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

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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.

References

  1. Thompson, D., Easton, D.F. & Goldgar, D.E. A full-likelihood method for the evaluation of causality of sequence variants from family data. Am. J. Hum. Genet. 73, 652–655 (2003).

    Article  CAS  Google Scholar 

  2. Carvalho, M.A., Couch, F.J. & Monteiro, A.N. Functional assays for BRCA1 and BRCA2. Int. J. Biochem. Cell Biol. 39, 298–310 (2007).

    Article  CAS  Google Scholar 

  3. Sharan, S.K. et al. Embryonic lethality and radiation hypersensitivity mediated by Rad51 in mice lacking Brca2. Nature 386, 804–810 (1997).

    Article  CAS  Google Scholar 

  4. Ramirez-Solis, R., Liu, P. & Bradley, A. Chromosome engineering in mice. Nature 378, 720–724 (1995).

    Article  CAS  Google Scholar 

  5. Oddoux, C. et al. The carrier frequency of the BRCA2 6174delT mutation among Ashkenazi Jewish individuals is approximately 1%. Nat. Genet. 14, 188–190 (1996).

    Article  CAS  Google Scholar 

  6. Freedman, M.L. et al. Common variation in BRCA2 and breast cancer risk: a haplotype-based analysis in the Multiethnic Cohort. Hum. Mol. Genet. 13, 2431–2441 (2004).

    Article  CAS  Google Scholar 

  7. Wu, K. et al. functional evaluation and cancer risk assessment of brca2 unclassified variants. Cancer Res. 65, 417–426 (2005).

    CAS  PubMed  Google Scholar 

  8. Szabo, C., Masiello, A., Ryan, J.F. & Brody, L.C. The breast cancer information core: database design, structure, and scope. Hum. Mutat. 16, 123–131 (2000).

    Article  CAS  Google Scholar 

  9. Wong, J.M., Ionescu, D. & Ingles, C.J. Interaction between BRCA2 and replication protein A is compromised by a cancer-predisposing mutation in BRCA2. Oncogene 22, 28–33 (2003).

    Article  CAS  Google Scholar 

  10. Goldgar, D.E. et al. Integrated evaluation of DNA sequence variants of unknown clinical significance: application to BRCA1 and BRCA2. Am. J. Hum. Genet. 75, 535–544 (2004).

    Article  CAS  Google Scholar 

  11. Easton, D.F. et al. A systematic genetic assessment of 1,433 sequence variants of unknown clinical significance in the BRCA1 and BRCA2 breast cancer-predisposition genes. Am. J. Hum. Genet. 81, 873–883 (2007).

    Article  CAS  Google Scholar 

  12. Atanassov, B.S., Barrett, J.C. & Davis, B.J. Homozygous germ line mutation in exon 27 of murine Brca2 disrupts the Fancd2-Brca2 pathway in the homologous recombination-mediated DNA interstrand cross-links' repair but does not affect meiosis. Genes Chromosom. Cancer 44, 429–437 (2005).

    Article  CAS  Google Scholar 

  13. Donoho, G. et al. Deletion of Brca2 exon 27 causes hypersensitivity to DNA crosslinks, chromosomal instability and reduced life span in mice. Genes Chromosom. Cancer 36, 317–331 (2003).

    Article  CAS  Google Scholar 

  14. Mazoyer, S. et al. A polymorphic stop codon in BRCA2. Nat. Genet. 14, 253–254 (1996).

    Article  CAS  Google Scholar 

  15. Marple, T., Kim, T.M. & Hasty, P. Embryonic stem cells deficient for Brca2 or Blm exhibit divergent genotoxic profiles that support opposing activities during homologous recombination. Mutat. Res. 602, 110–120 (2006).

    Article  CAS  Google Scholar 

  16. Godthelp, B.C. et al. Cellular characterization of cells from the Fanconi anemia complementation group, FA-D1/BRCA2. Mutat. Res. 601, 191–201 (2006).

    Article  CAS  Google Scholar 

  17. Patel, K.J. et al. Involvement of Brca2 in DNA repair. Mol. Cell 1, 347–357 (1998).

    Article  CAS  Google Scholar 

  18. Morimatsu, M., Donoho, G. & Hasty, P. Cells deleted for Brca2 COOH terminus exhibit hypersensitivity to γ-radiation and premature senescence. Cancer Res. 58, 3441–3447 (1998).

    CAS  PubMed  Google Scholar 

  19. Yuan, S.-S.F. et al. BRCA2 is required for ionizing radiation–induced assembly of Rad51 complex in vivo. Cancer Res. 59, 3547–3551 (1999).

    CAS  Google Scholar 

  20. Khanna, K.K. & Jackson, S.P. DNA double-strand breaks: signaling, repair and the cancer connection. Nat. Genet. 27, 247–254 (2001).

    Article  CAS  Google Scholar 

  21. Yang, Y., Swaminathan, S., Martin, B.K. & Sharan, S.K. Aberrant splicing induced by missense mutations in BRCA1: clues from a humanized mouse model. Hum. Mol. Genet. 12, 2121–2131 (2003).

    Article  CAS  Google Scholar 

  22. Fackenthal, J.D., Cartegni, L., Krainer, A.R. & Olopade, O.I. BRCA2 T2722R is a deleterious allele that causes exon skipping. Am. J. Hum. Genet. 71, 625–631 (2002).

    Article  CAS  Google Scholar 

  23. Yang, H. et al. BRCA2 function in DNA binding and recombination from a BRCA2–DSS1-ssDNA structure. Science 297, 1837–1848 (2002).

    Article  CAS  Google Scholar 

  24. Deffenbaugh, A.M., Frank, T.S., Hoffman, M., Cannon-Albright, L. & Neuhausen, S.L. Characterization of common BRCA1 and BRCA2 variants. Genet. Test. 6, 119–121 (2002).

    Article  CAS  Google Scholar 

  25. Sharan, S.K. et al. BRCA2 deficiency in mice leads to meiotic impairment and infertility. Development 131, 131–142 (2004).

    Article  CAS  Google Scholar 

  26. Court, D.L. et al. Mini-lambda: a tractable system for chromosome and BAC engineering. Gene 315, 63–69 (2003).

    Article  CAS  Google Scholar 

  27. Yang, Y. & Sharan, S.K. A simple two-step, 'hit and fix' method to generate subtle mutations in BACs using short denatured PCR fragments. Nucleic Acids Res. 31, e80 (2003).

    Article  Google Scholar 

  28. Scudiero, D.A. et al. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res. 48, 4827–4833 (1988).

    CAS  PubMed  Google Scholar 

  29. Kuznetsov, S. et al. RAD51C deficiency in mice results in early prophase I arrest in males and sister chromatid separation at metaphase II in females. J. Cell Biol. 176, 581–592 (2007).

    Article  CAS  Google Scholar 

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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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Authors

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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