Mapping intronic mutational hotspots by in silico mutagenesis enables single antisense oligonucleotide correction of multiple variants
Year: 2026
Type: preprint
Abstract: Deep intronic variants remain an understudied class of pathogenic variation, primarily due to their absence from standard exome and gene panel datasets and the complexity of non-coding genome interpretation. We hypothesized that pathogenic deep intronic variants are not randomly distributed but instead cluster within intronic “hotspots” inherently prone to pseudoexon activation, and that mapping such regions could improve molecular diagnosis. Importantly, such intronic hotspots nominate targets for antisense oligonucleotide (ASO) therapy. Using X-linked Alport syndrome as a proof-of-concept model and based on our previous work, we screened unsolved patients across multiple European diagnostic centers for variants within a defined region of COL4A5 intron 6. We identified eight independent variants in more than 35 affected individuals from ten unrelated families, which led to two pseudoexon inclusion events, both using the same strong cryptic splice donor site. In all, RNA sequencing and/or minigene assays confirmed aberrant splicing, even when prediction tools were discordant or fell below clinical thresholds. A single ASO targeting the shared donor site restored normal COL4A5 mRNA and α5(IV) collagen protein expression in patient-derived cells regardless of the causative variant. Extending this analysis gene-wide using the AlphaGenome sequence-to-function model, we confirmed intron 6 as one of the most critical COL4A5 splicing hotspot and identified additional potential hotspots harboring novel predicted spliceogenic variants. This gene-agnostic framework establishes a systematic strategy for identifying intronic mutational hotspots and matching patients to scalable, mutation-agnostic ASO-based precision therapies. Significance Statement Deep 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.
Source: medRxiv
Authors: Hassan Saei, Béatrice Ardin, Nicolas Kaiser, Mouad Ouahmane, Olivier Gribouval, Vincent Moriniere, Manon Mautret-Godefroy, Florian J. Wopperer, Korbinian M. Riedhammer, Daniel P. Gale, Omid Sadeghi-Alavijeh, Claire Goursaud, Olivier Grunewald, Chloe Prosper, Louis Lebreton, Marion Rabant, Carsten Bergmann, Raphael Kormann, Stephane Decramer, Bertrand Knebelmann, Corinne Antignac, Michael S. Wiesener, Guillaume Dorval
Institutions: Inserm, Université Paris Cité, Institut des Maladies Génétiques Imagine, Friedrich-Alexander-Universität Erlangen-Nürnberg, Universitätsklinikum Erlangen, Hôpital Necker-Enfants Malades, Assistance Publique – Hôpitaux de Paris, TUM Klinikum, Institut für Humangenetik, Technical University of Munich, Boston Children's Hospital, Harvard University, University College London, The Francis Crick Institute, Hospices Civils de Lyon, Centre Hospitalier Universitaire de Lille, Centre Hospitalier Universitaire de Bordeaux, Centre National de la Recherche Scientifique, Institut Necker Enfants Malades, Centre Hospitalier Régional et Universitaire de Nancy, Université Toulouse III - Paul Sabatier, Centre Hospitalier Universitaire de Toulouse, Hôpital Purpan
Language: English
Topic: Genomics and Rare Diseases
Subfield: Genetics
Domain: Life Sciences
Open Access status: green
Funders: DeepMind, Deutsche Forschungsgemeinschaft
OpenAlex ID: W7213325327