Targeted next-generation sequencing of the highly repetitive and purine-rich ORF15 region of RPGR, a hotspot for missing heritability in X-linked retinitis pigmentosa, a target for gene therapy

Abstract

Purpose : There has been a surge in clinical trials investigating gene therapy for inherited retinal diseases (IRD). An example is X-linked retinitis pigmentosa (XLRP), caused by mutations in RPGR in ~75% of cases. Currently, three clinical trials are investigating RPGR gene therapy. Establishing a molecular diagnosis of RPGR-disease can be challenging as RPGR contains a large exon ORF15, harbouring 60% of the mutations but characterized by a highly repetitive and purine-rich sequence region (~75% of ORF). This complicates genetic testing of XLRP and is a cause of missing heritability in IRD. We aimed to accelerate the molecular diagnosis of XLRP by developing an NGS-based test targeting XLRP genes, including the mutation hotspot in ORF15.

Methods : Primers were designed using in-house software to cover the entire coding region of RPGR exons 1-14 and ORF15. Amplification was performed using an optimized singleplex PCR for enrichment of exon ORF15. Six primer sets were designed to cover the hotspot region. Library preparation via a modified Nextera XT protocol was followed by NGS using short-read sequencing (MiSeq, Illumina). Data-analysis was performed using CLC Genomics Workbench, variant calling and annotation with VEP (Ensembl) and Alamut.

Results : Our targeted NGS strategy was first validated in 13 patients with XLRP and allowed us to identify ORF15 variants located in and surrounding the highly repetitive region (chrX(GRCh38):38285535-38286845). All changes could be confirmed by Sanger sequencing. Sequencing quality of the targeted regions was evaluated by coverage-based analysis. Coverage of ORF15 and the repeat-rich region could be improved to an average coverage of 387x for the most repetitive region, compared to 0-37x in whole exome sequencing data. We could obtain an average coverage of 2490x for the detected variants in the highly repetitive region.

Conclusions : To cover the highly repetitive and purine-rich region of ORF15, we propose an optimized NGS-based test targeting low-depth genomic regions such as ORF15. As RPGR is a target for gene therapy, identification of all (likely) pathogenic RPGR variants is of utmost importance. Finally, our approach can be extrapolated to other repeat-rich regions of disease genes and improve the overall diagnostic yield of IRD.

This is a 2020 ARVO Annual Meeting abstract.