Identification of numerous novel disease-causing variants in patients with inherited retinal diseases, combining careful clinical-functional phenotyping with systematic, broad NGS panel-based genotyping.

Purpose: The widespread consensus is that genotyping is essential for patients with inherited retinal disease (IRD). Given the numerous ongoing gene therapy clinical trials for IRDs, identifying the pathogenic mutation in these patients has potential important therapeutic implications. In this study, we demonstrate how we identified with a high degree of confidence numerous novel disease-causing mutations, deletions, and duplications in a large consecutive IRD case series by using a judicious combination of careful, in-depth clinical-functional phenotyping to guide and integrate our genotyping approach. Methods: We conducted a retrospective analysis of data between November 2016 and March 2018 from the Duke Center for Retinal Degenerations and Ophthalmic Genetic Diseases IRD patient database, which encompassed 378 IRD cases that had not yet been previously genotyped. With the exception of some patients who presented with classical clinical-functional phenotypes that allowed for targeted gene testing, all other subjects systematically underwent next-generation sequencing-based broad, IRD-focused panel testing. Most cases were also tested for parental allele phase. Results were reviewed vis-à-vis the clinical-functional phenotypes for reconciliation and potential addition of supplemental testing such as deletion/duplication microarrays or copy number variant (CNV) analysis. Supplemental testing was driven by an IRD specialist-laboratory consensus, and decisions were clinically or genetically driven or both. Results: By judiciously using this two-way approach and leveraging to its full potential the benefits of careful, in-depth clinical-functional phenotyping by an experienced IRD specialist, more than 80% of the cases in this series were successfully genotyped. We also identified with a high degree of confidence 52 novel disease-causing mutations, deletions, and duplications. Conclusions: The combination of meticulous, expert clinical-functional phenotyping studies with systematic next-generation sequencing panel-based genotyping and microarray deletion/duplication testing or CNV analysis as applicable in accordance with the above-mentioned consensus was extremely effective at the diagnostic end, reduced costs, and saved time. IRD specialist-laboratory two-way interactions and case discussions would augment the efficacy of this approach and improve the diagnostic yield in successfully solving and genotyping IRD cases.