ABCMdb

PMID: 15744523

Clain J, Lehmann-Che J, Girodon E, Lipecka J, Edelman A, Goossens M, Fanen P
A neutral variant involved in a complex CFTR allele contributes to a severe cystic fibrosis phenotype.
Hum Genet. 2005 May;116(6):454-60. Epub 2005 Mar 3., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC7 p.Ser912Leu ORIGINAL INVESTIGATION Je´ roˆ me Clain Æ Jacqueline Lehmann-Che Emmanuelle Girodon Æ Joanna Lipecka Aleksander Edelman Æ Michel Goossens Pascale Fanen A neutral variant involved in a complex CFTR allele contributes to a severe cystic fibrosis phenotype Received: 1 September 2004 / Accepted: 12 December 2004 / Published online: 3 March 2005 Ó Springer-Verlag 2005 Abstract In order to further elucidate the contribution of complex alleles to the wide phenotypic variability of cystic fibrosis (CF), we investigated the structure-function relationships of a severe CF-associated complex allele [p.S912L;p.G1244V]. Login to comment 2 ABCC7 p.Ser912Leu ABCC7 p.Gly1244Val Both p.G1244V and [p.S912L;p.G1244V] mutants had normal protein processing but markedly decreased ClÀ channel activity compared with wild-type. Login to comment 3 ABCC7 p.Gly1244Val Notably, the double mutant displayed a dramatic decrease in ClÀ channel activity compared with p.G1244V (P<0.001). Login to comment 4 ABCC7 p.Ser912Leu p.S912L had normal protein processing and no detectable impact on CFTR function. Login to comment 5 ABCC7 p.Arg709* ABCC7 p.Ser912Leu In other respects, the p.S912L variation was identified in compound heterozygosity with p.R709X in a healthy fertile man. Login to comment 6 ABCC7 p.Ser912Leu Together, these data strongly support the view that p.S912L in isolation should be considered as a neutral variant but one that might significantly impair CFTR function when inherited in cis with another CFTR mutation. Login to comment 12 ABCC7 p.Arg334Trp ABCC7 p.Arg347His ABCC7 p.Arg347His ABCC7 p.Asp1270Asn ABCC7 p.Arg74Trp ABCC7 p.Arg1158* ABCC7 p.Arg553Gln The combination of two missense mutations on the same chromo some has been described clinically to lessen ([p.R553Q;p.F508del], [p.R334W;p.R1158X]; Dork et al. 1991; Duarte et al. 1996) or worsen ([p.R74W;p.D1270N], [p.R347H;p.D979A]; Casals et al. 1995; Hojo et al. 1998) the phenotype of CF patients with regard to the commonest mutation alone (p.F508del, p.R1158X, p.D1270N, p.R347H). Login to comment 14 ABCC7 p.Ser912Leu The [p.S912L;p.G1244V] double-mutant allele is associated with severe CF (Savov et al. 1995). Login to comment 16 ABCC7 p.Arg709* ABCC7 p.Ser912Leu ABCC7 p.Ser912Leu In vitro experiments and identification of a healthy fertile man bearing the [p.R709X]+[p.S912L] genotype both indicate that the p.S912L mutation should be considered to be a polymorphism. Login to comment 17 ABCC7 p.Ser912Leu ABCC7 p.Gly1244Val ABCC7 p.Gly1244Val Surprisingly, the combination of p.S912L and p.G1244V decreases J. Clain Æ J. Lehmann-Che Æ E. Girodon M. Goossens Æ P. Fanen (&) Service de Biochimie et Ge´ ne´ tique, Hoˆ pital Henri Mondor, Institut National de la Sante´ et de la Recherche Me´ dicale U.468, AP-HP, 94010 Cre´ teil, France E-mail: pascale.fanen@im3.inserm.fr Tel.: +33-1-49812854 Fax: +33-1-48993345 J. Lipecka Æ A. Edelman Faculte´ de Me´ decine Necker, Institut National de la Sante´ et de la Reche0rche Me´ dicale U.467, 156 Rue de Vaugirard, 75015 Paris, France Hum Genet (2005) 116: 454-460 DOI 10.1007/s00439-004-1246-z cAMP-dependent ClÀ channel activity compared with p.G1244V, highlighting the deleterious effect exerted by polymorphisms on the phenotype when combined in cis with a mutation. Login to comment 54 ABCC7 p.Arg709* ABCC7 p.Ser912Leu The diagnosis of CF was confirmed in the fetus by the presence of two allelic mutations, 711+1G fi T, a splice mutation of intron 5 inherited from the mother, and p.R709X, a nonsense mutation of exon 13 inherited from the father; the father carried the p.S912L variant (not transmitted to the fetus) on the other chromosome. Login to comment 56 ABCC7 p.Ser912Leu ABCC7 p.Ser912Leu ABCC7 p.Gly1244Val Results Processing of CFTR mutants To analyse the processing and ClÀ channel activity of p.S912L, p.G1244V and [p.S912L;p.G1244V] mutant proteins, wild-type and mutant CFTR alleles were expressed in HeLa cells. Login to comment 63 ABCC7 p.Ser912Leu ABCC7 p.Ser912Leu ABCC7 p.Gly1244Val The kinetics of core-glycosylated and mature forms of mutant CFTRs were nearly identical to those of the wild-type, indicating that p.S912L, p.G1244V and [p.S912L;p.G1244V] mutations did not affect maturation of the CFTR protein. Login to comment 75 ABCC7 p.Ser912Leu p.S912L- transfected cells displayed a similar proportion of responsive cells to wild-type cells (92% of responsive cells vs 83%; P>0.2; Fig.3), suggesting that ClÀ channel function was not affected by this mutation. Login to comment 76 ABCC7 p.Ser912Leu ABCC7 p.Ser912Leu ABCC7 p.Gly1244Val ABCC7 p.Gly1244Val Unlike p.S912L, the p.G1244V and [p.S912L; p.G1244V] CFTR transfected cells showed a marked decrease in cAMP-stimulated ClÀ conductance compared with wild-type (43% and 2.4% of responsive cells, respectively vs. Login to comment 79 ABCC7 p.Ser912Leu ABCC7 p.Ser912Leu ABCC7 p.Gly1244Val Surprisingly, whereas the p.S912L mutation had no detectable impact on CFTR function, the [p.S912L;p.G1244V] complex allele showed an almost 20-fold reduction in cAMP-dependent conductive pathway compared with the p.G1244V deleterious mutant (2.4% of responsive cells vs. Login to comment 81 ABCC7 p.Ser912Leu ABCC7 p.Ser912Leu ABCC7 p.Gly1244Val Discussion Clinical data suggest that the combination of p.S912L and p.G1244V mutations in cis ([p.S912L;p.G1244V] complex allele) produces a severe CF phenotype similar to that of p.F508del homozygotes. Login to comment 83 ABCC7 p.Ser912Leu Thus, the poor ClÀ channel activity measured in vitro (2.4% of the wild-type) and the severity of the associated disease observed in vivo (pancreatic insufficiency, severe lung disease and elevated sweat electrolytes) both indicate that the [p.S912L;p.G1244V] complex allele should be considered to be a severe allele. Login to comment 85 ABCC7 p.Ser912Leu ABCC7 p.Ser912Leu ABCC7 p.Ser912Leu ABCC7 p.Gly1244Val In this study, we have Fig. 2 a-d Pulse-chase experiments showing the turnover of the immature and mature forms of wild-type and the mutant CFTR proteins (representative of two independent experiments) Table 1 Summary of MEQ fluorescence assay results from mock-transfected, wild-type and mutant cells Statistic DFstim-DFbasal Mock Wild-type p.S912L p.G1244V [p.S912L;p.G1244V] n 28 64 36 53 42 Mean 0.002 0.023 0.036 0.005 0.001 SEM 0.001 0.017 0.028 0.005 0.001 Range 0-0.005 0-0.340 0-0.140 0-0.034 0-0.006 provided functional and clinical evidence that the p.S912L mutation is not a disease-causing mutation on the basis of the following data. Login to comment 86 ABCC7 p.Ser912Leu (1) In vitro experiments have shown that the p.S912L mutation has no detectable impact on CFTR processing and ClÀ channel function. Login to comment 89 ABCC7 p.Arg709* ABCC7 p.Ser912Leu Notably, the healthy father of a CF fetus carrying the p.S912L mutation has been identified in our laboratory in the framework of prenatal diagnosis, whereas the CF-associated p.R709X mutation (Bonizzato et al. 1995) has been detected on his other allele. Login to comment 91 ABCC7 p.Ser912Leu Together, these data strongly support the view that the p.S912L mutation should be considered to be a neutral variant. Login to comment 94 ABCC7 p.Gly1244Glu At the same location, the severe CF-associated p.G1244E mutation (Devoto et al. 1991) exhibits a defect in the open state probability of the CFTR protein (Anderson and Welsh 1992). Login to comment 95 ABCC7 p.Gly1244Val Taken together, these data support the view that the p.G1244V mutation is a disease-causing mutation. Login to comment 96 ABCC7 p.Ser912Leu ABCC7 p.Gly1244Val Surprisingly, the p.G1244V and [p.S912L;p.G1244V] alleles affect in vitro ClÀ channel function in different ways. Login to comment 97 ABCC7 p.Ser912Leu ABCC7 p.Gly1244Val ABCC7 p.Gly1244Val This might indicate that p.S912L and p.G1244V mutations occurring on the same allele cause severe structural alterations worsening the effect of the p.G1244V mutation on CFTR function. Login to comment 98 ABCC7 p.Ser912Leu However, we cannot exclude the possibility that the p.S912L mutation may have a slight, but undetectable, effect on CFTR function and that a threshold is reached when these two mutations are combined in cis. Login to comment 99 ABCC7 p.Ser912Leu ABCC7 p.Gly1244Val An important consequence of our observation is that when a patient or an asymptomatic relative carries the p.S912L mutation, the p.G1244V mutation should be sought in order to exclude a CF allele; this is important for genetic counselling. Login to comment 100 ABCC7 p.Ser912Leu These results have important implications for CF because they suggest that ''neutral`` missense mutations, such as p.S912L, might significantly alter CFTR function when inherited in cis with another mutation. Login to comment 103 ABCC7 p.Arg117His ABCC7 p.Arg117His The length of a polythymidine tract in the 3' splice site of intron 8 modulates the clinical presentation of the p.R117H missense mutation because of a dramatic decrease in the amount of p.R117H-CFTR transcript (Kiesewetter et al. 1993). Login to comment 105 ABCC7 p.Ser549Arg The sequence change À102T fi A attenuates the effects of the severe p.S549R (T fi G) missense mutation by causing a significant increase in CFTR expression in vitro. Login to comment 106 ABCC7 p.Ser549Arg ABCC7 p.Ser549Arg This explains the striking differences in genotype-phenotype correlations between patients carrying mutation p.S549R alone, who presented severe disease, and patients carrying the complex allele [À102T fi A; p.S549R], who exhibit milder forms of CF (Romey et al. 2000). Login to comment 118 ABCC7 p.Leu436Phe The complex allele [p.R201C;p.L436F] has recently been reported (Caciotti et al. 2003); expression studies have revealed a deleterious effect of the p.L436F polymorphism on the known p.R201C mutation resulting in a late infantile subtype of type-II GM1, instead of the juvenile form classically associated with p.R201C. Login to comment