A functional analysis to differentiate pathogenic from benign variants identified in clinical diagnostic panels for breast cancer

Genetic testing for susceptibility genes through next‑generation sequencing (NGS) has become a widely used technique. Using this, a number of genetic variants have been identified, several of which are variants of unknown significance (VUS). These VUS can either be pathogenic or benign. However, since their biological effect remains unclear, functional assays are required to classify their functional nature. As the use of NGS becomes more mainstream as a diagnostic tool in clinical practice, the number of VUS is expected to increase. This necessitates their biological and functional classification. In the present study, a VUS was identified in the BRCA1 gene (NM_007294.3:c.1067A>G) in two women at risk for breast cancer, for which no functional data has been reported. Therefore, peripheral lymphocytes were isolated from the two women and also from two women without the VUS. DNA from all samples were sequenced by NGS of a breast cancer clinical panel. Since the BRCA1 gene is involved in DNA repair and apoptosis, the functional assays chromosomal aberrations, cytokinesis‑blocked micronucleus, comet, γH2AX, caspase and TUNEL assays were then conducted on these lymphocytes after a genotoxic challenge by ionizing radiation or doxorubicin to assess the functional role of this VUS. The micronucleus and TUNEL assays revealed a lower degree of DNA induced‑damage in the VUS group compared with those without the VUS. The other assays showed no significant differences between the groups. These results suggested that this BRCA1 VUS is likely benign, since the VUS carriers were apparently protected from deleterious chromosomal rearrangements, subsequent genomic instability and activation of apoptosis.publishersversionpublishe

Functional Characterization of Variants of Unknown Significance in Familial Breast Cancer

Familial breast cancer (BC) cases account for 5-10 % of all BC cases and are mainly associated with inherited mutations in BRCA1 and BRCA2 genes. Many other genes related with BC development have already been identified and are mostly related with Homologous Recombination (HR) repair system, one of the main pathways that repair DNA double-strand breaks (DSBs). Genetic testing for BC has become standard and with more widespread genetic testing, an increased detection of variants of unknown significance (VUS) as either benign or pathogenic will occur. Functional analyses on VUS may identify pathogenicity, and clearly categorize their mutational status. We carried-out a proof-of concept in vitro functional analysis in peripheral blood lymphocytes of VUS-harboring individuals and controls assessing the cellular response to -radiation. Six samples were collected, two BC patient with a pathogenic ATM mutation, two BRCA1 VUS carriers, and two controls. Several methodologies were selected to evaluate the cellular response to genetic lesions induced by -radiation (2Gy): chromosomal aberrations (CA), micronuclei (MN) and comet assay. The CA assay results present no statistical difference between samples. In the MN assay the carriers show lower amount of binucleated cells with MN when compared to control samples, which is possibly due to cellular death events. The comet assay results show a clear increase in sensitivity to ionizing radiation, possibly associated with deficiency in repair, of samples from carrying a pathogenic mutation in the ATM gene and those with the BRCA1 VUS. Overall, except for the CA assay, the results show an increased susceptibility to ionizing radiation in pathogenic ATM mutation carriers and BRCA1 VUS carriers. However, some additional studies should be performed to completely understand the results obtained, and the impact of alterations in cancer risk