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

Authors

Svobodova, E., Soucek, P., Reblova, K., Kubat, Z., Kratka, M., Freiberger, T., Kajan Grodecka, L.

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.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 21 Oct 2026.

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