TY - JOUR

T1 - Functional annotation of variants of the BRCA2 gene via locally haploid human pluripotent stem cells

AU - Li, Hanqin

AU - Bartke, Rebecca

AU - Zhao, Lei

AU - Verma, Yogendra

AU - Horacek, Anna

AU - Rechav Ben-Natan, Alma

AU - Pangilinan, Gabriella R.

AU - Krishnappa, Netravathi

AU - Nielsen, Rasmus

AU - Hockemeyer, Dirk

N1 - Publisher Copyright: © 2023, The Author(s).

PY - 2024

Y1 - 2024

N2 - 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.

AB - 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.

U2 - 10.1038/s41551-023-01065-7

DO - 10.1038/s41551-023-01065-7

M3 - Journal article

C2 - 37488236

AN - SCOPUS:85165564203

VL - 8

SP - 165

EP - 176

JO - Nature Biomedical Engineering

JF - Nature Biomedical Engineering

SN - 2157-846X

IS - 2

ER -