A multi-omics approach increases the diagnostic yield ‘beyond the exome’ in unsolved patients with inherited eye diseases

Abstract

Purpose : Exome sequencing (ES)-based testing in patients with inherited eye diseases (IED) has an overall diagnostic yield of ~60%. To overcome the missing heritability in molecularly undiagnosed ES-tested IED patients, we used a multi-omics approach, combining genomics, transcriptomics, gene-centric functional assays and reverse phenotyping.

Methods : Short-read genome sequencing (GS) was performed in 185 IED cases, with 111 cases (103 probands) analyzed using Franklin (Genoox) and Seqplorer (in-house). Coding single nucleotide variants (SNVs) in known IED genes were prioritized, followed by non-coding variants assessed for an effect on splicing (SpliceAI, Alamut) or cis-regulation (RegRet), and by structural variants (SVs) (Franklin). Candidate genes were selected based on single-cell expression profiles (CellxGene, Spectacle) and function. GS data directed whole-blood RNA-seq and/or in vitro minigene and reporter assays.

Results : A likely genetic diagnosis was obtained for 44.5% of probands. Genomic data combined with tailored functional assessment pinpointed likely pathogenic non-coding variants in 18.5%, predominantly affecting splicing (16.5%). First, OPA1 variant c.1608+622A>G, leading to pseudo-exon inclusion (PEI), explained optic atrophy in 11 individuals. Next, integrated GS, RNA-seq and minigene assays supported the interpretation of a novel OPA1 variant, leading to out-of-frame PEI. A first deep-intronic ALMS1 variant and a novel deep-intronic RPGRIP1 variant, both identified in monoallelic cases, were also shown to lead to PEI using minigene assays. In vitro reporter assays demonstrated a reduced (40%) regulatory activity of novel promotor variant RPGRIP1 c.-152A>C, predicted to disrupt OTX2 binding. Variants in novel candidate genes, such as SAMD7 and GPATCH11, were identified in 14.5%, while coding SNVs (8.5%) and SVs (3%) impacting known IED genes were found in 11.5%.

Conclusions : A multi-omics approach solved missing heritability in 44.5% of molecularly undiagnosed IED cases, mainly attributed to non-coding deep-intronic splicing variants and variants in novel genes. Overall, its added diagnostic value urges its implementation in standard-of-care genetic testing 'beyond the exome’ of IED.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.