Retinitis pigmentosa (RP) is the most common form of inherited retinal degeneration affecting around 1:3,000 individuals worldwide. The majority of the X-linked RP, associated with a severe phenotype, is caused by mutations in the RPGR gene. All known mutations causing RPGR-related retinal dystrophies are found to affect the RPGR ORF15 isoform, which contains a unique Cterminal 567-aa exon called ORF15.
ORF15 is a mutational hotspot for RPGR-associated RP, accounting for two thirds of all disease-causing mutations. The exon ORF15, however, includes a highly repetitive, purine-rich sequence, which generally performs poorly in next-generation sequencing (NGS) -based assays. To address the clinical importance of the RPGR ORF15 and the lack of high quality NGS-based diagnostics, we have developed a novel test to detect variants in the ORF15 region.
Blueprint Genetics’ WES assay shows improved coverage in the exon ORF15 of RPGR compared to other NGS technology. A custom Sanger sequencing assay was developed for the 400-600-bp difficult-to-sequence region. This diagnostic test combines NGS analysis with Illumina NovaSeq 6000 platform and Sanger sequencing. Diagnostic assay was validated with seven samples with a known variant in the ORF15 region and showed 100% sensitivity to detect variants in this region.
Clinical validation with 16 patients with a clinical suspicion of XLRP and a negative result from a previous genetic testing revealed a truncating variant in ORF15 in
25% (4/16) of the patients. In an unselected set of 386 patients analyzed with the BpG 266-gene Retinal Dystrophy Panel, the RPGR molecular diagnosis was found in 6.6% of the patients.
Keywords: X-linked retinitis pigmentosa (XLRP), RPGR gene, ORF15 isoform, difficult-to-sequence region, NGS diagnostics, Sanger sequencing
Authors: Johanna Känsäkoski, Johanna Sistonen, Kati Kämpjärvi, Miika Mehine, Miko Valori, Pertteli Salmenperä, Eeva-Marja Sankila, Eveliina Salminen, Tiia Maria Luukkonen, Samuel Myllykangas, Tero-Pekka Alastalo, Juha Koskenvuo, Sari Tuupanen