Functional evaluation and clinical classification of BRCA2 variants

• Published in: Nature (London) 2025-02, Vol.638 (8050), p.528-537
• Main Authors: Huang, Huaizhi, Hu, Chunling, Na, Jie, Hart, Steven N., Gnanaolivu, Rohan David, Abozaid, Mohamed, Rao, Tara, Tecleab, Yohannes A., Pesaran, Tina, Lyra, Paulo Cilas Morais, Karam, Rachid, Yadav, Siddhartha, Nathanson, Katherine L., Domchek, Susan M., de la Hoya, Miguel, Robson, Mark, Mehine, Miika, Bandlamudi, Chaitanya, Mandelker, Diana, Monteiro, Alvaro N. A., Iversen, Edwin S., Boddicker, Nicholas, Chen, Wenan, Richardson, Marcy E., Couch, Fergus J.
• Format: Article
• Language: English
• Subjects: 13; 13/106; 45; 45/70; 631/67/1347; 631/67/68; 692/699/67/1347; Bayes Theorem; BRCA2 Protein - genetics; Breast Neoplasms - genetics; Breast Neoplasms - pathology; Cell Line; CRISPR-Cas Systems - genetics; Exons - genetics; Female; Gene Editing; Genetic Predisposition to Disease - genetics; Humanities and Social Sciences; Humans; Loss of Function Mutation - genetics; multidisciplinary; Mutation, Missense; Ovarian Neoplasms - genetics; Polymorphism, Single Nucleotide - genetics; Reproducibility of Results; Science; Science (multidisciplinary)
• ISSN: 0028-0836; 1476-4687; 1476-4687
• DOI: 10.1038/s41586-024-08388-8

Germline BRCA2 loss-of function variants, which can be identified through clinical genetic testing, predispose to several cancers 1 , 2 , 3 , 4 – 5 . However, variants of uncertain significance limit the clinical utility of test results. Thus, there is a need for functional characterization and clinical classification of all BRCA2 variants to facilitate the clinical management of individuals with these variants. Here we analysed all possible single-nucleotide variants from exons 15 to 26 that encode the BRCA2 DNA-binding domain hotspot for pathogenic missense variants. To enable this, we used saturation genome editing CRISPR–Cas9-based knock-in endogenous targeting of human haploid HAP1 cells 6 . The assay was calibrated relative to nonsense and silent variants and was validated using pathogenic and benign standards from ClinVar and results from a homology-directed repair functional assay 7 . Variants (6,959 out of 6,960 evaluated) were assigned to seven categories of pathogenicity based on a VarCall Bayesian model 8 . Single-nucleotide variants that encode loss-of-function missense variants were associated with increased risks of breast cancer and ovarian cancer. The functional assay results were integrated into models from ClinGen, the American College of Medical Genetics and Genomics, and the Association for Molecular Pathology 9 for clinical classification of BRCA2 variants. Using this approach, 91% were classified as pathogenic or likely pathogenic or as benign or likely benign. These classified variants can be used to improve clinical management of individuals with a BRCA2 variant. Results from a comprehensive evaluation of the function of BRCA2 variants, particularly variants of uncertain significance, provide a useful resource to improve the clinical management of individuals who carry such genetic variants.