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Phenotyping of Rare CFTR Mutations Reveals Distinct Trafficking and Functional Defects
Marjolein Ensinck1, Liesbeth De Keersmaecker1, Lise Heylen1
1Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, 3000 Leuven, Flanders, Belgium.
Insights
Researchers studied rare cystic fibrosis transmembrane conductance regulator (CFTR) mutations, finding that a combination of assays is needed to understand their diverse cellular defects and guide treatment development.
Area of Science:
- Molecular biology
- Genetics
- Pharmacology
Background:
- The common CFTR mutation F508del causes multiple cellular defects.
- Defects caused by rarer CFTR mutations are less understood.
- This study investigates four rare CFTR mutations: E60K, G85E, E92K, and A455E.
Purpose of the Study:
- To characterize the cellular defects of rare CFTR mutations.
- To evaluate the response of these mutations to CFTR modulators VX-809 and VX-770.
- To compare rare mutations with well-characterized mutations F508del and G551D.
Main Methods:
- Utilized HEK293T stable cell lines with complementary assays.
- Assessed CFTR maturation (Western blotting), trafficking (flow cytometry), and function (YFP quenching).
- Validated findings in intestinal organoids using forskolin-induced swelling assays.
Main Results:
- VX-809 treatment restored maturation, localization, and function for E60K and E92K mutations.
- VX-809 improved CFTR traffic efficiency for E60K and E92K, but not A455E.
- G85E mutation showed resistance to both VX-809 and VX-770.
Conclusions:
- No single assay can fully elucidate all CFTR variant defects.
- A combination of phenotypic assays provides rapid insights into CFTR variant defects.
- This approach aids in understanding diverse mutations and potential therapeutic strategies.
Abstract:
Background. The most common CFTR mutation, F508del, presents with multiple cellular defects. However, the possible multiple defects caused by many rarer CFTR mutations are not well studied. We investigated four rare CFTR 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. Methods. 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. Results. 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. Conclusions. 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.
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