Caratterizzazione di varianti di possibile rilevanza patologica nelle regioni codificanti e regolatorie di geni coinvolti nell'insorgenza di carcinoma familiare della mammella e/o ovaio

Mutational analysis screening, for patient with hereditary breast and/or ovarian cancer is limited to the coding regions and intron-exon junctions of BRCA1/2 genes. Those clinical limitations are precluding the investigation not only of the non-coding and regulatory regions of those two genes, but also other high/moderate and low penetrating genes that could potentially influence the pathological outcomes. Cancer risk prediction is further complicated by the identification of variants with unknown functional significance (VUS). To tackle the problem, this thesis aims to identify VUSs in coding and non-coding regions of 12 high/moderate and low-penetrance genes and in the regulatory regions of BRCA1/2 by developing of clinically useful assays. NGS technologies and enrichment protocols have allowed us to identify 45 rare variants, classified as VUS, testing samples derived from 120 patients. Two VUS, located in PTEN 3’UTR, were prioritized and they are going to be tested using the Dual luciferase assay. This assay was set up to verify alterations in the miRNA-mediated regulation, predicted by the online tools. We are going to exploit this technology to address the putative biological function of the variants found in the non-coding region. To summarize, mutational screening has once again confirmed the genetic complexity underlying the onset of breast and/or ovarian cancer as well as the need for reliable functional testing, to cope with VUS classification