Functional evaluation of variants of unknown significance in the BRCA2 gene identified in genetic testing.

ABSTRACT: Heterozygous germline BRCA2 mutations predispose to breast, ovarian, pancreatic and other types of cancer. The presence of a pathogenic mutation in patients or their family members warrants close surveillance or prophylactic surgery. Besides clearly pathogenic mutations, variants leading only to a single amino acid substitution are often identified. The influence of such variants on cancer risk is often unknown, making their presence a major clinical problem. When genetic methods are insufficient to classify these variants, functional assays with various cellular models are performed. We developed and applied a new syngeneic model of human cancer cells to test all variants of unknown significance in exon 18 identified by genetic testing of high-risk cancer patients in the Czech Republic, via introduction of constructs containing each of these variants into the wild-type allele of BRCA2-heterozygous DLD1 cells (BRCA2wt/?ex11). We found unaffected DNA repair function of BRCA2 in cell lines BRCA27997G>C/?ex11, BRCA28111C>T/?ex11, BRCA28149G>T/?ex11, BRCA28182G>A/?ex11, and BRCA28182G>T/?ex11, whereas the cell line BRCA28168A>G/?ex11 and the nonsense mutation carrying line BRCA28305G>T/?ex11 did affect protein function. Targeting the BRCA2 wild-type allele with a construct carrying the variant c.7988A> G resulted in incorporation exclusively into the already defective allele in all viable clones, strongly suggesting a detrimental phenotype. Our model thus offers a valuable tool for the functional evaluation of unclassified variants in the BRCA2 gene and provides a stable and distributable cellular resource for further research.

SUBMITTER: Heczkova M 

PROVIDER: S-EPMC6606029 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature