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

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.

[1]  B. Ponder,et al.  Involvement of Brca2 in DNA repair. , 1998, Molecular cell.

[2]  F. Couch,et al.  Functional evaluation and cancer risk assessment of BRCA2 unclassified variants. , 2005, Cancer research.

[3]  G E Tomlinson,et al.  BRCA2 is required for ionizing radiation-induced assembly of Rad51 complex in vivo. , 1999, Cancer research.

[4]  P. Hasty,et al.  Embryonic stem cells deficient for Brca2 or Blm exhibit divergent genotoxic profiles that support opposing activities during homologous recombination. , 2006, Mutation research.

[5]  Wen-Hwa Lee,et al.  BRCA2 function in DNA binding and recombination from a BRCA2-DSS1-ssDNA structure. , 2002, Science.

[6]  Janice P. Evans,et al.  BRCA2 deficiency in mice leads to meiotic impairment and infertility , 2004, Development.

[7]  M. Stratton,et al.  A polymorphic stop codon in BRCA2 , 1996, Nature Genetics.

[8]  Kenneth Offit,et al.  The carrier frequency of the BRCA2 6174delT mutation among Ashkenazi Jewish individuals is approximately 1% , 1996, Nature Genetics.

[9]  D. Scudiero,et al.  Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. , 1988, Cancer research.

[10]  Olufunmilayo I Olopade,et al.  BRCA2 T2722R is a deleterious allele that causes exon skipping. , 2002, American journal of human genetics.

[11]  L. Shulman,et al.  A Systematic Genetic Assessment of 1,433 Sequence Variants of Unknown Clinical Significance in the BRCA1 and BRCA2 Breast Cancer–Predisposition Genes , 2008 .

[12]  A. Bradley,et al.  Chromosome engineering in mice , 1995, Nature.

[13]  P. Hasty,et al.  Cells deleted for Brca2 COOH terminus exhibit hypersensitivity to gamma-radiation and premature senescence. , 1998, Cancer research.

[14]  F. Couch,et al.  Functional assays for BRCA1 and BRCA2. , 2007, The international journal of biochemistry & cell biology.

[15]  L. Cannon-Albright,et al.  Characterization of common BRCA1 and BRCA2 variants. , 2002, Genetic testing.

[16]  S. Sharan,et al.  Aberrant splicing induced by missense mutations in BRCA1: clues from a humanized mouse model. , 2003, Human molecular genetics.

[17]  D. Court,et al.  Mini-lambda: a tractable system for chromosome and BAC engineering. , 2003, Gene.

[18]  P. V. van Buul,et al.  Cellular characterization of cells from the Fanconi anemia complementation group, FA-D1/BRCA2. , 2006, Mutation research.

[19]  Douglas F Easton,et al.  A full-likelihood method for the evaluation of causality of sequence variants from family data. , 2003, American journal of human genetics.

[20]  G. Eichele,et al.  Embryonic lethality and radiation hypersensitivity mediated by Rad51 in mice lacking Brca2 , 1997, Nature.

[21]  D. Altshuler,et al.  Common variation in BRCA2 and breast cancer risk: a haplotype-based analysis in the Multiethnic Cohort. , 2004, Human molecular genetics.

[22]  David J. Chen,et al.  Deletion of Brca2 exon 27 causes hypersensitivity to DNA crosslinks, chromosomal instability, and reduced life span in mice , 2003, Genes, chromosomes & cancer.

[23]  Csilla Szabo,et al.  The Breast Cancer Information Core: Database design, structure, and scope , 2000, Human mutation.

[24]  S. Sharan,et al.  A simple two-step, 'hit and fix' method to generate subtle mutations in BACs using short denatured PCR fragments. , 2003, Nucleic acids research.

[25]  C. Ingles,et al.  Interaction between BRCA2 and replication protein A is compromised by a cancer-predisposing mutation in BRCA2 , 2003, Oncogene.

[26]  F. Couch,et al.  Integrated evaluation of DNA sequence variants of unknown clinical significance: application to BRCA1 and BRCA2. , 2004, American journal of human genetics.

[27]  S. West,et al.  RAD51C deficiency in mice results in early prophase I arrest in males and sister chromatid separation at metaphase II in females , 2007, The Journal of cell biology.

[28]  K. Khanna,et al.  DNA double-strand breaks: signaling, repair and the cancer connection , 2001, Nature Genetics.

[29]  D. Court,et al.  Mini-λ: a tractable system for chromosome and BAC engineering , 2003 .

[30]  J. Barrett,et al.  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 , 2005, Genes, chromosomes & cancer.