.Arg297Gln Exon 8 1 1 1 2 0 0 R347P c.1040G>C p.Arg347Pro Exon 8 3 5 2 4 1 1 T338I c.1013C>T p.Thr338Ile Exon 8 1 1 1 2 1 1 DF508 c.1521_1523delCTT p.Phe508del Exon 11 130 195 172 345 88 (4) 92 DI507 c.1519_1521delATC p.Ile507del Exon 11 1 5 5 11 2 1 Q493R c.1478A>G p.Gln493Arg Exon 11 5 5 2 2 2 2 1717-1G-A c.1585-1G>A - Intron 11 6 10 9 18 6 8 G542X c.1624G>T p.Gly542X Exon 12 14 17 15 34 10 10 G551S c.1651G>A p.Gly551Ser Exon 12 1 1 1 2 1 1 G551D c.1652G>A p.Gly551Asp Exon 12 12 22 19 33 7 8 I556V c.1666A>G p.Ile556Val Exon 12 1 2 2 4 1 1 R553X c.1657C>T p.Arg553X Exon 12 3 4 2 4 0 0 R560T c.1679G>C p.Arg560Thr Exon 12 1 1 1 2 1 2 1898+1G-A c.1766 &#b1; 1G>A - Intron 13 1 1 1 2 1 1 2184delA c.2052delA p.Lys684AsnfsX38 Exon 14 1 1 0 0 0 0 G622D c.1865G>A p.Gly622Asp Exon 14 1 1 1 3 0 0 N703S c.2108A>G p.Asn703Ser Exon 14 1 2 2 3 2 2 S737F c.2210C>T p.Ser737Phe Exon 14 1 1 0 0 0 0 2622+1G-A c.2490 &#b1; 1G>A - Intron 14 1 5 5 13 1 1 2752-26A-G c.2620-26A>G - Intron 15 1 2 2 4 0 0 2789+5G-A c.2657 &#b1; 5G>A - Intron 16 3 5 4 8 0 0 3120G-A c.2988G>A - Exon 18 2 2 1 2 1 0 3067-72del c.3067_3072del p.Ile1023_Val1024del Exon 19 1 1 1 1 0 0 I1027T c.3080T>C p.Ile1027Thr Exon 19 1 1 1 1 0 0 L997F c.2991G>C p.Leu997Phe Exon 19 1 2 2 4 1 (1) 0 M1028R c.3083T>G p.Met1028Arg Exon 19 1 1 1 2 1 2 F1052V c.3154T>G p.Phe1052Val Exon 20 1 1 0 0 0 0 Y1092X c.3276C>A p.Tyr1092X Exon 20 1 2 1 2 1 1 A1136T c.3406G>A p.Ala1136Thr Exon 21 1 2 1 2 1 0 D1152H c.3454G>C p.Asp1152His Exon 21 3 7 7 15 1 1 3659 del C c.3528delC p.Lys1177SerfsX15 Exon 22 2 4 3 7 3 3 R1162X c.3484C>T p.Arg1162X Exon 22 1 3 2 5 2 2 S1235R c.3705T>G p.Ser1235Arg Exon 22 2 3 3 5 2 1 3849+10kbC>T c.3717 &#b1; 12191C>T - Intron 22 2 4 4 5 0 0 W1282X c.3846G>A p.Trp1282X Exon 23 15 20 20 42 11 11 N1303K c.3909C>G p.Asn1303Lys Exon 24 9 12 11 24 4 5 Q1352H c.4056G>C p.Gln1352His Exon 25 1 1 1 1 1 1 Total 265 404 345 685 172 (6a ) 175 Values are n unless otherwise stated. Login to comment

[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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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
71 This was expected, as the CFTR mutations tested include known or putative CF-causing mutations, as well as CFTR mutations associated with varying clinical consequences (e.g., R668C, F1052V, D1152H) or complex CFTR alleles that may modify disease severity (e.g., S1235R) (www.CFTR2.org) [8,16]. 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
92 Mutant CFTR forms that did not significantly respond to ivacaftor under the experimental conditions used in this study were generally associated with severe defects in CFTR processing A B C D E F 0 100 200 300 400 -9 -8 -7 -6 -5 -4 0 S1235R D1152H F1052V D1270N ivacaftor [Log M] 0 100 200 300 400 -9 -8 -7 -6 -5 -4 0 R668C K1060T R74W R117H ivacaftor [Log M] 0 100 200 300 400 -9 -8 -7 -6 -5 -4 0 E193K A1067T L997F R1070Q ivacaftor [Log M] Chloride Transport ( &#b5;A/cm 2 ) Chloride Transport ( &#b5;A/cm 2 ) Chloride Transport ( &#b5;A/cm 2 ) Chloride Transport ( &#b5;A/cm 2 ) Chloride Transport ( &#b5;A/cm 2 ) Chloride Transport ( &#b5;A/cm 2 ) Chloride Transport ( &#b5;A/cm 2 ) Chloride Transport ( &#b5;A/cm 2 ) Chloride Transport ( &#b5;A/cm 2 ) 0 100 200 300 400 -9 -8 -7 -6 -5 -4 0 D110E D579G D110H R1070W ivacaftor [Log M] 0 100 200 300 400 -9 -8 -7 -6 -5 -4 0 F1074L E56K P67L A455E ivacaftor [Log M] 0 100 200 300 400 -9 -8 -7 -6 -5 -4 0 R347H S945L L206W S977F ivacaftor [Log M] 0 100 200 300 400 -8 -6 -4 0 T338I R1066H R117C R352Q ivacaftor [Log M] 0 100 200 300 400 -9 -8 -7 -6 -5 -4 0 F508del R334W H1054D E92K ivacaftor [Log M] 0 5 10 15 20 -9 -8 -7 -6 -5 -4 0 F508del R334W H1054D E92K R1066H T338I ivacaftor [Log M] G H I Fig. 3. Login to comment

[hide] Defining the disease liability of variants in the ... Nat Genet. 2013 Oct;45(10):1160-7. doi: 10.1038/ng.2745. Epub 2013 Aug 25. Sosnay PR, Siklosi KR, Van Goor F, Kaniecki K, Yu H, Sharma N, Ramalho AS, Amaral MD, Dorfman R, Zielenski J, Masica DL, Karchin R, Millen L, Thomas PJ, Patrinos GP, Corey M, Lewis MH, Rommens JM, Castellani C, Penland CM, Cutting GR
Defining the disease liability of variants in the cystic fibrosis transmembrane conductance regulator gene.
Nat Genet. 2013 Oct;45(10):1160-7. doi: 10.1038/ng.2745. Epub 2013 Aug 25., [PMID:23974870]

Abstract [show]
Allelic heterogeneity in disease-causing genes presents a substantial challenge to the translation of genomic variation into clinical practice. Few of the almost 2,000 variants in the cystic fibrosis transmembrane conductance regulator gene CFTR have empirical evidence that they cause cystic fibrosis. To address this gap, we collected both genotype and phenotype data for 39,696 individuals with cystic fibrosis in registries and clinics in North America and Europe. In these individuals, 159 CFTR variants had an allele frequency of l0.01%. These variants were evaluated for both clinical severity and functional consequence, with 127 (80%) meeting both clinical and functional criteria consistent with disease. Assessment of disease penetrance in 2,188 fathers of individuals with cystic fibrosis enabled assignment of 12 of the remaining 32 variants as neutral, whereas the other 20 variants remained of indeterminate effect. This study illustrates that sourcing data directly from well-phenotyped subjects can address the gap in our ability to interpret clinically relevant genomic variation.

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137 In addition to these ten variants, c.1210-12(7) (legacy name 7T) had already been reported to be non-penetrant48 and was identified as a second variant in numerous fathers, and a twelfth variant, p.Ile1027Thr, was deemed 159 variants ࣙ0.01% frequency in CFTR2 127 variants meet clinical and functional criteria Clinical and functional analysis 13 variants meet neither criteria 14 variants 5 variants 7 variants 6 variants Evidence of non-penetrance No evidence of non-penetrance 19 variants meet clinical or functional criteria 127 variants are CF causing 12 variants are non CF causing 20 variants are indeterminate p.Arg117HisߤC p.Arg75Gln p.Gly576Alaߤ p.Arg668Cys ߤ p.Met470Val C p.IIe1027Thr ߤC p.Val754Met ߤC p.IIe148Thr ߤC p.Arg31Cys C p.Ser1235Arg ߤ p.Leu997Phe ߤ p.Arg1162Leu p.Leu227Arg F p.Gln525* F p.Leu558SerC p.Asp614Gly C c.2657+2_2657+3insA C c.1418delG F c.1210-12(7) ߤ p.Arg1070Gln C p.Asp1270Asn ߤC p.[Gln359Lys; Thr360Lys] p.Gly1069Argߤ p.Asp1152His p.Phe1052Val c.1210-12(5) p.Arg74Trpߤ p.IIe1234Val ߤC p.Arg1070Trp ߤF p.Ser977Phe F p.Asp579Gly C p.Tyr569Asp F Penetrance analysis Figure 4ߒ Assignment of disease liability to the 159 most frequent CFTR variants using three criteria. Login to comment
183 Therefore, we are more confident that more frequent variants such as p.Gly551Asp are fully penetrant than we are for variants such as p.[Gln359Lys; Thr360Lys], p.Phe1052Val and p.Gly1069Arg, which were seen with an allele frequency of less than 0.0002. Login to comment

[hide] In vivo readout of CFTR function: ratiometric meas... PLoS One. 2013 Oct 24;8(10):e77114. doi: 10.1371/journal.pone.0077114. eCollection 2013. Wine JJ, Char JE, Chen J, Cho HJ, Dunn C, Frisbee E, Joo NS, Milla C, Modlin SE, Park IH, Thomas EA, Tran KV, Verma R, Wolfe MH
In vivo readout of CFTR function: ratiometric measurement of CFTR-dependent secretion by individual, identifiable human sweat glands.
PLoS One. 2013 Oct 24;8(10):e77114. doi: 10.1371/journal.pone.0077114. eCollection 2013., [PMID:24204751]

Abstract [show]
To assess CFTR function in vivo, we developed a bioassay that monitors and compares CFTR-dependent and CFTR-independent sweat secretion in parallel for multiple (~50) individual, identified glands in each subject. Sweating was stimulated by intradermally injected agonists and quantified by optically measuring spherical sweat bubbles in an oil-layer that contained dispersed, water soluble dye particles that partitioned into the sweat bubbles, making them highly visible. CFTR-independent secretion (M-sweat) was stimulated with methacholine, which binds to muscarinic receptors and elevates cytosolic calcium. CFTR-dependent secretion (C-sweat) was stimulated with a beta-adrenergic cocktail that elevates cytosolic cAMP while blocking muscarinic receptors. A C-sweat/M-sweat ratio was determined on a gland-by-gland basis to compensate for differences unrelated to CFTR function, such as gland size. The average ratio provides an approximately linear readout of CFTR function: the heterozygote ratio is ~0.5 the control ratio and for CF subjects the ratio is zero. During assay development, we measured C/M ratios in 6 healthy controls, 4 CF heterozygotes, 18 CF subjects and 4 subjects with 'CFTR-related' conditions. The assay discriminated all groups clearly. It also revealed consistent differences in the C/M ratio among subjects within groups. We hypothesize that these differences reflect, at least in part, levels of CFTR expression, which are known to vary widely. When C-sweat rates become very low the C/M ratio also tended to decrease; we hypothesize that this nonlinearity reflects ductal fluid absorption. We also discovered that M-sweating potentiates the subsequent C-sweat response. We then used potentiation as a surrogate for drugs that can increase CFTR-dependent secretion. This bioassay provides an additional method for assessing CFTR function in vivo, and is well suited for within-subject tests of systemic, CFTR-directed therapeutics.

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102 PS2 M F508del/R117H, 5T 92.5 2 38 0 0% 2.16 0.000 0.000 0.00% - - PS4 F F508del/3849+10 kb CRT 90 2 59 2 3% 2.42 0.000 0.000 0.00% 51% MRSA, Pa (muc, non-m) PS5 M F508del/R117H 83 3 51 0 0% 4.20 0.000 0.000 0.00% 66% Pa(muc and non), SA PS6 F F508del/3849+10 kb CRT 70 3 37 0 0% 1.23 0.000 0.000 0.00% 38% Pa (m) CFPS Mean 6 SD or sumsR 80612 12 228 2 1% 2.261.3 0.000 0.000 0.00% 49% CFTR-Related R1 M F508del/F1052V, nv 7T/9T 69 2 40 40 100% 6.76 0.265 0.038 14.16% - - R2 F M470V 44 3 57 57 100% 2.88 0.620 0.211 79.65% 81% A. xylosoxidans R3 F M470V, 7T/7T 71 3 34 13 38% 1.87 0.010 0.004 1.52% - - R4 M F508del/Unk 52 2 69 44 64% 4.03 0.021 0.005 1.98% 44% Pa (mucoid) positioned 1.7 cm above the skin to produce more diffuse light that worked well with the stained sweat droplets. 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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116 CFTR variant %Cases %Uctrls OR p-value %Cases w/N34S OR w/N34S p-value w/N34S F508C 0.5 0.3 1.58 0.21 0.0 0.00 0.67 R1162L 0.5 0.5 1.13 0.29 1.8 4.03 0.17 I1027T 0.5 0.3 1.99 0.17 0.0 0.00 0.70 R31C 0.3 0.7 0.42 0.088 0.0 0.00 0.52 I148T 0.3 0.4 0.75 0.27 0.0 0.00 0.63 R297Q 0.3 0.2 1.89 0.21 0.0 0.00 0.76 R74W 0.2 0.2 0.85 0.29 0.0 0.00 0.71 F1052V 0.1 0.2 0.63 0.27 0.0 0.00 0.76 I807M 0.1 0.1 1.26 0.30 0.0 0.00 0.83 R258G 0.1 0.1 1.26 0.30 0.0 0.00 0.83 G1069R 0.1 0.0 0.13 0.0 V201M 0.0 0.1 0.17 0.0 0.00 0.83 Of the 81 CFTR mutations tested in the cohort, 43 were observed at least once in cases or controls. Login to comment
269 67 SNPs (125GtoC, 1716G.A, 1717-1G.A, 1898+1G.A, 2183AA.G, 2184delA, 2789+5G.A, 3120+1G.A, 3659delC, 3849+10kbC.T, 621+ 1G.T, 711+5G.A, A455E, D110H, D1152H, D1270N, D443Y, D579G, F1052V, F1074L, F508C, F508del, G1069R, G1244E, G1349D, G178R, G542X, G551D, G551S, I1131L/V, I148T, I336K/T, I507del, I807M, IVS8T5, K1180T, L1065P, L967S, L997F, M1V, M470V, M952I, M952T, N1303K, P67L, Q1463Q, R1070Q, R1162X, R117C, R117H, R170H, R258G, R297Q, R31C, R352Q, R553X, R668C, R74W, R75Q, S1235R, S1255P, S485R, S977F, T338I, T854T, V201M, W1282X) were multiplexed into 6 wells; 14 SNPs (S492F, S945L, R74Q, R560T, R1162L, G85E, I1027T, R334W, R347P, G576A, 711+1G.T, 1001+11C.T, P1290P, 3199del6) were ascertained separately via TaqMan Gene Expression Assays, with repeat confirmation of all positive results. Login to comment

[hide] 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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31 Both methods used 5 &#b5;l of isolated DNA for the NGS assay. NGS assay for detection of CFTR mutations/variants CFTR mutations are described using both the international nomenclature of the Human Genome Variation Society Mutations that have varying consequences c.3454G>C (D1152H) c.3154T>G (F1052V) c.3208C>T (R1070W) c.2930C>T (S977F) - c.3808G>A (D1270N) c.3205G>A (G1069R) c.350G>A (R117H) PolyTG/ polyT - c.1736A>G (D579G) c.3209G>A (R1070Q) c.220C>T (R74W) - - Mutations still under evaluation c.2657ߙ+ߙ2_2657ߙ+ߙ3insA (2789ߙ+ߙ2insA) c.680T>G (L227R) c.1705T>G (Y569D) - - c.1841A>G (D614G) c.1673T>C (L558S) - - - c.3700A>G (I1234V) c. Login to comment

[hide] The improvement of the best practice guidelines fo... Eur J Hum Genet. 2015 May 27. doi: 10.1038/ejhg.2015.99. Girardet A, Viart V, Plaza S, Daina G, De Rycke M, Des Georges M, Fiorentino F, Harton G, Ishmukhametova A, Navarro J, Raynal C, Renwick P, Saguet F, Schwarz M, SenGupta S, Tzetis M, Roux AF, Claustres M
The improvement of the best practice guidelines for preimplantation genetic diagnosis of cystic fibrosis: toward an international consensus.
Eur J Hum Genet. 2015 May 27. doi: 10.1038/ejhg.2015.99., [PMID:26014425]

Abstract [show]
Cystic fibrosis (CF) is one of the most common indications for preimplantation genetic diagnosis (PGD) for single gene disorders, giving couples the opportunity to conceive unaffected children without having to consider termination of pregnancy. However, there are no available standardized protocols, so that each center has to develop its own diagnostic strategies and procedures. Furthermore, reproductive decisions are complicated by the diversity of disease-causing variants in the CFTR (cystic fibrosis transmembrane conductance regulator) gene and the complexity of correlations between genotypes and associated phenotypes, so that attitudes and practices toward the risks for future offspring can vary greatly between countries. On behalf of the EuroGentest Network, eighteen experts in PGD and/or molecular diagnosis of CF from seven countries attended a workshop held in Montpellier, France, on 14 December 2011. Building on the best practice guidelines for amplification-based PGD established by ESHRE (European Society of Human Reproduction and Embryology), the goal of this meeting was to formulate specific guidelines for CF-PGD in order to contribute to a better harmonization of practices across Europe. Different topics were covered including variant nomenclature, inclusion criteria, genetic counseling, PGD strategy and reporting of results. The recommendations are summarized here, and updated information on the clinical significance of CFTR variants and associated phenotypes is presented.European Journal of Human Genetics advance online publication, 27 May 2015; doi:10.1038/ejhg.2015.99.

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87 [Gln359Lys; Thr360Lys] L558S c.1673 T4C p.Leu558Ser Y569D c.1705 T4G p.Tyr569Asp D579G c.1736 A4G p.Asp579Gly D614G c.1841 A4G p.Asp614Gly S977F c.2930C4T p.Ser977Phe F1052V c.3154 T4G p.Phe1052Val G1069R c.3205G4A p.Gly1069Arg R1070Q c.3209G4A p.Arg1070Gln D1152H c.3454G4C p.Asp1152His I1234V c.3700 A4G p.Ile1234Val 5T c.1210 - 12[5] Examples of common not CF-causing variantsc R31C c.91C4T p.Arg31Cys R74W c.220C4T p.Arg74Trp R75Q c.224G4A p.Arg75Gln I148T c.443 T4C p.Ile148Thr M470V c.1408 A4G p.Met470Val G576A c.1727G4C p.Gly576Ala R668C c.2002C4T p.Arg668Cys V754M c.2260G4A p.Val754Met L997F c.2991G4C p.Leu997Phe I1027T c.3080 T4C p.Ile1027Thr R1070W c.3208C4T p.Arg1070Trp R1162L c.3485G4T p.Arg1162Leu Table 1 (Continued) HGVS nomenclature Legacy name cDNA nucleotide name Protein name S1235R c.3705 T4G p.Ser1235Arg D1270N c.3808G4A p.Asp1270Asn 7T c.1210-12[7] Abbreviation: HGVS, Human Genome Variation Society. Login to comment