Functional assessment of putative splice-altering variants detected in an Irish inherited retinal disease cohort

Abstract

Purpose : Aberrant RNA splicing has emerged as a significant cause of inherited retinal diseases (IRDs). Next generation sequencing of IRD patients living in Ireland and utilisation of in silico splice prediction tools have enabled identification of putative splice-altering variants in IRD-associated genes. The aim of this study was to experimentally validate several of these variants using in vitro splice assays.

Methods : Probands underwent target capture sequencing of exons and canonical/noncanonical splice site regions of 254 IRD-associated genes. Putative splice-altering variants were identified and prioritised for further analysis using in silico splice prediction tools, including SpliceAI and Alamut Visual software. Genomic segments containing the variant of interest or wild-type sequence and flanking exons were amplified, cloned into midigene plasmid vectors and transfected into HEK 293 cells. mRNA was isolated 48 hours post-transfection and transcripts were analysed via RT-PCR, gel electrophoresis and Sanger sequencing.

Results : Midigene splice assays were used to evaluate the consequences of OPA1 c.1681+1G>T (NM_130837.2) and FLVCR1 c.1307+5G>T (NM_014053.3), as well as previously unreported variants in TULP1 (NM_003322.4:c.999+5G>T) and CACNA1F (NM_005183.3: c.2576+4_2576+5delAG). Transfection of a wild-type OPA1 midigene resulted in a single mRNA product of expected length while the mutant construct exclusively produced a shorter fragment in which exon 17 was skipped, leading to a frameshift (p.Ile537Glyfs*10). A wild-type FLVCR1 midigene yielded a full-length transcript and an additional product lacking exon 6. In contrast, the mutant FLVCR1 midigene did not produce any detectable full-length transcript and appeared to increase skipping of exon 6, which causes an in-frame deletion of 37 amino acids. The novel TULP1 and CACNA1F variants tested appeared to induce complex aberrant splicing patterns and work is underway to elucidate the precise nature of these defects.

Conclusions : This study characterised splicing defects resulting from canonical and noncanonical splice site mutations in IRD-associated genes – providing new evidence in support of variant pathogenicity. Functional analysis of such variants is vital for accurate classification of pathogenicity and severity, and increasingly important for enabling access to emerging gene therapies.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.