Mapping intronic mutational hotspots by in silico mutagenesis enables single antisense oligonucleotide correction of multiple variants

The study focused on deep intronic variants, which are rarely studied and often missing from standard genetic tests. Researchers discovered that pathogenic intronic variants are not randomly distributed but clustered in so-called mutational hotspots prone to pseudoexon activation. They used X-linked Alport syndrome as a model and examined the COL4A5 gene. They identified eight independent variants in more than 35 affected individuals from ten unrelated families, leading to two pseudoexon inclusion events. A single antisense oligonucleotide targeting the shared donor site restored normal mRNA and collagen protein expression in patient cells regardless of the specific variant. Using the AlphaGenome model, they confirmed intron 6 as one of the most critical splicing hotspots in COL4A5 and identified additional potential hotspots. This approach represents a systematic strategy for identifying intronic mutational hotspots and matching patients to precision therapies based on antisense oligonucleotides.