Application of Whole-Exome and Retinal-Capture Next-Generation DNA Sequencing to Identify Disease-Causing Mutations in Families with a Diagnosis of Autosomal Dominant Retinitis Pigmentosa

Abstract

Purpose: To detect disease-causing mutations in families with a diagnosis of autosomal dominant retinitis pigmentosa (adRP), focusing on genes known to cause inherited retinal diseases, using whole-exome next-generation sequencing (NGS) and retinal-capture NGS.

Methods: Families were selected from a cohort of 260 with a diagnosis of adRP and 3 affected generations and affected females, or two generations with male-to-male transmission. The families were previously screened for common mutations by Sanger sequencing. Coding exons and flanking intron-exon junctions of all known RetNet genes and candidate genes were targeted for NGS. Testing was either 1.) whole-exome NGS using Agilent SureSelect liquid-capture probes and the Illumina platform, focusing analysis on 190 retinal disease genes, 2.) targeted liquid-capture and NGS of coding regions of 160 retinal disease genes, or 3.) both. Families tested included positive controls with known mutations and families without a previously-identified mutation. Mutations detected by NGS were confirmed by Sanger sequencing.

Results: Probands and additional family members from 87 families, including controls, were tested by one or both methods. Of 22 families tested by whole-exome NGS, mutations were found in 7 (32%). Of 18 control families with known mutations tested by retinal-capture NGS, mutations were found in 17. (A large deletion in PRPF31 was not detected). Of 69 families without known mutations tested by retinal-capture NGS, mutations were found in 10 (15%). In summary, of a total of 76 families whose mutations were not known prior to NGS, mutations were found in 17 (22%). Several of these mutations are in genes not currently associated with autosomal dominant disease.

Conclusions: Targeted retinal-gene capture or focused retinal-gene analysis following whole-exome NGS, are reliable, efficient methods for detecting disease-causing mutations in families with dominant retinitis pigmentosa. Based on a variety of methods, mutations in coding exons or intron-exon junctions of known retinal disease genes (RetNet genes) account for at least 72% of families in our cohort with dominant RP.

Keywords: 696 retinal degenerations: hereditary • 604 mutations • 537 gene screening