In silico analysis and theratyping of an ultra-rare CFTR genotype (W57G/A234D) in primary human rectal and nasal epithelial cells
Karina Kleinfelder, Virginia Lotti, Adriana Eramo, Felice Amato, Stefania Lo Cicero, Germana Castelli, Francesca Spadaro, Alessia Farinazzo, Daniele Dell’Orco, Sara Preato, Jessica Conti, L. F. Rodella, F. Tomba, Cerofolini Angelo, Elena Baldisseri, Marina Bertini, Sonia Volpi, Valeria Rachela Villella, Speranza Esposito, Immacolata Zollo, Giuseppe Castaldo, Carlo Laudanna, Eric J. Sorsher, Jeong S. Hong, Disha Joshi, G. Cutting, Marco Lucarelli, Paola Melotti, Claudio Sorio
Abstract
Mutation targeted therapy in cystic fibrosis (CF) is still not eligible for all CF subjects, especially for cases carrying rare variants such as the CFTR genotype W57G/A234D (c.169T>G/c.701C>A). We performed in silico analysis of the effects of these variants on protein stability, which we functionally characterized using colonoids and reprogrammed nasal epithelial cells. The effect of mutations on cystic fibrosis transmembrane conductance regulator (CFTR) protein was analyzed by western blotting, forskolin-induced swelling (FIS), and Ussing chamber analysis. We detected a residual CFTR function that increases following treatment with the CFTR modulators VX661±VX445±VX770, correlates among models, and is associated with increased CFTR protein levels following treatment with CFTR correctors. In vivo treatment with VX770 reduced sweat chloride concentration to non-CF levels, increased the number of CFTR-dependent sweat droplets, and induced a 6% absolute increase in predicted FEV1% after 27 weeks of treatment indicating the relevance of theratyping with patient-derived cells in CF.