Functional annotation of variants of the BRCA2 gene via locally haploid human pluripotent stem cells

Human pluripotent stem cells with one copy of the BRCA2 gene deleted can be used to annotate variants of the gene and to test their sensitivities to inhibition by the poly(ADP-ribose) polymerase. Mutations in the BRCA2 gene are associated with sporadic and familial cancer, cause genomic instability and sensitize cancer cells to inhibition by the poly(ADP-ribose) polymerase (PARP). Here we show that human pluripotent stem cells (hPSCs) with one copy of BRCA2 deleted can be used to annotate variants of this gene and to test their sensitivities to PARP inhibition. By using Cas9 to edit the functional BRCA2 allele in the locally haploid hPSCs and in fibroblasts differentiated from them, we characterized essential regions in the gene to identify permissive and loss-of-function mutations. We also used Cas9 to directly test the function of individual amino acids, including amino acids encoded by clinical BRCA2 variants of uncertain significance, and identified alleles that are sensitive to PARP inhibitors used as a standard of care in BRCA2-deficient cancers. Locally haploid human pluripotent stem cells can facilitate detailed structure-function analyses of genes and the rapid functional evaluation of clinically observed mutations.

Automated summary

By deleting one copy of the BRCA2 gene, human pluripotent stem cells can be used to annotate gene variants and test their sensitivity to poly(ADP-ribose) polymerase inhibition. Mutations in the BRCA2 gene are associated with sporadic and familial cancer, causing genomic instability and sensitizing cancer cells to poly(ADP-ribose) polymerase inhibition. The deletion of one copy of BRCA2 in human pluripotent stem cells allows for detailed characterization of essential regions in the gene and evaluation of clinical BRCA2 variants' sensitivity to poly(ADP-ribose) polymerase inhibitors.