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.

Keywords: BRCA2; PALB2; PALB2-BRCA2 interacting domain; VUS; breast cancer; breast cancer predisposition genes; functional analyses.

Figure 1

The BRCA2:c.91T>G (p.Trp31Gly) and the PALB2:c.3262C>T (p.Pro1088Ser) variants, and pedigrees of the two mutation carriers. (A) Electropherograms showing the BRCA2 and PALB2 sequence of the individuals carrying the two variants, and normal controls. (B) Family pedigrees 1 and 2 of the two Italians probands carrying the BRCA2:c.91T>G (p.Trp31Gly;) and the PALB2:c.3262C>T (p.Pro1088Ser) variants, respectively. Probands are indicated by arrow. Cancer type, age at diagnosis and age of death are reported when known. Age of healthy individuals, if known, was annotated at date of genetic counseling. Events occurred after genetic counseling, if known, are annotated. Additional relatives carrying the variants are indicated by +/–. Cancer type is reported as follows: Bl, bladder cancer; Br, breast cancer; CR, colorectal cancer; Le, leukemia; Lu, lung cancer.

Figure 2

Family pedigree of the additional proband carrying the PALB2:c.3262C>T (p.Pro1088Ser) variant. The proband had large multi gene panel test (67 genes) due to a family history of cancer. The analysis was performed at the Ambry Genetics using Next Generation Sequencing and Array CGA (https://www.ambrygen.com/clinician/genetic-testing/28/oncology/cancernext-expanded). Proband is indicated by arrow. Cancer type, age at diagnosis and age of death are reported when known. Age of healthy individuals, if known, was annotated at date of genetic counseling. Events occurred after genetic counseling, if known, are annotated. Cancer type is reported as follows: BC, basal cells cancer; Bl, bladder cancer; Me, melanoma; Pa, pancreatic cancer; Pr, prostate cancer; Un, unknown.

Figure 3

Detection of the BRCA2-PALB2 interaction. (A) Schematic representation of the specific domains mediating the BRCA2-PALB2 binding and of the location of variants analyzed in the present study. (B) GFP-reassembly in vitro assay. Fluorescence was recovered, under long-wave UV light (365 nm), after 24 h of growth at 30°C followed by 3 days of incubation at room temperature. The variants PALB2 L939W or M1022A and BRCA2 I27V were included as positive controls. The variants PALB2 A1025R and BRCA2 W31C or W31R were included as negative controls. (C) IMAC purification. The BRCA2-PALB2 reassembled complexes were purified from the co-transformed E. coli BL21 (DE3) cells, subjected to SDS-PAGE and visualized by Western blotting using a polyclonal anti-GFP antibody. (D) Analysis of expression of BRCA2-NfrGFP and PALB2-CfrGFP normal and mutant forms. Whole cell extracts from the co-transformed E. coli BL21 (DE3) cells were subjected to SDS-PAGE and visualized by Western blotting using a polyclonal anti-GFP antibody. Images of (C, D) were selected from original autoradiographic films shown in Supplementary Figure 1.