Improved Diagnosis of Inherited Retinal Dystrophies by High-Fidelity PCR of ORF15 followed by Next-Generation Sequencing

Abstract

Retinitis pigmentosa (RP) is the most common form of retinal dystrophy. The disease is characterized by the progressive degeneration of photoreceptors, ultimately leading to blindness. The exon ORF15 of RP GTPase regulator (RPGR) is a mutation hot spot for X-linked RP and one form of cone dystrophy. However, accurate molecular testing of ORF15 is challenging because of a large segment of highly repetitive purine-rich sequence in this exon. ORF15 performs poorly in next-generation sequencing-based panels or whole exome sequencing analysis, whereas Sanger sequencing of ORF15 requires special reagents and PCR conditions with multiple pairs of overlapping primers that often do not provide a clean sequence. Because of these technical difficulties, molecular analysis of ORF15 is performed mostly in research laboratories without validation for clinical application. Herein, we report the development of a single step of high-fidelity PCR followed by next-generation sequencing for accurate mutation detection, which is easily integrated into routine clinical practice. Our approach has improved coverage depth of ORF15 with the ability to detect single-nucleotide variants and deletions/duplications. Using this method, we were able to identify ORF15 pathogenic variants in approximately 31% of undiagnosed RP patients. Our results underline the clinical importance of complete and accurate sequence analysis of ORF15 for patients with retinal dystrophies.