Functional studies of rare missense mutations in CFTR facilitate interpretation of genotype-phenotype relationships

摘要

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 have “severe” pancreatic insufficient cystic fibrosis (CF) and R1070W have “mild” pancreatic sufficient CF or congenital bilateral absence of the vas deferens. Intriguingly, CFTR bearing each of these mutations is functional when expressed in non-polarized cells. To determine whether R1070 mutations cause disease by affecting CFTR localization, we created polarized 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. Re-analysis 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 of 5 patients with R1070Q had “mild” disease, thereby reconciling the apparent discrepancy between the functional studies of R1070Q and the phenotype of patients bearing this mutation. Our results emphasize that localization studies in relevant model systems are needed for the interpretation of the disease-causing potential of rare missense mutations.