Two Missense Variants Detected in Breast Cancer Probands Preventing BRCA2-PALB2 Protein Interaction

Abstract

PALB2 (partner and localizer of BRCA2) was initially identified as a binding partner of BRCA2. It interacts also with BRCA1 forming a complex promoting DNA repair by homologous recombination. Germline pathogenic variants in BRCA1, BRCA2 and PALB2 DNA repair genes are associated with high risk of developing breast cancer. Mutation screening in these breast cancer predisposition genes is routinely performed and allows the identification of individuals who carry pathogenic variants and are at risk of developing the disease. However, variants of uncertain significance (VUSs) are often detected and establishing their pathogenicity and clinical relevance remains a central challenge for the risk assessment of the carriers and the clinical decision-making process. Many of these VUSs are missense variants leading to single amino acid substitutions, whose impact on protein function is uncertain. Typically, VUSs are rare and due to the limited genetic, clinical, and pathological data the multifactorial approaches used for classification cannot be applied. Thus, these variants can only be characterized through functional analyses comparing their effect with that of normal and mutant gene products used as positive and negative controls. The two missense variants BRCA2:c.91T >G (p.Trp31Gly) and PALB2:c.3262C >T (p.Pro1088Ser) were detected in two breast cancer probands originally ascertained at Breast Cancer Units of Institutes located in Milan and Bergamo (Northern Italy), respectively. These variants were located in the BRCA2-PALB2 interacting domains, were predicted to be deleterious by in silico analyses, and were very rare and clinically not classified. Therefore, we initiate to study their functional effect by exploiting a green fluorescent protein (GFP)-reassembly in vitro assay specifically designed to test the BRCA2-PALB2 interaction. This functional assay proved to be easy to develop, robust and reliable. It also allows testing variants located in different genes. Results from these functional analyses showed that the BRCA2:p.Trp31Gly and the PALB2:p.Pro1088Ser prevented the BRCA2-PALB2 binding. While caution is warranted when the interpretation of the clinical significance of rare VUSs is based on functional studies only, our data provide initial evidences in favor of the possibility that these variants are pathogenic.

Screening of BRCA1/2 genes mutations and copy number variations in patients with high risk for hereditary breast and ovarian cancer syndrome (HBOC)

“…Both sisters carrying the variant suffered from cancer while their third sister who didn't harbor the variant was healthy. This variant was reported by Laura Caleca et al [63]. In her study, Caleca proved that this variant affects the binding of BRCA2 protein with the PALB2 protein.…”

Molecular Mechanisms of PALB2 Function and Its Role in Breast Cancer Management

“…Moreover, p.P8L (c.23C>T), p.K18R (c.53A>G), p.L24S (c.71T>C), p.Y28C (c.83A>G), and p.R37H (c.110G>A) compromise HR activity of PALB2 and are suggested to be pathogenic (106)(107)(108). In the PALB2 C-terminus, p.W912G, p.G937R, p.L939W, p.I944N (c.2831T>A), p.L947F (c.2841G>T), p.L947S (c.2840T>C), p.L961P, p.L972Q, p.A1025R, p.T1030I (c.3089C>T), p.I1037T, p.G1043D, p.L1070P (c.3209T>C), p.P1088S (c.3262C>T), p.W1140G (c.3418T>C), p.L1143P, and p.L1172P promoted a decrease in the HR activity of PALB2 (20,(107)(108)(109)112). Among these mutations, p.L939W, p.A1025R, p.T1030I, p.P1088S, and p.L1143P disrupt BRCA2-PALB2 interaction; p.W912G, p.G937R, p.I944N, p.L961P, p.L972Q, p.T1030I, p.I1037T, p.G1043D, and p.L1172P are associated with PALB2 protein instability; and p.I944N, p.L947F, p.L947S, p.T1030I, p.L1070P, and p.W1140G result in the mislocalization of PALB2 to the cytoplasm.…”

Variants of uncertain clinical significance in hereditary breast and ovarian cancer genes: best practices in functional analysis for clinical annotation

“…After the development of a large number of functional assays for high-risk genes ( BRCA1 , BRCA2 and TP53 ),36 37 40 significant attention has been focused on developing assays for other high-risk/moderate-risk genes such as PALB2 , ATM and CHEK2 41–46. However, here we will focus on functional assays for missense variants of BRCA1 and BRCA2 as exemplars from which we have derived general guidelines.…”