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

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

The Genetic Basis of Pericentral Retinitis Pigmentosa—A Form of Mild Retinitis Pigmentosa

Pericentral retinitis pigmentosa (RP) is an atypical form of RP that affects the near-peripheral retina first and tends to spare the far periphery. This study was performed to further define the genetic basis of this phenotype. We identified a cohort of 43 probands with pericentral RP based on a comprehensive analysis of their retinal phenotype. Genetic analyses of DNA samples from these patients were performed using panel-based next-generation sequencing, copy number variations, and whole exome sequencing (WES). Mutations provisionally responsible for disease were found in 19 of the 43 families (44%) analyzed. These include mutations in RHO (five patients), USH2A (four patients), and PDE6B (two patients). Of 28 putatively pathogenic alleles, 15 (54%) have been previously identified in patients with more common forms of typical RP, while the remaining 13 mutations (46%) were novel. Burden testing of WES data successfully identified HGSNAT as a cause of pericentral RP in at least two patients, suggesting it is also a relatively common cause of pericentral RP. While additional sequencing might uncover new genes specifically associated with pericentral RP, the current results suggest that genetically pericentral RP is not a separate clinical entity, but rather is part of the spectrum of mild RP phenotypes