# Generation of functional noncanonical donor splice sites by +2T variants in breast cancer susceptibility genes: impact on clinical interpretation

## TL;DR

This study shows how certain genetic variants in breast cancer genes can create functional splicing sites, leading to normal protein production and affecting how these variants are clinically interpreted.

## Contribution

The study identifies specific +2T variants in ATM, BRCA1, and PALB2 that generate functional noncanonical splice sites, impacting clinical classification.

## Key findings

- Four +2T > C variants and ATM c.6347+2T > A/G produced functional full-length transcripts.
- Two variants were reclassified from likely pathogenic to variant of uncertain significance.
- Functional noncanonical splice sites may preserve gene function despite initial pathogenic predictions.

## Abstract

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.