Functional analysis of variants of uncertain significance in the breast cancer predisposition gene PALB2

• Grant number: 17/17451-6
• Support Opportunities: Scholarships in Brazil - Master
• Start date: December 01, 2017
• End date: February 28, 2019
• Field of knowledge: Biological Sciences - Genetics - Human and Medical Genetics
• Principal Investigator: Edenir Inêz Palmero
• Grantee: Ariane Stéfani Pereira
• Host Institution: Hospital do Câncer de Barretos. Fundação Pio XII (FP). Barretos , SP, Brazil

Abstract

Breast cancer is the more frequent tumor type in women, about 5-10% of the cases are hereditary. The main genes associated with a higher breast cancer predisposition are BRCA1, BRCA2, TP53, PTEN, CDH1, STK11 and, more recently, PALB2, a tumor suppressor gene involved in several cellular functions, mainly in the DNA Double-Strand Break (DSB) repair. The genetic variants found in these genes can be classified as pathogenic, likely pathogenic, variants of uncertain significance (VUS), likely benign or benign. The management of patients VUS carriers is difficult, because the impact of those variants is not clear to the patient or their relatives. Previous work from our group identified, at the PALB2 gene, VUS and new missense variants (never before reported in databases), classified as likely pathogenic or pathogenic by in silico prediction tools. Therefore, in this study we intend, through functional assays using two cell lines of breast cancer (T47D and HS578T), to help on the understanding of the impact of these PALB2 variants. For that, the endogenous PALB2 gene will be knocked out with CRISPR/Cas9 system. Next, the c.2816T>G, c.2981T>G and c.1042C>T (positive control) variants will be inserted into the gene PALB2 through the site-directed mutagenesis assay. Finally, functional analysis will be performed through assays like cellular proliferation (BrdU), cellular viability (MTS), apoptosis (flow citometry), proficiency of the homologous recombination system, cellular localization and co-immunoprecipitation. All assays will have positive and negative controls. We expect that the functional analysis help us in the definition of the biological and clinical impact of these alterations in the gene PALB2. (AU)