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

Hanqin Li, Rebecca Bartke, Lei Zhao, Yogendra Verma, Anna Horacek, Alma Rechav Ben-Natan, Gabriella R. Pangilinan, Netravathi Krishnappa, Rasmus Nielsen, Dirk Hockemeyer*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

2 Citations (Scopus)
4 Downloads (Pure)

Abstract

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.

Original languageEnglish
JournalNature Biomedical Engineering
Volume8
Issue number2
Pages (from-to)165-176
DOIs
Publication statusPublished - 2024

Bibliographical note

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

Cite this