Development of a High Throughput in Vitro Protein Localization Assay to Characterize Potentially Pathogenic Genetic Variants Associated With Retinitis Pigmentosa

Abstract

Retinitis pigmentosa is a disease comprised of a group of retinal degenerations (RD) that affects about 1/4,000 people worldwide (Wang, 2014). Currently, there is no cure for RP, and studying the disease is difficult because it is so genetically and phenotypically heterogeneous and because investigation of the retina in vivo is necessarily invasive. The advance of next generation sequencing (NGS) has allowed for great inroads into the genetic investigation of this disease. Here we describe the development of a flexible in vitro assay based on protein surface expression to identify potentially pathogenic genetic variants of unknown significance (VUS) discovered via NGS. The assay relies on the methods of plasmid transfection, immunofluorescence, fluorescence activated cell sorting (FACS) and NGS. By creating a library of Rhodopsin (RHO) variants to test our system, this work serves as proof of concept that we are able to efficiently pool and then deconvolute a mixture of cells transfected with different variants into categories based on protein localization. This assay could theoretically be used to test VUS in a variety of genes related to RP and beyond, thus facilitating not only the characterization of VUS discovered through NGS, but also influencing the development of possible future therapies.