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.

Findings from a Genotyping Study of over 1000 People with Inherited Retinal Disorders in Ireland

“…A previously reported sequencing strategy of RPGR was employed in order to cover the highly repetitive sequences of ORF15, a major cause of X-linked RP [19]. Primers used to probe this region can be found in Figure S2.…”

“…All of these variants, with the exception of c.5351T>C Pathogenic variants in RPGR (MIM: 312610) were the most frequent cause of X-Linked RP, explaining over 88% (23/26) of sequenced pedigrees ( Figure 3). Employment of a bespoke sequencing strategy of ORF15 has enhanced the success of sequencing this region, which typically presents a diagnostic challenge due to low coverage and poor variant detection [19].…”

Targeted deletion of Nmnat1 in mouse retina leads to early severe retinal dystrophy

“…Sequencing studies have recently found that mutations in NMNAT1 can cause LCA and these constitute approximately 10% of unsolved cases (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47). The majority of NMNAT1 mutations found in LCA patients are missense, with a small portion are nonsense and frameshift.…”

Clinically-guided mutation screening of two families with hereditary retinal disease

“…In the HRD field, the molecular diagnosis of disease cases also benefits a lot from a variety of NGS-based methods, including whole exome sequencing and targeted capture sequencing. Correspondingly, the molecular diagnosis rate of HRD cases has been significantly improved [4][5][6][7][8][9][10][11][12][13] , and a series of novel HRD-associated genes have been identified [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33] .…”