Digenic inheritance of heterozygous RP1L1 and C2orf71 variants in syndromic retinitis pigmentosa

Abstract

Purpose: To identify the genetic cause for a syndrome identified in an isolated case, which consisted of retinitis pigmentosa (RP), hearing loss, ataxia and cerebellar atrophy.

Methods: The presence of copy number variants (CNVs) were investigated using a genome-wide CGH array and by MLPA analysis of OPA1 and WFS1. Candidate disease genes (CEP290, GJB2, OPA1, WFS1, ZCD2) and mitochondrial DNA variants were analysed by Sanger sequencing. Allele-specific primer extension analysis was performed using microarrays containing variants implicated in Leber congenital amaurosis, autosomal recessive and dominant RP, and Usher syndrome. The exome of the proband was enriched using the SureSelect Human All Exon v4 Kit (50 Mb), and sequenced on a 5500XL platform. Morpholino oligonucleotides (MOs) and CRISPR gRNAs were designed for zebrafish genes rp1l1 and C2orf71-like for knockdown and knockout, respectively. MOs or CRISPR gRNAs plus Cas9 RNA were microinjected into the yolk of zebrafish embryos at 1-8 cell stage, and convergent extension phenotype was examined at 10 somite stage. At 5 days post fertilization, lateral images of zebrafish larvae were taken to measure eye size. Larvae were then analysed by histoimmunostaining.

Results: No CNVs, mutations in known disease genes or mitochondrial DNA defects were found that could account for the phenotype under an autosomal recessive paradigm. However, exome sequencing revealed a complex heterozygous protein-truncating mutation in RP1L1, p.[(K111Qfs*27; Q2373*)], and a heterozygous nonsense mutation in C2orf71, p.(S512*) on the other allele. Mutations in both genes have been implicated previously in autosomal recessive nonsyndromic RP, raising the possibility of a digenic model in this family. Functional testing for two key phenotypes of the patient in zebrafish showed that the combinatorial suppression of rp1l1 and c2orf71l induced discrete pathology in terms of reduction of eye size with concomitant loss of rhodopsin in the photoreceptors, and disorganization of the cerebellum.

Conclusions: We propose that the combination of heterozygous loss-of-function mutations in these genes drives syndromic retinitis pigmentosa, likely through the genetic interaction of at least two loci, whereas haploinsuffiency of each is insufficient to induce overt pathology.