Higher Throughput Assays for Understanding the Pathogenicity of Variants of Unknown Significance in the RPE65 Gene.

Purpose: RPE65 is a key enzyme in the visual cycle that regenerates 11-cis-retinal. Mutations in RPE65 cause a retinal dystrophy that is treatable with an approved gene therapy. Variants of unknown significance (VUS) on genetic testing can prevent patients from obtaining a firm genetic diagnosis and accessing gene therapy. Because most RPE65 mutations have a low protein expression level, this study developed and validated multiple methods for assessing the expression level of RPE65 variants. This functional evidence is expected to aid in reclassifying RPE65 VUS as pathogenic, which can improve the diagnosis and treatment of RPE65 patients. Methods: Thirty different variants of RPE65 (12 pathogenic, 13 VUS, five benign) were cloned into lentiviral expression vectors. Protein expression levels were measured after transient transfection or in stable cell lines, using western blots and immunostaining with flow cytometry. Then, a pooled, high-throughput, fluorescence-activated cell sorting (FACS) assay with a next-generation sequencing-based readout was used to assay pools of RPE65 variants. Results: A high correlation was observed between protein levels measured by western blot, flow cytometry, and the pooled FACS assay. Using these assays, we confirmed and extended RPE65 variant data, including that Pro111Ser has a low, pathogenic expression level. There was a high correlation between RPE65 expression and previously reported enzyme activity levels; further development of a high-throughput enzymatic activity assay would complement these expression data. Conclusions: This scalable approach can be used to solve patient pedigrees with VUS in RPE65, facilitating treatment and providing RPE65 structure-function information.