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Mouse embryonic stem cell–based functional assay to evaluate mutations in BRCA2

Nature Medicine volume 14, pages 875–881 (2008)Cite this article

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Abstract

Individuals with mutations in breast cancer susceptibility genes BRCA1 and BRCA2 have up to an 80% risk of developing breast cancer by the age of 70. Sequencing-based genetic tests are now available to identify mutation carriers in an effort to reduce mortality through prevention and early diagnosis. However, lack of a suitable functional assay hinders the risk assessment of more than 1,900 BRCA1 and BRCA2 variants in the Breast Cancer Information Core database that do not clearly disrupt the gene product. We have established a simple, versatile and reliable assay to test for the functional significance of mutations in BRCA2 using mouse embryonic stem cells (ES cells) and bacterial artificial chromosomes and have used it to classify 17 sequence variants. The assay is based on the ability of human BRCA2 to complement the loss of endogenous Brca2 in mouse ES cells. This technique may also serve as a paradigm for functional analysis of mutations found in other genes linked to human diseases.

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Figure 1: BRCA2 mutation analysis in mouse embryonic stem cells.
Figure 2: Mutant phenotypes of Y3308X and other BRCA2 variants in ES cells.
Figure 3: Analysis of BRCA2 mutants affecting splicing or domain structure.
Figure 4: Evaluation of BRCA2 variants.

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Change history

  • 11 July 2008

    In the version of this article initially published online, the online publication date was listed in correctly as 6 July 2007 on the PDF. The error has been corrected for all versions of this article.

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Acknowledgements

We thank J. Acharya, K. Biswas, S. Chang, I. Daar, G. Merlino, A. Nussenzweig, S. Philip and L. Tessarollo for helpful discussions and critical review of the manuscript. We also thank A. Deffenbaugh for providing epidemiological data; B. Martin, S. Burkett, L. North and S. Stauffer for technical assistance; L. Cleveland for help with DNA sequencing; R. Frederickson and A. Kane for illustrations; D. Du (US National Cancer Institute–Frederick) for the Rosa26 genomic construct; K. Biswas (US National Cancer Institute–Frederick) for the Brca1 targeting construct; S. West (Cancer Research UK) for RAD51 antibody; and M. Lewandoski for helpful discussions. We thank C. Ware (University of Washington, Seattle) for providing human ES cell pellet and J. Collins for help with computer modeling. The research was sponsored by the Center for Cancer Research, National Cancer Institute, US National Institutes of Health.

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Author notes

  1. Pentao Liu

    Present address: Current address: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1HH, UK.,

Authors and Affiliations

  1. Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute at Frederick, 1050 Boyles Street, Frederick, 21702, Maryland, USA

    Sergey G Kuznetsov, Pentao Liu & Shyam K Sharan

Authors
  1. Sergey G Kuznetsov
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Contributions

S.G.K. conducted all of the experiments and wrote the manuscript. P.L. helped with initial concept and generated selection marker cassettes. S.K.S. conceived the idea, generated the conditional ES cell line, supervised the study and wrote the manuscript.

Corresponding author

Correspondence to Shyam K Sharan.

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Supplementary Figs. 1–6 and Supplementary Table 1 (PDF 581 kb)

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Kuznetsov, S., Liu, P. & Sharan, S. Mouse embryonic stem cell–based functional assay to evaluate mutations in BRCA2. Nat Med 14, 875–881 (2008). https://doi.org/10.1038/nm.1719

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