Decoding pseudoexon activation by deep intronic variants using a high-throughput minigene assay in the NF1 gene

Abstract

Deep intronic variants can cause aberrant splicing, often manifested as pseudoexon inclusion, which represents a significant challenge in clinical diagnostics. Pseudoexon activations have been repeatedly described in the NF1 gene, making it a promising genomic context for studying underlying mechanisms. Here, we developed a specialized minigene assay by combining high-throughput mutagenesis and next-generation sequencing to evaluate the splicing effect of 501 deep intronic variants within NF1 intron 3. We identified and validated eleven pseudoexon-activating variants, including seven novel ones, which clustered into four activation-prone hotspots. Additionally, we detected a rare, out-of-frame wild-type pseudoexon whose inclusion was significantly enhanced by three specific mutations. Using this experimentally validated dataset, we benchmarked SpliceAI performance and ESRseq scores to refine variant prioritization. This integrated high-throughput framework offers a robust starting point for the systematic functional prioritization of deep intronic variation, providing a scalable model for variant annotation in other disease-associated genes.