ABCMdb

PMID: 22678879

El-Seedy A, Girodon E, Norez C, Pajaud J, Pasquet MC, de Becdelievre A, Bienvenu T, des Georges M, Cabet F, Lalau G, Bieth E, Blayau M, Becq F, Kitzis A, Fanen P, Ladeveze V
CFTR mutation combinations producing frequent complex alleles with different clinical and functional outcomes.
Hum Mutat. 2012 Nov;33(11):1557-65. doi: 10.1002/humu.22129. Epub 2012 Jul 2., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC7 p.Gly149Arg ABCC7 p.Gly149Arg ABCC7 p.Arg668Cys ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr To elucidate the clinical significance of complex alleles involving p.Gly149Arg, p.Asp443Tyr, p.Gly576Ala, and p.Arg668Cys, we performed a collaborative genotype-phenotype correlation study, collected epidemiological data, and investigated structure-function relationships for single and natural complex mutants, p.[Gly576Ala;Arg668Cys], p.[Gly149Arg; Gly576Ala;Arg668Cys], and p.[Asp443Tyr;Gly576Ala; Arg668Cys]. Login to comment 3 ABCC7 p.Gly149Arg Among 153 patients carrying at least one of these mutations, only three had classical CF and all carried p.Gly149Arg in the triple mutant. Login to comment 4 ABCC7 p.Gly149Arg Sixty-four had isolated infertility and seven were healthy individuals with a severe mutation in trans, but none had p.Gly149Arg. Login to comment 5 ABCC7 p.Gly149Arg ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr Functional studies performed on all single and natural complex mutants showed that (1) p.Gly149Arg results in a severe misprocessing defect; (2) p.Asp443Tyr moderately alters CFTR maturation; and (3) p.Gly576Ala, a known splicing mutant, and p.Arg668Cys mildly alter CFTR chloride conductance. Login to comment 6 ABCC7 p.Gly149Arg ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr Overall, the results consistently show the contribution of p.Gly149Arg to the CF phenotype, and suggest that p.[Arg668Cys], p.[Gly576Ala;Arg668Cys], and p.[Asp443Tyr;Gly576Ala;Arg668Cys] are associated with CFTR-related disorders. Login to comment 20 ABCC7 p.Arg668Cys ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala Although some variants have been considered neutral because they were identified in the non-CF allele of the parents of CF patients, they seem to be involved in moderate forms or syndromes of late onset, as reported for c.2002C>T, p.Arg668Cys (R668C) and c.1727G>C, p.Gly576Ala (G576A) [Fanen et al., 1992]. Login to comment 21 ABCC7 p.Gly149Arg ABCC7 p.Gly149Arg ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr These C 2012 WILEY PERIODICALS, INC. mutations have been further described in isolation, in association within complex alleles, and together with c.1327G>T, p.Asp443Tyr (D443Y) or c.445G>A, p.Gly149Arg (G149R) as triple mutants, notably in infertile patients with a congenital bilateral absence of the vas deferens (CBAVD) but also in patients with CF [Abramowicz et al., 2000; Bienvenu et al., 1997; Chillon et al., 1995a; Costes et al., 1995; Mercier et al., 1995; Pignatti et al., 1995; Ratbi et al., 2007; CFMD]. Login to comment 24 ABCC7 p.Gly149Arg ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr We thus implemented a collaborative study through the FrenchCFLaboratoryNetworktocollectallpatientsandindividuals carrying p.Asp443Tyr, p.Gly576Ala, p.Arg668Cys, and p.Gly149Arg, either in isolation or in complex alleles, and gathered epidemiological data on these mutations from the general population of France. Login to comment 26 ABCC7 p.Gly149Arg ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr Materials and Methods Patients and Healthy Individuals Patients and healthy individuals known to the French CF Laboratory Network before 1st January 2009, who were heterozygous for the p.Asp443Tyr, p.Gly576Ala, p.Arg668Cys, and p.Gly149Arg mutations, either in isolation or in a complex allele, were included. Login to comment 31 ABCC7 p.Gly149Arg ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr Epidemiological Study in the French General Population The allelic prevalences of the p.Asp443Tyr, p.Gly576Ala, p.Arg668Cys, and p.Gly149Arg mutations, either in isolation or in a complex allele, were determined by allele counting in a sample of healthy adult individuals from the French general population. Login to comment 44 ABCC7 p.Gly149Arg ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr Specific substitutions observed either in isolation (p.Gly149Arg, p.Asp443Tyr, p.Gly576Ala, and p.Arg668Cys) or in different combinations in patients were introduced into the WT CFTR plasmid using the Gene tailor site-directed mutagenesis kit (Invitrogen) and the designed primers (available upon request), in accordance with the manufacturer`s protocol (Fig. 1). Login to comment 91 ABCC7 p.Gly149Arg ABCC7 p.Gly149Arg ABCC7 p.Arg668Cys ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr Results Phenotype of Patients Carrying p.Asp443Tyr, p.Gly576Ala, p.Arg668Cys, and/or p.Gly149Arg in Various Combinations A total of 153 patients and healthy individuals carrying at least one of the alleles p.Asp443Tyr, p.Gly576Ala, p.Arg668Cys, and/or p.Gly149Arg, either in isolation or in a complex allele, were identified (Table 1). Login to comment 95 ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr Phenotype and Genotype Data of Patients Carrying At Least One of the CFTR Gene Mutations p.Gly549Arg, p.Asp443Tyr, p.Gly576Ala, and p.Arg668Cys Phenotype CFTR genotype No. Login to comment 96 ABCC7 p.Gly149Arg ABCC7 p.Gly149Arg of patients Main diagnosis Additional information Age at diagnosis Sweat test (Cl-,mmol/L) Allele 1 Allele 2 2 CF P, PI 3 m1/2, 5 m NA p.[Gly149Arg;Gly576Ala;Arg668Cys] p.Phe508del 1 CF P, PI 6 y 92 p.[Gly149Arg;Gly576Ala;Arg668Cys] p.Phe508del 4 CF? Login to comment 97 ABCC7 p.Gly576Ala GI, cholestasis 2 m,1 y, 4 y, NA NA p.[Gly576Ala;Arg668Cys] NI 1 CF? Login to comment 98 ABCC7 p.Gly576Ala ABCC7 p.Arg75Glu Bronchitis, ENT 16 y 55, 58 p.[Gly576Ala;Arg668Cys] p.Arg75Glu 1 CF? Login to comment 99 ABCC7 p.Gly576Ala P 59 y 80 p.[Gly576Ala;Arg668Cys] NI 1 CF? Login to comment 100 ABCC7 p.Arg668Cys P, PI 27 y 50 p.Arg668Cys c.1766+73T>G 1 CF? Login to comment 101 ABCC7 p.Gly576Ala Failure to thrive 2 y 20-59 p.[Gly576Ala;Arg668Cys] p.Phe508del 1 CF? Login to comment 102 ABCC7 p.Gly576Ala P 1 y 36-48 p.[Gly576Ala;Arg668Cys] p.Phe508del 1 CF? Login to comment 103 ABCC7 p.Arg668Cys P NA 40-59 p.Arg668Cys NI 1 DB PI 78 y 41 c. Login to comment 104 ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr ABCC7 p.Leu997Phe ABCC7 p.Glu92Asn [2002C>T;3718-2477C>T] (3849+10kbC>T) p.Glu92Asn 2 DB 60 y, 71 y NA p.[Gly576Ala;Arg668Cys] p.Phe508del 1 DB Pa infections 20 y 67 p.[Asp443Tyr;Gly576Ala;Arg668Cys] p.[Gly576Ala;Arg668Cys] 1 DB 17 y <40 p.[Gly576Ala;Arg668Cys] NI 1 DB 26 y 23-71 p.[Gly576Ala;Arg668Cys] p.Leu997Phe 1 DB Pa infections 72 y 34-60 p.[Gly576Ala;Arg668Cys] NI 1 DB Azoospermia NA NA p.Arg668Cys NI 1 DB 66 y 80-87 p.[Asp443Tyr;Gly576Ala;Arg668Cys] c.262_263delTT (394delTT) 1 CSD ENT 19 y NA p.[Asp443Tyr;Gly576Ala;Arg668Cys] p.Phe508del 1 CSD Bronchitis 48 y NA p.[Gly576Ala;Arg668Cys] p.Phe508del 1 CSD Sinusitis, bronchiolitis 72 y NA p.[Gly576Ala;Arg668Cys] p.Phe508del 1 CSD Nasal polyposis 18 y >60 c. Login to comment 105 ABCC7 p.Arg117His ABCC7 p.Arg347His ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys ABCC7 p.Ser549Asn ABCC7 p.Thr338Ile ABCC7 p.Asp1152His ABCC7 p.Arg668Cys ABCC7 p.Arg668Cys ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Arg31Cys ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr ABCC7 p.Thr1246Ile ABCC7 p.Met1137Val ABCC7 p.Glu379Lys [2002C>T;3718-2477C>T] p.Gln689X 2 CSD Nasal polyposis 14 y,16 y NA, 29 p.[Gly576Ala;Arg668Cys] NI 3 IP 35-39 y NA p.[Asp443Tyr;Gly576Ala;Arg668Cys] NI 1 IP Bronchitis 49 y NA p.[Gly576Ala;Arg668Cys] p.PheF508del 1 IP 42 y NA p.[Gly576Ala;Arg668Cys] p.Arg668Cys 1 IP NA NA p.[Gly576Ala;Arg668Cys] c.1210_34TG[12]T[5] 4 IP 19-69 y NA p.[Gly576Ala;Arg668Cys] NI 1 Cholestasis 60 y NA p.[Gly576Ala;Arg668Cys] c.1584G>A 33 CBAVD 27-50 y 9-82 p.[Asp443Tyr;Gly576Ala;Arg668Cys] p.Phe508del 2 CBAVD 30 y,36 y NA p.[Asp443Tyr;Gly576Ala;Arg668Cys] c.2051_2052delAAinsG 1 CBAVD 34 y 72 p.[Asp443Tyr;Gly576Ala;Arg668Cys] p.Trp1282X 1 CBAVD NA NA p.[Asp443Tyr;Gly576Ala;Arg668Cys] p.Asn1303Lys 1 CBAVD 35 y 65-66 p.[Asp443Tyr;Gly576Ala;Arg668Cys] p.Ser549Asn 1 CBAVD NA NA p.[Asp443Tyr;Gly576Ala;Arg668Cys] c.3605delA 1 CBAVD 30 y 41-69 p.[Asp443Tyr;Gly576Ala;Arg668Cys] p.Gln1411X 1 CBAVD 31 y NA p.[Asp443Tyr;Gly576Ala;Arg668Cys] p.Arg347His 3 CBAVD 29 y, 34 y, NA NA p.[Asp443Tyr;Gly576Ala;Arg668Cys] p.Gly542X 1 CBAVD 35 y NA p.[Asp443Tyr;Gly576Ala;Arg668Cys] c.946delT 1 CBAVD 26 y NA p.[Asp443Tyr;Gly576Ala;Arg668Cys] c.4242_4242+1delGGinsT 1 CBAVD 41 y 31 p.[Asp443Tyr;Gly576Ala;Arg668Cys] p.Arg117His 1 CBAVD 32 y NA p.[Asp443Tyr;Gly576Ala;Arg668Cys] p.Thr338Ile 1 CBAVD NA NA p.[Asp443Tyr;Gly576Ala;Arg668Cys] p.Glu379Lys 1 CBAVD NA NA p.[Asp443Tyr;Gly576Ala;Arg668Cys] p.Met1137Val 1 CBAVD NA NA p.[Asp443Tyr;Gly576Ala;Arg668Cys] p.Thr1246Ile 2 CBAVD NA NA p.[Asp443Tyr;Gly576Ala;Arg668Cys] NI 1 CBAVD 34 NA p.[Gly576Ala;Arg668Cys] p.Asn1303Lys 8 CBAVD 30-42 y NA p.[Gly576Ala;Arg668Cys] NI 1 CBAVD 27 y NA p.Arg668Cys p.Phe508del 1 CBAVD 30 y NA p.Arg668Cys NI 1 CUAVD NA NA p.[Asp443Tyr;Gly576Ala;Arg668Cys] p.Phe508del 1 CUAVD NA NA p.[Gly576Ala;Arg668Cys] NI 1 CUAVD Renal agenesis NA NA p.[Gly576Ala;Arg668Cys] NI 1 Hypofertility (not CBAVD) CF carrier`s partner NA NA p.[Gly576Ala;Arg668Cys] p.Asp1152His 1 FBA Mild CF considered possible, 2 older brothers with the same genotype, one with a very mild phenotype, the other being asymptomatic 22 wg NA p.[Asp443Tyr;Gly576Ala;Arg668Cys] p.Asn1303Lys 1 FBA TOP for de novo chromosomal translocation; not CF 21 wg NA p.[Asp443Tyr;Gly576Ala;Arg668Cys] p.Arg31Cys 1 FBA Not CF at birth 28 wg <30 p.[Gly576Ala;Arg668Cys] p.Phe508del 1 FBA Unknown outcome 23 wg NA p.[Gly576Ala;Arg668Cys] p.Phe508del 1 FBA Not CF at birth 21 wg <30 p.[Gly576Ala;Arg668Cys] p.Trp846X (Continued) Table 1. Login to comment 107 ABCC7 p.Ser1235Arg ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr ABCC7 p.Ser912Leu ABCC7 p.Leu137Arg of patients Main diagnosis Additional information Age at diagnosis Sweat test (Cl-,mmol/L) Allele 1 Allele 2 1 FBA Fetal death 20 wg NA p.[Gly576Ala;Arg668Cys] p.Ser1235Arg 1 FBA Unknown outcome p.Arg668Cys p.Phe508del 1 FBA Not CF at birth 38 wg NA p.[Asp443Tyr;Gly576Ala;Arg668Cys] NI 1 FBA Not CF at birth 28 wg NA p.[Gly576Ala;Arg668Cys] NI 1 Healthy CF patient`s mother NA NA p.[Asp443Tyr;Gly576Ala;Arg668Cys] p.Phe508del 1 Healthy Newborn, elevated IRT but normal ST (not CF) Birth <30 p.[Gly576Ala;Arg668Cys] p.Phe508del 1 Healthy Mother of a CF fetus (p.[Phe508del]+ [phe508del]) NA NA p.[Gly576Ala;Arg668Cys] p.Phe508del 1 Healthy Mother of a fetus with FBA but not affected with CF NA NA p.[Gly576Ala;Arg668Cys] p.Phe508del 2 Healthy Mother of a fetus with FBA but not affected with CF NA NA p.[Gly576Ala;Arg668Cys] NI 1 Healthy CF patient`s mother 59 y NA p.[Gly576Ala;Arg668Cys] p.Phe508del 1 Healthy CF patient`s mother NA NA p.[Gly576Ala;Arg668Cys] p.[Ser912Leu;Asn1303Lys] 1 Healthy CF patient`s mother 32 y NA p.[Gly576Ala;Arg668Cys] p.Leu137Arg 1 Healthy CF carrier`s partner NA NA p.[Gly576Ala;Arg668Cys] c. Login to comment 108 ABCC7 p.Arg668Cys ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr ABCC7 p.Ser519Gly [3705T>G;1210-13T[5]] 1 Healthy CF carrier`s partner NA NA p.[Ser519Gly;Gly576Ala;Arg668Cys] NI 2 Healthy CF carrier`s partner 37 y, NA NA p.[Asp443Tyr;Gly576Ala;Arg668Cys] NI 20 Healthy CF carrier`s partner 24-42 y NA p.[Gly576Ala;Arg668Cys] NI 1 Healthy CF carrier`s partner 32 y NA p.Gly576Ala NI 1 Healthy CF patient`s mother 37 y NA p.Arg668Cys p.Arg792X 1 Healthy CF relative (p.Gly745X) 22 y NA p.Arg668Cys NI 1 Healthy General population NA NA p.[Gly576Ala;Arg668Cys] NI CF, cystic fibrosis; CF?, suspicion of cystic fibrosis; CBAVD, congenital bilateral absence of vas deferens; CSD, chronic sinus disease; DB, disseminated bronchiectasis; ENT, ear, nose, and throat symptoms; FBA, fetal bowel anomaly; GI, gastrointestinal symptoms; IP, idiopathic pancreatitis; IRT, immunoreactive trypsinemia; m, months; NA, not available; NI, not identified; P, pulmonary symptoms; Pa: Pseudomonas aeruginosa; PI, pancreatic insufficiency; wg, weeks of gestation; ST, sweat test; TOP, termination of pregnancy; y, years. Login to comment 110 ABCC7 p.Gly149Arg The three classical CF patients carried the p.[Gly149Arg;Gly576Ala;Arg668Cys] complex allele in combination with a severe CF mutation in the other allele. Login to comment 111 ABCC7 p.Gly149Arg In contrast to the other mutations, no genotypes involving p.Gly149Arg and a severe CF mutation in trans were found in any other patient with mild disease or in any of the healthy individuals. Login to comment 112 ABCC7 p.Gly149Arg This raised the hypothesis that the major deleterious effect of the complex allele was attributable to p.Gly149Arg. Login to comment 113 ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr The most frequent phenotype associated with combinations of p.Asp443Tyr, p.Gly576Ala, and p.Arg668Cys was CBAVD. Login to comment 117 ABCC7 p.Asp443Tyr Outcome was known for seven out of nine cases: (1) one baby, with the triple mutant p.[Asp443Tyr;Gly576Ala;Arg668Cys], was considered possibly affected with mild CF or CFTR-RD at the time of analysis. Login to comment 120 ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr Of the 37 healthy individuals, seven had a severe CF mutation in trans; one carried the triple mutant p.[Asp443Tyr;Gly576Ala;Arg668Cys]; five carried the double mutant p.[Gly576Ala;Arg668Cys]; and one carried p.Arg668Cys in isolation. Login to comment 121 ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr These observations in patients carrying varied combinations of p.Arg668Cys, p.Gly576Ala, p.Asp443Tyr in trans with a classical CF mutation argue against as considering them as CF defects, but as CFTR-RD-associated mutations. Login to comment 122 ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr ABCC7 p.Ser519Gly Epidemiological Data from the French General Population Of the 1,423 healthy individuals screened, 26 were heterozygous for p.[Gly576Ala;Arg668Cys] (allelic frequency 0.91%, 95% CI 0.60-1.33%); four for p.[Asp443Tyr;Gly576Ala;Arg668Cys] (allelic frequency 0.14%, 95% CI 0.04-0.36%); two for p.Arg668Cys; two for p.Gly576Ala (allelic frequency for each 0.07%, 95% CI 0.0080.25%); and one for p.[Ser519Gly;Gly576Ala;Arg668Cys] (allelic frequency 0.04%, 95% CI 0.0009-0.20%). Login to comment 124 ABCC7 p.Gly149Arg Of the subset of 791 individuals who had undergone complete scanning of the coding regions, none were found to carry p.Gly149Arg (allelic frequency <0.20%). Login to comment 125 ABCC7 p.Gly149Arg ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr Processing of CFTR Mutants To evaluate the contribution of each mutation to the phenotype, we first studied the maturation of CFTR in HeLa cells that had been transiently transfected with cDNA encoding the WT and mutated CFTR proteins p.Gly149Arg, p.Asp443Tyr, p.Gly576Ala, and p.Arg668Cys in different combinations. Login to comment 132 ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr ABCC7 p.Gly576Arg A: Western blot analysis of the CFTR expression: 1, WT; 2, p.Gly576Ala; 3, p.Arg668Cys; 4, p.[Gly576Arg;Arg668Cys]; 5, p.[Asp443Tyr;Gly576Ala;Arg668Cys]; 6, pTracer; 7, p.Asp443Tyr. Login to comment 136 ABCC7 p.Gly149Arg ABCC7 p.Gly149Arg C: Western blot analysis of the CFTR expression: 1, p.Phe508del; 2, p.[Gly149Arg;Gly576Ala;Arg668Cys]; 3, p.Gly149Arg; 4, WT. Login to comment 140 ABCC7 p.Arg668Cys ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala The relative amount of fully glycosylated protein (band C) did not differ between p.Gly576Ala, p.Arg668Cys, or p.[Gly576Ala; Arg668Cys] and WT CFTR. Login to comment 141 ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr Lower amounts of mature p.Asp443Tyr and p.[Asp443Tyr;Gly576Ala;Arg668Cys] proteins were detected, with a decrease in proportion of band C (ratio band C/(band B + band C): 56.9% vs. 83.5 for the WT (Fig. 2A). Login to comment 144 ABCC7 p.Gly149Arg [Gly149Arg;Gly576Ala;Arg668Cys]proteins, whichwasindicativeofablockadeintheERcompartment(Fig.2C). Login to comment 146 ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr As shown in Figure 3, p.Gly576Ala, p.Arg668Cys, p.Asp443Tyr, p.[Gly576Ala;Arg668Cys], and p.[Asp443Tyr;Gly576Ala;Arg668Cys] were targeted to the plasma membrane (Figs. 3B-F). Login to comment 147 ABCC7 p.Asp443Tyr The p.Asp443Tyr mutants (single ortriple)alsoshowedanintenseperinuclearstainingcomparedwith the WT CFTR protein (Figs. 3D and 3F). Login to comment 148 ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr Thus, p.Asp443Tyr seemed to be more perinuclear than p.[Asp443Tyr;Gly576Ala;Arg668Cys], and this result is consistent with the Western blotting results and a partial block in CFTR trafficking. Login to comment 149 ABCC7 p.Gly149Arg ABCC7 p.Gly149Arg In contrast, the p.Gly149Arg and p.[Gly149Arg;Gly576Ala;Arg668Cys] mutants exhibited no cell surface staining but did exhibit intense perinuclear staining (Figs. 3H and 3I). Login to comment 150 ABCC7 p.Gly149Arg These experiments showed that p.Gly149Arg proteins were restricted to intracellular compartments, thus confirming the presence of a processing defect. Login to comment 151 ABCC7 p.Gly149Arg ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr Functional Analysis of Chloride Channel Function in CFTR Mutants To determine the impact of p.Gly149Arg, p.Asp443Tyr, p.Gly576Ala, and p.Arg668Cys in single, double, or triple mutants on CFTR chloride channel function, we expressed full-length WT and mutant proteins in HeLa cells, and measured chloride channel activity using single-cell fluorescence imaging and the potential-sensitive probe DiSBAC2(3)(Molecular Probes). Login to comment 152 ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr The CFTR Cl- channel conductance was detected at the plasma membrane in WT CFTR and mutants containing p.Asp443Tyr, p.Gly576Ala, and p.Arg668Cys, whereas no conductance was observed in cells transfected with either GFP negative control or p.Phe508del protein (Figs. 4A and 4B). Login to comment 154 ABCC7 p.Asp443Tyr The activity of the p.Asp443Tyr and WT channels was similar. Login to comment 155 ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala In contrast, p.Gly576Ala and p.Arg668Cys mutants exhibited a slight but significant reduction of levels of CFTR conductance (P < 0.05) (Fig. 4B). Login to comment 158 ABCC7 p.Gly149Arg ABCC7 p.Gly149Arg In contrast, Cl- channel conductance was not detected in the cells transfected with p.Gly149Arg or p.[Gly149Arg;Gly576Ala;Arg668Cys]. Login to comment 160 ABCC7 p.Gly149Arg ABCC7 p.Gly149Arg ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr Discussion In the present study, we investigated genotype-phenotype correlations and the in vitro consequences of CFTR mutations: four in isolation (i.e., p.Gly149Arg, p.Asp443Tyr, p.Gly576Ala, and p.Arg668Cys) and three complex alleles observed in patients or healthy individuals (i.e., p.[Gly576Ala;Arg668Cys], p.[Asp443Tyr;Gly576Ala;Arg668Cys], and the rarer p.[Gly149Arg; Gly576Ala;Arg668Cys]). Login to comment 165 ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr ABCC7 p.Asp443Tyr A: Wild-type CFTR; B: p.Gly576Ala; C: p.Arg668Cys; D: p.Asp443Tyr; E: p.[Gly576Ala;Arg668Cys], F: p.[Asp443Tyr;Gly576Ala;Arg668Cys] are targeted at least to the plasma membrane. Login to comment 166 ABCC7 p.Gly149Arg ABCC7 p.Gly149Arg Mutants (G) p.Phe508del, (H) p.Gly149Arg, (I) p.[Gly149Arg;Gly576Ala;Arg668Cys] were not targeted to the plasma membrane. Login to comment 170 ABCC7 p.Gly149Arg ABCC7 p.Gly149Arg Classification of Mutants with Regard to Clinical and Functional Data Classical CF was only observed in patients carrying p.Gly149Arg within the context of the complex allele p.[Gly 149Arg;Gly576Ala;Arg668Cys] in trans with a severe CF mutation. Login to comment 173 ABCC7 p.Gly149Arg ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr In contrast, genotypes combining mutants other than p.Gly149Arg, namely p.Arg668Cys, p.[Gly576Ala;Arg668Cys], and p.[Asp443Tyr;Gly576Ala;Arg668Cys], in trans with a CF mutation, were not observed in patients with classical CF, although they were observed in patients with moderate phenotypes, in particular CBAVD. Login to comment 175 ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr This observation is also consistent with the results of the functional studies, which demonstrated residual CFTR function, with p.Asp443Tyr having an effect on protein maturation, and p.Gly576Ala and p.Arg668Cys having an effect on Cl- channel activity. Login to comment 177 ABCC7 p.Gly576Ala The epidemiological data provided further evidence that p.[Gly576Ala;Arg668Cys] is a frequent variant in the general population. Login to comment 178 ABCC7 p.Gly149Arg ABCC7 p.Asp443Tyr ABCC7 p.Ser519Gly This was corroborated by the observation that additional, different mutations occurred on this haplotype (p.Asp443Tyr, p.Gly149Arg, and p.Ser519Gly, the latter being observed only once in the sample from the general population). Login to comment 179 ABCC7 p.Gly149Arg ABCC7 p.Gly149Arg ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr These results have substantial implications for diagnostic and genetic counseling, as they classify p.[Gly149Arg;Gly576Ala;Arg668Cys] or p.Gly149Arg (even if no CF patient was detected with only this mutation) as a CF-causing mutation, and p.Arg668Cys, p.[Gly576Ala;Arg668Cys], and p.[Asp443Tyr;Gly576Ala;Arg668Cys] as CFTR-RD mutations. Login to comment 180 ABCC7 p.Gly149Arg Therefore, CF carrier testing in relatives and prenatal diagnosis should be offered or discussed only when p.Gly149Arg is present. Login to comment 184 ABCC7 p.Gly576Ala Likewise, it has been documented that p.Gly576Ala causes partial exon 12 skipping [Pagani et al., 2003]. Login to comment 186 ABCC7 p.Gly576Ala [Gly576Ala;Arg668Cys]and 4. Login to comment 190 ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr Statistical significance was set at *** P < 0.001; ** P < 0.01; * P < 0.05; ns, nonsignificant difference (P > 0.05).B-D:Histogramsreportthemeansoftherelativefluorescencecollectedfromseparateexperiments.B:HeLacellstransfectedwithWTCFTRas positive control, p.F508del or GFP as a negative controls, and CFTR mutants as p.Asp443Tyr, p.Gly576Ala, p.Arg668Cys. Login to comment 191 ABCC7 p.Gly149Arg ABCC7 p.Gly149Arg ABCC7 p.Asp443Tyr ABCC7 p.Gly576Arg ABCC7 p.Gly576Arg D: HeLa cells transfected with WT CFTR as positive control, p.F508del as a negative control, and CFTR mutants as p.[Gly576Arg;Arg668Cys], [p.Asp443Tyr;Gly576Ala;Arg668Cys], p.[Gly149Arg; Gly576Arg;Arg668Cys] and p.Gly149Arg. Login to comment 192 ABCC7 p.Asp443Tyr p.[Asp443Tyr;Gly576Ala;Arg668Cys] (personal data). Login to comment 196 ABCC7 p.Gly149Arg In addition to clinical data, the present study provides molecular and functional evidence that the CFTR p.Gly149Arg mutation should be classified as a misprocessing, class II mutation [Welsh and Smith, 1993]. Login to comment 200 ABCC7 p.Gly149Arg Interestingly, p.Gly149Arg is located in the first intracellular loop of MSD1,andwethereforehypothesizethatp.Gly149Argcouldmodify the interaction between MSD1 and NBD1, thus leading to a severe class II defect. Login to comment