In vitro pathogenicity evaluation of deep intronic variants for recessive genetic retinal diseases.

PURPOSE: Deep intronic variants (DIVs) that activate cryptic exons (CEs) cause ∼20% of inherited retinal disease (IRD) cases, yet their pathogenicity remains difficult to predict. We measured the splicing effects of rare DIVs in patients with recessive IRD who each carry one confirmed pathogenic allele.

METHODS

We tested 640 rare DIVs from 76 patients using a high-throughput splicing assay (HTSA): a split-GFP minigene separated by an SMN1 gene intron into which 270-bp sequences flanking each DIV were cloned. The plasmid library was transfected into HEK293T cells, minigene transcripts were amplified by RT-PCR and sequenced, and intron sequences spliced between the two GFP exons were identified and quantified.

RESULTS

Ninety-eight variants activated a CE more than 100-fold relative to the matched reference oligo and were classified as pathogenic in this experimental setting, with validation in a longer genomic context; 26 variants were classified as VUS. Only 6 of 90 variants (6.6%) were predicted to be pathogenic by SpliceAI. The results confirmed the diagnosis for 50 of 78 patients (64%).

CONCLUSION

Overall, 19.4% of tested DIVs caused CE activation, indicating that a substantial portion of undiagnosed patients with recessive IRD carry a pathogenic intronic variant that current prediction algorithms miss.