Hidden Splicing Variants in Inherited Retinal Degeneration: Discovery and Functional Insight

To enhance the molecular diagnosis of inherited retinal degeneration (IRD) by systematically identifying pathogenic splicing variants and characterizing their transcript-level consequences.

We analyzed 738 IRD families who underwent targeted gene panel sequencing. A splicing variant detection pipeline, integrating two computational algorithms (SpliceAI and dbscSNV_ADA) with functional validation via minigene assays, was implemented to detect splice-disrupting variants beyond canonical sites.

Splicing variants accounted for 14% of genetically diagnosed families. Of these, 4% were newly identified through our combined computational and experimental platform. Notably, 28% of all splice-disrupting variants, located in noncanonical, exonic, or deep-intronic regions, would likely have been missed by conventional analysis pipelines, which typically prioritize protein-coding changes and canonical splice sites, and often lack systematic evaluation of splicing effects beyond these regions. Five recurrent splice-disrupting variants were observed across multiple families, including EYS:c.5644+5G>A, which caused exon truncation and was found in 11 unrelated families. Functional assays confirmed aberrant splicing, and the associated phenotypes were consistent with known disease presentations.

This study demonstrates the utility of a combined splicing variant detection platform in uncovering hidden pathogenic variants and improving IRD diagnostic yield. These findings have implications for refining genetic testing and guiding the development of splicing-targeted therapies.