High-throughput splicing assay assesses pathogenicity of deep intronic variants in inherited retinal disease

A preprint posted to bioRxiv presents results from a high-throughput splicing assay (HTSA) designed to evaluate the pathogenicity of deep intronic variants in genes associated with recessively inherited retinal diseases (IRDs). Deep intronic variants — sequence changes located far from canonical splice sites — are increasingly recognised as a significant cause of Mendelian disorders because they can activate cryptic exons and thereby disrupt normal pre-mRNA splicing. The authors estimate that such variants may account for approximately 20% of IRD cases.

The research team tested 640 rare deep intronic variants drawn from 76 patients who carried one confirmed pathogenic variant but lacked a second identifiable hit on the same gene — a common diagnostic impasse in recessive disease. Each candidate variant was placed in trans (on the opposite chromosome copy) with the known pathogenic allele, and splicing outcomes were measured using the HTSA system.

The authors report that the assay successfully classified a substantial proportion of the tested variants, identifying those that activate cryptic exons and those that do not affect splicing. The work is directly relevant to variant classification pipelines used in clinical genetics laboratories and has implications for the interpretation of variants of uncertain significance (VUS) in diagnostic whole-genome sequencing. The manuscript has not yet undergone peer review; results should be interpreted accordingly.