Splicing Predictions, Splicing Assays, and Variant Classification Using ACMG/AMP Guidelines: Challenges Observed with...

Area of Science:

• Genetics
• Oncology
• Molecular Biology

Background:

• Germline variants in BRCA1 and BRCA2 genes are key drivers of hereditary breast and ovarian cancers.
• Aberrant splicing is a common mechanism, but classifying spliceogenic variants is challenging.
• Many variants remain of uncertain significance due to limited experimental data and interpretation framework limitations.

Purpose of the Study:

• To investigate the splicing outcomes of 17 specific variants in BRCA1 and BRCA2 genes.
• To determine the pathogenicity of these variants in families with suspected hereditary breast and/or ovarian cancer.
• To refine variant classification and improve genetic risk assessment.

Main Methods:

• Examined splicing outcomes of 10 BRCA1 and 7 BRCA2 variants using carrier-derived mRNA or splicing-reporter minigene assays.
• Applied the PVS1_(RNA) criterion based on ACMG/AMP guidelines.
• Combined experimental findings with existing data for variant interpretation.

Main Results:

• Eight variants demonstrated aberrant splicing, while nine showed no spliceogenic effect.
• Full application of the PVS1_(RNA) criterion was sometimes precluded by residual full-length transcripts or in-frame mis-spliced isoforms.
• Classified variants into pathogenic, likely pathogenic, benign, likely benign, and uncertain significance categories.

Conclusions:

• Classified 4 variants as pathogenic/likely pathogenic, 10 as benign/likely benign, and 3 as uncertain significance.
• Demonstrated the value of combining experimental and computational evidence for variant interpretation.
• Refined the clinical classification of BRCA1 and BRCA2 variants, enhancing hereditary cancer risk assessment.