Expression of human BRCA1 variants in mouse ES cells allows functional analysis of BRCA1 mutations
Suhwan Chang, … , Stacey Stauffer, Shyam K. Sharan
Suhwan Chang, … , Stacey Stauffer, Shyam K. Sharan
Published September 21, 2009
Citation Information: J Clin Invest. 2009;119(10):3160-3171. https://doi.org/10.1172/JCI39836.
View: Text | PDF
Technical Advance

Expression of human BRCA1 variants in mouse ES cells allows functional analysis of BRCA1 mutations

Abstract

To date, inheritance of a mutant BRCA1 or BRCA2 gene is the best-established indicator of an increased risk of developing breast cancer. Sequence analysis of these genes is being used to identify BRCA1/2 mutation carriers, though these efforts are hampered by the high frequency of variants of unknown clinical significance (VUSs). Functional evaluation of such variants has been restricted due to lack of a physiologically relevant assay. In this study we developed a functional assay using mouse ES cells to study variants of BRCA1. We introduced BAC clones with human wild-type BRCA1 or variants into Brca1-null ES cells and confirmed that only wild-type and a known neutral variant rescued cell lethality. The same neutral variant was also able to rescue embryogenesis in Brca1-null mice. A test of several BRCT domain mutants revealed all to be deleterious, including a VUS. Furthermore, we used this assay to determine the effects of BRCA1 variants on cell cycle regulation, differentiation, and genomic stability. Importantly, we discovered that ES cells rescued by S1497A BRCA1 exhibited significant hypersensitivity after γ-irradiation. Our results demonstrate that this ES cell–based assay is a powerful and reliable method for analyzing the functional impact of BRCA1 variants, which we believe could be used to determine which patients may require preventative treatments.

Authors

Suhwan Chang, Kajal Biswas, Betty K. Martin, Stacey Stauffer, Shyam K. Sharan

×

Figure 4

Role of S1497 residue of BRCA1 in irradiation-induced DNA damage repair.

Options: View larger image (or click on image) Download as PowerPoint
Role of S1497 residue of BRCA1 in irradiation-induced DNA damage repair....
(A) Relative survival after γ-irradiation (IR) of 2 independent clones expressing S1497A compared with ES cells expressing no transgene (PL2F8), WT BRCA1, and S1423/1524A double mutant. *P < 0.006 for WT and S1497A-1. Error bars represent ± SEM. *P < 0.006 (B) Western blot analysis of 3 clones expressing S1497A and 1 with WT BRCA1 using phospho-S1497 antibody (upper panel). The lower panel shows expression of BRCA1. Rabbit IgG was used as negative control (NC); HeLa cells were used as positive control (PC). (C) Clonogenic survival assay to examine hypersensitivity of S1497A ES cells in response to 0.2 and 0.5 Gy of IR. (D) Quantitation of the number of colonies in C. (E) Quantitation of TUNEL-positive ES cells expressing S1497A BRCA1 after IR (2 Gy). ES cells expressing WT BRCA1 were used as a control. Con, control, before IR. Error bars represent ± SEM. (F) Phosphorylation of the S1497 residue affects the hyperphosphorylation of BRCA1 after IR. Wild-type or S1497A cells were left untreated or pretreated with an inhibitor of Cdk2 kinases. After IR, BRCA1 phosphorylation was analyzed by IP–Western blotting with a BRCA1 antibody and a phospho-S1497–specific antibody. Densitometric quantitation of the S1497 residue is indicated below the left panel. HEK293 cells were used as a control to show the difference in mobility of phosphorylated and unphosphorylated BRCA1. (G) Quantitation of chromosomal abnormalities in response to IR in ES cells (n = 100) expressing WT or S1497A BRCA1. (H) Representative picture of metaphase spreads of WT and S1497A cells after IR. Inset shows magnified view of 3 chromosomal aberrations. The scale bar on the left applies to the insets; the scale bar on the right applies to the larger images.