Splicing is controlled by a large set of regulatory elements (SREs) including splicing enhancers and silencers, which are involved in exon recognition. Variants at these motifs may dysregulate splicing and trigger loss‐of‐function transcripts associated with disease. Our goal here was to study the alternatively spliced exons 8 and 10 of the breast cancer susceptibility gene CHEK2 . For this purpose, we used a previously published minigene with exons 6–10 that produced the expected minigene full‐length transcript and replicated the naturally occurring events of exon 8 [Δ(E8)] and exon 10 [Δ(E10)] skipping. We then introduced 12 internal microdeletions of exons 8 and 10 by mutagenesis in order to map SRE‐rich intervals by splicing assays in MCF‐7 cells. We identified three minimal (10‐, 11‐, 15‐nt) regions essential for exon recognition: c.863_877del [ex8, Δ(E8): 75%] and c.1073_1083del and c.1083_1092del [ex10, Δ(E10): 97% and 62%, respectively]. Then 87 variants found within these intervals were introduced into the wild‐type minigene and tested functionally. Thirty‐eight of them (44%) impaired splicing, four of which (c.883G>A, c.883G>T, c.884A>T, and c.1080G>T) induced negligible amounts (A, c.886G>T, c.1075G>A, c.1075G>T, c.1076A>T, and c.1078G>T) showed significantly strong impacts (20–50% of the minigene full‐length transcript). Thirty‐three of the 38 spliceogenic variants were annotated as missense, three as nonsense, and two as synonymous, underlying the fact that any exonic change is capable of disrupting splicing. Moreover, c.883G>A, c.883G>T, and c.884A>T were classified as pathogenic/likely pathogenic variants according to ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)‐based criteria. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Background/Objectives: The non-canonical GC-5′ splice sites (5′ss) are the most common exception (~1%) to the classical GT/AG splicing rule. They constitute weak 5′ss and can be regulated by splicing factors, so they are especially sensitive to genetic variations inducing the misrecognition of their respective exons. We aimed to investigate the GC-5′ss of the breast/ovarian cancer susceptibility genes, ATM (exon 50), BRIP1 (exon 1), and PALB2 (exon 12), and their dysregulation induced by DNA variants. Methods: Splicing assays of the minigenes, mgATM_49-52, mgBRIP1_1-2, and mgPALB2_5-12, were conducted to study the regulation of the indicated GC-5′ss. Results: A functional map of the splicing regulatory elements (SRE) formed by overlapping exonic microdeletions revealed three essential intervals, ATM c.7335_7344del, PALB2 c.3229_3258del, and c.3293_3322del, which are likely targets for spliceogenic SRE-variants. We then selected 14 ATM and 9 PALB2 variants (Hexplorer score < −40) located at these intervals that were assayed in MCF-7 cells. Nine ATM and three PALB2 variants affected splicing, impairing the recognition of exons 50 and 12, respectively. Therefore, these variants likely disrupt the active SREs involved in the inclusion of both exons in the mature mRNA. DeepCLIP predictions suggested the participation of several splicing factors in exon recognition, including SRSF1, SRSF2, and SRSF7, involved in the recognition of other GC sites. The ATM spliceogenic variants c.7336G>T (p.(Glu2446Ter)) and c.7340T>A (p.(Leu2447Ter)) produced significant amounts of full-length transcripts (55–59%), which include premature termination stop codons, so they would inactivate ATM through both splicing disruption and protein truncation mechanisms. Conclusions: ATM exon 50 and PALB2 exon 12 require specific sequences for efficient recognition by the splicing machinery. The mapping of SRE-rich intervals in minigenes is a valuable approach for the identification of spliceogenic variants that outperforms any prediction software. Indeed, 12 spliceogenic SRE-variants were identified in the critical intervals.

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 mgATM_4-9 (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 mgATM_4-9 significantly increased △(E7) (57–96%), revealing their critical role for exon 7 inclusion. Forty-eight candidate variants (HEXplorer, △HZEI score < -40) within these SRE-rich segments were functionally assayed, 34 of which (71%) impaired exon 7 recognition. Nineteen variants presented strong impacts with high expression of △(E7)-transcripts (69–96%), of which c.668A > 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.

Pathogenic germline variants in the ATM gene are associated with a 20–30% lifetime risk of breast cancer. Crucially, a relevant fraction of loss-of-function variants in breast cancer susceptibility genes disrupts pre-mRNA splicing. We aimed to perform splicing analysis of ATM splice-site variants identified in the large-scale sequencing project BRIDGES (Breast Cancer After Diagnostic Gene Sequencing). To this end, we bioinformatically selected 47 splice-site variants across 17 exons that were genetically engineered into three minigenes and assayed in MCF-7 cells. Aberrant splicing was observed in 38 variants. Of these, 30 variants, including 7 missense, yielded no or negligible expression of the minigene full-length (mgFL) transcript. A total of 69 different transcripts were characterized, 48 of which harboured a premature termination codon. Some variants, such as c.2922-1G>A, generated complex patterns with up to 10 different transcripts. Alternative 3′ or 5′ splice-site usage was the predominant event. Integration of ATM minigene read-outs into the ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based specifications for the ATM gene enabled the classification of 30 ATM variants as pathogenic or likely pathogenic and 9 as likely benign. Overall, splicing assays provide key information for variant interpretation and the clinical management of patients.

Splicing dysregulation is a relevant mechanism of pathogenicity for variants in disease susceptibility genes. Variants affecting the critical intronic +1 and +2 GT nucleotides of the 5’ splice sites (5'ss) are generally strong indicators of pathogenicity. However, some +2 T variants create functional noncanonical 5'ss that generate wildtype transcripts, hampering accurate variant interpretation and genetic counseling. We previously showed that variants PALB2 c.108+2T > C and ATM c.1898+2T > G generated significant levels of full‐length (FL) transcripts by creating functional atypical GC and GG donor sites, respectively. In this study, we aimed to investigate the splicing impact of +2T variants in the breast cancer susceptibility genes ATM , BRCA1 , and PALB2 . For this purpose, five minigenes encompassing 29 exons of ATM , BRCA1 , and PALB2 were employed. A total of 30 +2T > C/G/A variants were introduced into these constructs by site‐directed mutagenesis and analyzed in MCF‐7 cells. Four +2T > C variants ( ATM c.6347+2T > C, BRCA1 c.5193+2T > C and c.5277+2T > C, and PALB2 c.2748+2T > C) and ATM variants c.6347+2T > A/G produced FL‐transcripts (4%–81% of the overall expression). All +2T > C leaky variants conserved a central core of 6 nucleotides (AGgcaa). Variants were assessed according to the ClinGen specifications of the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) interpretation guidelines. Two variants ( ATM c.6347+2T > C and BRCA1 c.5193+2T > C) were classified as likely benign, consistent with predictions based on their respective ACMG/AMP‐based gene specifications. Conversely, two variants ( ATM c.6347+2T > G and BRCA1 c.4675+2T > C), initially predicted as likely pathogenic, were reclassified as variant of uncertain significance (VUS). In conclusion, a significant proportion of +2T variants can create functional noncanonical 5'ss, resulting in the production of FL‐transcripts that may preserve gene function. Variant‐splicing assays provide essential data for accurate clinical classification and for the development of effective clinical management strategies for patients and their families. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.