Next-generation Sequencing Of All Known Genes For RP And Allied Diseases Identifies The Causative Mutations In The Majority Of Patients

Abstract

Purpose: : Retinitis pigmentosa (RP), Leber congenital amaurosis (LCA) and Stargardt disease (STGD) are genetically heterogeneous, making comprehensive genetic testing difficult. However, knowing the causative mutations is desirable for disease management and genetic counseling. We have developed a diagnostic next-generation sequencing (NGS) approach for these entities. To identify Usher syndrome type 1 (USH1) patients in individuals with congenital deafness before onset of visual symptoms, we included the USH1 genes in NGS for hearing impairment.

Methods: : Genomic DNA of 32 patients (group 1: 27 patients with autosomal recessive RP (ARRP), sporadic RP or STGD; group 2: 5 LCA patients) was fragmented (Covaris S2 AFA system) and ligated to barcoded adaptors for multiplexing. Samples were hybridized to a customized in-solution capture library (NimbleGen), targeting all coding exons of 31 ARRP genes (413 exons), 23 ADRP genes (248 exons) and 15 LCA genes LCA (196 exons). Post-capture amplified enriched DNA was subjected to emulsion PCR and subsequent NGS on a Roche 454 GS FLX platform. NGS data were analysed using an in-house bioinformatic pipeline and JSI Medical Systems SeqNext-Software (vs 3.5). Sequence variants of interest were verified by Sanger sequencing.

Results: : 90% of the target exons were covered more than 15-fold, with an average coverage of 50-70-fold per sample. Mutations were identified in 20 out of the 27 group 1 patients: ABCA4, RP1, CRB1 and PDE6B showed biallelic mutations in 2 - 4 patients each (13 in total), whereas biallelic mutations in C2ORF71, EYS, MERTK and RDH5 were each identified in single individuals. In one RP patient, a single heterozygous truncating mutation was identified in SAG. Two patients turned out to be affected by ADRP that appeared sporadic due to incomplete penetrance of the underlying monoallelic PRPF31 mutations. In 2 of the 5 group 2 patients (LCA), causative biallelic mutations were identified in CEP290 and CRB1, respectively. NGS of all deafness genes in a cohort of hearing-impaired children identified a child with a homozygous MYO7A mutation that is known to cause USH1.

Conclusions: : NGS targeting the coding exons of the known RP, STGD and LCA genes unraveled the genetic basis of disease in 69% of cases (73% of RP cases). PRPF31 mutations in 2 patients with sporadic and thus presumably recessive RP demonstrate the need to consider PRPF31 in such cases. Although 4 genes - ABCA4, RP1, CRB1 and PDE6B - had a higher prevalence, the involvement of 11 genes in total demonstrates the need for comprehensive analysis. NGS of all known deafness genes can identify Usher patients before onset of visual problems, allowing for early and specific medical follow-up.

Keywords: retinitis • genetics • degenerations/dystrophies