Phenotyping of Rare CFTR Mutations Reveals Distinct Trafficking and Functional Defects

Abstract

<i>Background.</i> The most common <i>CFTR</i> mutation, F508del, presents with multiple cellular defects. However, the possible multiple defects caused by many rarer <i>CFTR</i> mutations are not well studied. We investigated four rare <i>CFTR</i> mutations E60K, G85E, E92K and A455E against well-characterized mutations, F508del and G551D, and their responses to corrector VX-809 and/or potentiator VX-770. <i>Methods.</i> Using complementary assays in HEK293T stable cell lines, we determined maturation by Western blotting, trafficking by flow cytometry using extracellular 3HA-tagged CFTR, and function by halide-sensitive YFP quenching. In the forskolin-induced swelling assay in intestinal organoids, we validated the effect of tagged versus endogenous CFTR. <i>Results.</i> Treatment with VX-809 significantly restored maturation, PM localization and function of both E60K and E92K. Mechanistically, VX-809 not only raised the total amount of CFTR, but significantly increased the traffic efficiency, which was not the case for A455E. G85E was refractory to VX-809 and VX-770 treatment. <i>Conclusions.</i> Since no single model or assay allows deciphering all defects at once, we propose a combination of phenotypic assays to collect rapid and early insights into the multiple defects of CFTR variants.