Functional Analysis of Human Breast Cancer Susceptibility Gene Variants

Sharan, Shyam

Abstract

To evaluate the functional significance of human BRCA1 and BRCA2 variants and to carry out functional dissection of these proteins, we have developed a simple, reliable and physiologically relevant functional assay using mouse embryonic stem (ES) cells. The assay is based on the fact that Brca1 and Brca2-null ES cells cannot survive. Therefore we engineered ES cells to express a conditional allele of Brca1 or Brca2. Desired mutations are generated in the human BRCA1 or BRCA2 gene in bacterial artificial chromosomes (BAC) and introduced into the ES cells. Functional analysis is performed after deletion of the conditional allele. The ability of and the extent to which specific variants complement the lethality of ES cells associated with Brca1 or Brca2 deficiency is used to evaluate their functional significance. Variants that can rescue lethality are then screened for a defect in the known functions of BRCA1 and BRCA2.

Funding Agency

• Agency: National Institute of Health (NIH)
• Institute: National Cancer Institute (NCI)
• Type: Investigator-Initiated Intramural Research Projects (ZIA)
• Project #: 1ZIABC011181-05
• Application #: 8763384
• Support Year: 5
• Fiscal Year: 2013
• Total Cost: $647,584

Institution

• Name: National Cancer Institute Division of Basic Sciences

Publications

Toland, Amanda Ewart; Forman, Andrea; Couch, Fergus J et al. (2018) Clinical testing of BRCA1 and BRCA2: a worldwide snapshot of technological practices. NPJ Genom Med 3:7

Meyer, Stefan; Stevens, Adam; Paredes, Roberto et al. (2017) Acquired cross-linker resistance associated with a novel spliced BRCA2 protein variant for molecular phenotyping of BRCA2 disruption. Cell Death Dis 8:e2875

Thirthagiri, Eswary; Klarmann, Kimberly D; Shukla, Anil K et al. (2016) BRCA2 minor transcript lacking exons 4-7 supports viability in mice and may account for survival of humans with a pathogenic biallelic mutation. Hum Mol Genet 25:1934-1945

Bakker, Janine L; Thirthagiri, Eswary; van Mil, Saskia E et al. (2014) A novel splice site mutation in the noncoding region of BRCA2: implications for Fanconi anemia and familial breast cancer diagnostics. Hum Mutat 35:442-6

Chang, Suhwan; Sharan, Shyam K (2013) The role of epigenetic transcriptional regulation in BRCA1-mediated tumor suppression. Transcription 4:24-8

Biswas, Kajal; Stauffer, Stacey; Sharan, Shyam K (2012) Using recombineering to generate point mutations:galK-based positive-negative selection method. Methods Mol Biol 852:121-31

Chang, Suhwan; Stauffer, Stacey; Sharan, Shyam K (2012) Using recombineering to generate point mutations: the oligonucleotide-based ""hit and fix"" method. Methods Mol Biol 852:111-20

Biswas, Kajal; Das, Ranabir; Eggington, Julie M et al. (2012) Functional evaluation of BRCA2 variants mapping to the PALB2-binding and C-terminal DNA-binding domains using a mouse ES cell-based assay. Hum Mol Genet 21:3993-4006

Chang, Suhwan; Sharan, Shyam K (2012) Epigenetic control of an oncogenic microRNA, miR-155, by BRCA1. Oncotarget 3:5-6

Chang, Suhwan; Sharan, Shyam K (2012) BRCA1 and microRNAs: emerging networks and potential therapeutic targets. Mol Cells 34:425-32