title: Functional variant analyses (FVAs) predict pathogenicity in the BRCA1 DNA double-strand break repair pathway.
abstract: Heritable mutations in the BRCA1 and BRCA2 and other genes in the DNA double-strand break (DSB) repair pathway disrupt binding of the encoded proteins, transport into the nucleus and initiation of homologous recombination, thereby increasing cancer risk [Scully, R., Chen, J., Plug, A., Xiao, Y., Weaver, D., Feunteun, J., Ashley, T. and Livingston, D.M. (1997) Association of BRCA1 with Rad51 in mitotic and meiotic cells. Cell, 88, 265-275, Chen, J., Silver, D.P., Walpita, D., Cantor, S.B., Gazdar, A.F., Tomlinson, G., Couch, F.J., Weber, B.L., Ashley, T., Livingston, D.M. et al. (1998) Stable interaction between the products of the BRCA1 and BRCA2 tumor suppressor genes in mitotic and meiotic cells. Mol. Cell, 2, 317-328]. To meet the challenge of correct classification, flow cytometry-based functional variant analyses (FVAs) were developed to determine whether variants in DSB repair genes disrupted the binding of BRCA1 to BARD1, PALB2, BRCA2 and FANCD2, phosphorylation of p53 or BRCA1 nuclear localization in response to DNA damage caused by diepoxybutane, mitomycin C and bleomycin. Lymphoblastoid cells from individuals with BRCA1 pathogenic mutations, benign variants, and variants of uncertain significance or with known BRCA2, FANCC or NBN mutations were tested. Mutations in BRCA1 decreased nuclear localization of BRCA1 in response to individual or combination drug treatment. Mutations in BRCA1 reduced binding to co-factors, PALB2 and FANCD2 and decreased phosphorylation of p53. Mutations in BRCA2, FANCC and NBN decreased nuclear localization of BRCA1 in response to drug treatment, cofactors binding and p53 phosphorylation. Unsupervised cluster analysis of all and as few as two assays demonstrated two apparent clusters, high-risk BRCA1 mutations and phenocopies and low-risk, fully sequenced controls and variants of uncertain significance (VUS). Thus, two FVA assays distinguish BRCA1 mutations and phenocopies from benign variants and categorize most VUS as benign. Mutations in other DSB repair pathway genes produce molecular phenocopies. FVA assays may represent an adjunct to sequencing for categorizing VUS or may represent a stand-alone measure for assessing breast cancer risk.
authors: Department of Pathology and.
authors: Johnny
authors: J
authors: Loke
authors: Department of Pathology and.
authors: Alexander
authors: A
authors: Pearlman
authors: Department of Pathology and.
authors: Kinnari
authors: K
authors: Upadhyay
authors: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA and.
authors: Lydia
authors: L
authors: Tesfa
authors: Division of Biostatistics, New York University School of Medicine, New York, NY 10016, USA.
authors: Yongzhao
authors: Y
authors: Shao
authors: Department of Pathology and harry.ostrer@einstein.yu.edu.
authors: Harry
authors: H
authors: Ostrer
mesh_terms: D019313:BRCA1 Protein / Q000235:genetics* / Q000378:metabolism; D001943:Breast Neoplasms / Q000235:genetics; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000378:metabolism; D000076164:Fanconi Anemia Complementation Group N Protein; D005260:Female; D006801:Humans; D058977:Molecular Sequence Annotation; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D059767:Recombinational DNA Repair; D025521:Tumor Suppressor Proteins / Q000378:metabolism
chemical_list: D019313:BRCA1 Protein; C492913:BRCA1 protein, human; C499905:FANCD2 protein, human; D052236:Fanconi Anemia Complementation Group D2 Protein; D000076164:Fanconi Anemia Complementation Group N Protein; D009687:Nuclear Proteins; C512790:PALB2 protein, human; D025521:Tumor Suppressor Proteins