Whole genome analysis of inherited retinal disease patients reveals mutations intractable to other detection strategies

Abstract

Purpose : To identify and analyse pathogenic variants in whole genome sequencing (WGS) data from a cohort of patients with inherited retinal disease (IRD).

Methods : Patients were recruited for WGS studies from the inherited eye disease clinics at Moorfields Eye Hospital, London, UK. 599 singletons and 250 families (trios or duos) underwent WGS using the Illumina Truseq PCR-free kit on an Illumina Hiseq 2500 generating a minimum coverage of 15x for >95% of the genome. The Illumina Isaac pipeline including CANVAS and MANTA algorithms were used for single nucleotide (SNV), indel and structural variant (SV) calling.

Candidate pathogenic non-coding variants were investigated in patients unsolved by prior IRD gene exonic variant analysis and focusing on those patients in whom there was evidence for a single gene (eg. suggestive clinical phenotype, carrier of single mutation in a recessive gene). Candidate intronic variants underwent in silico cryptic splicing analysis and likely pathogenic alleles were selected for functional testing.

Results : 225/599 families remained unsolved after exonic SNV and SV analysis in a panel of 224 known and candidate IRD genes and together harbored 91,758 ultra-rare variants (MAF <0.0005) across the entire gene panel.

Likely pathogenic intronic variants identified included a CRB1 variant (c.3879-1203C>G) found in trans with a previously reported coding mutation, in a family of three affected siblings with clinically suspected CRB1-retinopathy. A second similarly affected proband was found to harbor the same deep intronic mutation in trans with a different reported mutation. Splice prediction analysis indicates this variant may activate a cryptic splice donor site leading to a 559bp pseudoexon.

Additional likely pathogenic intronic variants were found in ABCA4, USH2A, PRPF31 and a deletion spanning the untranslated first exon of BEST1 (chr11:61711370_61719807del) undetected by prior screening and in trans with a coding mutation in a recessive family.

Conclusions : WGS enables interrogation of variants on a scale never before possible. We report potential pathogenic mutations in IRD genes intractable to other mutation detection strategies. In silico and in vitro functional investigation (where possible) confirms the pathogenicity of these candidate mutations.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.