PO-020 Functional characterisation of variant of unknown significate in familial breast cancer

Introduction Familial breast cancer (BC) cases account for 5%–10% of all BC cases, and are essentially correlated with the prevalence of pathogenic BRCA1 and BRCA2 mutations in cancer patients. Recent advances in next generation sequencing (NGS) have enabled panel gene testing, or simultaneous testing for mutations in multiple genes for a clinical condition. With more widespread genetic testing, an increased detection of alleles with moderate penetrance without established clinical guidelines and of variants of unknown significance (VUS) as either benign or disease-causing will occur. Functional analyses on VUS may identify pathogenicity, and clearly categorise their mutational status. We carried-out a proof-of concept in vitro functional analysis in peripheral blood lymphocytes of VUS-harbouring individuals and healthy controls assessing the cellular response to γ-radiation. Material and methods Four samples were collected, one BC patient with a pathogenic ATM mutation; one BRCA2 VUS detected without diagnosed disease, and two controls (A, B, no variant detected). All samples were previously sequenced by NGS to confirm the presence of pathogenic mutations or VUS detected in a genetic testing panel. Several methodologies were selected to evaluate the cellular response to genetic lesions induced by γ-radiation (2Gy): chromosomal aberrations (CA), micronuclei (MN) and comet assay. Results and discussions Comet assay results revealed a higher DNA damage in the patient with a pathogenic ATM mutation (14% DNA in tail) after irradiation, which might be related with the non-optimal function of the ATM protein. The BRCA2 VUS sample also showed a higher%DNA in tail (6.5%), contrasting with the damage quantified for control samples (2.8% and 0.9% DNA in tail for control A and B, respectively). These results revealed that the presence of such variants might be correlated with their biological function, being crucial to categorise the mutational status. Regarding CA and MN, these assays were not sensitive enough to discriminate pathogenic from control samples. Conclusion Results obtained for comet so far suggest a significant difference between variant carriers and controls, which may indicate an increased susceptibility to ionising radiation. However, more sensitive assays (e.g. H2AX) will be necessary allowing more informative results. As this study is a proof-of concept approach, in the near future the sample size will be incremented.