Functional evaluation of BRCA2 variants mapping to the PALB2-binding and C-terminal DNA-binding domains using a mouse ES cell-based assay

• Published in: Human molecular genetics Vol. 21; no. 18; pp. 3993 - 4006
• Main Authors: BISWAS, Kajal, DAS, Ranabir, PRUSS, Dmitry, BOWLES, Karla R, ROA, Benjamin B, HUNTER, Neil, TESSAROLLO, Lino, WENSTRUP, Richard J, BYRD, R. Andrew, SHARAN, Shyam K, EGGINGTON, Julie M, QIAO, Huanyu, NORTH, Susan L, STAUFFER, Stacey, BURKETT, Sandra S, MARTIN, Betty K, SOUTHON, Eileen, SIZEMORE, Scott C
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 15.09.2012
• Subjects: Amino Acid Sequence; Animals; Biological and medical sciences; BRCA2 Protein - chemistry; BRCA2 Protein - genetics; Breast Neoplasms - genetics; Cell Cycle Proteins; Cell physiology; Cell Survival; Cell transformation and carcinogenesis. Action of oncogenes and antioncogenes; Cells, Cultured; Chromosome Mapping; Conserved Sequence; DNA Breaks, Double-Stranded; DNA Repair; DNA-Binding Proteins; Embryonic Stem Cells - drug effects; Embryonic Stem Cells - metabolism; Embryonic Stem Cells - physiology; Fanconi Anemia Complementation Group N Protein; Female; Fundamental and applied biological sciences. Psychology; Genetic Association Studies; Genetics of eukaryotes. Biological and molecular evolution; Humans; Likelihood Functions; Male; Mice; Mice, Transgenic; Mitomycin - pharmacology; Models, Molecular; Molecular and cellular biology; Mutagens - pharmacology; Mutation; Nuclear Proteins - metabolism; Protein Binding; Protein Interaction Domains and Motifs - genetics; Protein Structure, Quaternary; Structural Homology, Protein; Tumor Suppressor Proteins - metabolism; Cartography; Genetic variability; Rodentia; Genotype; Binding site; C terminal-Sequence; Variant; Vertebrata; Mammalia; Mouse; BRCA2 gene; Animal; DNA; Genetics; Tumor suppressor gene
• ISSN: 0964-6906; 1460-2083
• DOI: 10.1093/hmg/dds222

Single-nucleotide substitutions and small in-frame insertions or deletions identified in human breast cancer susceptibility genes BRCA1 and BRCA2 are frequently classified as variants of unknown clinical significance (VUS) due to the availability of very limited information about their functional consequences. Such variants can most reliably be classified as pathogenic or non-pathogenic based on the data of their co-segregation with breast cancer in affected families and/or their co-occurrence with a pathogenic mutation. Biological assays that examine the effect of variants on protein function can provide important information that can be used in conjunction with available familial data to determine the pathogenicity of VUS. In this report, we have used a previously described mouse embryonic stem (mES) cell-based functional assay to characterize eight BRCA2 VUS that affect highly conserved amino acid residues and map to the N-terminal PALB2-binding or the C-terminal DNA-binding domains. For several of these variants, very limited co-segregation information is available, making it difficult to determine their pathogenicity. Based on their ability to rescue the lethality of Brca2-deficient mES cells and their effect on sensitivity to DNA-damaging agents, homologous recombination and genomic integrity, we have classified these variants as pathogenic or non-pathogenic. In addition, we have used homology-based modeling as a predictive tool to assess the effect of some of these variants on the structural integrity of the C-terminal DNA-binding domain and also generated a knock-in mouse model to analyze the physiological significance of a residue reported to be essential for the interaction of BRCA2 with meiosis-specific recombinase, DMC1.