openRxiv

Significance StatementDeep intronic variants are an underrecognized cause of genetic disease, undetectable to standard exome and gene-panel testing and difficult to interpret. Using X-linked Alport syndrome as an exemplar, we identified a recurrent deep intronic mutational hotspot in which independent pathogenic variants from unrelated families converge on the same cryptic splice donor site to activate pseudoexon inclusion. A single antisense oligonucleotide therapy corrected this shared splicing defect and restored normal collagen IV protein production in patient cells, regardless of the underlying causative variant. Extending this analysis gene-wide using an AlphaGenome sequence-to-function splicing model, we uncovered additional deep intronic hotspots across the gene, establishing a gene-agnostic strategy for systematically identifying hidden pathogenic variants and matching patients to mutation-agnostic, RNA-based precision therapies in Mendelian disorders.