Sequencing-based functional assays for classification of BRCA2 variants in mouse ESCs

Area of Science:

• Genetics
• Oncology
• Molecular Biology

Background:

• Genetic sequencing of BRCA1 and BRCA2 genes is crucial for individuals with a history of breast or ovarian cancer.
• Over 17,000 BRCA2 variants have been identified, but most lack known functional or clinical significance, termed variants of uncertain clinical significance (VUSs).

Purpose of the Study:

• To develop and validate a high-throughput, next-generation sequencing (NGS)-based assay for the functional classification of BRCA2 variants.
• To evaluate the clinical utility of this new assay by assessing a large set of BRCA2 variants from ClinVar.

Main Methods:

• Utilized a previously established mouse embryonic stem cell (mESC)-based assay for functional variant classification.
• Developed a novel NGS-based approach to functionally evaluate pooled mESCs, each expressing multiple (10-25) BRCA2 variants from specific exons.
• Applied this method to functionally assess 223 BRCA2 variants listed in the ClinVar database.

Main Results:

• Successfully implemented an NGS-based functional assay for evaluating numerous BRCA2 variants simultaneously.
• The functional classification of BRCA2 variants using this method demonstrated high concordance with existing classifications in ClinVar and other orthogonal assays.
• This approach provides a scalable and reliable method for assessing the functional impact of genetic variants.

Conclusions:

• The developed NGS-based mESC assay is effective for the functional classification of BRCA2 variants.
• This method aids in clarifying the clinical significance of variants of uncertain clinical significance (VUSs), potentially improving genetic risk assessment for hereditary cancers.
• The findings support the use of this assay for large-scale functional variant analysis in clinical genetics settings.