Experimental Mis-Splicing Assessment and ACMG/AMP-Guided Classification of 47 ATM Splice-Site Variants

Area of Science:

• Genetics
• Molecular Biology
• Cancer Research

Background:

• Pathogenic germline variants in the ATM gene confer a 20-30% lifetime risk of breast cancer.
• A significant proportion of loss-of-function variants in breast cancer susceptibility genes are known to disrupt pre-mRNA splicing.

Purpose of the Study:

• To conduct splicing analysis of ATM splice-site variants identified in the BRIDGES (Breast Cancer After Diagnostic Gene Sequencing) project.
• To determine the functional impact of ATM splice-site variants on gene expression and splicing patterns.

Main Methods:

• Bioinformatically selected 47 ATM splice-site variants across 17 exons.
• Engineered variants into three minigenes and assayed them in MCF-7 cells.
• Characterized transcript variants using sequencing and bioinformatic analysis.

Main Results:

• Aberrant splicing was observed in 38 out of 47 variants.
• 30 variants, including 7 missense, resulted in no or negligible minigene full-length (mgFL) transcript expression.
• 69 different transcripts were identified, with 48 containing a premature termination codon; alternative 5' or 3' splice-site usage was common.

Conclusions:

• Splicing assays are crucial for interpreting the pathogenicity of ATM variants.
• Integration of minigene assay data with ACMG/AMP guidelines enabled classification of 30 variants as pathogenic/likely pathogenic and 9 as likely benign.
• These findings improve the clinical management of patients with potential ATM-related breast cancer risk.