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

摘要

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 (BRCA2(wt/Delta ex11)). We found unaffected DNA repair function of BRCA2 in cell lines BRCA2(7997G>C/Delta ex11), BRCA2(8111C>T/Delta ex11), BRCA2(8149G>T/Delta ex11), BRCA2(8182G>A/Delta ex11), and BRCA2(8182G>T/Delta ex11), whereas the cell line BRCA2(8168A>G/Delta ex11) and the nonsense mutation carrying line BRCA2(8305G>T/Delta 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.