摘要：

Cystic Fibrosis (CF) is a common, life-limiting autosomal recessive disorder caused by a mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. CF patients suffer from chronic lung infections, pancreatic insufficiency, abnormal sweat chloride levels, and difficulties in digestion and weight gain. Though treatments have improved in recent years, patients succumb to respiratory failure by their mid-30s.;Certain classes of CFTR mutations cause protein mislocalization due to structural alteration or disruption of apical membrane targeting signals such as those located at the N- and C-termini of CFTR. Since localization signals have been identified in the cytoplasmic loops of several ion channels, the CFTR cytoplasmic loops were inspected for putative trafficking signals. The studies revealed high cross-species conservation of human CFTR compared to that of 50 diverse species, with cytoplasmic loop 4 being the most highly conserved in terms of amino acid identity. Of the 68 amino acids in this loop, 36 have been found to be mutated in humans, and 12 sites have multiple missense mutations at the same amino acid.;Curiously, different substitutions in the same cytoplasmic loop 4 residue can have variable disease consequences. In particular, patients with CFTR R1070P and R1070Q mutations have severe CF, but those with R1070W mutations have mild CF. To determine whether R1070 mutations cause disease by affecting CFTR localization, CFTR wild-type and CFTR mutant stable MDCK cell lines were created using the Flp-InRTM System, which allowed for the creation of isogenic single site integration clones, that could be directly compared without concern of variable copy number or incidental integration effects.;Confocal microscopy studies using the stable MDCK-FRT-CFTR cell lines revealed that R1070P was cytoplasmic, R1070Q congregated near the apical membrane, and R1070W was apical. Quantitative biotinylation studies indicated that R1070P was present in trace amounts inside the cell and absent from the apical surface, R1070Q was highly expressed and inserted into the apical membrane, and R1070W had a low level of expression but was detected in partially and fully glycosylated forms on the apical membrane.;Although the localization of CFTR R0170P and R1070W were distinctly different from wild-type CFTR and consistent with their proposed deleterious roles in CF patients, the profile of CFTR R1070Q was inconsistent with a severe CF phenotype. Re-analysis of 16 patients with detailed clinical histories and the R1070Q mutation revealed that 11 carried an in cis nonsense CFTR mutation, S466X. Discovery of the in cis S466X mutation reconciles the apparent discrepancy between localization studies of R1070Q and the phenotype of patients bearing this mutation. These results demonstrate that substitutions of evolutionarily conserved amino acids are not necessarily deleterious and emphasize that localization and protein processing studies in relevant model systems are valuable for the interpretation of the disease-causing potential of rare missense mutations.

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