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This is a In Vitro Study study.

September 21, 2025Open Access

Unproductive alternative splicing of ATM exon 7: mapping of critical regulatory elements and identification of 34 spliceogenic variants

ILInés Llinares‐BurguetUniversidad de Valladolid LSLara Sanoguera‐MirallesInstituto de Biomedicina y Genética Molecular de Valladolid AGAlicia García‐ÁlvarezInstituto de Biomedicina y Genética Molecular de Valladolid

Key Points

ATM exon 7 skipping represents a form of gene-expression regulation via unproductive splicing.
Thirty-four out of 48 tested spliceogenic variants impaired exon 7 inclusion, potentially increasing cancer risk.
The study utilized an in silico/minigene approach to examine critical splicing regulatory elements affecting variant impact.
Eight variants were classified as likely pathogenic, which could inform genetic counseling and risk assessment.

Abstract

Abstract ATM loss-of-function variants are significantly associated with increased breast cancer risk. ATM exon 7 skipping (△ (E7) ) is a naturally-occurring alternative splicing event, which introduces a premature termination codon and represents a form of gene-expression regulation via unproductive splicing. Disruption of splicing regulatory elements (SRE) by variants can lead to mis-splicing, potentially contributing to disease susceptibility. To study the regulatory mechanisms of △ (E7) and the impact of exonic variants on splicing, a combined in silico/minigene approach was employed, using the construct mgATM₄-9 (exons 4–9) that recapitulates this splicing event. HEXplorer analysis of ATM exon 7 predicted two splicing enhancer-rich regions (c. 665–681 and c. 867–898). Deletions of these intervals in mgATM₄-9 significantly increased △ (E7) (57–96%), revealing their critical role for exon 7 inclusion. Forty-eight candidate variants (HEXplorer, △HZEI score T, c. 680C > A and c. 680C > T exhibited particularly strong effects (4–13% full-length transcripts). DeepCLIP analysis suggested that SR proteins SRSF7 and SRSF10 play a positive regulatory role in exon 7 inclusion. Eight variants were classified as likely pathogenic according to ACMG/AMP-based guidelines. Furthermore, nine missense and two synonymous variants with strong impacts (16–29% full-length transcripts) might represent intermediate risk alleles. This work collectively demonstrates the intricate nature of ATM exon 7 recognition, regulated by cis -acting SREs, emphasizing the value of in silico predictions for initial variant filtering and minigene assays for dissecting splicing regulation and clinical interpretation of variants. Key Messages Frameshift ATM exon 7 skipping is an unproductive alternative splicing event that may represent a form of gene-expression regulation. Two SRE-rich intervals are involved in exon 7 recognition. Thirty-four out of 48 tested variants located at these intervals disrupted exon 7 inclusion. Nineteen variants showed strong impacts on splicing. All variant types can impair splicing: 23 missense, 7 nonsense, and 4 synonymous variants upregulate exon 7 skipping. Eight variants were classified as likely pathogenic.

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