Decoding pseudoexon activation by deep intronic variants using a high-throughput minigene assay in the NF1 gene
- 1Molecular Genetics Laboratory, Centre for Cardiovascular Surgery and Transplantation
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.
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BibTeX
@misc{svobodova2026decoding,
title = {{Decoding pseudoexon activation by deep intronic variants using a high-throughput minigene assay in the NF1 gene}},
author = {E. Svobodova and P. Soucek and K. Reblova and Z. Kubat and M. Kratka and T. Freiberger and L. Kajan Grodecka},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.20.760351},
url = {https://www.biorxiv.org/content/10.64898/2026.10.20.760351v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Decoding pseudoexon activation by deep intronic variants using a high-throughput minigene assay in the NF1 gene
AU - E. Svobodova
AU - P. Soucek
AU - K. Reblova
AU - Z. Kubat
AU - M. Kratka
AU - T. Freiberger
AU - L. Kajan Grodecka
PY - 2026
DA - 2026/10/20
PB - bioRxiv
DO - 10.64898/2026.10.20.760351
UR - https://www.biorxiv.org/content/10.64898/2026.10.20.760351v1
AB - 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.
ER -