Proceedings of Oncobiology Symposiums
Proceedings of the XV Oncobiology Symposium

INTRODUCTION AND OBJECTIVES: PALB2 is a tumor suppressor gene that encodes for a homonymous protein that works as a scaffold connecting BRCA1 and BRCA2 proteins. The BRCA1-PALB2-BRCA2 complex plays an important role in genome integrity maintenance and in homologous recombination repair, a DNA repair pathway. The interaction between PALB2 and BRCA2 forms a stable heterodimer mediated by the interaction of PALB2 WD40 domain and BRCA2 N-terminal region. Germline mutations in the PALB2 increase the risk of breast and pancreatic cancers. In recent years, the identification of PALB2 genetic variants in the population has increased significantly.However, the classification of variants according to their association with cancer is still a clinical challenge. Missense mutations (the exchange of a single amino acid residue) constitute a particularly group of difficult interpretation, usually being classified as variants of uncertain significance (VUS). In this work, our goal is to functionally evaluate PALB2 missense VUS located in the WD40 coding region. MATERIAL AND METHODS: We performed a literature curation to identify all the PALB2 VUS already described in the population (510 variants) and nearly all of them without information regarding protein function or pathogenicity. Variants were analyzed using the Align GVGD in silico predictor. We selected 10 PALB2 variants with a prediction score equal to or greater than C25 (moderate risk). Variants were functionally investigated based on their ability to interact with the C-terminal of BRCA2 protein, evaluated through a mammalian two-hybrid assay using BRCA2 as the bait and PALB2 as the prey protein. All variants were generated by site-directed mutagenesis strategies using PrimeSTAR® Max DNA polymerase and specific primers with the mutation of interest. All variants were confirmed by automatic sequencing using the Sanger method. The two-hybrid mammalian assay was performed in HEK293FT cells co-transfected with constructs encoding N-terminal BRCA2 fused to the DNA binding domain of GAL4 (GAL4:DBD-BRCA2) and wild-type PALB2 C-terminal or one of the analyzed variants fused to VP16 transcriptional activator (VP16-PALB2), in addition to the reporter system constructs (pG5Luc, encoding the luciferase reporter gene; pGR-TK, internal transfection control). Protein profile was evaluated by immunoblotting. RESULTS AND CONCLUSION: Our data demonstrated an intermediate interaction activity with BRCA2 for 7 out of the 10 variants (V919L; C933G; Q958E; V991A; M992K; P1009L; G1021E) suggesting a non-pathogenic behavior. The other three variants (E943K; G1028D and G1028V) showed a reduced activity, an indicative of pathogenic behavior. As a matter of fact, complementary functional information is needed for a final classification. The functional analysis of VUS is critical to built information on genetic variants behavior and corroborate to risk classification (pathogenicity) and hereditary susceptibility to cancer.