The importance of Sanger sequencing of exon ORF15 of RPGR at the time of next-generation sequencing

Abstract

Purpose: Mutations in RPGR with a hotspot region ORF15 are believed to represent common causes for X-linked rod-cone dystrophies (Xl-RCD). However, RPGR mutations are not restricted to RCD but may also cause cone dystrophy (CD) or cone-rod dystrophy (CRD). Phenotypic variability in association with this gene defect in the same family and female carriers showing a phenotype have also been documented. Next generation sequencing (NGS) of all genes underlying inherited retinal disorders is commonly used to identify the cause of patients with RCD, CD and CRD. However, due to its repeat region, the majority of ORF15 sequence is not targeted by this approach. The purpose of this study was to Sanger-sequence ORF15 prior to targeted NGS in patients, for whom X-linked mode of inheritance cannot be excluded. <br />

Methods: Patients of a French cohort underwent a full ophthalmic examination. Informed consent was obtained from each patient and unaffected family member. The study protocol adhered to the tenets of the Declaration of Helsinki and was approved by the local ethics committee. The DNA of more than 390 index patients with a initial diagnosis of sporadic, “autosomal recessive”, “autosomal dominant” or X-linked rod-cone dystrophy or cone or cone-rod dystrophy was extracted and screened for ORF15 mutation by Sanger sequencing as previously described [Neidhardt et al., 2008].<br />

Results: Twenty-nine of the Sanger-sequenced patients revealed an ORF15 mutation, of which eleven were novel, representing frameshift and nonsense mutations and one deletion lacking the complete ORF15 sequence. Provisional diagnosis for eighteen cases was sporadic or “autosomal recessive” RCD, for two cases “autosomal dominant” RCD and for nine cases Xl-RCD.<br />

Conclusions: Provisional diagnosed patients with sporadic or “autosomal recessive”, with “autosomal dominant” and Xl-RCD or CRD harbored 6%, 6% and 28% ORF15 mutations, respectively. In total, we detected in ~7% of the screened patients a mutation in ORF15, which would have been missed by targeted NGS approaches. These results highlight the importance to screen this region prior to targeted NGS applying Sanger sequencing in male patients with various retinal dystrophies of all mode of inheritance, except in clear autosomal dominant cases showing male-to-male transmission.<br />