Cost-effective identification of causal variants in 113 genes underlying retinitis pigmentosa and Leber congenital amaurosis

Abstract

Purpose : The identification of genetic variants underlying retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) is challenging as 108 genes are involved. Whole exome sequencing is often used in diagnostic facilities to identify causal variants explaining these phenotypes, but the costs are still prohibiting global applicability. An efficient and cost-effective targeted sequencing method is therefore needed.

Methods : We sequenced the exons and splice sites, and pseudo-exons due to pathogenic deep-intronic variants of 113 genes, and the RP17 autosomal dominant RP locus, using 16,812 smMIPs designed by Molecular Loop Biosciences. Sequencing libraries for 2,000 probands and control cases were prepared in series of 384 samples and analyzed using Illumina NovaSeq 6000. Single nucleotide variants (SNVs) were annotated with an in-house pipeline and prioritized for ≤1.0% allele frequency. Variants with a Franklin-ACMG classification of class 3, 4, or 5 variants were selected for further interpretation.

Results : Across five sequencing runs an average read count per nucleotide of 662x was achieved. All 19 SNVs and 22 copy number variants (CNVs) in control cases were reliably detected. All prioritized variants were evaluated in the first 480 probands. 248 RP and LCA probands (53%) were solved in the initial data analysis encompassing homozygous CNVs, homozygous and compound heterozygous SNVs in genes associated with autosomal recessive phenotypes, and heterozygous variants in genes associated with autosomal dominant or X-linked phenotypes. The 5 most frequently mutated genes were USH2A (12.5%), ABCA4 (11.3%), EYS (10.9%), CRB1 (4.9%), and RPGR (4.1%).

Conclusions : We showed that the smMIPs panel utilized in this study can be used to effectively sequence RP and LCA genes and loci. While the initial solve rate for this cohort of partially prescreened RP/LCA cases is 53%, further analysis will include the analysis of duplications and heterozygous deletions as well as an in-depth analysis of potential splice-site altering variants, adding to this diagnostic yield. We encourage professionals from low- and middle-income countries to contact us for potential patient inclusion.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.