Germ line inactivating mutations in BRCA1 confer susceptibility for breast and ovarian cancer. However, the relevance of the many missense changes in the gene for which the effect on protein function is unknown remains unclear. Determination of which variants are causally associated with cancer is important for assessment of individual risk. We used a functional assay that measures the transactivation activity of BRCA1 in combination with analysis of protein modeling based on the structure of BRCA1 BRCT domains. In addition, the information generated was interpreted in light of genetic data. We determined the predicted cancer association of 22 BRCA1 variants and verified that the common polymorphism S1613G has no effect on BRCA1 function, even when combined with other rare variants. We estimated the specificity and sensitivity of the assay, and by meta-analysis of 47 variants, we show that variants with <45% of wild-type activity can be classified as deleterious whereas variants with >50% can be classified as neutral. In conclusion, we did functional and structure-based analyses on a large series of BRCA1 missense variants and defined a tentative threshold activity for the classification missense variants. By interpreting the validated functional data in light of additional clinical and structural evidence, we conclude that it is possible to classify all missense variants in the BRCA1 COOH-terminal region. These results bring functional assays for BRCA1 closer to clinical applicability.
Eukaryotic cells have evolved an intricate system to resolve DNA damage to prevent its transmission to daughter cells. This system, collectively known as the DNA damage response (DDR) network, includes a large number of proteins responsible for detection of DNA damage, promotion of repair, and coordination with cell cycle progression. Because defects in this network can lead to cancer, this network constitutes a barrier against tumorigenesis. The BRCT domain is a modular protein domain critical for relaying signals in the DDR. We performed a systematic analysis of protein-protein interactions involving tandem BRCT domains (tBRCT) in the DDR by combining literature curation, yeast two hybrid (Y2H) screens, and tandem affinity purification coupled to mass spectrometry (TAP-MS). We identified one previously unrecognized BRCT protein and generated human protein-protein interaction network for this type of modular domain. This study also reveals several novel components in DNA damage signaling such as COMMD1 and mTORC2. Additionally, integration of tBRCT domain interactions with DDR phosphoprotein studies and analysis of kinase-substrate interactions revealed signaling subnetworks that may aid in understanding the involvement of tBRCT in disease and DNA repair.
Germline mutations in the tumor suppressor gene BRCA1 confer an estimated lifetime risk of 56–80% for breast cancer and 15–60% for ovarian cancer. Since the mid 1990’s when BRCA1 was identified, genetic testing has revealed over 3,000 unique germline variants. However, for a significant number of these variants, the effect on protein function is unknown making it difficult to infer the consequences on risks of breast and ovarian cancers. Thus, many individuals undergoing genetic testing for BRCA1 mutations receive test results reporting a variant of uncertain clinical significance (VUS), leading to issues in risk assessment, counseling, and preventive care. Here we describe functional assays for BRCA1 to directly or indirectly assess the impact of a variant on protein conformation or function and how these results can be used to complement genetic data to classify a VUS as to its clinical significance. Importantly, these methods may provide a framework for genome-wide pathogenicity assignment.
Variants of Uncertain Significance (VUS) are genetic variants whose association with a disease phenotype has not been established. They are a common finding in sequencing-based genetic tests and pose a significant clinical challenge. The objective of this study was to assess the use of functional data to classify variants according to pathogenicity. We conduct functional analysis of a large set of BRCA1 VUS combining a validated functional assay with VarCall, a Bayesian hierarchical model to estimate the likelihood of pathogenicity given the functional data. The results from the functional assays were incorporated into a joint analysis of 214 BRCA1 VUS to predict their likelihood of pathogenicity (breast cancer). We show that applying the VarCall model (1.0 sensitivity; lower bound of 95% confidence interval (CI) = 0.75 and 1.0 specificity; lower bound of 95% CI = 0.83) to the current set of BRCA1 variants, use of the functional data would significantly reduce the number of VUS associated with the C-terminal region of the BRCA1 protein by ~ 87%. We extend this work developing yeast-based functional assays for two other genes coding for BRCT domain containing proteins, MCPH1 and MDC1. Analysis of missense variants in MCPH1 and MDC1 shows that structural inference based on the BRCA1 data set can aid in prioritising variants for further analysis. Taken together our results indicate that systematic functional assays can provide a robust tool to aid in clinical annotation of VUS. We propose that well-validated functional assays could be used for clinical annotation even in the absence of additional sources of evidence.
While biallelic mutations in the PALB2 tumor suppressor cause Fanconi anemia subtype FA-N, monoallelic mutations predispose to breast and familial pancreatic cancer. Although hundreds of missense variants in PALB2 have been identified in patients to date, only a few have clear functional and clinical relevance. Herein, we investigate the effects of 44 PALB2 variants of uncertain significance found in breast cancer patients and provide detailed analysis by systematic functional assays. Our comprehensive functional analysis reveals two hotspots for potentially deleterious variations within PALB2, one at each terminus. PALB2 N-terminus variants p.P8L [c.23C>T], p.Y28C [c.83A>G], and p.R37H [c.110G>A] compromised PALB2-mediated homologous recombination. At the C-terminus, PALB2 variants p.L947F [c.2841G>T], p.L947S [c.2840T>C], and most strikingly p.T1030I [c.3089C>T] and p.W1140G [c.3418T>C], stood out with pronounced PARP inhibitor sensitivity and cytoplasmic accumulation in addition to marked defects in recruitment to DNA damage sites, interaction with BRCA2 and homologous recombination. Altogether, our findings show that a combination of functional assays is necessary to assess the impact of germline missense variants on PALB2 function, in order to guide proper classification of their deleteriousness.
Galectin-3 (gal-3) is a β-galactoside binding protein related to many tumoral aspects, e.g. angiogenesis, cell growth and motility and resistance to cell death. Evidence has shown its upregulation upon hypoxia, a common feature in solid tumors such as glioblastoma multiformes (GBM). This tumor presents a unique feature described as pseudopalisading cells, which accumulate large amounts of gal-3. Tumor cells far from hypoxic/nutrient deprived areas express little, if any gal-3. Here, we have shown that the hybrid glioma cell line, NG97ht, recapitulates GBM growth forming gal-3 positive pseudopalisades even when cells are grafted subcutaneously in nude mice. In vitro experiments were performed exposing these cells to conditions mimicking tumor areas that display oxygen and nutrient deprivation. Results indicated that gal-3 transcription under hypoxic conditions requires previous protein synthesis and is triggered in a HIF-1α and NF-κB dependent manner. In addition, a significant proportion of cells die only when exposed simultaneously to hypoxia and nutrient deprivation and demonstrate ROS induction. Inhibition of gal-3 expression using siRNA led to protein knockdown followed by a 1.7–2.2 fold increase in cell death. Similar results were also found in a human GBM cell line, T98G. In vivo, U87MG gal-3 knockdown cells inoculated subcutaneously in nude mice demonstrated decreased tumor growth and increased time for tumor engraftment. These results indicate that gal-3 protected cells from cell death under hypoxia and nutrient deprivation in vitro and that gal-3 is a key factor in tumor growth and engraftment in hypoxic and nutrient-deprived microenvironments. Overexpression of gal-3, thus, is part of an adaptive program leading to tumor cell survival under these stressing conditions.
The deoxyribonucleic acid (DNA) damage response (DDR) is a major feature in the maintenance of genome integrity and in the suppression of tumorigenesis. PALB2 (Partner and Localizer of Breast Cancer 2 (BRCA2)) plays an important role in maintaining genome integrity through its role in the Fanconi anemia (FA) and homologous recombination (HR) DNA repair pathways. Since its identification as a BRCA2 interacting partner, PALB2 has emerged as a pivotal tumor suppressor protein associated to hereditary cancer susceptibility to breast and pancreatic cancers. In this review, we discuss how other DDR proteins (such as the kinases Ataxia Telangiectasia Mutated (ATM) and ATM- and Rad3-Related (ATR), mediators BRCA1 (Breast Cancer 1)/BRCA2 and effectors RAD51/DNA Polymerase η (Polη) interact with PALB2 to orchestrate DNA repair. We also examine the involvement of PALB2 mutations in the predisposition to cancer and the role of PALB2 in stimulating error-free DNA repair through the FA/HR pathway.
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