Combining a prioritization strategy and functional studies nominates 5'UTR variants underlying inherited retinal disease

Background5 untranslated regions (5UTRs) are essential modulators of protein translation. Predicting the impact of 5UTR variants is challenging and typically not performed in routine diagnostics. Here, we present a combined approach of a comprehensive prioritization strategy and subsequent functional assays to evaluate 5UTR variation in two large cohorts of patients with inherited retinal diseases (IRDs). MethodsWe performed an isoform-level re-analysis of retinal RNA-seq data to identify the protein-coding transcripts of 378 IRD genes with highest expression in retina. We evaluated the coverage of these 5UTRs by different whole exome sequencing (WES) capture kits. The selected 5UTRs were analyzed in whole genome sequencing (WGS) and WES data from IRD sub-cohorts from the 100,000 Genomes Project (n = 2,417 WGS) and an in-house database (n = 1,682 WES), respectively. Identified variants were annotated for 5UTR-relevant features and classified into 7 distinct categories based on their predicted functional consequence. We developed a variant prioritization strategy by integrating population frequency, specific criteria for each category, and family and phenotypic data. A selection of candidate variants underwent functional validation using diverse experimental approaches. ResultsIsoform-level re-quantification of retinal gene expression revealed 76 IRD genes with a non-canonical retina-enriched isoform, of which 20 display a fully distinct 5UTR compared to that of their canonical isoform. Depending on the probe-design 3-20% of IRD genes have 5UTRs fully captured by WES. After analyzing these regions in both IRD cohorts we prioritized 11 (likely) pathogenic variants in 10 genes (ARL3, MERTK, NDP, NMNAT1, NPHP4, PAX6, PRPF31, PRPF4, RDH12, RD3), of which 8 were novel. Functional analyses further supported the pathogenicity of 2 variants. The MERTK:c.-125G>A variant, overlapping a transcriptional start site, was shown to significantly reduce both luciferase mRNA levels and activity. The RDH12:c.-123C>T variant was found in cis with the reported hypomorphic RDH12:c.701G>A (p.Arg234His) variant in 11 patients. This 5UTR variant, predicted to introduce an upstream open reading frame, was shown to result in reduced RDH12 protein but unaltered mRNA levels. ConclusionsThis study demonstrates the importance of 5UTR variants implicated in IRDs and provides a systematic approach for 5UTR annotation and validation that is applicable to other inherited diseases.