ABCMdb

ABCC7 p.Arg1070Trp

Admin's notes:	Class II-III (maturation defect, gating defect) Veit et al.
ClinVar:	c.3209G>C , p.Arg1070Pro ? , not provided c.3209G>A , p.Arg1070Gln D , Pathogenic/Likely pathogenic, not provided c.3208C>T , p.Arg1070Trp ? , not provided
CF databases:	c.3209G>A , p.Arg1070Gln ? , Varying clinical consequence ; CFTR1: This missense mutation was found in one Italian CF patient. The nucleotide change was G->A at position 3341 of exon 17b leading to R 1070 Q amino acid change. It was found once using DGGE screening and DNA sequencing among 50 Italian CF chromosomes. c.3208C>T , p.Arg1070Trp ? , Varying clinical consequence ; CFTR1: Teh R1070W mutation was detected on 1 US Caucasian chromosome out of 48 screened. ASO analysis of 100 non-CF Caucasian chromosomes did not reveal this mutation on any of the tested chromosomes. The 15 months old CBAVD patient carries the [delta]F508 mutation on the other allele. c.3209G>C , p.Arg1070Pro (CFTR1) ? , This 26 year old individual of Polish extraction with mild CF presented at age 11 with nasal polyps. He had noted salt crystals on his skin in warm weather, but did not have a chronic cough or gastrointestinal complaints. Pulmonary function tests and chest X-ray were normal. Sweat chloride was 121 mMol/L (repeat value was 104 mMol/L). No formal pancreatic function testing was performed. Most recent pulmonary function tests show mild obstructive airways disease. This individual is a compound heterozygote for the 2143delT CF mutations. R1070P was originally detected by SSC/HA and can be detected by virtue of the creation of a Sau96I or destruction of a BslI site. Mutation R1070P was also reported by DÃ¶rk T, Hughes D, Dworniczak B, Stuhrmann M (Jan 30, (NL#69)) in a CF patient from Northern Ireland who carried R1070P on his paternal and [delta]F508 on his maternal allele.
Predicted by SNAP2:	A: N (66%), C: D (53%), D: D (85%), E: D (75%), F: D (91%), G: D (71%), H: N (57%), I: D (63%), K: N (66%), L: D (63%), M: D (66%), N: D (63%), P: D (71%), Q: D (75%), S: D (53%), T: D (63%), V: D (63%), W: D (95%), Y: D (71%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: D, G: D, H: N, I: D, K: N, L: D, M: N, N: N, P: D, Q: N, S: N, T: N, V: D, W: D, Y: N,

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

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

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96 L206W/- 5T/9T 1 L206W/3121-1G→A 7T/9T 1 L206W/1949del84 7T/9T 1 transrectal ultrasonography was significantly smaller in∆E115/S50P 7T/7T 1 2869insG/R1070W 7T/7T 1 CBAVD than in CUAVD (F ϭ 8.1, P ϭ 0.005). Login to comment

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

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

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47 a sweat chloride level of Ͼ70 mmol/l did not show any pulmonary Except for R1070W in exon 17b (four out of 47 ϭ 8.5%)or gastrointestinal symptoms of CF. Login to comment
50 total, ∆F508, T5 allele, R117H, R1070W and L375F representNone had undergone abdominal ultrasonography or excretory 83% of the mutation types in our cohort. Login to comment
54 The three men with CUAVD wereTotal genomic DNA was isolated from the patients` peripheral blood cells and analysed for mutations in the whole CFTR region and splice compound heterozygotes (G542X/R1070W, ∆F508/R117H, junctions. Login to comment
60 Tworegions (1, 2, 5, 6a, 6b, 7, 10, 11, 12, 14a, 14b, 15, 16, 17a, 17b, 18, 20, 22, 23, 24) were amplified with a GC-clamp primer and six exons mutations were found in 31.9% of patients, 31.9% had one Table I. Summary of the clinical and biological findings of a population of men with congenital bilateral absence of the vas deferens (CBAVD, n ϭ 37), congenital unilateral absence of the vas deferens (CUAVD, n ϭ 3) and obstructive azoospermia (Obs A, n ϭ 7) Patient Phenotype Surgical Age Weight Height Sweat test Other clinical CFTR exploration (years) (kg) (m) (Cl- mEq/l) manifestation genotype 1 CBAVD ϩ 40 63 1.72 72 ∆F508/T5 2 CBAVD ϩ 31 66 1.76 40 L1227S/3272-26A→G 3 CBAVD ϩ 29 ∆F508/T5 4 CBAVD 29 sinusitis -/- 5 CBAVD 32 50 1.60 ∆F508/T5 6 CBAVD 35 64 1.66 ∆F508/T5 7 CBAVD ϩ 28 ∆F508/R117H 8 CBAVD ϩ 34 69 1.80 24 ∆F508/R117H 9 CBAVD ϩ 35 65 1.70 R117H/T5 10 CBAVD ϩ 32 50 1.70 31 asthma ∆F508/T5 11 CBAVD ϩ 26 left hydrocele T5/- 12 CBAVD ϩ 23 left varicocele, G551D/T5 asthma, anosmia 13 CBAVD ϩ 29 ∆F508/T5 14 CBAVD ϩ 36 63 1.64 52 ∆F508/R117H 15 CBAVD ϩ 37 60 1.76 ∆F508/T5 16 CBAVD ϩ 34 70 1.65 24 ∆F508/A1067V 17 CBAVD 35 61 1.73 42 ∆F508/R117H 18 CBAVD 25 72 1.82 86 2183AA→G/T5 19 CBAVD 28 88 1.76 7 -/- 20 CBAVD ϩ 29 ∆F508/T5 21 CBAVD 31 48 epididymite -/- 22 CBAVD 28 ∆F508/T5 23 CBAVD ϩ 32 68 1.76 36 flatulence ∆F508/R1070W 24 CBAVD ϩ 31 64 1.76 39 R1162X/T5 25 CBAVD 30 17 asthma R117H/L375F 26 CBAVD ϩ 36 62 1.70 ∆F508/R1070W 27 CBAVD 30 6 -/- 28 CBAVD 35 85 1.70 R1070W/- 29 CBAVD 39 bronchectasis -/- 30 CBAVD ϩ 29 ∆F508/- 31 CBAVD 31 bronchectasis, -/- deafness 32 CBAVD ϩ 26 asthma, otitis -/- 33 CBAVD ϩ 28 allergy -/- 34 CBAVD 37 36 R117H/- 35 CBAVD 33 -/- 36 CBAVD ϩ 30 64 1.68 R117H/T5 37 CBAVD ϩ 37 71 1.78 31 pancreatitis, 621ϩ1G→T/I980K alcoholism 38 CUAVD 43 62 1.68 40 allergy G542X/R1070W 39 CUAVD ϩ 35 allergy ∆F508/R117H 40 CUAVD ϩ 34 hydrocele L375F/G551D 41 Obs A ϩ 32 26 T5/- 42 Obs A 23 60 sinusitis ∆F508/2789ϩ2insA 43 Obs A ϩ 25 80 sinusitis, chronic ∆F508/4428insGA 44 Obs A ϩ 30 bronchitis -/- anosmia 45 Obs A 29 50 -/- 46 Obs A 29 75 1.77 ∆F508/T5 47 Obs A ϩ 30 82 1.66 -/- mutation and the T5 allele, 10.7% had only one mutation and clinical palpation. Login to comment
72 In addition, he had an elevated sweat test (80 mEq/l)39 ∆F508/R117H (TG)10T9/(TG)11T7 23 ∆F508/R1070W (TG)10T9/(TG)10T7 and sinusitis. Login to comment
73 This mutation creates a stop codon 43 nucleotides 26 ∆F508/R1070W (TG)10T9/(TG)10T7 downstream leading to the deletion of 33 C-terminus amino 16 ∆F508/A1067V (TG)10T9/(TG)10T7 acids of the CFTR protein including the TRL-COOH domain.42 ∆F508/2789ϩ2insA (TG)10T9/(TG)10T7 43 ∆F508/4428insGA (TG)10T9/(TG)11T7 This highly conserved proteic site is a perfect match for the 25 R117H/L375F (TG)10T7/(TG)10T7 binding consensus domain of the Naϩ-Hϩ exchanger regulatory 38 G542X/R1070W (TG)10T9/(TG)11T7 factor (NHE-RF), a cytoplasmic phosphoprotein that may play40 L375F/G551D (TG)10T7/(TG)10T7 37 621ϩ1G→T/I980K (TG)10T9/(TG)10T9 an important regulatory role in CFTR function (Wang et al., 2 L1227S/3272-26A→G (TG)10T9/(TG)12T7 1998). Login to comment
79 Under these circumstances, polymorphisms could 28 R1070W/- (TG)11T7/(TG)10T7 be ruled out. Login to comment

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

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

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

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

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

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64 Spectrum of the CFTR Mutations Identified in the Cohorts from Brittany, Dublin Centre, and Cork Area Nucleotide Amino acid change * change Exon Number Frequency Number Frequency Number Frequency 211delG 2 1 0.1% 310G>T E60X 3 5 0.6% 4 0.3% 347C>A A72D 3 1 0.1% 368G>A W79X 3 1 0.1% 386G>A G85E 3 2 0.3% 3 0.2% 403G>A G91R 3 2 0.3% 482G>A R117H 4 4 0.5% 38 3.0% 4 1.4% 498T>A Y122X 4 1 0.1% 574delA 4 1 0.1% 577G>A G149R 4 1 0.1% 621+1G>T int 4 5 0.6% 21 1.7% 790C>T Q220X 6a 1 0.1% 875+1G>C int 6a 1 0.4% 905delG 6b 1 0.1% 1065C>G F311L 7 2 0.3% 1078delT 7 28 3.6% 1132C>T R334W 7 1 0.1% 1172G>A R347H 7 5 0.6% 1172G>T R347L 7 1 0.1% 1172G>C R347P 7 1 0.1% 1187G>A R352Q 7 3 0.2% 2 0.7% 1208A>G Q359R 7 1 0.1% 1154insTC 7 2 0.2% 1221delCT 7 2 0.3% 1248+1G>A int 7 1 0.1% 1249-27delTA int 7 1 0.4% 1334G>A W401X 8 1 0.1% 1461ins4 9 5 0.4% 1471delA 9 2 0.2% 1607C>T S492F 10 2 0.3% 1609C>T Q493X 10 1 0.1% 1648_1653delATC I507del 10 3 0.4% 10 0.8% 1 0.4% 1652_1655del 3 bp F508del 10 582 74.8% 966 76.5% 226 81.3% 1690G>T V520F 10 4 0.3% 1717-1G>A int 10 8 1.0% 9 0.7% 1756G>T G542X 11 5 0.6% 8 0.6% 1779T>G S549R 11 1 0.1% 1784G>A G551D 11 29 3.7% 82 6.5% 27 9.7% 1789C>G R553G 11 1 0.1% 1789C>T R553X 11 3 0.4% 1 0.1% 1806delA 11 1 0.1% 1811G>A R560K 11 2 0.3% 1811G>C R560T 11 30 2.4% 2 0.7% 1819T>A Y563N 12 1 0.1% 1853C>A P574H 12 1 0.1% 1898+1G>A int 12 1 0.1% 2184delA 13 1 0.1% 1 0.1% 2184insA 13 1 0.1% 2622+1G>A int 13 1 0.1% 2 0.2% 2622+1G>T int 13 1 0.1% 2623-2A>G ** int 13 1 0.1% 2670G>A W846X2 14a 8 1.0% 2752-1G>T int 14a 1 0.1% 2752-26A>G int 14a 2 0.2% 2789+5G>A int 14b 6 0.8% 2966C>T S945L 15 2 0.3% 3007delG 15 4 0.3% 3040G>C G970R 15 1 0.1% 3062C>T S977F 16 1 0.1% 3120+1G>A int 16 1 0.1% 3272-26A>G int 17a 4 0.5% 2 0.2% 2 0.7% 3320dupli(CTATG) 17b 1 0.1% 3329G>A R1066H 17b 1 0.1% 3340C>T R1070W 17b 1 0.1% 3408C>A Y1092X 17b 7 0.9% 3442G>T E1104X 17b 1 0.1% 3446T>G ** M1105R 17b 1 0.1% 3586G>C D1152H 18 1 0.1% 3601-17T>C + 1367delC int 18 + 9 1 0.1% 3616C>T R1162X 19 1 0.1% 2 0.2% 3659delC 19 2 0.2% 3832A>G I1234V 19 2 0.3% 3849+4A>G int 19 1 0.1% 3849+10kbC>T int 19 3 0.2% 3877G>A G1249R 20 1 0.1% 3884G>A S1251N 20 1 0.1% 3898insC 20 1 0.1% 3905insT 20 2 0.3% 3978G>A W1282X 20 3 0.4% 4005+1G>A int 20 6 0.8% 4016insT 21 1 0.1% 4041C>G N1303K 21 11 1.4% 5 0.4% 4136T>C L1335P 22 1 0.1% 1 0.4% 4279insA 23 1 0.1% Unidentified Unidentified - 3 0.4% 41 3.2% 11 4.0% Total 778 100.0% 1262 100.0% 278 100.0% * All nucleotide changes correspond to cDNA numbering. Login to comment

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

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

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8 R117H, D1152H, L206W, 3272-26A>G, S1235R, G149R, R1070W, S945L, and the poly-T tract variation commonly called IVS8-5T were also observed. Login to comment
44 Table 1 : Genotypes and Phenotypes of Patients with Normal or BordIerline Sweat Tests Patient Age at diagnosis (years) CFTR GENOTYPE* Allele 1 Allele 2 SWEAT CL- MEAN (MMOL/L) PHENOTYPE 1 0.2 F508del G149R 38 P+PI, neonatal hypertrypsinemia, 2 0.3 G551D R117H-7T 31 neonatal hypertrypsinemia 3 0.4 F508del R1070W 30.5 neonatal hypertrypsinemia 4 0.4 F508del R117H-7T 52 P 5 0.6 F508del 3849+10kbC>T 48 P 6 0.11 F508del S945L 58 P+PI 7 1 F508del 5T 40 P+CBAVD 8 2 F508del L206W 53 P 9 2 W1282X 5T 42.5 P 10 5 F508del 3849+10kbC>T 55.5 P 11 5 F508del L206W 55 P 12 5 G91R 5T 47.5 P 13 6 G551D S1235R+5T 49.5 P, neonatal hypertrypsinemia 14 7 F508del 3849+10kb 50 P, nasal popyposis 15 13 F508del R117H-7T 58 P, nasal polyposis 16 18 F508del 5T 60.5 P 17 20 G542X 3849+10kbC>T 52 P+PI 18 21 I507del 3849+10kbC>T 54 P, bronchiectasis 19 30 R347P 3849+10kbC>T 43 P, Pseudomonas colonisation 20 30 I507del L206W 57.5 CBAVD, chronic cough 21 31 F508del R117H-7T 60 CBAVD 22 32 G542X 3849+10kbC>T 30 P, Pseudomonas colonisation 23 34 F508del 3272-26A>G 64 P, CBAVD 24 37 R1070Q D1152H 56 CBAVD, bronchectasis 25 46 F508del D1152H 43 P 26 55 F508del D1152H 48 P, Pseudomonas colonisation 27 56 I507del S1235R 53 P 28 >18 F508del D1152H 60 P+PI 29 >20 F508del 3849+10kbC>T 18 P, bronchiectasis 30 >20 F508del 3272-26A>G 61 P *All mutations are named in accordance with the numbering used in the CFTR Mutation Database: http://www.genet.sickkids.on.ca/cftr/. Login to comment
101 The other mutations observed in trans of severe mutations were G149R, R1070W, S945L and S1235R. Login to comment
102 G149R and R1070W mutations have been previously described in CBAVD patients [Mercier et al., 1995; Jezequel et al., 2000] and S1235R have been described associated with variable pulmonary symptoms and occasionally borderline sweat tests [Monagham et al., 2000]. Login to comment

[hide] Mutations of the CFTR gene in Turkish patients wit... Hum Reprod. 2004 May;19(5):1094-100. Epub 2004 Apr 7. Dayangac D, Erdem H, Yilmaz E, Sahin A, Sohn C, Ozguc M, Dork T
Mutations of the CFTR gene in Turkish patients with congenital bilateral absence of the vas deferens.
Hum Reprod. 2004 May;19(5):1094-100. Epub 2004 Apr 7., [PMID:15070876]

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

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44 1 (1.0) DoÈrk et al. 1997b M952I Exon 15 G®C at 2988 Amino acid substitution 1 (1.0) Girodon et al. 1995 3120+1G®A Intron 16 G®A at 3120+1 Aberrant splicing 1 (1.0) Macek et al. 1997 3272-26A®G Intron 17a A®G at 3272±26 Aberrant splicing 1 (1.0) Fanen et al. 1992 R1070W Exon 17b C®T at 3340 Amino acid substitution 1 (1.0) Macek et al. 1993* G1130A Exon 18 G®C at 3521 Amino acid substitution 1 (1.0) This study Mutations were designated following the recommended nomenclature (Beaudet and Tsui, 1993) except for the IVS8-(T)n alleles which are named according to the number of their residual thymidines (Chu et al., 1993). Login to comment
47 *The following mutations were previously reported as personal communications to the CF Genetic Analysis Consortium (http://www.genet.sickkids.on.ca): 359insT by Claustres M, Desgeorges M, Romey M-C; R334Q by FeÂrec C, Quere I, Verlingue C, Raguenes O, AudreÂzet M-P, Mercier B; T388M by Zielenski J, Markiewicz D, Tsui L-C, Rawashdeh M, Khateeb M; E831X by FeÂrec C, Quere I, Audrezet MP, Verlingue C, Guillermit H, Mercier B; M952I by Girodon E, Costes B, Cazeneuve C, Ghanem N, Goossens M; R1070W by Macek M Jr, Sedriks S, Kiesewetter S, Cutting GR; D1152H by Highsmith WE Jr, Burch L, Friedman KJ, Wood BM, Spock A, Silverman LM, Knowles MR. Login to comment
72 CFTR genotypes in 51 patients with congenital bilateral absence of the vas deferens Mutation genotypes IVS8-(TG)mTn M470V n (%) Two mutations detected: D1152H/D1152H (TG)11 7T/ (TG)11 7T V/V 5 (9.8) IVS8-5T/IVS8-5T (TG)13 5T/ (TG)13 5T M/M 2 (3.9) (TG)12 5T/ (TG)13 5T M/V 1 (1.9) (TG)12 5T/ (TG)12 5T V/V 1 (1.9) IVS8-5T/D1152H (TG)12 5T/ (TG)11 7T V/V 2 (3.9) IVS8-5T/DF508 (TG)12 5T/ (TG)10 9T M/V 2 (3.9) IVS8-5T/2789+5G®A (TG)12 5T/ (TG)10 7T M/V 2 (3.9) IVS8-5T/365insT (TG)13 5T/ (TG)11 7T M/V 1 (1.9) IVS8-5T/D110H (TG)12 5T/ (TG)11 7T M/V 1 (1.9) IVS8-5T/E585X (TG)12 5T/ (TG)10 7T M/V 1 (1.9) IVS8-5T/2752-15C®G (TG)12 5T/ (TG)11 7T V/V 1 (1.9) IVS8-5T/M952I (TG)12 5T/ (TG)10 7T M/V 1 (1.9) IVS8-5T/3120+1G®A (TG)12 5T/ (TG)11 7T V/V 1 (1.9) D1152H/A349V (TG)10 7T/ (TG)11 7T M/V 1 (1.9) D1152H/2789+5G®A (TG)10 7T/ (TG)11 7T M/V 1 (1.9) D1152H/G1130A (TG)10 7T/ (TG)11 7T M/V 1 (1.9) CFTRdele2(ins186)/ IVS8-6T (TG)13 6T/ (TG)11 7T M/V 1 (1.9) CFTRdele2(ins186)/D110H (TG)11 7T/ (TG)11 7T V/V 1 (1.9) E831X/D110H (TG)11 7T/ (TG)11 7T V/V 1 (1.9) E831X/1677delTA (TG)11 7T/ (TG)11 7T V/V 1 (1.9) R334Q/R347H (TG)11 7T/ (TG)11 7T V/V 1 (1.9) 1767del6/1767del6 (TG)11 7T/ (TG)11 7T V/V 1 (1.9) 3041-15T®G/3041-15T®G (TG)12 7T/ (TG)12 7T M/M 1 (1.9) 3041-13del7/3041-13del7 (TG)10 7T/ (TG)10 7T M/M 1 (1.9) R1070W/3272-26A®G (TG)10 7T/ (TG)11 7T M/V 1 (1.9) I853F/L997F (TG)11 7T/ (TG)10 9T V/V 1 (1.9) One mutation detected: L997F/? Login to comment

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

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

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58 C-D565G II DF508 D1507 S549R S549I S549N S549R S945D S945L H1054D G1061R L1065P R1066C R1066M L1077P H1085R N1303K G85E III G551D S492F V520F R553G R560T R560S Y569D IV R117H, R117C, R117P, R117L D1152H, L88S, G91R, E92K, Q98R, P205S, L206W, L227R, F311L, G314E, R334W, R334Q, I336K, T338I, L346P, R347C, R347H, R347L, R347P, L927P, R1070W, R1070Q V 3849 þ 10 kb C ! Login to comment

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

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

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72 The two deletions Table 2 Atypical CF or nonclassic patients in whom extensive sequencing revealed two CFTR mutations Patient Genotype Phenotype Sex Age (years) Sweat chloride concentration (mmol/l) 1 p.S912L/DF508 Chronic lung and sinus disease F 52 Not done 2 p.R1070W/p.N1303K Recurrent respiratory infections F 4.5 2X intermediate 3 p.G551D/c.2789+2 InsA Pancreatic insufficiency, little lung involvement F 50 92, 96 4 c.3849+10kb C>T/p.L732X Failure to thrive, chronic cough, chronic sinusitis M 5.5 70,73 5 p.W1282X/p.R170H Chronic pancreatitis, CBVAD M 44 Borderline (c.1641 AG>T, and c.2949-2953 del TACTC) are expected to be severe disease-associated mutations, since they change the CFTR reading frame; the two patients harboring these novel deletions had a diagnosis of CF with elevated sweat chloride levels and carried a second, previously described, CF mutation. Login to comment

[hide] Atomic model of human cystic fibrosis transmembran... Cell Mol Life Sci. 2008 Aug;65(16):2594-612. Mornon JP, Lehn P, Callebaut I
Atomic model of human cystic fibrosis transmembrane conductance regulator: membrane-spanning domains and coupling interfaces.
Cell Mol Life Sci. 2008 Aug;65(16):2594-612., [PMID:18597042]

Abstract [show]
We describe herein an atomic model of the outward-facing three-dimensional structure of the membrane-spanning domains (MSDs) and nucleotide-binding domains (NBDs) of human cystic fibrosis transmembrane conductance regulator (CFTR), based on the experimental structure of the bacterial transporter Sav1866. This model, which is in agreement with previous experimental data, highlights the role of some residues located in the transmembrane passages and directly involved in substrate translocation and of some residues within the intracellular loops (ICL1-ICL4) making MSD/NBD contacts. In particular, our model reveals that D173 ICL1 and N965 ICL3 likely interact with the bound nucleotide and that an intricate H-bond network (involving especially the ICL4 R1070 and the main chain of NBD1 F508) may stabilize the interface between MSD2 and the NBD1F508 region. These observations allow new insights into the ATP-binding sites asymmetry and into the molecular consequences of the F508 deletion, which is the most common cystic fibrosis mutation.

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259 Among these, several missense mutations have been observed for the critical R1070 residue (R1070W, R1070Q, R1070P; http:// www.genet.sickkids.on.ca/cftr/), but no functional data are available for them. Login to comment

[hide] Localization studies of rare missense mutations in... Hum Mutat. 2008 Nov;29(11):1364-72. Krasnov KV, Tzetis M, Cheng J, Guggino WB, Cutting GR
Localization studies of rare missense mutations in cystic fibrosis transmembrane conductance regulator (CFTR) facilitate interpretation of genotype-phenotype relationships.
Hum Mutat. 2008 Nov;29(11):1364-72., [PMID:18951463]

Abstract [show]
We have been investigating the functional consequences of rare disease-associated amino acid substitutions in the cystic fibrosis transmembrane conductance regulator (CFTR). Mutations of the arginine residue at codon 1070 have been associated with different disease consequences; R1070P and R1070Q with "severe" pancreatic insufficient cystic fibrosis (CF) and R1070W with "mild" pancreatic sufficient CF or congenital bilateral absence of the vas deferens. Intriguingly, CFTR bearing each of these mutations is functional when expressed in nonpolarized cells. To determine whether R1070 mutations cause disease by affecting CFTR localization, we created polarized Madin Darby canine kidney (MDCK) cell lines that express either wild-type or mutant CFTR from the same genomic integration site. Confocal microscopy and biotinylation studies revealed that R1070P was not inserted into the apical membrane, R1070W was inserted at levels reduced from wild-type while R1070Q was present in the apical membrane at levels comparable to wild-type. The abnormal localization of CFTR bearing R1070P and R1070W was consistent with deleterious consequences in patients; however, the profile of CFTR R1070Q was inconsistent with a "severe" phenotype. Reanalysis of 16 patients with the R1070Q mutation revealed that 11 carried an in cis nonsense mutation, S466X. All 11 patients carrying the complex allele R1070Q-S466X had severe disease, while 4 out of 5 patients with R1070Q had "mild" disease, thereby reconciling the apparent discrepancy between the localization studies of R1070Q and the phenotype of patients bearing this mutation. Our results emphasize that localization studies in relevant model systems can greatly assist the interpretation of the disease-causing potential of rare missense mutations.

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1 Mutations of the arginine residue at codon 1070 have been associated with different disease consequences; R1070P and R1070Q with ''severe`` pancreatic insufficient cystic fibrosis (CF) and R1070W with ''mild`` pancreatic sufficient CF or congenital bilateral absence of the vas deferens. Login to comment
4 Confocal microscopy and biotinylation studies revealed that R1070P was not inserted into the apical membrane, R1070W was inserted at levels reduced from wild-type while R1070Q was present in the apical membrane at levels comparable to wild-type. Login to comment
5 The abnormal localization of CFTR bearing R1070P and R1070W was consistent with deleterious consequences in patients; however, the profile of CFTR R1070Q was inconsistent with a ''severe`` phenotype. Login to comment
27 Patients with R1070W (c.3208C4T; p.Arg1070Trp) have pancreatic sufficient CF or congenital bilateral absence of the vas deference (CBAVD) and a normal life span, whereas patients with R1070Q (c.3209G4A; p.Arg1070Gln) and R1070P (c3209G4C; p.Arg1070Pro) have classic CF with significant clinical features of lung disease, pancreatic insufficiency, and elevated sweat chloride levels [Mickle et al., 2000]. Login to comment
28 Previous studies revealed that the biogenesis and function of CFTR bearing R1070W and R1070P is substantially altered, consistent with their pathologic role. Login to comment
29 However, CFTR with R1070Q had a less severe channel gating abnormality than R1070W and either no defect [Seibert et al., 1996; Mickle et al., 2000] or a milder defect than R1070W upon protein processing [Seibert et al., 1996]. Login to comment
30 These functional studies indicate that the R1070Q mutation should cause a milder phenotype than R1070W, rather than the more severe phenotype observed. Login to comment
36 While CFTR bearing R1070W and R1070P displayed localization defects consistent with their associated phenotypes, R1070Q was difficult to distinguish from wild-type CFTR. Login to comment
85 RESULTS Wild-Type CFTR Is Localized toApical Membranes of MDCK Cells To determine the effect of R1070 mutations on CFTR localization, we expressed heterologous CFTR (wild-type or 1 of 3 CFTR mutants-R1070P, R1070Q, or R1070W) from an FRT integration site in MDCK type II cell lines (Fig. 1A). Login to comment
104 CFTR bearing R1070W, a mutation associated with mild disease, localized to the apical surface of MDCK cells (see overlap with apical surface marker in Fig. 2; R1070W, left column) but was also present in other locations in the cell. Login to comment
105 The absence of colocalization with Na1 /K1 ATPase basolateral marker is consistent with presence of CFTR R1070W in the cytoplasm. Login to comment
117 CFTR with R1070W was present in both mature (C band) and immature (B band) forms (Fig. 3A; lane 3), although the ratio of C band to B band and the ratio of C band to actin control (Fig. 3A; lane 3, lower panel) was lower than seen for wild-type CFTR. Login to comment
118 Biotinylation revealed that CFTR R1070W is inserted into the apical membrane, albeit at very low levels when compared to wild-type (Fig. 3B; lane 5). Login to comment
119 These results indicate that R1070W is able to process into mature apically-localized protein but with decreased efficiency compared to wild-type. Login to comment
120 As shown by confocal microscopy, these biochemical assays are consistent with only a fraction of R1070W at the apical membrane. Login to comment
121 There was a small amount of B band in the apical biotinylated fraction of R1070W, implying that some immature CFTR protein bearing this mutation may be inserted into the apical membrane. Login to comment
127 Each panel shows MDCK-FRT cells expressing either wild-type, R1070P, R1070W, or R1070Q CFTR. Login to comment
133 Altered Function of CFTR Bearing R1070W and R1070P Is ConsistentWith Phenotype of Patients CarryingThese Mutations A worldwide survey identified 29 patients who carried the R1070W mutation (24 of whom had detailed clinical information); 26 patients with R1070Q (16 of whom had detailed data), and 2 patients with R1070P (1 of whom had detailed data). Login to comment
136 Out of 24 patients who carry the R1070W mutation, 16 had F508del (c.1521_1523delCTT; p.Phe508del) on the other allele and all were diagnosed with either pancreatic-sufficient CF or CBAVD (Table 1). Login to comment
137 Pancreatic status was unavailable for 6 out of the 16 patients with the R1070W and F508del genotype. Login to comment
140 As for the remaining R1070W patients (8/24 with detailed clinical information), five individuals carried a CFTR mutation associated with pancreatic insufficiency (2869insG (c.2737insG, p.Tyr913fs); G542X (c.1624G4 T, p.Gly542X); R668C-K710X (c. Login to comment
144 The one remaining individual with the R1070W/R668C-K710X genotype had pancreatic-insufficient CF. Login to comment
145 Overall, the R1070W mutation was associated with either pancreatic-sufficient CF or CBAVD when in trans with a CFTR mutation that confers pancreatic insufficiency, indicating that the R1070W mutation generally conferred mild disease. Login to comment
152 A: Western blotting of MDCK cell lysates expressing wild-type, R1070Q, R1070W, or R1070P CFTR. Login to comment
156 CFTR R1070W has a lower amount of stable protein expressed and is readily visible as both C and B bands (lane 3). Login to comment
159 B:Western blotting of the apical biotinylated fraction (odd-numbered lanes) or total lysates (even-numbered lanes) of MDCK cells expressing wild-type, R1070Q, R1070W, or R1070P CFTR.Wild-type CFTR (lanes 1 and 2) and CFTR R1070Q (lanes 3 and 4) have a 205-kDa band in the biotinylated fraction that is consistent with the presence of mature protein in the apical membrane.The fraction of R1070Q inserted into the apical membrane is similar to wild-type.CFTR R1070W (lanes 5 and 6) has a faint 205-kDa band and a very faint 175-kDa band, suggesting that mature and some immature protein is inserted in the apical membrane.CFTR R1070P (lanes 7 and 8) has no bands visible in the biotinylated fraction and only a 175-kDa band is seen in the total lysate, indicating that this mutant protein is not present in the apical membrane.The lower panel is an immunoblot of each apical fraction and each total lysate after incubation with an actin antibody. Login to comment
162 Summarized Clinical Information on R1070 Patients Patient mutations R1070W R1070P R1070Q R1070Q in cis S466X Number of patientsa 24 2 5 11 Second mutaiton dF508 16 1 0 7 other 8 1 5 4 Disease diagnosis CBACD (infertility) 15 0 3 0 Nonclassic CF 9 1 1 0 Classic CF 1 1 1 11 Pancreatic status Su/cient 9 0 1 0 Insu/cient 4a 1 1 10 Not reported 11b 1 3b 1 Sweat chloride levels Normal or low 12 0 1 0 Elevated460 mmol/L 4 1 1 10 Not reported 8b 1 2b 1 a One patient has classic CF; the other three have normal sweat chloride levels and high FVC values. Login to comment
187 Thus, functional studies were informative for all three R1070 mutations, whereby R1070P and R1070W revealed processing defects that are consistent with their role in disease, while the properties of CFTR bearing R1070Q provoked a reevaluation of the established genotype-phenotype relationship that led to a more plausible explanation for the pathology observed in patients with a glutamine substitution at codon 1070. Login to comment
194 Although CFTR R1070W is primarily associated with male infertility (CBAVD), previously published reports and the current study showed that the tryptophan mutation consistently has decreased protein expression levels and incomplete apical localization. Login to comment
195 CFTR R1070W transiently expressed in nonpolarized HEK-293 cells [Mickle et al., 2000], COS-1 cells [Seibert et al., 1996], and the stable expression in isogenic MDCK cell lines shown here all demonstrated lower levels than wild-type CFTR. Login to comment
197 Partial mislocalization of CFTR R1070W may be due to alteration in the rate of membrane recycling, as has previously been observed with N287Y, a CFTR cytoplasmic loop 2 mutant [Silvis et al., 2003]. Login to comment

[hide] Molecular models of the open and closed states of ... Cell Mol Life Sci. 2009 Nov;66(21):3469-86. Epub 2009 Aug 26. Mornon JP, Lehn P, Callebaut I
Molecular models of the open and closed states of the whole human CFTR protein.
Cell Mol Life Sci. 2009 Nov;66(21):3469-86. Epub 2009 Aug 26., [PMID:19707853]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR), involved in cystic fibrosis (CF), is a chloride channel belonging to the ATP-binding cassette (ABC) superfamily. Using the experimental structure of Sav1866 as template, we previously modeled the human CFTR structure, including membrane-spanning domains (MSD) and nucleotide-binding domains (NBD), in an outward-facing conformation (open channel state). Here, we constructed a model of the CFTR inward-facing conformation (closed channel) on the basis of the recent corrected structures of MsbA and compared the structural features of those two states of the channel. Interestingly, the MSD:NBD coupling interfaces including F508 (DeltaF508 being the most common CF mutation) are mainly left unchanged. This prediction, completed by the modeling of the regulatory R domain, is supported by experimental data and provides a molecular basis for a better understanding of the functioning of CFTR, especially of the structural features that make CFTR the unique channel among the ABC transporters.

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33 Of note, recent work has also shown that the R1070P and R1070W mutations lead, respectively, to the absence or to reduced levels of insertion of the protein into the apical membrane of polarized epithelial cells [23], suggesting that these mutations may, as DF508, affect the inter-domain interactions of CFTR. Login to comment

[hide] Association of cystic fibrosis genetic modifiers w... Fertil Steril. 2010 Nov;94(6):2122-7. Epub 2010 Jan 25. Havasi V, Rowe SM, Kolettis PN, Dayangac D, Sahin A, Grangeia A, Carvalho F, Barros A, Sousa M, Bassas L, Casals T, Sorscher EJ
Association of cystic fibrosis genetic modifiers with congenital bilateral absence of the vas deferens.
Fertil Steril. 2010 Nov;94(6):2122-7. Epub 2010 Jan 25., [PMID:20100616]

Abstract [show]
OBJECTIVE: To investigate whether genetic modifiers of cystic fibrosis (CF) lung disease also predispose to congenital bilateral absence of the vas deferens (CBAVD) in association with cystic fibrosis transmembrane conductance regulator (CFTR) mutations. We tested the hypothesis that polymorphisms of transforming growth factor (TGF)-beta1 (rs 1982073, rs 1800471) and endothelin receptor type A (EDNRA) (rs 5335, rs 1801708) are associated with the CBAVD phenotype. DESIGN: Genotyping of subjects with clinical CBAVD. SETTING: Outpatient and hospital-based clinical evaluation. PATIENT(S): DNA samples from 80 subjects with CBAVD and 51 healthy male controls from various regions of Europe. This is one of the largest genetic studies of this disease to date. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Genotype analysis. RESULT(S): For single nucleotide polymorphism (SNP) rs 5335, we found increased frequency of the CC genotype among subjects with CBAVD. The difference was significant among Turkish patients versus controls (45.2% vs. 19.4%), and between all cases versus controls (36% vs. 15.7%). No associations between CBAVD penetrance and polymorphisms rs 1982073, rs 1800471, or rs 1801708 were observed. CONCLUSION(S): Our findings indicate that endothelin receptor type A polymorphism rs 5335 may be associated with CBAVD penetrance. To our knowledge, this is the first study to investigate genetic modifiers relevant to CBAVD.

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68 Portuguese CFTR alleles Spanish CFTR alleles Turkish CFTR alleles 5T 22 F508del 11 5T 20 F508del 14 5T 9 D1152H 14 R334W 5 D443Ya 3 D110H 3 R117H 3 G576Aa 3 F508del 2 S1235R 3 R668Ca 3 3041-11del7 2 N1303K 2 G542X 2 1767del6 2 P205S 2 R117H 2 2789þ5G>A 2 D614G 2 V232D 2 CFTRdele2(ins186) 2 G542X 1 L997F 1 3120þ1G>A 1 L206W 1 H609R 1 G1130A 1 V562I 1 N1303H 1 M952I 1 I507del 1 L206W 1 365insT 1 3272-26A>G 1 3272-26A/G 1 E585X 1 2789þ5G>A 1 L15P 1 2752-15C>G 1 G576Aa 1 R347H 1 R334Q 1 R668Ca 1 2689insG 1 R347H 1 CFTRdele2,3 1 R1070W 1 E831X 1 L1227S 1 I 1027T 1 R1070W 1 E831X 1 3272-26A>G 1 L997F 1 I853F 1 A349V 1 6T 1 Note: CFTR ¼ cystic fibrosis transmembrane conductance regulator. Login to comment

[hide] Restoration of domain folding and interdomain asse... FASEB J. 2010 Aug;24(8):3103-12. Epub 2010 Mar 16. He L, Aleksandrov LA, Cui L, Jensen TJ, Nesbitt KL, Riordan JR
Restoration of domain folding and interdomain assembly by second-site suppressors of the DeltaF508 mutation in CFTR.
FASEB J. 2010 Aug;24(8):3103-12. Epub 2010 Mar 16., [PMID:20233947]

Abstract [show]
Deletion of PHE508 (DeltaF508) from the first nucleotide-binding domain (NBD1) of CFTR, which causes most cystic fibrosis, disrupts the folding and assembly of the protein. Although the folding pathways and yield of isolated NBD1 are altered, its global structure is not, and details of the changes in the rest of the protein remain unclear. To gain further insight into how the whole mutant protein is altered, we have determined the influence of known second-site suppressor mutations in NBD1 on the conformation of this domain and key interfaces between domains. We found that the suppressors restored maturation of only those processing mutations located in NBD1, but not in other domains, including those in the C-terminal cytoplasmic loop of the second membrane-spanning domain, which forms an interface with the NBD1 surface. Nevertheless, the suppressors promoted the formation of this interface and others in the absence of F508. The suppressors restored maturation in a DeltaF508 construct from which NBD2 was absent but to a lesser extent than in the full-length, indicating that DeltaF508 disrupts interactions involving NBD2, as well as other domains. Rescue of DeltaF508-CFTR by suppressors required the biosynthesis of the entire full-length protein in continuity, as it did not occur when N- and C-terminal "halves" were coexpressed. Simultaneous with these interdomain perturbations, DeltaF508 resulted in suppressor reversed alterations in accessibility of residues both in the F508-containing NBD1 surface loop and in the Q loop within the domain core. Thus, in the context of the full-length protein, DeltaF508 mutation causes detectable changes in NBD1 conformation, as well as interdomain interactions.

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95 Indeed, some substitutions on the CL4 side of this interface such as R1070W have been reported to improve ⌬F508-CFTR maturation (23). Login to comment

[hide] Measurement of nasal potential difference in young... Thorax. 2010 Jun;65(6):539-44. Sermet-Gaudelus I, Girodon E, Roussel D, Deneuville E, Bui S, Huet F, Guillot M, Aboutaam R, Renouil M, Munck A, des Georges M, Iron A, Thauvin-Robinet C, Fajac I, Lenoir G, Roussey M, Edelman A
Measurement of nasal potential difference in young children with an equivocal sweat test following newborn screening for cystic fibrosis.
Thorax. 2010 Jun;65(6):539-44., [PMID:20522854]

Abstract [show]
BACKGROUND: A challenging problem arising from cystic fibrosis (CF) newborn screening is the significant number of infants with hypertrypsinaemia (HIRT) with sweat chloride levels in the intermediate range and only one or no identified CF-causing mutations. OBJECTIVES: To investigate the diagnostic value for CF of assessing CF transmembrane conductance regulator (CFTR) protein function by measuring nasal potential difference in children with HIRT. METHODS: A specially designed protocol was used to assess nasal potential difference (NPD) in 23 young children with HIRT (3 months-4 years) with inconclusive neonatal screening. Results were analysed with a composite score including CFTR-dependent sodium and chloride secretion. Results were correlated with genotype after extensive genetic screening and with clinical phenotype at follow-up 3 years later. RESULTS: NPD was interpretable for 21 children with HIRT: 13 had NPD composite scores in the CF range. All 13 were finally found to carry two CFTR mutations. At follow-up, nine had developed a chronic pulmonary disease consistent with a CF diagnosis. The sweat test could be repeated in nine children, and six had sweat chloride values >or=60 mmol/l. Of the eight children with normal NPD scores, only two had two CFTR mutations, both wide-spectrum mutations. None had developed a CF-like lung disease at follow-up. The sweat test could be reassessed in five of these eight children and all had sweat chloride values <60 mmol/l. CF diagnosis was ruled out in six of these eight children. CONCLUSION: Evaluation of CFTR function in the nasal epithelium of young children with inconclusive results at CF newborn screening is a useful diagnostic tool for CF.

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130 Table 3 Genotypes of the children with HIRT according to the diagnostic score cut-off in the 21 patients with reliable NPD tests; results after extensive genetic analysis CFTR genotypes Diagnosis score >0.27 (8 patients) £0.27 (13 patients) A/A 0 F508del/621+3A/G F508del/Q1291R A/AB F508del/R347H F508del/R117H;T7 W846X/R117C n¼2 F508del/R1070W 2183AA/G/L206W F508del/3272-26A/G F508del/R117H;T7; n¼4 A/D 0 F508del/R933G G551D/R352Q B/D G622D/3849+45G/A 0 A/0 F508del/0 n¼2 0 0/0 3 0 0, no identified mutation; A, CF-causing mutation; B, mutation associated with cystic CFTR-related disorders; C, mutation with no clinical consequence ; D, mutation of unknown or uncertain clinical relevance; AB, mutation that is associated with a wide phenotypic spectrum that might belong to either group A or B. CFTR, cystic fibrosis transmembrane conductance regulator; HIRT, hypertrypsinaemia; NPD, nasal potential difference. Login to comment

[hide] Clinical phenotype and genotype of children with b... Am J Respir Crit Care Med. 2010 Oct 1;182(7):929-36. Epub 2010 Jun 10. Sermet-Gaudelus I, Girodon E, Sands D, Stremmler N, Vavrova V, Deneuville E, Reix P, Bui S, Huet F, Lebourgeois M, Munck A, Iron A, Skalicka V, Bienvenu T, Roussel D, Lenoir G, Bellon G, Sarles J, Macek M, Roussey M, Fajac I, Edelman A
Clinical phenotype and genotype of children with borderline sweat test and abnormal nasal epithelial chloride transport.
Am J Respir Crit Care Med. 2010 Oct 1;182(7):929-36. Epub 2010 Jun 10., 2010-10-01 [PMID:20538955]

Abstract [show]
RATIONALE: The diagnosis of cystic fibrosis (CF) is based on a characteristic clinical picture in association with a sweat chloride (Cl(-)) concentration greater than 60 mmol/L or the identification of two CF-causing mutations. A challenging problem is the significant number of children for whom no definitive diagnosis is possible because they present with symptoms suggestive of CF, a sweat chloride level in the intermediate range between 30 and 60 mmol/L, and only one or no identified CF-causing mutation. OBJECTIVES: To investigate the function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in the airways of children with intermediate sweat tests and inconclusive genetic findings in correlation with clinical phenotype and genotype. METHODS: We developed a composite nasal potential difference (NPD) diagnostic score to discriminate patients with CF from non-CF patients. We tested NPD in 50 children (age, 6 mo to 18 yr) with equivocal diagnoses and correlated the NPD diagnostic score with clinical phenotypes and genotypes. MEASUREMENTS AND MAIN RESULTS: Fifteen of the 50 children had NPD scores in the CF range. Eight of the 15 carried two CFTR mutations compared with only 5 of the 35 children with normal NPD scores (P = 0.01). They were significantly younger at evaluation and had recurrent lower respiratory tract infections, chronic productive coughs, and chronic Staphylococcus aureus colonization significantly more often than the 35 children with normal NPD results. CONCLUSIONS: Evaluation of CFTR function in the nasal epithelium of children with inconclusive CF diagnoses can be a useful diagnostic tool and help clinicians to individualize therapeutic strategy.

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162 CLINICAL CHARACTERISTICS OF CHILDREN WITH EQUIVOCAL DIAGNOSES AND NASAL POTENTIAL DIFFERENCE DIAGNOSTIC SCORE <0.27 Pt Mutation Age (yr) NPD Score Sweat Cl2 Chronic CF Pulmonary Disease CF Pathogens Airway Obstruction CF Lung Imaging FEV1 (%) BMI Others 1 F508del/S977F A-D 8 0.181 43 RLRTI, chronic productive cough S. aureus No Bronchiectasis 80 14.5 No Bronchial thickening Atelectasis 2 0/0 4 0.121 43 No S. aureus Yes Air trapping NA 13 Pancreatic extracts 0-0 Bronchial thickening 3 0/0 15 20.032 46 RLRTI S. aureus, P. aeruginosa Yes Air trapping 74 14 Polyposis 0-0 Bronchiectasis 4 F508del/0 2 20.249 57 RLRTI P. aeruginosa Yes Air trapping NA 16 No A-0 5 N1303K/(TG12)T5 11.8 20.263 47 RLRTI S. aureus, P. aeruginosa No Bronchial thickening ND 20 No A-B 6 F508del/L206W 5.9 20.278 40 RLRTI S. aureus No Bronchial thickening 115 22 Chronic pancreatitis A-AB 7 R668C/0 15 20.403 40 RLRTI None Yes Bronchiectasis 112 20 No B-0 Air trapping 8 F508del/L997F A-B 1 20.594 38 RLRTI, chronic productive cough P. aeruginosa No Bronchial thickening NA 16 CF hepatopathy 9 G576A;R668C/S1235R 8 20.659 31 0 None Wheezing Normal 100 20 No B-B 10 G542X/0 5 20.718 49 RLRTI, chronic productive cough S. aureus No Bronchial thickening NA 18 No A-0 11 0/0 7 20.742 37 RLRTI None No Normal 106 18 No 0-0 12 F508del/D110E 16 20.777 50 No S. aureus No No 100 21 No A-AB 13 F508del/R1070W 7 21.006 40 RLRTI S. aureus Wheezing Bronchial thickening 110 14 No A-AB 14 F508del-L467F/0 12 21.897 55 RLRTI, chronic productive cough S. aureus No Bronchiectasis 109 17 Pansinusitis A-0 15 F508del/H1054D 9 22.327 59 RLRTI, chronic productive cough S. aureus No Bronchial thickening 100 20 DIOS A-D Definition of abbreviations: A, B, AB, and D: A 5 CF-causing mutation; B 5 mutation that results in a CFTR-RD (clinical entities associated with CFTR mutations that do not meet the current diagnostic criteria for CF); AB 5 wide-spectrum mutation that may belong to either group A or group B; D 5 mutation of uncertain clinical relevance; BMI 5 body mass index; CF 5 cystic fibrosis; CFTR 5 gene encoding cystic fibrosis transmembrane conductance regulator; DIOS 5 distal intestinal obstructive syndrome; NA 5 not applicable; ND 5 not determined; NPD 5 nasal potential difference; P. aeruginosa 5 Pseudomonas aeruginosa; Pt 5 patient; RLRTI 5 recurrent lower respiratory tract infection; S. aureus 5 Staphylococcus aureus. Login to comment

[hide] The cystic fibrosis-causing mutation deltaF508 aff... J Biol Chem. 2010 Nov 12;285(46):35825-35. Epub 2010 Jul 28. Thibodeau PH, Richardson JM 3rd, Wang W, Millen L, Watson J, Mendoza JL, Du K, Fischman S, Senderowitz H, Lukacs GL, Kirk K, Thomas PJ
The cystic fibrosis-causing mutation deltaF508 affects multiple steps in cystic fibrosis transmembrane conductance regulator biogenesis.
J Biol Chem. 2010 Nov 12;285(46):35825-35. Epub 2010 Jul 28., 2010-11-12 [PMID:20667826]

Abstract [show]
The deletion of phenylalanine 508 in the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator is directly associated with >90% of cystic fibrosis cases. This mutant protein fails to traffic out of the endoplasmic reticulum and is subsequently degraded by the proteasome. The effects of this mutation may be partially reversed by the application of exogenous osmolytes, expression at low temperature, and the introduction of second site suppressor mutations. However, the specific steps of folding and assembly of full-length cystic fibrosis transmembrane conductance regulator (CFTR) directly altered by the disease-causing mutation are unclear. To elucidate the effects of the DeltaF508 mutation, on various steps in CFTR folding, a series of misfolding and suppressor mutations in the nucleotide binding and transmembrane domains were evaluated for effects on the folding and maturation of the protein. The results indicate that the isolated NBD1 responds to both the DeltaF508 mutation and intradomain suppressors of this mutation. In addition, identification of a novel second site suppressor of the defect within the second transmembrane domain suggests that DeltaF508 also effects interdomain interactions critical for later steps in the biosynthesis of CFTR.

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228 The substitution of R1070W had little effect on the maturation of wild type CFTR but measurably promoted trafficking of ⌬F508 CFTR (Fig. 6B). Login to comment
230 To evaluate the potential mechanisms by which the R1070W mutation rescued ⌬F508, this mutation was also introduced into the ⌬F508-3M and F508K backgrounds (Fig. 6, C and D). Login to comment
232 The combination of the -3M mutations with the R1070W mutation increased ⌬F508 Band C production, as compared with ⌬F508-3M and ⌬F508/ R1070W alone. Login to comment
234 In this regard, the R1070W mutation induced the formation of Band C in the F508K mutant predicted to disrupt the interdomain interaction, an effect not seen for low temperature or with the -3M mutations (Fig. 6D). Login to comment
314 C, the R1070W and -3M suppressor mutations were evaluated for their ability to rescue the ⌬F508 mutation either independently or in combination. Login to comment
315 The inclusion of the -3M and R1070W mutations in combination rescued more ⌬F508 CFTRthaneithersuppressorsetalone.Cellswereculturedat37 °CandevaluatedbyWesternblottingusingthe M3A7 antibody. Login to comment
316 D, trafficking of the F508K missense protein was evaluated with the R1070W mutation. Login to comment
317 Trafficking of F508K was partially rescued by the R1070W mutation. Login to comment
325 The R1070W mutant in TMD2 suppresses ⌬F508 by promoting the interactions between NBD1 and ICL4 as required for maturation. Login to comment
326 By relieving the QC-NBD interactions, the -3M and R1070W mutations promote CFTR maturation; upper pathway. Login to comment
339 Although both the Arg-1070 and -3M suppressors rescue ⌬F508, suppression by the R1070W mutation is likely accomplished by independent mechanisms. Login to comment
341 Thus, the R1070W mutation putatively promotes appropriate domain-domain associations by increasing hydrophobic interactions (affinity) at the NBD1-ICL4 domain-domain interaction surface. Login to comment
342 The relatively hydrophobic surface proximal to the Phe508 position could potentially accommodate the hydrophobicity and volume of the R1070W substitution. Login to comment
344 Maturation of the F508K CFTR molecule was potentially facilitated by interactions between the indole side chain from R1070W and the NBD1 surface. Login to comment
346 Intriguingly, the R1070W mutation, which rescues ⌬F508 CFTR, has been identified in patients with mild disease (congenital bilateral absence of the vas deferens, pancreatic sufficient CF). Login to comment
347 Previous studies suggest that in the wild type background the R1070W mutation alters protein expression, localization, and function (44). Login to comment

[hide] p.Ser1235Arg should no longer be considered as a c... Eur J Hum Genet. 2011 Jan;19(1):36-42. Epub 2010 Aug 18. Rene C, Paulet D, Girodon E, Costa C, Lalau G, Leclerc J, Cabet-Bey F, Bienvenu T, Blayau M, Iron A, Mittre H, Feldmann D, Guittard C, Claustres M, Georges M
p.Ser1235Arg should no longer be considered as a cystic fibrosis mutation: results from a large collaborative study.
Eur J Hum Genet. 2011 Jan;19(1):36-42. Epub 2010 Aug 18., [PMID:20717170]

Abstract [show]
Among the 1700 mutations reported in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, a missense mutation, p.Ser1235Arg, is a relatively frequent finding. To clarify its clinical significance, we collected data from 104 subjects heterozygous for the mutation p.Ser1235Arg from the French CF network, addressed for various indications including classical CF, atypical phenotypes or carrier screening in subjects with or without a family history. Among them, 26 patients (5 having CF, 10 CBAVD (congenital bilateral absence of the vas deferens) and 11 with CF-like symptoms) and 14 healthy subjects were compound heterozygous for a second CFTR mutation. An exhaustive CFTR gene analysis identified a second mutation in cis of p.Ser1235Arg in all CF patients and in 81.8% CBAVD patients. Moreover, epidemiological data from >2100 individuals found a higher frequency of p.Ser1235Arg in the general population than in CF or CBAVD patients. These data, added to the fact that in silico analysis and functional assays suggest a benign nature of this substitution, give several lines of evidence against an association of p.Ser1235Arg with CF or CBAVD.

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69 Of these, eight were compound heterozygous for p.Phe508del, and two for mutations associated with CF-related diseases,3,27 (p.Arg117His;T7) and p.Arg1070Trp (http://www.genet.sickkids.on.ca/cftr/). Login to comment
95 of subjects Allele 1 Allele 2 p.Ser1235Arg;p.Arg785X p.Phe508del Severe CF 2 p.Ser1235Arg;p.Arg785X NAa Severe CF 1 p.Ser1235Arg;875+1G4A (c.743+1C4A) 3629delT (c.3497delT) Severe CF 1 p.Ser1235Arg;p.Arg785X p.Gly542X Severe CF 1 p.Ser1235Arg;(TG)13(T)5 p.Gly551Asp Mild CF 1 p.Ser1235Arg;(TG)13(T)5 p.Phe508del CBAVD 6 p.Ser1235Arg;(TG)13(T)5 p.Arg1070Trp CBAVD 1 p.Ser1235Arg;(TG)13(T)5 p.Arg117His; (T)7 CBAVD 1 p.Ser1235Arg p.Phe508del CBAVD 1 p.Ser1235Arg - CBAVD 1 p.Ser1235Arg;(TG)13(T)5 p.Phe508del CUAVD 1 Suspicion CF/mild phenotype: p.Ser1235Arg - Genital symptoms 5 p.Ser1235Arg - Respiratory symptoms 16 p.Ser1235Arg;(TG)13(T)5 p.Phe508del Respiratory symptoms 2 p.Ser1235Arg 406-6T4C (c.274-6T4C) Respiratory symptoms 1 p.Ser1235Arg p.Tyr1092X Respiratory symptoms 1 p.Ser1235Arg p.Glu831X Respiratory symptoms 1 p.Ser1235Arg p.Gln493X Respiratory symptoms 1 p.Ser1235Arg p.Ile507del Respiratory symptoms 1 p.Ser1235Arg - Digestive symptoms 13 p.Ser1235Arg p.Gly542X Digestive symptoms 1 p.Ser1235Arg - Hyperechogenic fetal bowel 5 p.Ser1235Arg p.Arg668Cys; p.Arg576Ala Hyperechogenic fetal bowel 1 p.Ser1235Arg p.Val920Met Hyperechogenic fetal bowel 1 p.Ser1235Arg p.Phe508del Hyperechogenic fetal bowel 1 aNA: not available; we could only test the mother and a healthy sister (the patient was deceased and the father`s DNA was not available). Login to comment
115 Genotype Familial information Allele 1 Allele 2 1 p.Ser1235Arg; (TG)13(T)5 p.Phe508del Brother of CUAVD (no parental project) 2 p.Ser1235Arg; (TG)13(T)5 p.Phe508del Sister of CUAVD 3 p.Ser1235Arg; (TG)13(T)5 p.Arg1070Trp Sister of CBAVD 4 p.Ser1235Arg p.Gly542X Father of CF [p.Phe508del]+ [p.Gly542X] and healthy children [p.Ser1235Arg]+[(TG)11(T)5] 5 p.Ser1235Arg p.Gln493X Father of CF [p.Phe508del]+ [p.Gln493X] 6 p.Ser1235Arg p.Phe508del Uncle of CF (no parental project) 7 p.Ser1235Arg p.Phe508del Mother of CF [p.Phe508del]+ [1717-1G4A (c.1585-1G4A)] 8 p.Ser1235Arg 2347delG (c.2215delG) Mother of CF [p.Phe508del]+ [2347delG (c.2215delG)] 9 p.Ser1235Arg (TG)11(T)5 Mother of non-CF fetus with hyperechogenic fetal bowel [p.Phe508del]+[(TG)11(T)5] 10 p.Ser1235Arg p.Phe508del Sister of CBAVD 11 p.Ser1235Arg p.Phe508del Sister of CBAVD 12 p.Ser1235Arg p.Glu831X Sister of CF-like patient 13 p.Ser1235Arg p.Phe508del First-cousin of CF-like patient 14 p.Ser1235Arg p.Phe508del A 18-month-old child with prenatal diagnostic based on familial history of CF Table 2 Comparison of the allelic frequencies of p.Ser1235Arg and p.Phe508del in the general population, CF and CBAVD patients Populations Significance (P-values) General population, this study CF patients30 (n¼7420) CBAVD patients30 (n¼1626) General population vs CF General population vs CBAVD p.Phe508del 0.82% (n¼1950) 67.2% 21.6% S (Po0.05) S (Po0.05) p.Ser1235Arg 0.76% (n¼4228) 0.11% 0.43% S (Po0.05) NS (P¼0.17) Significance (P-values) p.Phe508del vs p.Ser1235Arg NS (P¼0.91) S (Po0.05) S (Po0.05) Abbreviations: N, number of unrelated tested chromosomes; S, significant difference; NS, no significant difference. Login to comment

[hide] [R74W;R1070W;D1270N]: a new complex allele respons... J Cyst Fibros. 2010 Dec;9(6):447-9. Epub 2010 Sep 28. de Prada Merino A, Butschi FN, Bouchardy I, Beckmann JS, Morris MA, Hafen GM, Fellmann F
[R74W;R1070W;D1270N]: a new complex allele responsible for cystic fibrosis.
J Cyst Fibros. 2010 Dec;9(6):447-9. Epub 2010 Sep 28., [PMID:20880762]

Abstract [show]
Since the beginning of population screening for CF carriers, it has become apparent that complex CFTR alleles are not uncommon. Deciphering their impact in disease pathogenesis remains a challenge for both clinicians and researchers. We report the observation of a new complex allele p.[R74W+R1070W+D1270N] found in trans with a type 1 mutation and associated with clinical diagnosis of cystic fibrosis in a one year-old Moroccan patient. This case underlines the difficulties in counseling patients with uncommon mutations and the necessity of functional studies to evaluate the structure-function relationships, since the association of several variations in cis can dramatically alter CFTR function.

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5 Keywords: Complex allele; R74W; D1270N; R1070W; CFTR; Cystic fibrosis 1. Login to comment
59 To our knowledge, R1070W has never been described within a complex allele. Login to comment
60 R1070W is considered a mutation of "mild" pancreatic-sufficient CF or of CFTR-related disease including CBAVD [1,16]; functional studies have revealed abnormal localization of CFTR bearing R1070W [16]. Login to comment

[hide] Small molecule correctors of F508del-CFTR discover... J Comput Aided Mol Des. 2010 Dec;24(12):971-91. Epub 2010 Oct 26. Kalid O, Mense M, Fischman S, Shitrit A, Bihler H, Ben-Zeev E, Schutz N, Pedemonte N, Thomas PJ, Bridges RJ, Wetmore DR, Marantz Y, Senderowitz H
Small molecule correctors of F508del-CFTR discovered by structure-based virtual screening.
J Comput Aided Mol Des. 2010 Dec;24(12):971-91. Epub 2010 Oct 26., [PMID:20976528]

Abstract [show]
Folding correctors of F508del-CFTR were discovered by in silico structure-based screening utilizing homology models of CFTR. The intracellular segment of CFTR was modeled and three cavities were identified at inter-domain interfaces: (1) Interface between the two Nucleotide Binding Domains (NBDs); (2) Interface between NBD1 and Intracellular Loop (ICL) 4, in the region of the F508 deletion; (3) multi-domain interface between NBD1:2:ICL1:2:4. We hypothesized that compounds binding at these interfaces may improve the stability of the protein, potentially affecting the folding yield or surface stability. In silico structure-based screening was performed at the putative binding-sites and a total of 496 candidate compounds from all three sites were tested in functional assays. A total of 15 compounds, representing diverse chemotypes, were identified as F508del folding correctors. This corresponds to a 3% hit rate, ~tenfold higher than hit rates obtained in corresponding high-throughput screening campaigns. The same binding sites also yielded potentiators and, most notably, compounds with a dual corrector-potentiator activity (dual-acting). Compounds harboring both activity types may prove to be better leads for the development of CF therapeutics than either pure correctors or pure potentiators. To the best of our knowledge this is the first report of structure-based discovery of CFTR modulators.

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111 In support of this hypothesis, it has been demonstrated that the double mutant F508del/R1070W, predicted by our model to improve hydrophobic packing in this region (Fig. 7), improves the trafficking of F508del-CFTR (Philip J. Thomas, personal communication). Login to comment
134 a MOLCAD surface of the interface site. b Binding site residues (stick representation) and binding site interaction regions generated with SiteMap. Yellow hydrophobic region, blue H-bond donor region, and red H-bond acceptor region Y1073 F1074 R1070W W496 M498 V510 F1068 Fig. 7 In silico generation of the R1070W mutant in the F508del model suggests that a tryptophan in this position may restore interactions lost in the F508del mutant evaluate this large set of compounds, in vitro screening commenced with a high throughput iodide flux corrector assay [22] performed in the laboratory of Professor Luis Galietta (Advanced Biotechnology Center, Genova, Italy). Login to comment

[hide] Benzbromarone stabilizes DeltaF508 CFTR at the cel... Biochemistry. 2011 May 31;50(21):4393-5. Epub 2011 May 3. Loo TW, Bartlett MC, Clarke DM
Benzbromarone stabilizes DeltaF508 CFTR at the cell surface.
Biochemistry. 2011 May 31;50(21):4393-5. Epub 2011 May 3., 2011-05-31 [PMID:21520952]

Abstract [show]
Deletion of Phe508 from the first nucleotide-binding domain of the CFTR chloride channel causes cystic fibrosis because it inhibits protein folding. Indirect approaches such as incubation at low temperatures can partially rescue DeltaF508 CFTR, but the protein is unstable at the cell surface. Here, we show that direct binding of benzbromarone to the transmembrane domains promoted maturation and stabilized DeltaF508 CFTR because its half-life at the cell surface was ~10-fold longer than that for low-temperature rescue. Therefore, a search for small molecules that can rescue and stabilize DeltaF508 CFTR could lead to the development of an effective therapy for cystic fibrosis.

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14 It was recently reported that the stability of ΔF508 CFTR at the cell surface approached that of wild-type CFTR when NBDÀTMD2 interactions were restored.21 It was shown that introduction of a V510D mutation into NBD1 promoted the maturation and stability of ΔF508 CFTR by forming a a salt bridge with Arg1070 of TMD2.21 Similarly, maturation of ΔF508 CFTR was promoted by a R1070W suppressor mutation in TMD2.5 These suppressor mutation results suggested that direct binding of a compound to the TMDs may promote the maturation and stability of ΔF508 CFTR. Login to comment

[hide] The W232R suppressor mutation promotes maturation ... Biochemistry. 2011 Feb 8;50(5):672-85. Epub 2011 Jan 11. Loo TW, Bartlett MC, Clarke DM
The W232R suppressor mutation promotes maturation of a truncation mutant lacking both nucleotide-binding domains and restores interdomain assembly and activity of P-glycoprotein processing mutants.
Biochemistry. 2011 Feb 8;50(5):672-85. Epub 2011 Jan 11., 2011-02-08 [PMID:21182301]

Abstract [show]
ATP-binding cassette (ABC) proteins contain two nucleotide-binding domains (NBDs) and two transmembrane (TM) domains (TMDs). Interdomain interactions and packing of the TM segments are critical for function, and disruption by genetic mutations contributes to disease. P-glycoprotein (P-gp) is a useful model to identify mechanisms that repair processing defects because numerous arginine suppressor mutations have been identified in the TM segments. Here, we tested the prediction that a mechanism of arginine rescue was to promote intradomain interactions between TM segments and restore interdomain assembly. We found that suppressor W232R(TM4/TMD1) rescued mutants with processing mutations in any domain and restored defective NBD1-NBD2, NBD1-TMD2, and TMD1-TMD2 interactions. W232R also promoted packing of the TM segments because it rescued a truncation mutant lacking both NBDs. The mechanism of W232R rescue likely involved intradomain hydrogen bond interactions with Asn296(TM5) since only N296A abolished rescue by W232R and rescue was only observed when Trp232 was replaced with hydrogen-bonding residues. In TMD2, suppressor T945R(TM11) also promoted packing of the TM segments because it rescued the truncation mutant lacking the NBDs and suppressed formation of alternative topologies. We propose that T945R rescue was mediated by interactions with Glu875(TM10) since T945E/E875R promoted maturation while T945R/E875A did not.

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331 For example, the R1070W mutation was postulated to promote folding through hydrophobic interactions with NBD1 in ΔF508-CFTR (65). Login to comment

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

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

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52 Mutation 0.46-0.35 9 c.1078delT #, p.R347P # 8 p.G85V, c.621 + 1G > T #, p.S549R (T > G) #, p.R553X #, c.3849 + 10kbC > T # 7 p.R347H #, c.1812-1G > A, p.R709X 0.30-0.10 6 p.H199Y, p.P205S, 5 p.R117H #, p.G551D #, p.W1089X, p.Y1092X, CFTR50kbdel 4 c.296 + 3insT, c.1717-1G > A #, c.1949del84, c.3849 + 1G > A 3 p.E92K, c.936delTA, c.1717-8G > A, c.1341G > A, p.A561E, c.2603delT, p.G1244E, [p.D1270N; p.R74W] 2 p.Q2X, p.P5L, CFTRdele2,3, p.S50P, p.E60K, c.405 + 1G > A, c.1677delTA, p.L558S, p.G673X, p.R851X, p.Y1014C, p.Q1100P, p.M1101K, p.D1152H, CFTRdele19, p.G1244V, p.Q1281X, p.Y1381X <0,1 1 c.124del23bp, p.Q30X, p.W57X, c.406-1G > A, p.Q98R, p.E115del, c.519delT, p.L159S, c.711 + 3A > T, p.W202X, c.875 + 1G > A, p.E278del, p.W361R, c.1215delG, p.L365P, p.A399D, c.1548delG, p.K536X, p.R560G, c.1782delA, p.L571S, [p.G576A; p.R668C], p.T582R, p.E585X, c.1898 + 1G > A, c.1898 + 3A > G, c.2051delTT, p.E692X, p.R851L, c.2711delT, c.2751 + 3A > G, c.2752-26A > G, p.D924N, p.S945L, c.3121-1G > A, p.V1008D, p.L1065R, [p.R1070W; p.R668C], [p.F1074L; 5T], p.H1085R, p.R1158X, c.3659delC #, c.3667del4, c.3737delA, c.3860ins31, c.3905insT #, c.4005 + 1G > A, p.T1299I, p.E1308X, p.Q1313X, c.4095 + 2T > A, rearrangements study (n = 4) Mutations identified in CF families with mixed European origin: c.182delT, p.L1254X, c.4010del4. Login to comment
67 Seven other complex alleles were observed: [c.296 + 3insT; p.V754M], [p.F508del; p.I1027T], [p.S549R; -102T > A], [p.G576A; p.R668C], [p.R1070W; p.R668C], [p.D1270N; p.R74W] and [p.T1299I; p.I148T]. Login to comment

[hide] Fixing cystic fibrosis by correcting CFTR domain a... J Cell Biol. 2012 Oct 15;199(2):199-204. doi: 10.1083/jcb.201208083. Okiyoneda T, Lukacs GL
Fixing cystic fibrosis by correcting CFTR domain assembly.
J Cell Biol. 2012 Oct 15;199(2):199-204. doi: 10.1083/jcb.201208083., [PMID:23071149]

Abstract [show]
For cystic fibrosis (CF) patients most therapies focus on alleviating the disease symptoms. Yet the cellular basis of the disease has been well studied; mutations in the CF gene can impair folding, secretion, cell surface stability, and/or function of the CFTR chloride channel. Correction of these basic defects has been a challenge, but indicates that a deeper understanding of the molecular and cellular mechanism of mutations is a prerequisite for developing more efficient therapies.

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75 Stabilization of the NBD1-MSD2 interface by second site suppressor mutations (e.g., R1070W) largely restored the WT-like coupling efficiency between NBD1 stability and F508-CFTR folding (Rabeh et al., 2012). Login to comment
88 Likewise, reversing the NBD1-MSD2 interface instability by second site mutations (e.g., R1070W) only marginally rescued the F508-CFTR phenotype (Fig. 2 B). Login to comment

[hide] Thermal instability of DeltaF508 cystic fibrosis t... Biochemistry. 2012 Jun 26;51(25):5113-24. Epub 2012 Jun 15. Liu X, O'Donnell N, Landstrom A, Skach WR, Dawson DC
Thermal instability of DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) channel function: protection by single suppressor mutations and inhibiting channel activity.
Biochemistry. 2012 Jun 26;51(25):5113-24. Epub 2012 Jun 15., [PMID:22680785]

Abstract [show]
Deletion of Phe508 from cystic fibrosis transmembrane conductance regulator (CFTR) results in a temperature-sensitive folding defect that impairs protein maturation and chloride channel function. Both of these adverse effects, however, can be mitigated to varying extents by second-site suppressor mutations. To better understand the impact of second-site mutations on channel function, we compared the thermal sensitivity of CFTR channels in Xenopus oocytes. CFTR-mediated conductance of oocytes expressing wt or DeltaF508 CFTR was stable at 22 degrees C and increased at 28 degrees C, a temperature permissive for DeltaF508 CFTR expression in mammalian cells. At 37 degrees C, however, CFTR-mediated conductance was further enhanced, whereas that due to DeltaF508 CFTR channels decreased rapidly toward background, a phenomenon referred to here as "thermal inactivation." Thermal inactivation of DeltaF508 was mitigated by each of five suppressor mutations, I539T, R553M, G550E, R555K, and R1070W, but each exerted unique effects on the severity of, and recovery from, thermal inactivation. Another mutation, K1250A, known to increase open probability (P(o)) of DeltaF508 CFTR channels, exacerbated thermal inactivation. Application of potentiators known to increase P(o) of DeltaF508 CFTR channels at room temperature failed to protect channels from inactivation at 37 degrees C and one, PG-01, actually exacerbated thermal inactivation. Unstimulated DeltaF508CFTR channels or those inhibited by CFTR(inh)-172 were partially protected from thermal inactivation, suggesting a possible inverse relationship between thermal stability and gating transitions. Thermal stability of channel function and temperature-sensitive maturation of the mutant protein appear to reflect related, but distinct facets of the DeltaF508 CFTR conformational defect, both of which must be addressed by effective therapeutic modalities.

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5 Thermal inactivation of ΔF508 was mitigated by each of five suppressor mutations, I539T, R553M, G550E, R555K, and R1070W, but each exerted unique effects on the severity of, and recovery from, thermal inactivation. Login to comment
18 We identified unique functional signatures for five second-site mutations, four in NBD1 (I539T, G550E, R553M, and R555K) and one in the fourth intracellular loop (ICL4, R1070W), and also investigated the relation of thermal stability to variations in channel gating brought about by intracellular cAMP, CFTR potentiators, and CFTR inhibitors. Login to comment
150 Protection from thermal inactivation by mutations that increased the open probability of ΔF508 CFTR channels led us to examine the thermal behavior of R1070W/ΔF508 CFTR. Login to comment
151 The substitution of this aromatic side chain in ICL4 increased the open probability of the double mutant 7 and was proposed to act, at least in part, by repairing defective coupling between NBD1 and ICL4.6,7 Results depicted in Figure 7A show that pairing ΔF508 with R1070W improved the thermal stability of the double mutant, but did not restore wt-like thermal stability. Login to comment
154 Pairing R1070W and a second NBD1 suppressor, R555K, with ΔF508, however, resulted in thermal stability that was indistinguishable from that of wt CFTR (Figure 7B). Login to comment
155 In contrast, combining ΔF508 with R1070W and I539T resulted in channels that could not sustain the elevated conductance seen immediately after warming to 37 °C but were nevertheless able to sustain a substantial conductance at 37 °C (Figure 7C). Login to comment
168 Protection of ΔF508 CFTR from thermal inactivation by R1070W. Login to comment
169 (A) Following stimulation, an oocyte expressing R1070W/ ΔF508 CFTR (n = 4) was warmed to 37 °C (gray bar and circles) for 10 min twice in succession. After cooling to 22 °C, the oocyte was exposed to 10 μM CF172 (black bar and circles). Login to comment
170 (B) Following stimulation, an oocyte expressing R555K/R1070W/ΔF508 CFTR (n = 4) was warmed to 37 °C for 10 min twice. After cooling to 22 °C, the oocyte was exposed to 10 μM CF172. Login to comment
171 (C) Following stimulation, an oocyte expressing I539T/R1070W/ΔF508 CFTR (n = 4) was warmed to 37 °C for 10 min twice. After cooling to 22 °C, the oocyte was exposed to 10 μM CF172. Login to comment
247 Similarly, G550E, R555K, and R1070W; when combined individually with ΔF508, improved protein maturation at 37 °C to at most 18% of wt,4,6 but nevertheless significantly improved the thermal stability of the double mutant channels. Login to comment
265 The ICL4 mutation, R1070W, increased the thermal stability of ΔF508 CFTR channel function, although it did not fully restore the wt-like behavior at 37 °C. Login to comment
266 Like G550E, R553M, and R555K, this second-site mutation has been associated with increased open probability of the double mutant,7 an effect attributed to a partial improvement in the interaction between NBD1 and ICL4.29,57 Combining the ICL4 mutation with an NBD1 suppressor mutation on the ΔF508 background (R555K/R1070W/ΔF508), however, fully restored wt-like thermal stability at 37 °C, an "additive" effect similar to that reported by Mendoza et al 6 in their study of the effect of these mutations on NBD1 folding and ΔF508 CFTR protein yield. Login to comment
267 The rescue of wt thermal stability of channel function by the R553M mutation, however, indicates that the structural modifications introduced by combining ΔF508 with R1070W are not required for the thermal stabilization of channel gating. Login to comment
268 Mendoza et al.6 also reported that combining R1070W with I539T, which alone increased the cellular yield of NBD1 to about 80% of wt, resulted in a yield of the I539T/R1070W/ ΔF508 protein that was 76% of the wt level. Login to comment

[hide] Allosteric modulation balances thermodynamic stabi... J Mol Biol. 2012 May 25;419(1-2):41-60. Epub 2012 Mar 8. Aleksandrov AA, Kota P, Cui L, Jensen T, Alekseev AE, Reyes S, He L, Gentzsch M, Aleksandrov LA, Dokholyan NV, Riordan JR
Allosteric modulation balances thermodynamic stability and restores function of DeltaF508 CFTR.
J Mol Biol. 2012 May 25;419(1-2):41-60. Epub 2012 Mar 8., [PMID:22406676]

Abstract [show]
Most cystic fibrosis is caused by a deletion of a single residue (F508) in CFTR (cystic fibrosis transmembrane conductance regulator) that disrupts the folding and biosynthetic maturation of the ion channel protein. Progress towards understanding the underlying mechanisms and overcoming the defect remains incomplete. Here, we show that the thermal instability of human DeltaF508 CFTR channel activity evident in both cell-attached membrane patches and planar phospholipid bilayers is not observed in corresponding mutant CFTRs of several non-mammalian species. These more stable orthologs are distinguished from their mammalian counterparts by the substitution of proline residues at several key dynamic locations in first N-terminal nucleotide-binding domain (NBD1), including the structurally diverse region, the gamma-phosphate switch loop, and the regulatory insertion. Molecular dynamics analyses revealed that addition of the prolines could reduce flexibility at these locations and increase the temperatures of unfolding transitions of DeltaF508 NBD1 to that of the wild type. Introduction of these prolines experimentally into full-length human DeltaF508 CFTR together with the already recognized I539T suppressor mutation, also in the structurally diverse region, restored channel function and thermodynamic stability as well as its trafficking to and lifetime at the cell surface. Thus, while cellular manipulations that circumvent its culling by quality control systems leave DeltaF508 CFTR dysfunctional at physiological temperature, restoration of the delicate balance between the dynamic protein's inherent stability and channel activity returns a near-normal state.

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151 Thus, both the constant and varying temperature experiments demonstrated a requirement for a balance between thermal stability and channel activity in CFTR, consistent with considerations of such a relationship between stability and catalytic function of proteins in general.28 NBD1 stabilization restores an NBD1-CL4 interface In addition to destabilizing NBD1, deletion of F508 also disrupts interdomain contacts including the interface between the NBD1 surface and the cytoplasmic loop (CL)4 in MSD2 in which the residue normally participates.29 Specific second-site mutations on either side of this interface (e.g., R1070W or V510D) have been shown to promote maturation of ΔF508 CFTR.27,30,31 The current observations that the ΔF508 protein with NBD1 strongly stabilized by the proline and I539T substitutions had channel activity similar to the WT at physiological temperature suggested either that the NBD1-CL4 interface is not important for function or that it is adequately restored by NBD1 stabilization. Login to comment

[hide] Correction of both NBD1 energetics and domain inte... Cell. 2012 Jan 20;148(1-2):150-63. Rabeh WM, Bossard F, Xu H, Okiyoneda T, Bagdany M, Mulvihill CM, Du K, di Bernardo S, Liu Y, Konermann L, Roldan A, Lukacs GL
Correction of both NBD1 energetics and domain interface is required to restore DeltaF508 CFTR folding and function.
Cell. 2012 Jan 20;148(1-2):150-63., [PMID:22265408]

Abstract [show]
The folding and misfolding mechanism of multidomain proteins remains poorly understood. Although thermodynamic instability of the first nucleotide-binding domain (NBD1) of DeltaF508 CFTR (cystic fibrosis transmembrane conductance regulator) partly accounts for the mutant channel degradation in the endoplasmic reticulum and is considered as a drug target in cystic fibrosis, the link between NBD1 and CFTR misfolding remains unclear. Here, we show that DeltaF508 destabilizes NBD1 both thermodynamically and kinetically, but correction of either defect alone is insufficient to restore DeltaF508 CFTR biogenesis. Instead, both DeltaF508-NBD1 energetic and the NBD1-MSD2 (membrane-spanning domain 2) interface stabilization are required for wild-type-like folding, processing, and transport function, suggesting a synergistic role of NBD1 energetics and topology in CFTR-coupled domain assembly. Identification of distinct structural deficiencies may explain the limited success of DeltaF508 CFTR corrector molecules and suggests structure-based combination corrector therapies. These results may serve as a framework for understanding the mechanism of interface mutation in multidomain membrane proteins.

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132 Remarkably, NBD1, MSD1, and NBD2 were susceptible to comparable misfolding upon disrupting the NBD1-CL4 interface by the R1070W substitution in WT CFTR. Login to comment
136 R1070W or V510D substitutions at the interfaces restored the proximity of the DF508 NBD1 and CL4 as shown by Cys crosslinking. Login to comment
137 The NBD1-CL4 interface stabilization can be accomplished by filling the cavity created by the DF508 with the bulky hydrophobic side chain of R1070W or by salt bridge formation between V510D in NBD1 and R1070 in CL4 (Figure S6B) (He et al., 2010; Loo et al., 2008, 2010; Thibodeau et al., 2010). Login to comment
138 R1070W, similar to V510D, alone modestly increased the DF508 CFTR folding efficiency and cellular and PM expression (Figures 6A-6E), in part confirming previous reports (He et al., 2010; Thibodeau et al., 2010; Loo et al., 2010). Login to comment
140 At the individual domain level, the combination of R1S and R1070W, but not R1S alone, largely restored domain assembly, as indicated by the WT-like trypsin resistance of the MSD1, MSD2, and NBD2 in DF508-CFTR-R1S-R1070W (Figure 5C). Login to comment
141 Similarly, the DF508 CFTR folding and expression were synergistically rescued by combining the DF508-NBD1 stabilizing mutation DRI with R1070W or V510D (Figures 6F-6H and S7B), ruling out nonspecific effects of second-site mutations. Login to comment
142 Direct energetic stabilization of the DF508-NBD1-0S and -3S by the V510D mutation was marginal (Figure S5). Login to comment
144 Accordingly, substantially increased folding efficiency of DF508-CFTR-1218X was only achieved by synergistic stabilization of the NBD1-CL4 interface (R1070W or V510D) and NBD1 (3S, -3R, or -R1S) (Figures 7A- 7C, and S7C). Login to comment
148 Likewise, disrupting the NBD1-CL4 interface by the R1070W mutation compromised both WT CFTR and CFTR-1218X folding (Figures 6B, 6C, 6E, left panel, and 7C), as well as the NBD1, NBD2, and MSD1 conformation, which was partly rescued by stabilizing the NBD1 with 3S, R, or R1S (Figure S6A). Login to comment
153 Red and blue circles depict the combined effect of R1070W or V5010D interface and 3S, R, or R1S stabilizing mutations on the DF508 CFTR expression. Login to comment
158 resistance of NBD1, MSD1, and NBD2 (Figure S6A) as well as restored folding and processing upon NBD1 stabilization by 3S, R, R1S, or DRI in the CFTR-R1070W and CFTR-1218X- R1070W (Figures 6B, 6C, 6E, and 7C). Login to comment
166 (D and E) Correction of the DF508 CFTR gating defect by the combination of the 3S and R1070W mutations. Login to comment
174 WT-like domain assembly and stabilization of DF508 CFTR were achieved by a combination of NBD1 and NBD1-CL4 interface-stabilizing mutations (e.g., 3S and R1070W or V510D). Login to comment
175 M2* and red line depict the R1070W mutation. Login to comment
178 The 3S and R1070W mutations increased the DF508 CFTR Po by 27% and 54%, respectively. Login to comment
197 Although interdomain interactions likely stabilize NBD1 conformationally in WT CFTR, as suggested by the enhanced NBD1 protease susceptibility upon destabilizing the MSD1 or NBD2 by CF-causing point mutation (Cui et al., 2007; Du and Lukacs, 2009; Du et al., 2005; Xiong et al., 1997), or the NBD1-CL4 interface by R1070W (Figure S6A), these stabilizing factors are compromised due to coupled misfolding of MSD1, MSD2, and NBD2 in DF508 CFTR and other mutants (Cui et al., 2007; Du and Lukacs, 2009; Du et al., 2005; Xiong et al., 1997). Login to comment
203 S, R, or DRI mutations increased the WT channel folding efficiency (by 20%-80%) in proportion with NBD1 stabilization, a tendency that is also observed in the background of NBD1-CL4 destabilization in R1070W-CFTR (Figures 4B and 6B). Login to comment
204 Stabilization of the NBD1-CL4 interface by salt bridge formation between V510D and R1070 also improved WT CFTR biogenesis (Figure 6B). Login to comment
133 Remarkably, NBD1, MSD1, and NBD2 were susceptible to comparable misfolding upon disrupting the NBD1-CL4 interface by the R1070W substitution in WT CFTR. Login to comment
139 R1070W, similar to V510D, alone modestly increased the DF508 CFTR folding efficiency and cellular and PM expression (Figures 6A-6E), in part confirming previous reports (He et al., 2010; Thibodeau et al., 2010; Loo et al., 2010). Login to comment
145 Accordingly, substantially increased folding efficiency of DF508-CFTR-1218X was only achieved by synergistic stabilization of the NBD1-CL4 interface (R1070W or V510D) and NBD1 (3S, -3R, or -R1S) (Figures 7A- 7C, and S7C). Login to comment
149 Likewise, disrupting the NBD1-CL4 interface by the R1070W mutation compromised both WT CFTR and CFTR-1218X folding (Figures 6B, 6C, 6E, left panel, and 7C), as well as the NBD1, NBD2, and MSD1 conformation, which was partly rescued by stabilizing the NBD1 with 3S, R, or R1S (Figure S6A). Login to comment
154 Red and blue circles depict the combined effect of R1070W or V5010D interface and 3S, R, or R1S stabilizing mutations on the DF508 CFTR expression. Login to comment
159 resistance of NBD1, MSD1, and NBD2 (Figure S6A) as well as restored folding and processing upon NBD1 stabilization by 3S, R, R1S, or DRI in the CFTR-R1070W and CFTR-1218X- R1070W (Figures 6B, 6C, 6E, and 7C). Login to comment
167 (D and E) Correction of the DF508 CFTR gating defect by the combination of the 3S and R1070W mutations. Login to comment
176 M2* and red line depict the R1070W mutation. Login to comment
179 The 3S and R1070W mutations increased the DF508 CFTR Po by 27% and 54%, respectively. Login to comment
198 Although interdomain interactions likely stabilize NBD1 conformationally in WT CFTR, as suggested by the enhanced NBD1 protease susceptibility upon destabilizing the MSD1 or NBD2 by CF-causing point mutation (Cui et al., 2007; Du and Lukacs, 2009; Du et al., 2005; Xiong et al., 1997), or the NBD1-CL4 interface by R1070W (Figure S6A), these stabilizing factors are compromised due to coupled misfolding of MSD1, MSD2, and NBD2 in DF508 CFTR and other mutants (Cui et al., 2007; Du and Lukacs, 2009; Du et al., 2005; Xiong et al., 1997). Login to comment

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

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

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103 Table 1. Continued Mutations in 257 patients Allele counts of each mutation % of variant alleles (183) % of all alleles tested (514) R1070W 1 0.55 0.19 R1158X 1 0.55 0.19 R1438W 1 0.55 0.19 R334W 2 1.09 0.39 R352W 1 0.55 0.19 R553X 2 1.09 0.39 R668C 2 1.09 0.39 R74W 3 1.64 0.58 R75X 3 1.64 0.58 S1235R 2 1.09 0.39 S492F 2 1.09 0.39 S549N 1 0.55 0.19 S573CS573C 1 0.55 0.19 S945L 1 0.55 0.19 T351S 1 0.55 0.19 T501A 2 1.09 0.39 T604ST604S 1 0.55 0.19 V11I 1 0.55 0.19 V201 mol/L 1 0.55 0.19 V232D 2 1.09 0.39 V754 mol/L 1 0.55 0.19 W1089X 2 1.09 0.39 W1098C 1 0.55 0.19 W1204X 4 2.19 0.78 Y563N 1 0.55 0.19 Y913XY913X 1 0.55 0.19 85 different mutations 183 100.00 35.60 Novel variants are in boldface, mutations on the ACMG/ACOG panel are italicized. Login to comment
186 Table 3. Continued CFTR mutations Alleles Relative mutation frequency (%) (of 317) G567A 1 Ͻ1 S573C 1 Ͻ1 E585X 1 Ͻ1 T604S 1 Ͻ1 F693L 1 Ͻ1 V754 mol/L 1 Ͻ1 2108delA 1 Ͻ1 2184delA 1 Ͻ1 2215insG 1 Ͻ1 2585delT 1 Ͻ1 2752 - 6TϾC 1 Ͻ1 E831X 1 Ͻ1 D836Y 1 Ͻ1 Y913X 1 Ͻ1 S945L 1 Ͻ1 L967S 1 Ͻ1 3171delC 1 Ͻ1 3199del6 1 Ͻ1 3271 ϩ 8AϾG 1 Ͻ1 R1066H 1 Ͻ1 R1070W 1 Ͻ1 Y1092X 1 Ͻ1 W1098C 1 Ͻ1 3500 - 2AϾT 1 Ͻ1 4016insT 1 Ͻ1 4374 ϩ 13AϾG 1 Ͻ1 D1152H 1 Ͻ1 R1158X 1 Ͻ1 R1162X 1 Ͻ1 W1282X 1 Ͻ1 N1303K 1 Ͻ1 Q1313X 1 Ͻ1 P1372L 1 Ͻ1 R1438W 1 Ͻ1 Total 317 100 Table 3. Login to comment

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

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

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108 g D44G, 300delA, W57X, 405+1G>A, D110H, E116K, 541del4, 542del7, L137R, 621+2T>G, I175V, H199R, H199Y, C225X, V232D, Q290X, E292X, G314V, T338I, 1221delCT, W401X, Q452P, I502T, 1716+2T>C, G544S, R560S, A561E, V562I, Y569D, 1898+3A>G, 1898+5G>A, G628R(G>A), 2143delT, G673X, R851X, Q890X, S977F, 3129del4, 3154delG, 3271+1G>A, G1061R, R1066L, R1070W, 3601-17T>C, S1196X, 3732delA, G1249R, 3898insC, 4374+1G>A, del25kb. Login to comment
171 CFTR Mutation Genotypes Identified Both in Cystic Fibrosis (CF) and in Congenital Bilateral Absence of the Vas Deferens (CBAVD) CF CBAVD F508del/5T 3 143 F508del/2789+5G>A 53 1 F508del/3272-26A>G 17 4 F508del/R117H* 10 39 F508del/R117C 2 2 F508del/L206W 12 4 F508del/R347H 10 5 F508del/R347L 1 1 F508del/D443Y 1 5 F508del/Y569C 1 1 F508del/P574H 3 1 F508del/G628R(G>A) 2 1 F508del/V920M 1 1 F508del/R1070W 2 3 F508del/D1152H 6 8 F508del/S1235R 3 1 F508del/T1246I 1 1 F508del/D1270N+R74W 2 3 F508delN1303I 1 1 3659delC/R347H 1 1 G542X/T338I 2 2 R347H/R1066H 1 1 *The only case with CF whose alleles at IVS8(T)n were reported had mutation R117H associated with a 5T allele. Login to comment

[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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31 2 R1066S (C. Fe´rec, I. Quere, C. Verlingue, O. Raguenes, M.-P. Au- drezet, and B. Mercier, personal communication), F1074L (T. Casals, M. D. Ramos, J. Gime´nez, V. Nunes, and X. Estivill, personal communication), K1060T (T. Casals, M. Chillo´n, V. Nunes, J. Gime´nez, M. D. Ramos, and X. Estivill, personal communication), L1065R (T. Casals, M. D. Ramos, J. Gime´nez, V. Nunes, and X. Estivill, personal communication), T1086I (T. Bienvenu, S. Bousquet, C. Herbulot, C. Beldjord, and J. C. Kaplan, personal communication), and R1070W (M. Macek, S. Sedriks, S. Kiesewetter, and G. R. Cutting, personal communication). 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
82 A1067T, R1070W, and R1070Q CFTR had significantly lower efflux levels and significantly lower protein expression than wild-type CFTR in COS-1 cells. 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
128 B: छ, WT; E, G1069R; µ, R1070Q; Ç, R1070W; Ⅺ, control. Login to comment
132 Different substitutions at the same residue always produced the same effect, i.e. R1066C, R1066H, and R1066L, as well as M1101K and M1101R all inhibited maturation, whereas R1070W and R1070Q were both normally processed. 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
89 A1067T, R1070W, and R1070Q CFTR had significantly lower efflux levels and significantly lower protein expression than wild-type CFTR in COS-1 cells. 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
134 B: L, WT; E, G1069R; &#b5;, R1070Q; &#c7;, R1070W; M, control. Login to comment
138 Different substitutions at the same residue always produced the same effect, i.e. R1066C, R1066H, and R1066L, as well as M1101K and M1101R all inhibited maturation, whereas R1070W and R1070Q were both normally processed. 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] Neonatal screening for cystic fibrosis: result of ... Hum Genet. 1995 Nov;96(5):542-8. Ferec C, Verlingue C, Parent P, Morin JF, Codet JP, Rault G, Dagorne M, Lemoigne A, Journel H, Roussey M, et al.
Neonatal screening for cystic fibrosis: result of a pilot study using both immunoreactive trypsinogen and cystic fibrosis gene mutation analyses.
Hum Genet. 1995 Nov;96(5):542-8., [PMID:8530001]

Abstract [show]
We have evaluated a two-tier neonatal cystic fibrosis (CF) screening of immunoreactive trypsinogen (IRT) followed by CFTR gene mutation analysis using a systematic scanning of exons 7, 10, and 11, and, if necessary, by direct DNA sequencing. Over an 18-month period we screened 32,300 neonates born in the western part of Britanny. The first tier, involving IRT screening at 3 days of age, utilizes a low elevation of the trypsinogen level (600 ng/ml), which is highly sensitive. The second tier, which corresponds to the exhaustive screening for mutations in three exons of the gene, is highly specific for this population (Britanny). The false positive rate is very low, and no false negatives have been reported to date. This strategy has allowed the identification of five novel alleles (V322A, V317A, 1806 del A, R553G, G544S).

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82 {17bi DI507 [ Y569X W846X 2789+5G->A ,' $492F i ] i I G551D 2622+1 G->A Y1092X 1717-1 G->A E827X A1067T G542X 2183 AA->G R1066H R560K 2184 ins A 3320,ins 5 R553G R1070W 1806 del A & 4005+1G->A W1282X ] i "- Exons Fig.2 Distribution of the different mutations (except AF508) of the CFTR gene in Brittany Table 1 Mutations and genotypes in newborns Genotypes of newborns Number Sweat test AF508/AF508 7 + > 90 AF508/1806 del A 1 + > 90 R553G/G551D 1 Borderline (60) AF508/G551D 1 + > 90 AF508/R1070W 1 40 AF508/G542X 1 + > 90 AF508/G149R 1 45 Total 13 Mutations found in heterozygote newborns AF508 31 R560K 1 1078 del T 1 G544S l G542X 1 V317A 1 R347H 1 V322A 1 Total 38 gene. Login to comment

[hide] Mutations in the cystic fibrosis gene in patients ... N Engl J Med. 1995 Jun 1;332(22):1475-80. Chillon M, Casals T, Mercier B, Bassas L, Lissens W, Silber S, Romey MC, Ruiz-Romero J, Verlingue C, Claustres M, et al.
Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens.
N Engl J Med. 1995 Jun 1;332(22):1475-80., [PMID:7739684]

Abstract [show]
BACKGROUND: Congenital bilateral absence of the vas deferens (CBAVD) is a form of male infertility in which mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been identified. The molecular basis of CBAVD is not completely understood. Although patients with cystic fibrosis have mutations in both copies of the CFTR gene, most patients with CBAVD have mutations in only one copy of the gene. METHODS: To investigate CBAVD at the molecular level, we have characterized the mutations in the CFTR gene in 102 patients with this condition. None had clinical manifestations of cystic fibrosis. We also analyzed a DNA variant (the 5T allele) in a noncoding region of CFTR that causes reduced levels of the normal CFTR protein. Parents of patients with cystic fibrosis, patients with types of infertility other than CBAVD, and normal subjects were studied as controls. RESULTS: Nineteen of the 102 patients with CBAVD had mutations in both copies of the CFTR gene, and none of them had the 5T allele. Fifty-four patients had a mutation in one copy of CFTR, and 34 of them (63 percent) had the 5T allele in the other CFTR gene. In 29 patients no CFTR mutations were found, but 7 of them (24 percent) had the 5T allele. In contrast, the frequency of this allele in the general population was about 5 percent. CONCLUSIONS: Most patients with CBAVD have mutations in the CFTR gene. The combination of the 5T allele in one copy of the CFTR gene with a cystic fibrosis mutation in the other copy is the most common cause of CBAVD: The 5T allele mutation has a wide range of clinical presentations, occurring in patients with CBAVD or moderate forms of cystic fibrosis and in fertile men.

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74 OF PATIENTS POLYT GENOTYPE† ⌬F508/R668C ⌬F508/D1152H ⌬F508/D1270N ⌬F508/R75L ⌬F508/R117H ⌬F508/L206W ⌬F508/R258G ⌬F508/S1235R ⌬F508/R347H ⌬F508/R347H R117H/G1349D R117H/712-1G→T G149R/R668C R347H/R1066H R553X/R668C R1070W/2869insG ⌬F508/- G542X/- W1282X/- R334W/- K1060T/- R1162X/- N1303K/- A800G/- ⌬F508/- ⌬F508/- ⌬F508/- ⌬E115/- R117H/- R347H/- G542X/- R553X/- 1677delTA/- 2184delA/- 2789ϩ5G→Α/- S1235R/- W1282X/- -/- -/- -/- -/- 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 22 4 3 1 1 1 1 1 7 1 1 1 1 2 1 1 1 1 1 1 1 3 3 1 19 9T/7T 9T/7T 9T/7T 9T/7T 9T/7T 9T/9T 9T/7T 9T/7T 9T/7T 9T/9T 7T/7T 7T/9T 9T/7T 9T/7T 7T/7T 7T/7T 9T/5T 9T/5T 7T/5T 7T/5T 7T/5T 7T/5T 9T/5T 5T/5T 9T/7T 9T/9T 7T/7T 7T/7T 7T/7T 9T/7T 9T/7T 7T/7T 7T/7T 7T/7T 7T/7T 7T/9T 7T/7T 9T/5T 7T/5T 5T/5T 7T/7T -/- 3 7T/9T *Data were obtained from the Spanish population analyzed in this study. Login to comment

[hide] Extensive analysis of 40 infertile patients with c... Hum Genet. 1995 Feb;95(2):205-11. Casals T, Bassas L, Ruiz-Romero J, Chillon M, Gimenez J, Ramos MD, Tapia G, Narvaez H, Nunes V, Estivill X
Extensive analysis of 40 infertile patients with congenital absence of the vas deferens: in 50% of cases only one CFTR allele could be detected.
Hum Genet. 1995 Feb;95(2):205-11., [PMID:7532150]

Abstract [show]
Mutations in the cystic fibrosis (CF) conductance transmembrane regulator (CFTR) gene have been detected in patients with CF and in males with infertility attributable to congenital bilateral absence of the vas deferens (CBAVD). Thirty individuals with CBAVD and 10 with congenital unilateral absence of the vas deferens (CUAVD) were analyzed by single-strand conformation analysis and denaturing gradient gel electrophoresis for mutations in most of the CFTR gene. All 40 individuals were pancreatic sufficient, but twenty patients had recurrent or sporadic respiratory infections, asthma/asthmatic bronchitis, and/or rhino-sinusitis. Agenesia or displasia of one or both seminal vesicles was detected in 30 men and other urogenital malformations were present in six subjects. Among the 40 samples, we identified 13 different CFTR mutations, two of which were previously unknown. One new mutation in exon 4 was the deletion of glutamic acid at codon 115 (delta E115). A second new mutation was found in exon 17b, viz., an A --> C substitution at position 3311, changing lysine to threonine at codon 1060 (K1060T). CFTR mutations were detected in 22 out of 30 (73.3%) CBAVD patients and in one out of 10 (10%) CUAVD individuals, showing a significantly lower incidence of CFTR mutations in CBAVD/CUAVD patients (P << 0.0001), compared with that found in the CF patient population. Only three CBAVD patients were found with more than one CFTR mutation (delta F508/L206W, delta F508/R74W + D1270N, R117H/712-1G --> T), highlighting L206W, R74W/D1270N, and R117H as benign CF mutations. Sweat electrolyte values were increased in 76.6% of CBAVD patients, but three individuals without CFTR mutations had normal sweat electrolyte levels (10% of the total CBAVD patients), suggesting that factors other than CFTR mutations are involved in CBAVD. The failure to identify a second mutation in exons and their flanking regions of the CFTR gene suggests that these mutations could be located in introns or in the promoter region of CFTR. Such mutations could result in CFTR levels below the minimum 6%-10% necessary for normal protein function.

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88 A second change in exon 17b corresponded to R1070W (M. Macek Jr., personal communication to CFGAC), which has not previously been detected in Spanish CF chromosomes (Chill6n et al. 1994b). Login to comment
103 In 13 cases, the mutations are known to be associated with severe CF (AF508, G542X, Rl162X and 1677delTA), whereas in five cases, the phenotypic effect of the mutation is still unknown (AEll5, K1060T, R334W, R1070W, and 2789 + 5G---)A); in one case (Rll7H), the mutation is known to result in mild CE Of these mutations, R334W seems to cause pancreatic insufficiency with a variable age of onset (X. Estivill, in press), whereas mutation 2789 + 5G--)A (W. E. Jr. High- smith, personal communication to the CFGAC) has frequently been found in adult CF patients and is probably involved in a mild phenotype (T. Casals et al. unpublished). Login to comment

[hide] Obstructive azoospermia with agenesis of vas defer... Hum Reprod. 1995 Feb;10(2):338-41. Le Lannou D, Jezequel P, Blayau M, Dorval I, Lemoine P, Dabadie A, Roussey M, Le Marec B, Legall JY
Obstructive azoospermia with agenesis of vas deferens or with bronchiectasia (Young's syndrome): a genetic approach.
Hum Reprod. 1995 Feb;10(2):338-41., [PMID:7539448]

Abstract [show]
Two groups of infertile men with obstructive azoospermia were screened for cystic fibrosis (CF) gene mutations (delta F508, exons 3, 4, 7, 10, 11, 14a, 17b, 19, 20, 21). The first group was composed of 26 patients with congenital agenesis of vas deferens (CAVD). The second group was composed of 12 patients with obstructive azoospermia associated with chronic suppurating respiratory disease (Young's syndrome). Of the group with CAVD, 77% of patients showed at least one mutation in the CF transmembrane conductance regulator (CFTR) gene. The delta F508 mutation occurred most frequently (54%), and the second most frequent mutation to occur was R117H (27%). Six patients were double heterozygotes. In Young's syndrome, no CF mutations were detected. CAVD can be considered as an incomplete clinical form of CF. However, the differences observed in CF mutations between CF and CAVD suggest that they are different disorders resulting from mutations in the same gene. Young's syndrome is a very different clinical entity.

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43 Six patients were double heterozygotes: AF508/R117H (three cases), AF508/ R1070W (two cases), AF508/A1067V (one case). Login to comment
50 1 z 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Details of cystic fibrosis (CF) mutations identified congenital agenesis of vas deferens CF mutation AF5O8/ AF508/ AF508/ AF508/ AF508/ AF5O8/A1067V AF5O8/ AF508/R1070W R117H/ AF508/R1070W - AF508/ R117H/ 621 + 1 G->T7 AF508/RU7H R117H/ AF508/R117H AF5O8/ - AF508/R117H G551D/ R117H/ Respiratory disease - - asthma bronchiectasia - - asthma - - - - rhinitis - - - - - asthma - - in all patients with Sweat tests (mmol/ml of chloride) 72a 42 31 64a 24 - 30 39 17 - 6 - 36 11 31 24 - - 7 52 - - Patients with two results >60 mmol/ml were considered positive. Login to comment
71 an incomplete clinical form of CF. However, if AF508 is excluded, the most frequent mutations in the CAVD group (R117H and R1070W) were not observed in a group of 108 CF patients in a separate study, and these mutations seemed to be specific to CAVD. Login to comment

[hide] Independent origins of cystic fibrosis mutations R... Am J Hum Genet. 1994 Nov;55(5):890-8. Morral N, Llevadot R, Casals T, Gasparini P, Macek M Jr, Dork T, Estivill X
Independent origins of cystic fibrosis mutations R334W, R347P, R1162X, and 3849 + 10kbC-->T provide evidence of mutation recurrence in the CFTR gene.
Am J Hum Genet. 1994 Nov;55(5):890-8., [PMID:7526685]

Abstract [show]
Microsatellite analysis of chromosomes carrying particular cystic fibrosis mutations has shown different haplotypes in four cases: R334W, R347P, R1162X, and 3849 + 10kbC-->T. To investigate the possibility of recurrence of these mutations, analysis of intra- and extragenic markers flanking these mutations has been performed. Recurrence is the most plausible explanation, as it becomes necessary to postulate either double recombinations or single recombinations in conjunction with slippage at one or more microsatellite loci, to explain the combination of mutations and microsatellites if the mutations arose only once. Also in support of recurrence, mutations R334W, R347P, R1162X, and 3849 + 10kbC-->T involve CpG dinucleotides, which are known to have an increased mutation rate. Although only 15.7% of point mutations in the coding sequence of CFTR have occurred at CpG dinucleotides, approximately half of these CpG sites have mutated at least once. Specific nucleotide positions of the coding region of CFTR, distinct from CpG sequences, also seem to have a higher mutation rate, and so it is possible that the mutations observed are recurrent. G-->A transitions are the most common change found in those positions involved in more than one mutational event in CFTR.

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108 )-.T R347L Audrezet et al. 1993 G--S-C R347P Dean et al. 1990 1789 ......... C--.G R553G C. Ferec, personal communication CI-T R553X Cutting et al. 1990 1790 ......... G---A R553Q Dork et al.1991a 3328 ......... C-OT R1066C Fanen et al. 1992 3329 ......... G-.A R1066H Ferec et al. 1992 GT R1066L Mercier et al. 1993 3340 ......... CT R1070W M. Macek, Jr., unpublished data 3341 ......... G-A R1070Q Mercier et al. 1993 a This change is a polymorphism, not a disease mutation. Login to comment

[hide] Effects of cystic fibrosis and congenital bilatera... Am J Hum Genet. 2000 May;66(5):1485-95. Epub 2000 Apr 4. Mickle JE, Milewski MI, Macek M Jr, Cutting GR
Effects of cystic fibrosis and congenital bilateral absence of the vas deferens-associated mutations on cystic fibrosis transmembrane conductance regulator-mediated regulation of separate channels.
Am J Hum Genet. 2000 May;66(5):1485-95. Epub 2000 Apr 4., [PMID:10762539]

Abstract [show]
The protein defective in cystic fibrosis (CF), the CF transmembrane-conductance regulator (CFTR), functions as an epithelial chloride channel and as a regulator of separate ion channels. Although the consequences that disease-causing mutations have on the chloride-channel function have been studied extensively, little is known about the effects that mutations have on the regulatory function. To address this issue, we transiently expressed CFTR-bearing mutations associated with CF or its milder phenotype, congenital bilateral absence of the vas deferens, and determined whether mutant CFTR could regulate outwardly rectifying chloride channels (ORCCs). CFTR bearing a CF-associated mutation in the first nucleotide-binding domain (NBD1), DeltaF508, functioned as a chloride channel but did not regulate ORCCs. However, CFTR bearing disease-associated mutations in other domains retained both functions, regardless of the associated phenotype. Thus, a relationship between loss of CFTR regulatory function and disease severity is evident for NBD1, a region of CFTR that appears important for regulation of separate channels.

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50 Expression Analysis The mutations DF508, R1070W, D1270N, and G1349D were created in the vector pBQ4.7 containing CFTR cDNA (pBQ4.7 is a gift from J. Rommens and L. Login to comment
51 C. Tsui), by single-stranded mutagenesis (Youssoufian et al. 1995), and then were shuttled into pRSV-CFTR, a Rous sarcoma virus (RSV)-driven expression plasmid, by use of Kpn2I and HpaI (for DF508) or NcoI and SalI (for R1070W, D1270N, and G1349D) restriction sites common to both plasmids (Fulmer et al. 1995). Login to comment
68 Mutations at codon 1070 of TMD2 were selected, since two mutations (R1070P and R1070Q) have been associated with CF, whereas a third (R1070W) has been observed in men with CBAVD (table 1). Login to comment
69 The R1070W mutation was first reported by us to the Cystic Fibrosis Genetic Analysis Consortium. Login to comment
74 The latter mutation is predicted to change the amino acid at residue 1070 from arginine to tryptophan and is designated "R1070W." Login to comment
91 Thus, mutations R1070Q and R1070W altered but did not prohibit complex glycosylation. Login to comment
97 OF CASES PHENOTYPE Lung Status Pancreatic Status Sweat Cl2 Fertility Normala Abnormal Not Reported Sufficient Insufficient Not Reported Reported (Mean 5 SEM [mmol/literb ]) Not Reported Subfertilec Not Reported R1070W (7)d 5 0 2 5 0 2e 6 ( ) 50.2 5 13.4 1 6 1 CBAVD R1070P (2)f 0 1 1 0 1 1 1 (Positive) 1 0 2 CF R1070Q (14)g 0 7 7 0 7 7 7 (Positive) 7 2 12 CF D1270N (9)h 4 0 5 4 0 5 3 ( ) 77.5 5 16.7 6 3 6 CBAVD G1349D (3)i 0 0 3 0 0 3 ) 3 1 2 CF a No history of chronic lung disease. Login to comment
172 B, CBAVD(R1070W)- and CF(R1070P and R1070Q)-associated mutants in TMD2 had I-V plots similar to those of wild-type CFTR: outwardly rectified currents (blackened circles) that responded to DIDS (unblackened circles) and glibenclamide (crosses). Login to comment
180 Thus, the combination of I-V relationship and response to inhibitors allowed dissection of whole-cell Cl-currents into two components: outwardly rectified and DIDS sensitive, carried by separate channels such as ORCCs; and linear, DIDS insensitive, and gli- Table 2 Summary of Processing and Whole-Cell Function of CFTR Mutants DOMAIN AND MUTATION PHENOTYPE CFTR STATUS a Processingb Function Band A Band B Band C Cl2 Channel Regulatoryc Not applicable: Wild type Normal 2 2 111 111 1 NBF1:d A455Ee CFe 1 11 2 111 1 DF508 CF 1 11 2 1 2 G551D CF 2 2 111 1 2 TMD2: R1070W CBAVD 2 1 11 111 1 R1070P CF 1 11 2 111 1 R1070Q CF 2 1 11 111 1 NBF2: D1270N CBAVD 2 2 111 111 1 G1349D CF 2 2 111 111 1 a A minus sign (2) denotes absence; a single plus sign (1) denotes "low"; a double plus sign (11) denotes "intermediate"; and a triple plus sign denotes "high." Login to comment
188 .05 mutant (R1070W) and the CF mutants (R1070P and R1070Q) in TMD2 generated outwardly rectified Cl2 currents that were inhibited by DIDS (fig. 3B). Login to comment
230 Electronic-Database Information Accession numbers and URLs for data in this article are as follows: Cystic Fibrosis Genetic Analysis Consortium, http://www .genet.sickkids.on.ca/CFTR (for R1070W) Online Mendelian Inheritance in Man (OMIM), http://www .ncbi.nim.nih.gov/Omim (for CF [MIM 219700], CFTR [MIM 602421], and CBAVD [MIM 277180]) References Anderson MP, Rich DP, Gregory RJ, Smith AE, Welsh MJ (1991) Generation of cAMP-activated chloride currents by expression of CFTR. Login to comment

[hide] Requirements for efficient correction of DeltaF508... Cell. 2012 Jan 20;148(1-2):164-74. doi: 10.1016/j.cell.2011.11.023. Mendoza JL, Schmidt A, Li Q, Nuvaga E, Barrett T, Bridges RJ, Feranchak AP, Brautigam CA, Thomas PJ
Requirements for efficient correction of DeltaF508 CFTR revealed by analyses of evolved sequences.
Cell. 2012 Jan 20;148(1-2):164-74. doi: 10.1016/j.cell.2011.11.023., [PMID:22265409]

Abstract [show]
Misfolding of DeltaF508 cystic fibrosis (CF) transmembrane conductance regulator (CFTR) underlies pathology in most CF patients. F508 resides in the first nucleotide-binding domain (NBD1) of CFTR near a predicted interface with the fourth intracellular loop (ICL4). Efforts to identify small molecules that restore function by correcting the folding defect have revealed an apparent efficacy ceiling. To understand the mechanistic basis of this obstacle, positions statistically coupled to 508, in evolved sequences, were identified and assessed for their impact on both NBD1 and CFTR folding. The results indicate that both NBD1 folding and interaction with ICL4 are altered by the DeltaF508 mutation and that correction of either individual process is only partially effective. By contrast, combination of mutations that counteract both defects restores DeltaF508 maturation and function to wild-type levels. These results provide a mechanistic rationale for the limited efficacy of extant corrector compounds and suggest approaches for identifying compounds that correct both defective steps.

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146 The R1070W sterically clashes with the F508 position in the model (Figure 6A, top) a prediction consistent with fact that it is a CF-causing mutation (Krasnov et al., 2008) that inhibits CFTR maturation when F508 is present (open circle, Figure 6A, bottom). Login to comment
148 The ability of the R1070W mutation to counteract the ICL4-NBD1 interface defect caused by the DF508 mutation, allowed assessment of the quantitative effects of suppression of both defects concurrently. Login to comment
149 When NBD1 suppressor mutations were introduced on top of R1070W and DF508, the slope was restored (m = 0.77, R = 0.47) (Figure 6B). Login to comment
152 Similarly, the R1070W interface mutant produces an 7-fold increase in the slope, but only a modest improvement in CFTR maturation. Login to comment
158 Mirroring the maturation results, correction of either the NBD1 defect (I539T, R555K, red bars) or the ICL4-NBD1 defect (R1070W, white bar) alone provided only modest improvements of function. Login to comment
215 0.0 0.5 1.0 1.5 0 25 50 75 100 WT F WT F Relative Yield CFTR (ELISA) Current Density (pA/pF) F I 5 3 9 T R 5 5 5 K R 1 0 7 0 W I 5 3 9 T R 5 5 5 K W T R 1 0 7 0 W 0.0 0.5 0.0 0.5 1.0 1.5 2.0 2.5 Relative Conductance F F-R1070W D C 0.0 0.5 1.0 1.5 2.0 0 1 WT F Relative Yield NBD1 ( -gal.) 0.0 0.5 1.0 1.5 2.0 0 1 WT F Relative Yield NBD1 ( -gal.) Relative Yield CFTR (ELISA) A + Forskolin/IBMX + Inh 172 60 sec 500 pA + Forskolin/IBMX + Inh 172 B Relative Yield CFTR (ELISA) Figure 6. Login to comment
217 The R1070W (open triangle) mutation in ICL4 modestly improves the relative yield of DF508 CFTR maturation and decreases the relative yield for CFTR wild-type (open circle) consistent with the predicted steric clash with the F508 side chain (top) (&#b1;SEM). Login to comment
219 When R1070W is combined with mutations that improve DF508 NBD1 folding yield, I539T, G550E, R553M, R555K, and 3M (open triangles), the correlation between NBD1 folding and CFTR maturation in the wild-type protein is restored (m = 0.77, R = 0.47, black line) (&#b1;SEM). Login to comment
224 A representative trace of the corrected mutant, DF508-I539T-R1070W CFTR (cyan triangles) is more like wild-type (filled circles) than DF508 (filled triangles) (inset). Login to comment
241 On the WT or DF508 CFTR models, F508K, R1070, R1070W residues were modeled using the PyMOL (Schrodinger, 2010) mutagenesis function (Figures 1B, 2B, rightmost panel, 5, 6, and S1). Login to comment

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

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

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85 Notably, the W277 position is equivalent to the R1070 position in ICL4 (Mornon et al., 2008) that when mutated, R1070W, suppresses the ࢞F508 mutation (Thibodeau et al., 2010; Mendoza et al., 2012). Login to comment

[hide] Functional Rescue of F508del-CFTR Using Small Mole... Front Pharmacol. 2012 Sep 26;3:160. doi: 10.3389/fphar.2012.00160. eCollection 2012. Molinski S, Eckford PD, Pasyk S, Ahmadi S, Chin S, Bear CE
Functional Rescue of F508del-CFTR Using Small Molecule Correctors.
Front Pharmacol. 2012 Sep 26;3:160. doi: 10.3389/fphar.2012.00160. eCollection 2012., [PMID:23055971]

Abstract [show]
High-throughput screens for small molecules that are effective in "correcting" the functional expression of F508del-CFTR have yielded several promising hits. Two such compounds are currently in clinical trial. Despite this success, it is clear that further advances will be required in order to restore 50% or greater of wild-type CFTR function to the airways of patients harboring the F508del-CFTR protein. Progress will be enhanced by our better understanding of the molecular and cellular defects caused by the F508del mutation, present in 90% of CF patients. The goal of this chapter is to review the current understanding of defects caused by F508del in the CFTR protein and in CFTR-mediated interactions important for its biosynthesis, trafficking, channel function, and stability at the cell surface. Finally, we will discuss the gaps in our knowledge regarding the mechanism of action of existing correctors, the unmet need to discover compounds which restore proper CFTR structure and function in CF affected tissues and new strategies for therapy development.

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54 Substitution of the arginine at position 1070 with tryptophan (R1070W) in the context of the Wt-CFTR, introduces a bulky group on the face of the coupling helix that interacts with NBD1 and like the substitutions above, this leads to misprocessing. Login to comment
58 Introduction of R1070W or V510D in the F508del-CFTR protein partially corrects folding of the full-length protein, highlighting the idea that even in the absence of F508, assembly of the CFTR can be partially restored through structural changes at key loci in the protein (Thibodeau et al., 2010; Mendoza et al., 2012). Login to comment
64 However, in combination with R1070W, a mutation that reconstitutes a more Wt-like ICL4: NBD1 interface, the NBD1-"stabilizing" mutants mediate full correction and near normal processing. Login to comment

[hide] Pharmacological Correctors of Mutant CFTR Mistraff... Front Pharmacol. 2012 Oct 5;3:175. doi: 10.3389/fphar.2012.00175. eCollection 2012. Pedemonte N, Galietta LJ
Pharmacological Correctors of Mutant CFTR Mistrafficking.
Front Pharmacol. 2012 Oct 5;3:175. doi: 10.3389/fphar.2012.00175. eCollection 2012., [PMID:23060795]

Abstract [show]
The lack of phenylalanine 508 (DeltaF508 mutation) in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl(-) channel represents the most frequent cause of CF, a genetic disease affecting multiple organs such as lung, pancreas, and liver. DeltaF508 causes instability and misfolding of CFTR protein leading to early degradation in the endoplasmic reticulum and accelerated removal from the plasma membrane. Pharmacological correctors of mutant CFTR protein have been identified by high-throughput screening of large chemical libraries, by in silico docking of virtual compounds on CFTR structure models, or by using compounds that affect the whole proteome (e.g., histone deacetylase inhibitors) or a single CFTR-interacting protein. The presence of multiple defects of the CFTR protein caused by the DeltaF508 mutation and the redundancy of quality control mechanisms detecting DeltaF508-CFTR as a defective protein impose a ceiling to the maximal effect that a single compound (corrector) may obtain. Therefore, treatment of patients with the most frequent CF mutation may require the optimized combination of two drugs having additive or synergic effects.

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145 The second type of mutation, namely R1070W, improves the interaction of NBD1 with CL4 by providing an aromatic group that compensates for the lack of F508. Login to comment

[hide] Correctors of DeltaF508 CFTR restore global confor... FASEB J. 2013 Feb;27(2):536-45. doi: 10.1096/fj.12-216119. Epub 2012 Oct 26. He L, Kota P, Aleksandrov AA, Cui L, Jensen T, Dokholyan NV, Riordan JR
Correctors of DeltaF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.
FASEB J. 2013 Feb;27(2):536-45. doi: 10.1096/fj.12-216119. Epub 2012 Oct 26., [PMID:23104983]

Abstract [show]
Most cystic fibrosis is caused by the deletion of a single amino acid (F508) from CFTR and the resulting misfolding and destabilization of the protein. Compounds identified by high-throughput screening to improve DeltaF508 CFTR maturation have already entered clinical trials, and it is important to understand their mechanisms of action to further improve their efficacy. Here, we showed that several of these compounds, including the investigational drug VX-809, caused a much greater increase (5- to 10-fold) in maturation at 27 than at 37 degrees C (<2-fold), and the mature product remained short-lived (T(1/2) approximately 4.5 h) and thermally unstable, even though its overall conformational state was similar to wild type, as judged by resistance to proteolysis and interdomain cross-linking. Consistent with its inability to restore thermodynamic stability, VX-809 stimulated maturation 2-5-fold beyond that caused by several different stabilizing modifications of NBD1 and the NBD1/CL4 interface. The compound also promoted maturation of several disease-associated processing mutants on the CL4 side of this interface. Although these effects may reflect an interaction of VX-809 with this interface, an interpretation supported by computational docking, it also rescued maturation of mutants in other cytoplasmic loops, either by allosteric effects or via additional sites of action. In addition to revealing the capabilities and some of the limitations of this important investigational drug, these findings clearly demonstrate that DeltaF508 CFTR can be completely assembled and evade cellular quality control systems, while remaining thermodynamically unstable. He, L., Kota, P., Aleksandrov, A. A., Cui, L., Jensen, T., Dokholyan, N. V., Riordan, J. R. Correctors of DeltaF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.

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138 The R1070W substitution in CL4 may do so by contributing to interactions among a cluster of aromatic residues at the interface that is weakened by the absence of F508 from the NBD1 surface (11), whereas the V510D mutation was proposed to provide a salt bridge with R1070 (31). Login to comment
139 The V510D mutant on the NBD1 side of the interface is very sensitive to further enhancement of maturation by the compound, whereas that on the CL4 side (R1070W) responds only rather weakly (Fig. 5B). Login to comment
140 This difference may reflect the fact that the V510D substitution stabilizes isolated NBD1 (32) in the absence of the rest of CFTR, as well as influencing the interface, whereas R1070W has only the latter effect. Login to comment
141 One might speculate that the less substantial influence of VX-809 on R1070W/èc;F508 CFTR could possibly reflect similar effects of either the tryptophan residue or the aromatic small molecule to partially fill the void left by the absence of the F508 residue. Login to comment
142 Interestingly, as has already been observed by others (9), the influence of the combined V510D and R1070W substitutions is similar to that of V510D alone, which would not be expected if V510D were forming a salt bridge with R1070 but might be if V510D acted primarily to stabilize the NBD1 domain, as has been observed (32). Login to comment
149 B) èc;F508 with NBD1/CL4 interface substitutions R1070W and/or V510D. Login to comment
157 Interestingly, the patterns of enhancement by the compounds were remarkably similar for each of the three classes of NBD1 stabilizing mutations (èc;F/èc;RI, èc;F4S, and èc;F/4PT) with or without one of the interface substitutions (R1070W or V510D). Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Cold Spring Harb Perspect Med. 2013 Feb 1;3(2):a009514. doi: 10.1101/cshperspect.a009514. Hunt JF, Wang C, Ford RC
Cystic fibrosis transmembrane conductance regulator (ABCC7) structure.
Cold Spring Harb Perspect Med. 2013 Feb 1;3(2):a009514. doi: 10.1101/cshperspect.a009514., [PMID:23378596]

Abstract [show]
Structural studies of the cystic fibrosis transmembrane conductance regulator (CFTR) are reviewed. Like many membrane proteins, full-length CFTR has proven to be difficult to express and purify, hence much of the structural data available is for the more tractable, independently expressed soluble domains. Therefore, this chapter covers structural data for individual CFTR domains in addition to the sparser data available for the full-length protein. To set the context for these studies, we will start by reviewing structural information on model proteins from the ATP-binding cassette (ABC) transporter superfamily, to which CFTR belongs.

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255 A second-site R1070W mutation in the proximal region of the TMDs produces significant suppression of the defect (Thibodeau et al. 2010), presumably by increasing hydrophobic interactions with the altered conformation of residue V510 in F508del-NBD1. Login to comment

[hide] Novel pharmacological strategies to treat cystic f... Trends Pharmacol Sci. 2013 Feb;34(2):119-25. doi: 10.1016/j.tips.2012.11.006. Hanrahan JW, Sampson HM, Thomas DY
Novel pharmacological strategies to treat cystic fibrosis.
Trends Pharmacol Sci. 2013 Feb;34(2):119-25. doi: 10.1016/j.tips.2012.11.006., [PMID:23380248]

Abstract [show]
Cystic fibrosis (CF) is a lethal disease caused by mutations in the CFTR gene. The most frequent mutation is deletion of a phenylalanine residue (DeltaF508) that results in retention of the mutant, but otherwise functional, protein in the endoplasmic reticulum (ER). There have been recent advances in the identification of chemically diverse corrector compounds that allow DeltaF508-CFTR protein to traffic from the ER to the plasma membrane. The most studied correctors fall into two categories, pharmacological chaperones that bind to the mutant protein and circumvent its recognition by the cellular protein quality control systems and proteostasis regulators that modify the cellular pathways responsible for protein quality control and trafficking. This review focuses on recent advances in the field, strategies for the development of drugs from corrector compounds for the treatment of CF, and identification of their targets and mechanism(s) of action.

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145 The two-step folding hypothesis also suggests a rational way to test for new pharmacological chaperones that act at each step: pharmacological chaperones that correct NBD1 conformational stability should be additive, with mutations such as R1070W that restore contacts between NBD1 and MSD2 (CL4). Login to comment

[hide] Dynamics intrinsic to cystic fibrosis transmembran... Cold Spring Harb Perspect Med. 2013 Mar 1;3(3):a009522. doi: 10.1101/cshperspect.a009522. Chong PA, Kota P, Dokholyan NV, Forman-Kay JD
Dynamics intrinsic to cystic fibrosis transmembrane conductance regulator function and stability.
Cold Spring Harb Perspect Med. 2013 Mar 1;3(3):a009522. doi: 10.1101/cshperspect.a009522., [PMID:23457292]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) requires dynamic fluctuations between states in its gating cycle for proper channel function, including changes in the interactions between the nucleotide-binding domains (NBDs) and between the intracellular domain (ICD) coupling helices and NBDs. Such motions are also linked with fluctuating phosphorylation-dependent binding of CFTR's disordered regulatory (R) region to the NBDs and partners. Folding of CFTR is highly inefficient, with the marginally stable NBD1 sampling excited states or folding intermediates that are aggregation-prone. The severe CF-causing F508del mutation exacerbates the folding inefficiency of CFTR and leads to impaired channel regulation and function, partly as a result of perturbed NBD1-ICD interactions and enhanced sampling of these NBD1 excited states. Increased knowledge of the dynamics within CFTR will expand our understanding of the regulated channel gating of the protein as well as of the F508del defects in folding and function.

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98 Similar results are seen with an R1070W mutation, which might be expected to enhance the hydrophobic contacts at this interface or fill the void introduced by deletion of F508, or with a combined R1070D/V510R mutation, which would reverse the proposed salt bridge (Farinha et al. 2010; Loo et al. 2010). Login to comment

[hide] Managing the underlying cause of cystic fibrosis: ... Paediatr Drugs. 2013 Oct;15(5):393-402. doi: 10.1007/s40272-013-0035-3. Galietta LJ
Managing the underlying cause of cystic fibrosis: a future role for potentiators and correctors.
Paediatr Drugs. 2013 Oct;15(5):393-402. doi: 10.1007/s40272-013-0035-3., [PMID:23757197]

Abstract [show]
Cystic fibrosis (CF), a severe genetic disease, is caused by mutations that alter the structure and function of CFTR, a plasma membrane channel permeable to chloride and bicarbonate. Defective anion transport in CF irreversibly damages the lungs, pancreas, liver, and other organs. CF mutations cause loss of CFTR function in multiple ways. In particular, class 3 mutations such as p.Gly551Asp strongly decrease the time spent by CFTR in the open state (gating defect). Instead, class 2 mutations impair the maturation of CFTR protein and its transport from the endoplasmic reticulum to the plasma membrane (trafficking defect). The deletion of phenylalanine 508 (p.Phe508del), the most frequent mutation among CF patients (70-90 %), destabilizes the CFTR protein, thus causing both a trafficking and a gating defect. These two defects can be overcome with drug-like molecules generically called correctors and potentiators, respectively. The potentiator Kalydeco (also known as Ivacaftor or VX-770), developed by Vertex Pharmaceuticals, has been recently approved by the US FDA and the European Medicines Agency (EMA) for the treatment of CF patients carrying at least one CFTR allele with the p.Gly551Asp mutation (2-5 % of all patients). In contrast, the corrector VX-809, which significantly improves p.Phe508del-CFTR trafficking in vitro, is still under study in clinical trials. Because of multiple defects caused by the p.Phe508del mutation, it is probable that rescue of the mutant protein will require combined treatment with correctors having different mechanisms of action. This review evaluates the status of experimental and clinical research in pharmacotherapy for the CF basic defect.

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123 Other mutations, such as p.Arg1070Trp, improve the interaction between NBD1 and CL4 [60]. Login to comment

[hide] Corrector VX-809 stabilizes the first transmembran... Biochem Pharmacol. 2013 Sep 1;86(5):612-9. doi: 10.1016/j.bcp.2013.06.028. Epub 2013 Jul 5. Loo TW, Bartlett MC, Clarke DM
Corrector VX-809 stabilizes the first transmembrane domain of CFTR.
Biochem Pharmacol. 2013 Sep 1;86(5):612-9. doi: 10.1016/j.bcp.2013.06.028. Epub 2013 Jul 5., [PMID:23835419]

Abstract [show]
Processing mutations that inhibit folding and trafficking of CFTR are the main cause of cystic fibrosis (CF). A potential CF therapy would be to repair CFTR processing mutants. It has been demonstrated that processing mutants of P-glycoprotein (P-gp), CFTR's sister protein, can be efficiently repaired by a drug-rescue mechanism. Many arginine suppressors that mimic drug-rescue have been identified in the P-gp transmembrane (TM) domains (TMDs) that rescue by forming hydrogen bonds with residues in adjacent helices to promote packing of the TM segments. To test if CFTR mutants could be repaired by a drug-rescue mechanism, we used truncation mutants to test if corrector VX-809 interacted with the TMDs. VX-809 was selected for study because it is specific for CFTR, it is the most effective corrector identified to date, but it has limited clinical benefit. Identification of the VX-809 target domain will help to develop correctors with improved clinical benefits. It was found that VX-809 rescued truncation mutants lacking the NBD2 and R domains. When the remaining domains (TMD1, NBD1, TMD2) were expressed as separate polypeptides, VX-809 only increased the stability of TMD1. We then performed arginine mutagenesis on TM6 in TMD1. Although the results showed that TM6 had distinct lipid and aqueous faces, CFTR was different from P-gp as no arginine promoted maturation of CFTR processing mutants. The results suggest that TMD1 contains a VX-809 binding site, but its mechanism differed from P-gp drug-rescue. We also report that V510D acts as a universal suppressor to rescue CFTR processing mutants.

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180 To test if the V232D or H1085R mutants could be rescued by suppressor mutations in other domains, suppressor mutations in NBD1 (I539T), the NBD1-TMD2 interface (V510D), or TMD2 (R1070W) (only V232D) locations were introduced into the mutants. Login to comment
184 The I539T and R1070W mutations only rescued DF508 CFTR. Login to comment
185 We previously reported that the R1070W mutation also reduces the maturation efficiency of wild-type CFTR [32] while V510D does not [33]. Login to comment
237 (C) Extracts of cells expressing processing mutants DF508, V232D, or H1085R with or without the V510D, I539T, or R1070W suppressor mutations were subjected to immunoblot analysis. Login to comment

[hide] Revertants, low temperature, and correctors reveal... Chem Biol. 2013 Jul 25;20(7):943-55. doi: 10.1016/j.chembiol.2013.06.004. Farinha CM, King-Underwood J, Sousa M, Correia AR, Henriques BJ, Roxo-Rosa M, Da Paula AC, Williams J, Hirst S, Gomes CM, Amaral MD
Revertants, low temperature, and correctors reveal the mechanism of F508del-CFTR rescue by VX-809 and suggest multiple agents for full correction.
Chem Biol. 2013 Jul 25;20(7):943-55. doi: 10.1016/j.chembiol.2013.06.004., [PMID:23890012]

Abstract [show]
Cystic fibrosis is mostly caused by the F508del mutation, which impairs CFTR protein from exiting the endoplasmic reticulum due to misfolding. VX-809 is a small molecule that rescues F508del-CFTR localization, which recently went into clinical trial but with unknown mechanism of action (MoA). Herein, we assessed if VX-809 is additive or synergistic with genetic revertants of F508del-CFTR, other correctors, and low temperature to determine its MoA. We explored and integrated those various agents in combined treatments, showing how they add to each other to identify their complementary MoA upon correction of F508del-CFTR. Our experimental and modeling data, while compatible with putative binding of VX-809 to NBD1:ICL4 interface, also indicate scope for further synergistic F508del-CFTR correction by other compounds at distinct conformational sites/cellular checkpoints, thus suggesting requirement of combined therapies to fully rescue F508del-CFTR.

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16 The second F508del-associated defect impairs CFTR interdomain folding, namely, (1) the NBD1-NBD2 dimerization interface (critical for channel activation and accounting for the F508del-CFTR gating defect; Dalemans et al., 1991), which can be rescued by the G550E revertant, and (2) the interaction of NBD1 with the fourth intracellular loop (ICL4) of TMD2 (Serohijos et al., 2008), shown to be reverted by either V510D (Loo et al., 2010) or R1070W, which both fill the pocket left empty by F508del (Thibodeau et al., 2010). Login to comment
23 Herein, we explored the MoA of VX-809 by analyzing its synergistic/additive effect with those of previously characterized genetic revertants, which rescue F508del-CFTR by causing different effects: 4RK affecting traffic (Roxo-Rosa et al., 2006), G550E (Roxo-Rosa et al., 2006) and R555K increasing channel gating by strengthening the NBD1:NBD2 dimer interface, and R1070W (Serohijos et al., 2008) and V510D (Wang et al., 2007a; Loo et al., 2010) by filling the NBD1:ICL4 interface. Login to comment
36 VX-809 Adds to VRT-325 and Corr-4a to Rescue F508del-CFTR but Exhibits Variable Effects on Genetic Revertants In order to characterize the rescue mechanism of VX-809 on F508del-CFTR, we then tested the effect of incubating it together with VRT-325 and Corr-4a on BHK cells stably expressing this mutant alone or in cis with the following genetic revertants: (1) 4RK (where the four AFTs were simultaneously mutated to lysines), (2) G550E, (3) R1070W, (4) V510D, or (5) R555K (Figures 1A-1E). Login to comment
42 Moreover, whereas both VRT-325 and VX-809 add significantly to the effect of R1070W by 17% (p = 0.011) and 28% (p = 1.6 10 5 ), respectively, Corr-4a does not. Login to comment
46 These analyses were performed for three of the revertants with different F508del-CFTR effects (4RK, traffic; G550E, NBD1:NBD2 dimer interface; and R1070W, NBD1:ICL4 interface). Login to comment
57 Effect of Small Molecule Correctors on F508del-CFTR and Genetic Revertants (A-F) BHK cell lines stably expressing CFTR bearing F508del alone (A) or in cis with 4RK (B), G550E (C), R1070W (D), V510D (E), and R555K (F) were incubated for 24 hr with 6.7 mM VRT-325, 10 mM Corr-4a, or 3 mM VX-809 alone or in combination. Login to comment
72 We then assessed the effect of revertants G550E and R1070W by modeling (Figure 3F; Figures S2B and S2C), whereby G550E allows a salt bridge to form across the ATP binding site with Lys1250 and other residues from NBD2 (Figure 3F). Login to comment
73 By contrast, R1070W (Figure S2A) plausibly fills in the space left empty by absence of F508 at the NBD1:ICL4 interface as previously proposed (Thibodeau et al., 2010). Login to comment
87 Rescue of F508del-CFTR by Low Temperature Is Additive to Genetic Revertants To learn more about how low temperature rescues F508del-CFTR, we assessed its combined effect with that of the above genetic revertants: G550E, R1070W, 4RK, V510D, and R555K (Figures 4A and 4B). Login to comment
88 Results show that low temperature further increases processing levels of F508del-CFTR by the five genetic revertants, namely, V510D, G550E, R1070W, 4RK, and R555K, by an additional 35%, 65%, 38%, 27%, and 22%, respectively (compare gray and black bars in Figure 4B). Login to comment
101 Interestingly, these additive effects were observed not only for revertants promoting protein-autonomous folding (G550E, V510D, and R1070W) but also for the 4RK revertant, which bypasses the AFT-mediated ER retention. Login to comment
102 Combination of Different Genetic Revertants Is Also Additive Next, to assess the full potential for F508del-CFTR rescue, we combined the effects of folding and traffic revertants by producing stable BHK cell lines expressing F508del-G550E-CFTR, where 4RK, V510D, or R1070W were also added in cis, and analyzed processing (Figures 4C and 4D). Login to comment
103 Results in Figure 4C show that 4RK, V510D, and R1070W further increased processing of G550E-F508del-CFTR by another 12%, 59%, and 70%, respectively. Login to comment
104 In fact, the combined effects of G550E with either V510D or R1070W bring F508del-CFTR processing to z80%, i.e., close to levels of WT-CFTR, which can be further increased at 26 C reaching 88%. Login to comment
165 In contrast, the additive effect of VX-809 to R1070W and V510D is rather modest, thus suggesting that this corrector acts more similarly to R1070W/V510D than to G550E/R555K. Login to comment
166 VRT-325 in turn (and comparatively to its modest effect alone) significantly increases (by z29% versus VRT-325 alone) the rescue efficiency of R1070W and also V510D, suggesting effects at distinct sites. Login to comment
193 Furthermore, modeling also predicts that revertants G550E and R1070W act at different CFTR interdomain contacts disrupted by F508del: whereas G550E seems to restore the strength of the NBD1:NBD2 interface, R1070W rather promotes the NBD1:ICL4 interaction, as suggested previously (He et al., 2010; Serohijos et al., 2008) and in Figure S2A. Login to comment
195 Indeed, G550E, besides being able to promote rescue of F508del-CFTR (DeCarvalho et al., 2002), shows the largest combined effect with R1070W (or V510D). Login to comment
197 Moreover, the observed synergy of G550E or R555K with VX-809, but not VRT-325, is consistent with this model in which VRT-325 acts on the NBD1:NBD2 dimerization interface (also supported by the synergy between R1070W and VRT-325). Login to comment
198 Further modeling is, however, required to examine the observation that R1070W is still additive with VX-809 (albeit modestly). Login to comment
210 Our data also show that low temperature, similar to chemical correctors, further increases processing levels of F508del-CFTR by the five genetic revertants, although to variable levels: V510D (by an additional 35%), G550E (65%), R1070W (38%), 4RK (27%), and R555K (22%). Login to comment
225 Assessing the synergistic/additive effects of investigational drug VX-809, one of the most promising to rescue the F508del-CFTR-trafficking defect, with those of genetic revertants as well as other correctors (VRT-325 and Corr-4a) or low temperature pointed to major insights into its MoA: (1) VX-809 is additive to both VRT-325 and Corr-4a, suggesting that each compound operates by a different MoA; (2) VX-809 is additive to low temperature rescue of the mutant almost to WT-CFTR levels; (3) VX-809, VRT-325, and Corr-4a show variable additive effects with the genetic revertants tested (4RK, G550E, and R1070W), thus providing clues for their possible action being exerted at specific protein binding pockets: VX-809 at the NBD1:TMD2 interface (and VRT-325 at NBD1:NBD2) or acting unspecifically (Corr-4a); and (4) VX-809 does not rescue the diacidic code traffic mutant in contrast to low temperature, which seems to act at trafficking surveillance checkpoints. Login to comment
231 EXPERIMENTAL PROCEDURES Cells and Culture Conditions BHK cell lines expressing F508del-4RK (R29K/R516K/R555K/R716K)-, F508del-G550E-, F508del-R1070W-, F508del-V510D-, F508del-R555K-, F508del-V510D/G550E-, F508del-G550E/R1070W-, DAA (D567A)-, 4RK- DAA-, DD/AA (D565A, D567A)-, 4RK-DD/AA-, and R560T-CFTR were produced and cultured as previously described (Roxo-Rosa et al., 2006). 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
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
86 For example, the baseline level of chloride transport and ivacaftor response was higher for mutant CFTR forms associated with mild defects in CFTR processing (e.g., E56K, P67L, L206W, A455E, D579G, S945L, S977F, A1067T, R1070Q, R1070W, F1074L, and D1270N) than for those associated with severe defects in CFTR processing (e.g., F508del, H1054D, R1066H). Login to comment
89 For mutant CFTR forms that have multiple defects (e.g., R117H, F508del, S945L, R1070Q, A1067T, R1070W, and R347P), the relative impact of each defect is likely to affect the magnitude of the baseline chloride transport and ivacaftor response in vitro and in a clinical setting. 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

[hide] The cystic fibrosis V232D mutation inhibits CFTR m... Biochem Pharmacol. 2014 Mar 1;88(1):46-57. doi: 10.1016/j.bcp.2013.12.027. Epub 2014 Jan 9. Loo TW, Clarke DM
The cystic fibrosis V232D mutation inhibits CFTR maturation by disrupting a hydrophobic pocket rather than formation of aberrant interhelical hydrogen bonds.
Biochem Pharmacol. 2014 Mar 1;88(1):46-57. doi: 10.1016/j.bcp.2013.12.027. Epub 2014 Jan 9., [PMID:24412276]

Abstract [show]
Processing mutations that inhibit folding and trafficking of CFTR are the main cause of cystic fibrosis. Repair of CFTR mutants requires an understanding of the mechanisms of misfolding caused by processing mutations. Previous studies on helix-loop-helix fragments of the V232D processing mutation suggested that its mechanism was to lock transmembrane (TM) segments 3 and 4 together by a non-native hydrogen bond (Asp232(TM4)/Gln207(TM3)). Here, we performed mutational analysis to test for Asp232/Gln207 interactions in full-length CFTR. The rationale was that a V232N mutation should mimic V232D and a V232D/Q207A mutant should mature if the processing defect was caused by hydrogen bonds. We report that only Val232 mutations to charged amino acids severely blocked CFTR maturation. The V232N mutation did not mimic V232D as V232N showed 40% maturation compared to 2% for V232D. Mutation of Val232 to large nonpolar residues (Leu, Phe) had little effect. The Q207L mutation did not rescue V232D because Q207L showed about 50% maturation in the presence of corrector VX-809 while V232D/Q207A could no longer be rescued. These results suggest that V232D inhibits maturation by disrupting a hydrophobic pocket between TM segments rather than forming a non-native hydrogen bond. Disulfide cross-linking analysis of cysteines W356C(TM6) and W1145C(TM12) suggest that the V232D mutation inhibits maturation by trapping CFTR as a partially folded intermediate. Since correctors can efficiently rescue V232D CFTR, the results suggest that hydrophilic processing mutations facing a hydrophobic pocket are good candidates for rescue with pharmacological chaperones.

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169 For example, V510D promotes maturation of mutants with processing mutations in TMD1 (V232D), TMD2 (H1085R) and NBD1 (DF508) whereas other suppressors such as I539T and R1070W promote maturation of DF508 CFTR but not mutants V232D or H1085R [19]. Login to comment

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

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

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1442 Mutations located in NBD1, such as I539T, G550E, R553M/Q and R555K, as well as R1070W in CL4 of MSD2 promote Phe508del-CFTR maturation and trafficking to the cell surface and also restore channel activity (DeCarvalho et al., 2002; Teem et al., 1993, 1996; Thibodeau et al., 2010). Login to comment

[hide] VX-809 and related corrector compounds exhibit sec... Chem Biol. 2014 May 22;21(5):666-78. doi: 10.1016/j.chembiol.2014.02.021. Epub 2014 Apr 10. Eckford PD, Ramjeesingh M, Molinski S, Pasyk S, Dekkers JF, Li C, Ahmadi S, Ip W, Chung TE, Du K, Yeger H, Beekman J, Gonska T, Bear CE
VX-809 and related corrector compounds exhibit secondary activity stabilizing active F508del-CFTR after its partial rescue to the cell surface.
Chem Biol. 2014 May 22;21(5):666-78. doi: 10.1016/j.chembiol.2014.02.021. Epub 2014 Apr 10., [PMID:24726831]

Abstract [show]
The most common mutation causing cystic fibrosis (CF), F508del, impairs conformational maturation of CF transmembrane conductance regulator (CFTR), thereby reducing its functional expression on the surface of epithelia. Corrector compounds including C18 (VRT-534) and VX-809 have been shown to partially rescue misfolding of F508del-CFTR and to enhance its maturation and forward trafficking to the cell surface. Now, we show that there is an additional action conferred by these compounds beyond their role in improving the biosynthetic assembly. In vitro studies show that these compounds bind directly to the metastable, full-length F508del-CFTR channel. Cell culture and patient tissue-based assays confirm that in addition to their cotranslational effect on folding, certain corrector compounds bind to the full-length F508del-CFTR after its partial rescue to the cell surface to enhance its function. These findings may inform the development of alternative compounds with improved therapeutic efficacy.

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25 To date, however, there have been conflicting reports regarding the consequences of mutations at the NBD1-ICL4 interface (including R1070W) on the efficacy of VX-809 in in-cell maturation studies (He et al., 2013; Okiyoneda et al., 2013), prompting speculation that VX-809 may not bind directly to this site on the mutant protein. Login to comment

[hide] Synergy-based small-molecule screen using a human ... Mol Pharmacol. 2014 Jul;86(1):42-51. doi: 10.1124/mol.114.092478. Epub 2014 Apr 15. Phuan PW, Veit G, Tan J, Roldan A, Finkbeiner WE, Lukacs GL, Verkman AS
Synergy-based small-molecule screen using a human lung epithelial cell line yields DeltaF508-CFTR correctors that augment VX-809 maximal efficacy.
Mol Pharmacol. 2014 Jul;86(1):42-51. doi: 10.1124/mol.114.092478. Epub 2014 Apr 15., [PMID:24737137]

Abstract [show]
The most prevalent cystic fibrosis transmembrane conductance regulator (CFTR) mutation causing cystic fibrosis, DeltaF508, impairs folding of nucleotide binding domain (NBD) 1 and stability of the interface between NBD1 and the membrane-spanning domains. The interfacial stability defect can be partially corrected by the investigational drug VX-809 (3-[6-[[[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl]amino]-3-methy l-2-pyridinyl]-benzoic acid) or the R1070W mutation. Second-generation DeltaF508-CFTR correctors are needed to improve on the modest efficacy of existing cystic fibrosis correctors. We postulated that a second corrector targeting a distinct folding/interfacial defect might act in synergy with VX-809 or the R1070W suppressor mutation. A biochemical screen for DeltaF508-CFTR cell surface expression was developed in a human lung epithelium-derived cell line (CFBE41o(-)) by expressing chimeric CFTRs with a horseradish peroxidase (HRP) in the fourth exofacial loop in either the presence or absence of R1070W. Using a luminescence readout of HRP activity, screening of approximately 110,000 small molecules produced nine novel corrector scaffolds that increased cell surface F508-CFTR expression by up to 200% in the presence versus absence of maximal VX-809. Further screening of 1006 analogs of compounds identified from the primary screen produced 15 correctors with an EC50 < 5 microM. Eight chemical scaffolds showed synergy with VX-809 in restoring chloride permeability in F508-expressing A549 cells. An aminothiazole increased chloride conductance in human bronchial epithelial cells from a DeltaF508 homozygous subject beyond that of maximal VX-809. Mechanistic studies suggested that NBD2 is required for the aminothiazole rescue. Our results provide proof of concept for synergy screening to identify second-generation correctors, which, when used in combination, may overcome the "therapeutic ceiling" of first-generation correctors.

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2 The interfacial stability defect can be partially corrected by the investigational drug VX-809 (3-[6-[[[1-(2,2- difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl]amino]-3-methyl-2-pyridinyl]-benzoic acid) or the R1070W mutation. Login to comment
4 We postulated that a second corrector targeting a distinct folding/interfacial defect might act in synergy with VX-809 or the R1070W suppressor mutation. Login to comment
5 A biochemical screen for DF508-CFTR cell surface expression was developed in a human lung epithelium-derived cell line (CFBE41o2 ) by expressing chimeric CFTRs with a horseradish peroxidase (HRP) in the fourth exofacial loop in either the presence or absence of R1070W. Login to comment
35 Therefore, novel corrector molecules targeting either the NBD1 and/or its interface defects should be preferentially identified by screening in the background of VX-809 or the interface-stabilizing mutation R1070W. Login to comment
43 The cloning and characterization of 3HA-tagged variants of ƊF508-CFTR, R1070W- ƊF508-CFTR, and 3S-ƊF508-CFTR (containing the F494N, Q637R, and F429S NBD1 suppressor mutations) were described (Okiyoneda et al., 2013). Login to comment
58 In one set of assays, R1070W-ƊF508-CFTR-HRP (R1070W-HRP)-expressing CFBE41o2 cells were incubated with 100 ml medium containing 25 mM test compounds and 0.5 mg/ml doxycycline for 24 hours at 37&#b0;C. Login to comment
87 A second screen ("R1070W screen") (Fig. 1B) used CFBE41o2 cells transfected with DF508-CFTR-HRP containing a R1070W mutation (R1070W-HRP CFBE41o2 ). Login to comment
90 For the ƊF508 screen, cells were incubated with test compounds (at 25 mM) together with 2 mM VX-809; for the R1070W screen, cells were incubated with test compounds (at 25 mM) alone. Login to comment
96 Similarly, with the R1070W-HRP CFBE41o2 cells, VX-809 increased the signal maximally to approximately 220 a.u. over the DMSO control baseline of approximately 85 a.u., representing an approximately 2.5-fold signal increase (bar graphs in Fig. 1). Login to comment
98 Low-temperature rescue (27&#b0;C) of ƊF508-CFTR increased the HRP luminescence signal by approximately 2-fold (compared with 37&#b0;C) in ƊF508-HRP CFBE41o2 cells and approximately 3-fold in R1070W-HRP CFBE41o2 cells. Login to comment
100 EC50 values were 30 and 78 nM in the low temperature-rescued ƊF508-HRP and R1070W-HRP CFBE41o2 cells, respectively. Login to comment
101 Preferential correction of DF508-CFTR-3HA with the NBD1 stabilizing 3S mutations (F494N, Q637R, and F429S) compared with CFTR carrying the R1070W interface-stabilizing mutation has been taken as evidence that VX-809 preferentially stabilizes the interface between NBD1 and MSDs but not the NBD1 folding defect CFTR (Okiyoneda et al., 2013). Login to comment
103 The relative insensitivity of R1070W-HRP to VX-809 was used to identify correctors that act in synergy with VX-809. Login to comment
105 A total of 110,240 drug-like small synthetic molecules were tested in the ƊF508 and R1070W screens. Login to comment
108 For the R1070W screen, 25 active compounds were identified based on a .50% increase in the luminescence signal over that of DMSO. Login to comment
109 After retesting, nine compounds, grouped into six classes, were confirmed from the R1070W screen. Figure 2D shows structures of the six most active compounds (A-01, B-01, C-01, D-01, E-01, and F-01). Login to comment
110 Because different small-molecule collections were used for the ƊF508 and R1070W screens, we cross-tested all active correctors in both the ƊF508-HRP and R1070W-HRP CFBE41o2 cell lines (Supplemental Fig. 2). Login to comment
112 However, compounds D-01, E-01, and F-01, discovered from the R1070W screen, were not active in ƊF508-HRP CFBE41o2 cells. Login to comment
113 J-01, discovered from the ƊF508 screen, was not active in R1070W-HRP CFBE41o2 cells. Login to comment
117 We also measured the concentration-dependent activities of A-01, B-01, D-01, H-01, and K-01 (Fig. 3B) in R1070W-CFBE41o2 cells and found that D-01 is the most potent corrector, with an EC50 value of approximately 1.2 mM and a maximal signal that is approximately 65% of that produced by 2 mM VX-809. Login to comment
126 Similar compound potency and efficacy were found for class D analogs in R1070W-HRP CFBE41o2 cells (Fig. 4C). Login to comment
141 (B) Screening assay for R1070W-ƊF508-CFTR-HRP CFBE41o cells (top) showing incubation with 25 mM test compounds for 24 hours at 37&#b0;C. Login to comment
163 (C) Summary of primary findings of the R1070W-DF508-CFTR screen. Login to comment
170 To investigate whether any of the correctors identified here may target the NBD2 interface, their synergy with C4 was evaluated in R1070W-HRP CFBE41o2 cells using the HRP luminescence assay. Login to comment
191 (B) Concentration-dependence data of A-01, B-01, D-01, H-01, and K-01 in R1070W-ƊF508-CFTR-HRP CFBE41o2 cells (S.E., n = 3). Login to comment
197 (A) Concentration-dependence data of class D analogs in R1070W-ƊF508-CFTR-HRP CFBE41o2 cells (S.E., n = 3). Login to comment
212 A second screen was done in cells expressing R1070W-ƊF508-CFTR (in the absence of VX-809), since in the background of genetically stabilized ƊF508-NBD1, the R1070W mutation was necessary and sufficient to restore robust CFTR domain assembly and cell surface expression (Thibodeau et al., 2010; Mendoza et al., 2012; Rabeh et al., 2012). Login to comment
213 Screening was performed using a human lung epithelium-derived cell line (CFBE41o2 ) that was stably transfected with HRP-tagged ƊF508-CFTR or R1070W-ƊF508-CFTR. Login to comment
227 (C) Relative effect of corrector 4 (10 mM, 24 hours, 37&#b0;C) on the PM density of R1070W-HRP CFBE41o2 cells treated with A-01, B-01, C-01, D-01, H-01, J-01, K-01, or VX-809 at the indicated concentrations. Login to comment

[hide] Complement yourself: Transcomplementation rescues ... Biophys Rev. 2014 Mar 1;6(1):169-180. Cebotaru L, Guggino WB
Complement yourself: Transcomplementation rescues partially folded mutant proteins.
Biophys Rev. 2014 Mar 1;6(1):169-180., [PMID:24949105]

Abstract [show]
Cystic Fibrosis (CF) is an autosomal disease associated with malfunction in fluid and electrolyte transport across several mucosal membranes. The most common mutation in CF is an in-frame three-base pair deletion that removes a phenylalanine at position 508 in the first nucleotide-binding domain of the cystic fibrosis conductance regulator (CFTR) chloride channel. This mutation has been studied extensively and leads to biosynthetic arrest of the protein in the endoplasmic reticulum and severely reduced channel activity. This review discusses a novel method of rescuing DeltaF508 with transcomplementation, which occurs when smaller fragments of CFTR containing the wild-type nucleotide binding domain are co-expressed with the DeltaF508 deletion mutant. Transcomplementation rescues the processing and channel activity of DeltaF508 and reduces its rate of degradation in airway epithelial cells. To apply transcomplementation as a therapy would require that the cDNA encoding the truncated CFTR be delivered to cells. We also discuss a gene therapeutic approach based on delivery of a truncated form of CFTR to airway cells using adeno-associated viral vectors.

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89 Recent experiments have shown that certain amino acid substitutions, namely R1070W in combination with ƊF508 CFTR, can rescue ƊF508 CFTR trafficking, again pointing to the importance of domain-domain interactions in the mis-processing of ƊF508 (62). Login to comment

[hide] Restoration of NBD1 thermal stability is necessary... J Mol Biol. 2015 Jan 16;427(1):106-20. doi: 10.1016/j.jmb.2014.07.026. Epub 2014 Jul 30. He L, Aleksandrov AA, An J, Cui L, Yang Z, Brouillette CG, Riordan JR
Restoration of NBD1 thermal stability is necessary and sufficient to correct F508 CFTR folding and assembly.
J Mol Biol. 2015 Jan 16;427(1):106-20. doi: 10.1016/j.jmb.2014.07.026. Epub 2014 Jul 30., [PMID:25083918]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) (ABCC7), unique among ABC exporters as an ion channel, regulates ion and fluid transport in epithelial tissues. Loss of function due to mutations in the cftr gene causes cystic fibrosis. The most common cystic-fibrosis-causing mutation, the deletion of F508 (DeltaF508) from the first nucleotide binding domain (NBD1) of CFTR, results in misfolding of the protein and clearance by cellular quality control systems. The DeltaF508 mutation has two major impacts on CFTR: reduced thermal stability of NBD1 and disruption of its interface with membrane-spanning domains (MSDs). It is unknown if these two defects are independent and need to be targeted separately. To address this question, we varied the extent of stabilization of NBD1 using different second-site mutations and NBD1 binding small molecules with or without NBD1/MSD interface mutation. Combinations of different NBD1 changes had additive corrective effects on F508 maturation that correlated with their ability to increase NBD1 thermostability. These effects were much larger than those caused by interface modification alone and accounted for most of the correction achieved by modifying both the domain and the interface. Thus, NBD1 stabilization plays a dominant role in overcoming the DeltaF508 defect. Furthermore, the dual target approach resulted in a locked-open ion channel that was constitutively active in the absence of the normally obligatory dependence on phosphorylation by protein kinase A. Thus, simultaneous targeting of both the domain and the interface, as well as being non-essential for correction of biogenesis, may disrupt normal regulation of channel function.

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30 Furthermore, the combination of maximal NBD1 stabilization and interface modification by the paradoxical R1070W interface mutation disrupts the normal control of channel activity by phosphorylation. Login to comment
35 Rabeh et al. found that, while several of the solubilizing and suppressor mutations caused only a modest promotion of ƊF508 CFTR maturation, their effects were greater when they were combined with the NBD1/CL4 interface substitution R1070W or V510D [33], the latter also having been shown to have a direct effect on NBD1 thermostability [10]. Login to comment
55 In contrast, each of the interface substitutions, V510D (lane 8) and R1070W (lane 9), had much smaller effects. Login to comment
61 The rates of appearance of the mature products and the levels reached were increased further when the R1070W mutation was added to the combined NBD1 changes but not when V510D was added. Login to comment
84 The surface level of ƊF508 CFTR with the combined NBD1 stabilizing mutations (ƊF/combo) reached ~90% that of WT CFTR (Fig. 1c), and the level was somewhat further increased when V510D or R1070W was added to the combination (ƊF/combo/V510D and ƊF/combo/R1070W). Login to comment
87 Functional activity measured using this iodide efflux assay was similar for the ƊF508/combo and the ƊF508/4PT/R1070W, indicating the effectiveness of the combination of multiple NBD1 stabilizing mutations without interface modification in promoting ƊF508 CFTR channel activity. Login to comment
89 It was initially surprising to observe that the addition of R1070W to the ƊF508/combo apparently greatly diminished iodide efflux (Fig. 1d, bottom panel). Login to comment
91 This apparent inability of baby hamster kidney (BHK) cells expressing ƊF508/combo/R1070W CFTR to be effectively stimulated by forskolin cocktail suggested that the ƊF508/combo/R1070W CFTR channel might be constitutively active, a possibility that was subsequently confirmed at the single-channel level (see Fig. 5 below). Login to comment
111 The R1070W mutation in CL4 has different effects on the gating of WT and ƊF508 CFTR channels. Login to comment
112 (a) Western blot indicating enhancement of maturation of mutant R1070W and ƊF508 CFTRs by combined NBD1 stabilizing mutants (combo). Login to comment
113 (b and c) Single-channel recordings of WT (b) and ƊF508 CFTR (c) containing the R1070W mutant at 25 &#b0;C and 35 &#b0;C. All point histograms used to calculate single-channel parameters are shown to the left of each tracing. Login to comment
116 (d) WT and ƊF508/R1070W CFTR single-channel recordings during continuous temperature ramp of 1 &#b0;C/min from 30 to 35 &#b0;C, maintained at 30 &#b0;C for 3 min before initiating the ramp and held at 35 &#b0;C for 2 min. Login to comment
117 The moment of ƊF508/R1070W functional inactivation is shown by an arrow above the tracing and appeared after about 1 min at 35 &#b0;C. X-scale bar below trace is 60 s and Y-scale bar is 1 pA. 1.5 mM, reflecting low-affinity binding. Login to comment
126 The BEIA influence on the other interface substituted form of the protein, R1070W, was less pronounced, particularly at the lower concentrations of the compound. Login to comment
129 The disease-associated CL4 mutation R1070W differentially influences WT and ƊF508 CFTR R1070W is a relatively rare disease-associated mutation in the CL4 region of CFTR. Login to comment
131 Homology models of CFTR three-dimensional structure indicated that the R1070-containing segment of CL4 was proximal to F508 on the NBD1 surface [29-32] and Thomas et al. reasoned that the introduction of the aromatic tryptophan side chain by R1070W substitution might compensate for the loss of the phenylalanine side chain due to the absence of F508 at the interface between domains [8]. Login to comment
132 They showed that the R1070W mutation substantially improved the maturation of ƊF508 CFTR. Login to comment
135 Combined NBD1 stabilizing mutations promote maturation and function of R1070W CFTR in both ƊF508 and WT backgrounds. Login to comment
136 (a) Single-channel current tracings of the WT, ƊF508/Combo and ƊF508/Combo/R1070W CFTRs at 25 &#b0;C. All point histograms, single-channel conductances and open probabilities are shown as in Fig. 4. Login to comment
137 (b) Single-channel currents at 37 &#b0;C showing the influence of combined NBD1 stabilizing changes on ƊF508 and WT CFTR without and with the R1070W mutation. Login to comment
139 Combined NBD1 stabilizing mutations and R1070W result in phosphorylation-independent opening of ƊF508 CFTR channels. Login to comment
140 (a) Single-channel recordings at 37 &#b0;C of alkaline phosphatase treated ƊF508 CFTR containing combined NBD1 stabilizing mutations without (upper tracing) and with R1070W (middle tracing) as indicated. Login to comment
141 Lower tracing: single-channel recording at 37 &#b0;C of ƊF508/combo/R1070W with all 15 PKA phosphorylation sites mutated (15SA). Login to comment
145 Contrasting effects of R1070W on WT and ƊF508 CFTR is very evident at the level of biosynthetic processing as shown in Fig. 4a, where maturation of the WT is reduced while that of ƊF508 is promoted. Login to comment
148 While the opposite effects of R1070W on WT and ƊF508 CFTR maturation have been well documented, the corresponding functional consequences have not. Login to comment
150 The R1070W mutation alone resulted in very low channel open probabilities at both 25 &#b0;C and 35 &#b0;C (Fig. 4b). Login to comment
153 In contrast, robust channel gating was observed with the combined ƊF508/ R1070W variant with the expected elevation of Po as temperature was increased from 25 &#b0;C to 35 &#b0;C (Fig. 4c). Login to comment
156 Therefore, as with the much less active R1070W/WT form, this more active double mutant (ƊF508/R1070W) has a similar short functional lifetime at 35 &#b0;C confirming that it remains thermally unstable [15]. Login to comment
158 This behavior was especially evident when the single-channel activities of ƊF508 CFTR modified by combined NBD1 stabilizing changes with and without the R1070W interface mutation were compared with the unmodified WT at both 25 &#b0;C (Fig. 5a) and 37 &#b0;C (Fig. 5b). Login to comment
160 Strikingly, addition of the R1070W mutation to the NBD1 stabilized combination resulted in a very high open probability (0.91) even at this lower temperature. Login to comment
162 With the addition of R1070W, a nearly, completely locked open state was achieved with a Po of 0.98 (Fig. 5b, third tracing). Login to comment
164 The lower two tracings in Fig. 5b indicate that the combined stabilizing modifications of NBD1 also elevate the open probability of the WT channel with and without the R1070W mutation. Login to comment
165 Combined NBD1 stabilization and NBD1/CL4 interface modification result in constitutive ion channel activity of ƊF508 CFTR In iodide efflux experiments from whole cells expressing ƊF508/combo/R1070W CFTR, we had observed that the cells could not be efficiently loaded with the anion prior to the stimulation of cAMP production to activate protein kinase A (PKA) (Fig. 1d), suggesting the possibility that the apparent locked-open state might exist in the absence of the normally obligatory phosphorylation by PKA. Login to comment
168 In contrast, the NBD1 combo stabilized versions of the ƊF508 CFTR channels (Fig. 6a, middle tracing) and WT (Fig. 6b, middle tracing) responded very differently to addition of the R1070W mutation, the former channel becoming essentially locked open while the open probability of the latter was increased only modestly. Login to comment
169 To further verify that the high open probability state of ƊF508/combo/R1070W CFTR was reached independently of phosphorylation by PKA, we employed a construct in which serines or threonines at all 15 PKA phosphorylation sites were mutated to Ala (15SA) [39]. Login to comment
170 While the activity of the combo/ R1070W/WT CFTR was little changed by the 15SA modifications (Fig. 6b, lower tracing), the 15SA version of the ƊF508/combo/R1070W CFTR channel remained fully active even in the absence of PKA phosphorylation sites (Fig. 6a, lower tracing), confirming that the maximally NBD1 stabilized and NBD1/CL4 interface modified form of ƊF508 CFTR has lost normal regulatory control by phosphorylation. Login to comment
176 To further pursue this hypothesis here, we combined several of the most effective second-site changes in NBD1 with or without the interface stabilizing mutant R1070W and compared their effects on maturation, traffic to the cell surface and channel function. Login to comment
200 Turning to the impact of modifying the NBD1/CL4 interface, which may be a site of action of the VX-809 corrector [23,25], the R1070W mutation, shown by others to improve ƊF508 CFTR maturation [8], was the main tool we employed. Login to comment
202 Not only were their effects small compared to those of NBD1 stabilizing changes but R1070W also caused only a small increment above the correction caused by combined NBD1 stabilization (Fig. 1). Login to comment
203 From a functional perspective, most significant was the finding that, when modified by combined NBD1 mutations and R1070W, the efficiently assembled ƊF508 channel was constitutively active, not requiring phosphorylation by PKA (Fig. 6). Login to comment
204 Although the mechanism underlying this effect remains to be elucidated, it occurs in the stabilized mutant protein only when the NBD1/CL4 interface is altered by both the absence of F508 and the presence of R1070W (Figs. Login to comment
206 The disease-associated R1070W mutation alone is destabilizing of WT CFTR but does not cause it to be constitutively active without phosphorylation even when it contains all the NBD1 stabilizing changes (Fig. 6). Login to comment
208 These findings do not detract from the idea of therapeutically targeting the interface in different ways since it clearly plays a role in the propagation of stabilizing effects between domains as evidenced by the fact that impaired maturation caused by R1070W can be overcome by NBD1 stabilizing changes (Fig. 4a). Login to comment

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

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

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358 Some other CFTR mutations of varying clinical consequences, such as F1052 V, G1069R, and R1070W/R1070Q complete the list in this region. Login to comment

[hide] Cystic fibrosis genetics: from molecular understan... Nat Rev Genet. 2015 Jan;16(1):45-56. doi: 10.1038/nrg3849. Epub 2014 Nov 18. Cutting GR
Cystic fibrosis genetics: from molecular understanding to clinical application.
Nat Rev Genet. 2015 Jan;16(1):45-56. doi: 10.1038/nrg3849. Epub 2014 Nov 18., [PMID:25404111]

Abstract [show]
The availability of the human genome sequence and tools for interrogating individual genomes provide an unprecedented opportunity to apply genetics to medicine. Mendelian conditions, which are caused by dysfunction of a single gene, offer powerful examples that illustrate how genetics can provide insights into disease. Cystic fibrosis, one of the more common lethal autosomal recessive Mendelian disorders, is presented here as an example. Recent progress in elucidating disease mechanism and causes of phenotypic variation, as well as in the development of treatments, demonstrates that genetics continues to play an important part in cystic fibrosis research 25 years after the discovery of the disease-causing gene.

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55 Intriguingly, introduction of the disease-associated p.Arg1070Trp (legacy R1070W)29 variant in CL4 and correction of NBD1 misfolding using synthetic suppressor mutations could restore processing to F508delߛCFTR28,30,31 . 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
99 Missense variants R74W, R1070W, D1270N are classified as 'indeterminate` by Sosnay et al.,17 however, as they are frequently found in trans with a severe CF variant in asymptomatic individuals (including fertile fathers), they may not be sufficient to cause disease.19 Moreover, they are often associated within the same allele (eg in cis), forming various combinations ('complex alleles`) depending on individuals, so that their disease liability is questionable. Login to comment