BRCA1 and BRCA2 mutation detection by a Next Generation Sequencing approach: epidemiological study conducted in Southern Italy and analysis of novel mutations in hereditary breast and ovarian cancer women.

Hereditary breast and ovarian cancer (HBOCs) accounts for about 10% of all breast cancers and BRCA1 and BRCA2 are the most prevalent genes associated to this pathology. They play a role in the maintenance of genome stability, particularly in the homologous recombination (HR) pathway for double-strand DNA breaks repair (DSBR). BRCA1/BRCA2 germline mutations dramatically escalate the risk of developing HBOCs by up to 20 fold. Therefore, testing for BRCA gene mutations is important to improve the clinical management of high-risk patients and of their mutation-carrier family members. Here is reported the BRCA1/BRCA2 molecular screening of 300 patients with early-onset breast cancer (“under forty”), and/or with positive family history, carried out by using a next-generation sequencing (NGS)-based approach, in order to identify mutation carriers. In particular, all the BRCA1/BRCA2 coding regions were amplified by multiplex PCRs, using specific sequence tags able to univocally identify each patient. In this way, we were able to simultaneously analyze up to 60 different samples in a single NGS run. After sequence data analysis, 24 known BRCA1/BRCA2 predisposing mutations were identified in 27 unrelated patients.About 12.5% of analyzed patients, including some males, carried a causative mutation. Several novel variants were also identified: double mutations, which include 1 nonsense mutation in the BRCA1 gene thereby causing a premature stop codon; 2 synonymous variants, 1 missense variant predicted not to have clinical significance, and 1 missense variant predicted to be deleterious. In addition, 2 novel possibly pathogenetic variants were also identified and specific functional studies were performed to assess their role. The first is a splice variant for which bioinformatic predictions, performed with both Human Splice Finder and NetGene2 tools, suggested a possible deleterious effect, since it could cause the loss of a canonic donor splice site of a BRCA1 intron. This was experimentally demonstrated on the HBOC patient cDNA by PCR amplifications and enzymatic digestion. The other is a missense variant located on the BRCA2 DNA-binding site that could impair the BRCA2 DSBR functions by HR. The role of this variant was tested by cloning the site-directed-mutated BRCA2 cDNA into a pRc/CMV vector: plasmids were transfected in NIH-GS cells (DR-GFP stable clones) and subsequently transfected with plasmids codifying for I-SceI enzyme. Mutation effects were tested through DSBR assays and the extent of repair by HR was measured by counting GFP-positive cells by FACS: the novel variant resulted unable to bind DNA, probably hampering the normal DSBR pathway. Moreover, 26 variants with unknown clinical significance (VUSs) were also detected. Subsequent analysis of the mutation-carrier families resulted in the identification of at-risk subjects, including healthy male carriers, who were enrolled in surveillance healthcare programs. These results support the inclusion of an NGS-based approach for BRCA1/BRCA2 mutation detection in a diagnostic workflow and further suggest the use of direct in vitro assays to test for functional role of novel mutations found in these genes