Purpose To devise a comprehensive multi-platform genetic testing strategy for inherited retinal disease and describe its performance in 1,000 consecutive families seen by a single clinician. Methods The clinical records of all patients seen by a single retina specialist between January 2010 and June 2016 were reviewed and all patients who met the clinical criteria for a diagnosis of inherited retinal disease were included in the study. Each patient was assigned to one of 62 diagnostic categories and this clinical diagnosis was used to define the scope and order of the molecular investigations that were performed. The number of nucleotides evaluated in a given subject ranged from two (a multiplex allele-specific assay for the most common mutations in BBS1 and BBS10) to nearly 900,000 (the coding sequences, and splice junctions of 305 genes known to cause inherited retinal disease). Results Disease-causing genotypes were identified in 760 families (76%). These genotypes were distributed across 104 different genes. More than 70% of these 104 genes have coding sequences small enough to be efficiently packaged into an adeno-associated virus. Mutations in ABCA4 were the most common cause of disease in this cohort (173 families) while mutations in 80 genes caused disease in five or fewer families (i.e., 0.5% or less). Disease-causing genotypes were identified in 576 of the families without next generation sequencing (NGS). This included 23 families with mutations in the repetitive region of RPGR exon 15 that would have been missed by NGS. Whole exome sequencing of the remaining 424 families revealed mutations in an additional 182, and whole genome sequencing of four of the remaining 242 families revealed two additional genotypes that were invisible by the other methods. Performing the testing in a clinically-focused tiered fashion would be 6.1% more sensitive, 17.7% less expensive and have a significantly lower average false genotype rate than using whole exome sequencing to assess more than 300 genes in all patients (7.1 vs. 128%; p<0.001). Conclusions Genetic testing for inherited retinal disease is now more than 75% sensitive. A clinically-directed tiered testing strategy can increase sensitivity and improve statistical significance without increasing cost.

The human retinal pigment epithelium (RPE) and choroid are complex tissues that provide crucial support to the retina. Disease affecting either of these supportive tissues can lead to irreversible blindness in the setting of age-related macular degeneration. In this study, single-cell RNA sequencing was performed on macular and peripheral regions of RPE-choroid from 7 human donor eyes in 2 independent experiments. In the first experiment, total RPE/choroid preparations were evaluated and expression profiles specific to RPE and major choroidal cell populations were identified. As choroidal endothelial cells represent a minority of the total RPE/choroidal cell population but are strongly implicated in age-related macular degeneration (AMD) pathogenesis, a second single-cell RNA-sequencing experiment was performed using endothelial cells enriched by magnetic separation. In this second study, we identified gene expression signatures along the choroidal vascular tree, classifying the transcriptome of human choriocapillaris, arterial, and venous endothelial cells. We found that the choriocapillaris highly and specifically expresses the regulator of cell cycle gene (RGCC), a gene that responds to complement activation and induces apoptosis in endothelial cells. In addition, RGCC was the most up-regulated choriocapillaris gene in a donor diagnosed with AMD. These results provide a characterization of the human RPE and choriocapillaris transcriptome, offering potential insight into the mechanisms of choriocapillaris response to complement injury and choroidal vascular disease in age-related macular degeneration.

Retinitis pigmentosa (RP) is an inherited retinal degenerative disease with severe vision impairment leading to blindness. About 10–15% of RP cases are caused by mutations in the RPGR gene, with RPGR mutations accounting for 70% of X-linked RP cases. The mechanism by which RPGR mutations cause photoreceptor cell dysfunction is not well understood. In this study, we show that the two isoforms of RPGR (RPGR1−19 and RPGRORF15) interact with endogenous PDE6D, INPP5E, and RPGRIP1L. The RPGR1−19 isoform contains two PDE6D binding sites with the C-terminal prenylation site being the predominant PDE6D binding site. The C terminus of RPGR1−19 that contains the prenylation site regulates its interaction with PDE6D, INPP5E, and RPGRIP1L. Only the RPGR1−19 isoform localizes to cilia in cultured RPE1 cells. Missense variations found in RPGR patients disrupt the interaction between RPGR isoforms and their endogenous interactors INPP5E, PDE6D, and RPGRIP1L. We evaluated a RPGR missense variation (M58K) found in a family with X-linked retinitis pigmentosa (XLRP) and show that this missense variation disrupts the interaction of RPGR isoforms with their endogenous interactors. The M58K variation also disrupts the ciliary localization of the RPGR1−19 isoform. Using this assay, we also show that some of the RPGR missense variants reported in the literature might not actually be disease causing. Our data establishes an in vitro assay that can be used to validate the potential pathogenicity of RPGR missense variants.