Functional Analysis of Human Breast Cancer Susceptibility Gene Variants

Division Of Basic Sciences - Nci

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Abstract

Variants of unknown significance (VUS) limit the clinical usefulness of genetic information, particularly for cancer-predisposing DNA repair genes. To bridge this significant gap, functional assays are developed to assign a clinical significance to these VUSs. We previously developed the mouse embryonic stem cell-based functional assay to classify genetic variants of human BRCA2 (Kuznetsov et al., Nature medicine 2008) which led to the classification of over 400 variants to date. We generated variants in human BRCA2 cloned in a bacterial artificial chromosome (BAC) by recombineering and expressed them in mouse embryonic stem cells (mESC) to examine their functional impact. While this approach has proven to be reliable and resulted in functional classification of variants with a high level of concordance with functional assays developed by other labs, multiplexing a large number of variants is a limitation. We have developed a humanized mouse embryonic stem cell line (mESC) expressing a single copy of the human BRCA2 for a CRISPR-Cas9-based high-throughput functional assay (Sahu, PLoS genetics, 2023). We have used CRISPR-Cas9-based saturation genome editing approach to successfully classify 6551 variants in one of the key functional domain of BRCA2 spanning exons 15-26.