Multidisciplinary team directed analysis of whole genome sequencing reveals pathogenic non-coding variants in molecularly undiagnosed inherited retinal dystrophies

Abstract

Purpose: To identify, using genome sequencing (GS), likely pathogenic non-coding variants in inherited retinal dystrophy (IRD) genes Methods: Patients with IRD were recruited to the study and underwent comprehensive ophthalmological evaluation and GS. The results of GS were investigated through virtual gene panel analysis and plausible pathogenic variants and clinical phenotype evaluated by multi-disciplinary team (MDT) discussion. For unsolved patients in whom a specific gene was suspected to harbour a missed pathogenic variant, targeted re-analysis of non-coding regions was performed on GS data. Candidate variants were functionally tested including by mRNA analysis, minigene and luciferase reporter assays. Results: Previously unreported, likely pathogenic, non-coding variants, in 7 genes (PRPF31, NDP, IFT140, CRB1, USH2A, BBS10, and GUCY2D), were identified in 11 patients. These were shown to lead to mis-splicing (PRPF31, IFT140, CRB1, USH2A) or altered transcription levels (BBS10, GUCY2D). Conclusion: MDT-led, phenotype driven, non-coding variant re-analysis of GS is effective in identifying missing causative alleles.

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

“…Four of these variants were found to alter splicing in genes linked to autosomal dominant ( PRPF31 ), autosomal recessive ( NMNAT1 ), and X-linked ( NDP ) disease. The NDP :c.-70G>A and PRPF31 :c.-9+1G>A variants have recently been described by Daich Varela et al (2023) [ 49 ]. In particular, the authors of this study also predicted a deleterious effect on splicing for these variants, which could not be functionally validated as it was not possible to obtain fresh samples from the patients for further analysis.…”

“…Given the emerging role of non-coding variation underlying IRDs [ 46 , 49 , 52 – 55 ] and the essential regulatory function of 5’UTRs [ 2 , 6 ], we set out to evaluate their contribution to this heterogeneous group of disorders by analyzing, systematically annotating, filtering, and prioritizing 5’UTR variants in two large IRD cohorts in combination with different experimental approaches for functional validation.…”

“…Thus far, only a few studies have implicated 5’UTR variation in the molecular pathogenesis of IRD cases [ 49 , 53 , 54 ]. Our retrospective analysis of 5’UTR variants in IRD genes listed in the ClinVar database [ 67 ] revealed that as many as 58% of all variants have been classified as VUS.…”

“…Recent studies have revealed an important contribution of non-coding variation to IRDs, particularly with the identification of deep-intronic mis-splicing variants [ 39 , 42 – 51 ]. Moreover, there is a growing body of evidence supporting the emerging role of genetic variation affecting cis -regulatory regions in the molecular pathogenesis of IRDs [ 46 , 49 , 52 – 56 ]. However, thus far, only a few 5’UTR variants have been reported to be implicated in ocular diseases, including IRDs [ 49 , 53 , 54 , 57 – 59 ], but no large-scale studies have been performed yet.…”

PHYH c.678+5G>T Leads to In-Frame Exon Skipping and Is Associated With Attenuated Refsum Disease

“…All individuals and their genetic testing results were discussed in multidisciplinary team meetings, other candidate genes and variants were ruled out, and PHYH :c.678+5G>T was selected for further investigation. 26 …”