Higher throughput assays for understanding the pathogenicity of variants of unknown significance (VUS) in the RPE65 gene.

Purpose: is a key enzyme in the visual cycle that regenerates 11-cis retinal. Mutations in cause a retinal dystrophy that is treatable with an FDA-approved gene therapy. Variants of unknown significance (VUS) on genetic testing can prevent patients from obtaining a firm genetic diagnosis and accessing gene therapy. Since most mutations have a low protein expression level, this study developed and validated multiple methods for assessing the expression level of variants. This functional evidence is expected to aid in reclassifying VUS as pathogenic, which in turn can broaden the application of gene therapy for patients.

Methods: 30 different variants of (12 pathogenic, 13 VUS, 5 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 an NGS-based sequencing readout was used to assay pools of variants.

Results: There was a high correlation between protein levels measured by Western blot, flow cytometry, and the pooled FACS assay. Using these assays, we confirm and extend 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 this expression data.

Conclusion: This scalable approach can be used to solve patient pedigrees with VUS in , facilitating treatment and providing structure-function information.