BRCA1-associated RING Domain Research Articles

Functional Analysis of BARD1 Missense Variants on Homology-Directed Repair in Ovarian and Breast Cancers.

Women with germline BRCA1 mutations face an increased risk of developing breast and ovarian cancers. BARD1 (BRCA1 associated RING domain 1) is an essential heterodimeric partner of BRCA1, and mutations in BARD1 are also associated with these cancers. While BARD1 mutations are recognized for their cancer susceptibility, the exact roles of numerous BARD1 missense mutations remain unclear. In this study, we conducted functional assays to assess the homology-directed DNA repair (HDR) activity of all BARD1 missense substitutions identified in 55 breast and ovarian cancer samples, using the real-world data from the COSMIC and cBioPortal databases. Seven BARD1 variants (V85M, P187A, G491R, R565C, P669L, T719R, and Q730L) were confirmed to impair DNA damage repair. Furthermore, cells harboring these BARD1 variants exhibited increased sensitivity to the chemotherapeutic drugs, cisplatin, and olaparib, compared to cells expressing wild-type BARD1. These findings collectively suggest that these seven missense BARD1 variants are likely pathogenic and may respond well to cisplatin-olaparib combination therapy. This study not only enhances our understanding of BARD1's role in DNA damage repair but also offers valuable insights into predicting therapy responses in patients with specific BARD1 missense mutations.

Homologous recombination deficiency gene panel analysis results in synchronous endometrial and ovarian cancers.

The objective of this study was to analyze the genetic alterations of tumors within the scope of the homologous recombination deficiency gene panel in patients diagnosed with synchronous endometrial ovarian cancer who have been followed for over 5 years using next-generation sequencing. DNA was isolated from the patient's formalin-fixed, paraffin-embedded tissue blocks. Next-generation sequencing was performed using the Illumina capture-based sequencing method. Samples were sequenced using the Sophia HR Solution DNA Kit. Seven patients were included in this study. The ratios of likely pathogenic (LP)/pathogenic (P) somatic mutations in ATM (serine/threonine kinase or Ataxia-telangiectasia mutated gene), BRCA2 (breast cancer type 2 susceptibility gene), BARD1 (BRCA1 associated RING domain 1), TP53 (tumor protein p53), BIRP1 (BRCA1-interacting helicase 1 gene), PALB2 (partner and localizer of BRCA2), and CHECK2 were 21 (48.8%), 8 (18.6%), 5 (11.6%), 3 (6.9%), 2 (4.6%), 2 (4.6%), and 2 (4.6%), respectively, in endometrium, and the ratios of somatic mutations in ATM, BRCA2, TP53, BARD1, RAD54L (DNA repair/recombination protein like), BIRP1, and RAD51D (RAD51 recombinase paralog D) were 24 (60%), 6 (15%), 5 (12.5%), 2 (5%), 2 (5%), 1 (2.5%), and 1 (2.5%), respectively, in ovary. In endometrioid-synchronous endometrial ovarian cancer cases, P/LP mutations were observed in ATM and CHECK2 genes in endometrium and ATM, BRCA2, and TP53 genes in ovary. In two non-endometrioid-synchronous endometrial ovarian cancer cases, CHEK2 (checkpoint kinase 2) mutations were observed in endometrium and ATM and TP53 mutations in ovary, whereas in one case, P/LP mutations in ATM and TP53 genes were common in both tissues. Pathogenic variations confirming the diagnosis of synchronous endometrial ovarian cancer with genetic alterations were identified in all but one case. ATM gene mutation emerged as the most common alteration and has a potential association with a favorable prognosis.

Germline BARD1 variants predispose to mesothelioma by impairing DNA repair and calcium signaling

We report that ~1.8% of all mesothelioma patients and 4.9% of those younger than 55, carry rare germline variants of the BRCA1 associated RING domain 1 (BARD1) gene that were predicted to be damaging by computational analyses. We conducted functional assays, essential for accurate interpretation of missense variants, in primary fibroblasts that we established in tissue culture from a patient carrying the heterozygous BARD1V523A mutation. We found that these cells had genomic instability, reduced DNA repair, and impaired apoptosis. Investigating the underlying signaling pathways, we found that BARD1 forms a trimeric protein complex with p53 and SERCA2 that regulates calcium signaling and apoptosis. We validated these findings in BARD1-silenced primary human mesothelial cells exposed to asbestos. Our study elucidated mechanisms of BARD1 activity and revealed that heterozygous germline BARD1 mutations favor the development of mesothelioma and increase the susceptibility to asbestos carcinogenesis. These mesotheliomas are significantly less aggressive compared to mesotheliomas in asbestos workers.

Correlation of BARD1 gene polymorphisms with risk of neuroblastoma: a meta-analysis

BRCA1-associated RING domain protein 1 (BARD1) gene polymorphisms may be associated with neuroblastoma (NB) susceptibility. However, the results remain controversial. Relevant studies were identified by searching PubMed, Web of Science, Embase, China National Knowledge Infrastructure databases up to March 5, 2023. The strength of the association between BARD1 polymorphisms and susceptibility of NB was assessed by calculating odds ratios (ORs) and 95% confidence intervals (95% CIs) through the fixed- or random-effects model. Eight articles involving 12 studies were finally included. We found that rs6435862 T > G, rs3768716 A > G, rs17487792 C > T and rs7587476 C > T variant increase the risk of NB in allelic, dominant, recessive, homozygous and heterozygous genetic models, while rs7585356 G > A variant appeared protective against NB. When stratified by ethnicity, subgroup analysis indicated that the above association remained significant in Caucasian populations in all genetic models, except for rs7585356G > A polymorphism in Asians. In Asian populations, we found the similar results in the allelic and dominant model of rs6435862 T > G, rs3768716 A > G, rs17487792 C > T and rs7587476 C > T as in Caucasians, while there lacked a significant association in the other three model. In addition, rs7585356 G > A was not associated with an increased risk of NB in the Asian population. After Bonferroni correction, significant associations for rs7585356 G > A disappeared in both Asian and Caucasian populations, with no significant association found for rs7587476 in the allelic and dominant models among Asians. BARD1 polymorphisms might be significantly associated with NB susceptibility. It is crucial that these finding should be further confirmed through extensive and well-planned studies.

BARD1 deletion in a patient with suspected hereditary colorectal cancer

Deleterious germline variants in the BRCA1-associated ring domain (BARD1) gene moderately elevate breast cancer risk; however, their potential association with other neoplasms remains unclear. Here, we present the case of a 43-year-old female patient diagnosed with sigmoid colon adenocarcinoma whose maternal family members met the Amsterdam Criteria II for Lynch syndrome. Comprehensive multigene panel testing revealed a heterozygous BARD1 exon 3 deletion.

CellTool: An Open-Source Software Combining Bio-Image Analysis and Mathematical Modeling for the Study of DNA Repair Dynamics

Elucidating the dynamics of DNA repair proteins is essential to understanding the mechanisms that preserve genomic stability and prevent carcinogenesis. However, the measurement and modeling of protein dynamics at DNA lesions via currently available image analysis tools is cumbersome. Therefore, we developed CellTool—a stand-alone open-source software with a graphical user interface for the analysis of time-lapse microscopy images. It combines data management, image processing, mathematical modeling, and graphical presentation of data in a single package. Multiple image filters, segmentation, and particle tracking algorithms, combined with direct visualization of the obtained results, make CellTool an ideal application for the comprehensive analysis of DNA repair protein dynamics. This software enables the fitting of obtained kinetic data to predefined or custom mathematical models. Importantly, CellTool provides a platform for easy implementation of custom image analysis packages written in a variety of programing languages. Using CellTool, we demonstrate that the ALKB homolog 2 (ALKBH2) demethylase is excluded from DNA damage sites despite recruitment of its putative interaction partner proliferating cell nuclear antigen (PCNA). Further, CellTool facilitates the straightforward fluorescence recovery after photobleaching (FRAP) analysis of BRCA1 associated RING domain 1 (BARD1) exchange at complex DNA lesions. In summary, the software presented herein enables the time-efficient analysis of a wide range of time-lapse microscopy experiments through a user-friendly interface.

Circular RNA mediated gene regulation in human breast cancer: A bioinformatics analysis.

Circular RNAs (circRNAs) are a new acknowledged class of RNAs that has been shown to play a major role in several biological functions both in physiological and pathological conditions, operating as critical part of regulatory processes, like competing endogenous RNA (ceRNA) networks. The ceRNA hypothesis is a recently discovered molecular mechanism that adds a new key layer of post-transcriptional regulation, whereby various types of RNAs can reciprocally influence each other's expression competing for binding the same pool of microRNAs, even affecting disease development. In this study, we build a network of circRNA-miRNA-mRNA interactions in human breast cancer, called CERNOMA, that is a bipartite graph with one class of nodes corresponding to differentially expressed miRNAs (DEMs) and the other one corresponding to differentially expressed circRNAs (DEC) and mRNAs (DEGs). A link between a DEC (or DEG) and DEM is placed if it is predicted to be a target of the DEM and shows an opposite expression level trend with respect to the DEM. Within the CERNOMA, we highlighted an interesting deregulated circRNA-miRNA-mRNA triplet, including the up-regulated hsa_circRNA_102908 (BRCA1 associated RING domain 1), the down-regulated miR-410-3p, and the up-regulated ESM1, whose overexpression has been already shown to promote tumor dissemination and metastasis in breast cancer.

Abstract 868: Novel pathogenic mutations in BRCA1 gene that may influence the interaction of BRCA1 protein with BARD1 protein in breast cancer patients

Abstract Breast cancer (BC) contributes to 30% of all women suffering from cancer in the US. Despite enhanced survival rates and recovery rates, pathogenic mutation discovery remains indispensable for early detection and prognosis. BRCA1 pathogenic mutations increase the chance of BC to 55% by the age of 70. A crucial mechanism of mutations is affecting protein-protein interaction. BRCA1 interacts with the BARD1 protein forming a complex that contributes to DNA repair and plays a vital role in tumor suppression. In this study, we aimed to investigate mutations that contribute to the dysfunction of BRCA1 protein through sequencing and computational analysis and their impact on the active site interacting with BRCA1 associated Ring Domain 1 (BRAD1) of BRCA1, and hence its beneficial role in BC prognosis. Herein, we represent a study on 48 BC patients sequenced using Devyser BRCA NGS kit, according to the manufacturer’s instructions. The reagent kit V2, 500 Cycles PE, on the Illumina MiSeq System. Using the Illumina MiSeq System to detect pathogenic mutations. Bioinformatics analysis was carried out for variant calling and different databases, such as ClinVar and BIC, were searched to identify the novel mutations. P2RANK software was used to identify and confirm the active site protein sequences. For BRCA1, we studied three missense mutations present in the ring domain of the protein interacting with BRAD1. We used PDBsum server to retrieve protein structures predicted by the Artificial Intelligence (AI)-based AlphaFold software. Structures were further confirmed using Ramachandran plot. We used Dynamut server to study the stability and structural variability due to mutations. Docking was carried out using HDock software and significant poses were sorted out based on their RMSD values. For BRCA1, Cys47Ser and Cys39Arg caused destabilization of the protein represented as Gibb’s energy by -2.257 kcal/mol and -0.318 kcal/mol respectively. On the other hand, Cys39Tyr caused the stabilization of protein by 0.247 kcal/mol. The effect of mutations on rigidity was determined in terms of vibrational entropy where Cys47Ser caused an increase of protein flexibility with the energy of 0.262 kcal.mol−1.K−1. Cys39Arg and Cys39Tyr decreased the flexibility of the protein and caused rigidity by -0.164 kcal.mol−1.K−1 and -0.371 kcal.mol−1.K−1 respectively. In, conclusion, investigating the three selected pathogenic mutations showed a clear impact on the structure of the protein favored different types of bonds in the active site that may alter the interaction between BRCA1 and BARD1. This study has a beneficial role to investigate the impact of BRCA1 mutations on the structural stability of the protein and thereby the risk of BC development Citation Format: Yahia Ahmad Yahia, Nasra F. Abdel Fattah, Marwa A. Abdel Wahed, Fatma S S Hafez, Sara HA Arwa, Samah A. Loutfy. Novel pathogenic mutations in BRCA1 gene that may influence the interaction of BRCA1 protein with BARD1 protein in breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 868.

Abstract 6096: BRCA1-BARD1 ubiquitylates histones for genome maintenance

Abstract Background: Breast cancer type 1 susceptibility protein (BRCA1) is a tumor suppressor gene involved in DNA double strand break repair with well-known cancer implications. BRCA1 heterodimerizes with BRCA1 associated Ring domain 1 (BARD1) to form a complex with DNA binding and ubiquitin E3 ligase function capable of interacting with proteins of diverse biological processes, most notably homology-directed DNA repair. During DNA repair, BRCA1-BARD1 directly interfaces with nucleosomes and transfers mono ubiquitin (Ub) to lysine residues on the C-terminal tail of histone H2A. Although truncation of the enzymatic BRCA1-BARD1 RING-RING domain retains H2A ubiquitylating activity, full-length BRCA1-BARD1 binds more tightly with nucleosomes and displays higher H2A-Ub activity. However, the molecular basis and biological significance for this enhanced nucleosome binding and H2A-Ub activity is uncharacterized. Methods: Full length BRCA1-BARD1 or truncated mutants and histones were purified from E. coli. or insect cells. Nucleosomes were assembled for in vitro ubiquitylation reaction and binding assays. To determine the biological significance, mammalian cell lines that stably express wild type or mutant forms of BARD1 were established for cellular fractionation, foci analysis, and clonogenic survival studies alongside various DNA damage agents. Results: Our results show multiple interaction sites exist between BRCA1-BARD1 and nucleosomes which allow high-affinity chromatin binding and promote increased histone H2A ubiquitylation activity. Multivalent BARD1-nucleosome interactions, namely those using strong binding motifs located in the intrinsically disordered region (IDR) of BARD1, and the weak “kiss” interaction mediated by the RING domains of both BRCA1 and BARD1, are essential for H2A ubiquitylation by BRCA1-BARD1. Further, we isolated two types of specific histone binding and/or ubiquitylation-defective mutants of BARD1: a BARD1-IDR mutant with disrupted nucleosome binding withe retained H2A ubiquitylation ability, and a RING mutant that solely impairs H2A ubiquitylation. In both cases, we demonstrate that these mutants are hypersensitive to DNA damage agents, including polyADP-ribose polymerase (PARP) inhibitors, and demonstrate reduced capacity of BARD1 to associate with chromatin and foci formation owing to attenuated repair capacity. Conclusion: Our studies provide convincing evidence BRCA1-BARD1 interacts with nucleosomes and ubiquitylates histones via its E3 ligase activity. Further, it plays a critical role in DNA damage response and repair that contributes to genome stability, which when disrupted sensitizes them to DNA damage agents. Our results open new avenues towards understanding whether and how these mutations in BRCA1-BARD1 affect its tumor suppression functions and their implications clinically, ultimately with the goal to translate these findings for the benefit of cancer patients. Citation Format: Wenjing Li, Samuel R. Witus, Meiling Wang, Peter S. Brzovic, Rachel E. Klevit, Weixing Zhao. BRCA1-BARD1 ubiquitylates histones for genome maintenance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6096.

Germline heterozygous exons 8–11 pathogenic BARD1 gene deletion reported for the first time in a family with suspicion of a hereditary colorectal cancer syndrome: more than an incidental finding?

BackgroundColorectal cancer (CRC) is a highly prevalent disease in developed countries. Inherited Mendelian causes account for approximately 5% of CRC cases, with Lynch syndrome and familial adenomatous polyposis being the most prevalent forms. Scientific efforts are focused on the discovery of new candidate genes associated with CRC and new associations of phenotypes with well-established cancer-related genes. BRCA1-associated ring domain (BARD1) gene deleterious germline variants are associated with a moderate increase in the relative risk of breast cancer, but their association with other neoplasms, such as CRC, remains unclear.Case presentationWe present the case of a 49-year-old male diagnosed with rectal adenocarcinoma whose maternal family fulfilled Amsterdam clinical criteria for Lynch syndrome. Genetic test confirmed the presence in heterozygosis of a germline pathogenic deletion of exons 8–11 in BARD1 gene. The predictive genetic study of the family revealed the presence of this pathogenic variant in his deceased cancer affected relatives, confirming co-segregation of the deletion with the disease.ConclusionsTo the best of our knowledge, this is the first published work in which this BARD1 deletion is detected in a family with familial colorectal cancer type X (FCCTX) syndrome, in which the clinical criteria for Lynch syndrome without alteration of the DNA mismatch repair (MMR) system are fulfilled. Whether this incidental germline finding is the cause of familial colorectal aggregation remains to be elucidated in scientific forums. Patients should be carefully assessed in specific cancer genetic counseling units to account for hypothetical casual findings in other genes, in principle unrelated to the initial clinical suspicion, but with potential impact on their health.

Genetic variants in African-American and Hispanic patients with breast cancer.

Breast cancer is a disease with significant health disparity affecting mortality in minority women. The present study examined the genetic makeup of breast cancers in African-American and Hispanic/Latinx patients to determine specific genetic mutations associated with breast cancer in the minority population from South Los Angeles, United States. Whole-exome sequencing was performed on DNA extracted from breast cancer tumor biopsies collected from 13 African-American and 15 Hispanic women and 8 matched-normal samples for each ethnic category. The results were analyzed using Ensemble Variant Effect Predictor and Mutation Significance. Additionally, a comparative analysis with The Cancer Genome Atlas data was provided. Our data revealed somatic mutations in genes such as SET domain containing (lysine methyltransferase) 8, serine protease 1 and AT-rich interaction domain 1B (ARID1B) and known breast cancer genes, such as BRCA1/2, TP53 and the DNA damage response genes across all ethnicities. Additionally, Hispanic patients had BRCA1 associated RING domain 1B (BARD1) variants, while African-American patients had higher numbers of nonsynonymous variants in the RAD51 paralog B (RAD51B), ARID1B and X-ray repair cross complementing 3 (XRCC3) genes. In addition, our patients exhibited mutational signature enrichment that indicated DNA homologous recombination repair deficiencies. Therefore, African-American and Hispanic breast cancer samples showed considerable overlap in breast cancer genetic mutations. However, there are differences in specific genetic variants in TP53, BRCA1/2, BARD1 or ARID1B, which will require further study of their role in tumorigenesis.

Abstract 2597: Inhibition of BARD1-PLK1 axis enhances PARP inhibitor/platinum sensitivity in homologous repair proficient pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a grim prognosis. Though the overall 5-year survival rates have recently improved to 10%, there is still an unmet need for better therapies. PARP inhibitors (PARPi)/platinum therapies have demonstrated clinical efficacy in BRCA1/2 mutated PDAC and has recently led to the FDA approval of PARPi (olaparib) in the maintenance setting, offering the best personalized treatment approach for such patients. Although promising, the benefits of these therapeutic strategies are currently limited to only a few patients. We have recently established that BARD1 (BRCA1- Associated-Ring-Domain-1), the main binding partner of BRCA1 in regulating homologous recombination repair is upregulated in PDAC cells exposed to PARPi/platinum compounds, by a post-transcriptional RNA binding mechanism. We therefore hypothesized that inhibition of BARD1 will sensitize cells to these agents. We found that inhibition of BARD1 (both by siRNA/shRNA and CRISPR-KO) not only sensitized the cells to olaparib or oxaliplatin but enhanced DNA damage in cells, as analyzed by comet assays and γ-H2AX immunofluorescence staining. BARD1 inhibition led to a decrease in homologous repair efficiency of cells as analyzed by the well established pDRGFP assay. To further understand BARD1 dependent downstream signaling, we performed comparative transcriptomic analysis of BARD1 silenced cells compared to scramble control. RNA seq analysis revealed modulation of several cell cycle checkpoints and DNA damage response pathways. Notably, through gene set enrichment analysis (GSEA), we found that silencing BARD1 causes de-enrichment of pathway regulating Polo-Like-Kinase-1 (PLK1, a serine/threonine kinase) activity (p-val=0.05, FDR=0.07). PLK1 is frequently upregulated in PDAC cells and promotes progression of cells through mitosis. Based on these results, we found that a PLK1 specific inhibitor (onvansertib) synergized with olaparib to reduce cell survival of homologous repair proficient (HR-P) PDAC cells. Importantly, the mechanism of drug synergy involved induction of apoptosis and DNA damage, as assessed by cleaved caspase-3 and γ-H2AX protein analysis. These results are suggestive of targeting the BARD1-PLK1 axis in order to enhance sensitivity of PARPi/platinums in HR proficient cells. Our ongoing and future studies are aimed at understanding the interaction of BARD1 and PLK1 in PDAC cells and analyze the effects of onvansertib and olaparib on tumor growth in vivo. Accomplishment of these studies will establish novel therapeutic targets and synthetic lethal combinations that have not been explored yet for deadly pancreatic cancers, ultimately benefiting patient survival in the long run. Citation Format: Carolyn Coats, Anoohya Arkala, Eric Londin, Avinoam Nevler, Jonathan R. Brody, Charles J. Yeo, Aditi Jain. Inhibition of BARD1-PLK1 axis enhances PARP inhibitor/platinum sensitivity in homologous repair proficient pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2597.

Identified potential pathogenic variants of BARD1 in 1449 Chinese high-risk breast cancer patients.

e22525 Background: BARD1 (BRCA1-associated ring domain 1), nuclear partner of BRCA1, has been recognized as a breast cancer predisposition gene. Recently, two large-scale population-based case–control studies confirmed that germline loss-of-function mutations in BARD1 were associated with a risk of breast cancer. Since the germline mutation frequency of BARD1 is much less than that of BRCA1/2, the distinct mutation spectrum of BARD1 is still obscure, especially in Chinese breast cancer patients. To verify the utility of BARD1 genetic testing in Chinese population, we assessed the mutational frequency and spectrum of BARD1 in a sequential series of Chinese high-risk breast cancer patients. Methods: A cohort of high-risk breast cancer patients (n = 1449) were collected in Zhejiang Cancer Hospital from 2008 through 2020, including 608 familial breast cancer (BC) cases, 173 bilateral BC cases, 222 triple negative BC cases, 436 early-onset BC cases (≤40 years) and 10 male BC cases. The complete coding sequence and intron–exon boundaries of BARD1 were screened by a 98-gene panel sequencing assay. The SIFT, Polyphen2 and MutationTaster prediction programs were used for analyzing the effect of the variants of unknown significance (VUS) on the BARD1 protein function. Results: A total of three BARD1 pathogenic mutations (c.1348_1349delinsCAT, c.70_71insGT and c.373G > T) were identified, which had been all reported, and accounting for 0.2% (3/1449) in Chinese high-risk breast cancer patients. The frequencies were 0.33% (2/608) and 0.12% (1/841) in familial and non-familial breast cancer patients, respectively. In total, 19 VUSs including 17 missenses, one inframe deletion and one inframe insertion of BARD1 were identified in this study. Based on in silico analysis, nine BARD1 missense variants (c.76A > G, c.1912G > A, c.1693C > T, c.2191C > T, c.127C > A, c.1601C > T, c.443G > A, c.233G > A and c.1972C > T) were classified as potentially damaging. However, the two newly variants c.420_422del and c.68_69insTCCGGGAACGAGCCTCGTTCC still remain VUS, and the other eight variants were classified as benign. Conclusions: Our data presented the germline mutations of BARD1 in a large-scale Chinese high-risk breast cancer population, which accounted for 0.2% in our cohort. Moreover nine potential pathogenic variants were found based on in silico analysis. Clinically, these data may be helpful in genetic counseling of breast cancer patients with BARD1 germline mutation. While the further research on the VUS are needed.

BARD1 mystery: tumor suppressors are cancer susceptibility genes

The full-length BRCA1-associated RING domain 1 (BARD1) gene encodes a 777-aa protein. BARD1 displays a dual role in cancer development and progression as it acts as a tumor suppressor and an oncogene. Structurally, BARD1 has homologous domains to BRCA1 that aid their heterodimer interaction to inhibit the progression of different cancers such as breast and ovarian cancers following the BRCA1-dependant pathway. In addition, BARD1 was shown to be involved in other pathways that are involved in tumor suppression (BRCA1-independent pathway) such as the TP53-dependent apoptotic signaling pathway. However, there are abundant BARD1 isoforms exist that are different from the full-length BARD1 due to nonsense and frameshift mutations, or deletions were found to be associated with susceptibility to various cancers including neuroblastoma, lung, breast, and cervical cancers. This article reviews the spectrum of BARD1 full-length genes and its different isoforms and their anticipated associated risk. Additionally, the study also highlights the role of BARD1 as an oncogene in breast cancer patients and its potential uses as a prognostic/diagnostic biomarker and as a therapeutic target for cancer susceptibility testing and treatment.

Apparent regional differences in the spectrum of BARD1 pathogenic variants in Spanish population and importance of copy number variants

Only up to 25% of the cases in which there is a familial aggregation of breast and/or ovarian cancer are explained by germline mutations in the well-known BRCA1 and BRCA2 high-risk genes. Recently, the BRCA1-associated ring domain (BARD1), that partners BRCA1 in DNA repair, has been confirmed as a moderate-risk breast cancer susceptibility gene. Taking advantage of next-generation sequencing techniques, and with the purpose of defining the whole spectrum of possible pathogenic variants (PVs) in this gene, here we have performed a comprehensive mutational analysis of BARD1 in a cohort of 1946 Spanish patients who fulfilled criteria to be tested for germline pathogenic mutations in BRCA1 and BRCA2. We identified 22 different rare germline variants, being 5 of them clearly pathogenic or likely pathogenic large deletions, which account for 0.26% of the patients tested. Our results show that the prevalence and spectrum of mutations in the BARD1 gene might vary between different regions of Spain and expose the relevance to test for copy number variations.

BRCA1-associated RING domain-1 (BARD1) loss and GBP1 expression enhance sensitivity to DNA damage in Ewing sarcoma.

Ewing sarcoma is a fusion oncoprotein-driven primary bone tumor. A subset of patients (~10%) with Ewing sarcoma are known to harbor germline variants in a growing number of genes involved in DNA damage repair. We recently reported our discovery of a germline mutation in the DNA damage repair protein BARD1 (BRCA1-associated RING domain-1) in a patient with Ewing sarcoma. BARD1 is recruited to the site of DNA double stranded breaks via the poly(ADP-ribose) polymerase (PARP) protein and plays a critical role in DNA damage response pathways including homologous recombination. We thus questioned the impact of BARD1 loss on Ewing cell sensitivity to DNA damage and the Ewing sarcoma transcriptome. We demonstrate that PSaRC318 cells, a novel patient-derived cell line harboring a pathogenic BARD1 variant, are sensitive to PARP inhibition and by testing the effect of BARD1 depletion in additional Ewing sarcoma cell lines, we confirm that BARD1 loss enhances cell sensitivity to PARP inhibition plus radiation. Additionally, RNA-seq analysis revealed that loss of BARD1 results in the upregulation of GBP1 (guanylate-binding protein 1), a protein whose expression is associated with variable response to therapy depending on the adult carcinoma subtype examined. Here, we demonstrate that GBP1 contributes to the enhanced sensitivity of BARD1 deficient Ewing cells to DNA damage. Together, our findings demonstrate the impact of loss-of function mutations in DNA damage repair genes, such as BARD1, on Ewing sarcoma treatment response.

HuR Plays a Role in Double-Strand Break Repair in Pancreatic Cancer Cells and Regulates Functional BRCA1-Associated-Ring-Domain-1(BARD1) Isoforms.

Simple SummaryRNA binding proteins (RBPs) post-transcriptionally regulate and stabilize a variety of target mRNA transcripts. Human Antigen R (HuR) binds to several pro-survival mRNA transcripts and promotes tumor cell survival and chemotherapeutic resistance. Some of these targets are within the DNA damage repair pathway that help cells repair damaged DNA and allow them to proliferate. Homologous recombination repair is the preferred method of DNA repair in the cells. Here, we aim to understand if HuR regulates homologous recombination repair in pancreatic ductal adenocarcinoma (PDAC). Our analysis, using Ribonucleo-protein Immunoprecipitation (RNP-IP) coupled with microarray and RNA-sequencing studies, revealed that HuR binds BARD1 (BRCA-1 Associated Ring Domain 1) and regulates expression of BARD1 mRNA full length transcripts and alternative isoforms. Silencing BARD1 mRNA potentiated the DNA damage effects of clinically relevant drugs, olaparib and oxaliplatin, in PDAC cells. Together, this work underscores a transient regulatory DNA repair pathway (i.e., HuR—BARD1 mRNA), which is likely a PDAC therapeutic resistance mechanism.Human Antigen R (HuR/ELAVL1) is known to regulate stability of mRNAs involved in pancreatic ductal adenocarcinoma (PDAC) cell survival. Although several HuR targets are established, it is likely that many remain currently unknown. Here, we identified BARD1 mRNA as a novel target of HuR. Silencing HuR caused a >70% decrease in homologous recombination repair (HRR) efficiency as measured by the double-strand break repair (pDR-GFP reporter) assay. HuR-bound mRNAs extracted from RNP-immunoprecipitation and probed on a microarray, revealed a subset of HRR genes as putative HuR targets, including the BRCA1-Associated-Ring-Domain-1 (BARD1) (p < 0.005). BARD1 genetic alterations are infrequent in PDAC, and its context-dependent upregulation is poorly understood. Genetic silencing (siRNA and CRISPR knock-out) and pharmacological targeting of HuR inhibited both full length (FL) BARD1 and its functional isoforms (α, δ, Φ). Silencing BARD1 sensitized cells to olaparib and oxaliplatin; caused G2-M cell cycle arrest; and increased DNA-damage while decreasing HRR efficiency in cells. Exogenous overexpression of BARD1 in HuR-deficient cells partially rescued the HRR dysfunction, independent of an HuR pro-oncogenic function. Collectively, our findings demonstrate for the first time that BARD1 is a bona fide HuR target, which serves as an important regulatory point of the transient DNA-repair response in PDAC cells.

Metformin alleviates ionizing radiation-induced senescence by restoring BARD1-mediated DNA repair in human aortic endothelial cells.

Metformin is one of the most effective therapies for treating type 2 diabetes and has been shown to also attenuate aging and age-related disorders. In this study, we explored the relationship between metformin and DNA damage repair in ionizing radiation (IR)-induced damage of human aortic endothelial cells (HAECs). Metformin treatment suppressed IR-induced senescence phenotypes, such as increased senescent-associated β-galactosidase (SA β-gal) activity and decreased tube formation and proliferation. Moreover, metformin increased BRCA1-associated RING domain protein 1 (BARD1) and RAD51 expression in both aging and IR-exposed cells. Metformin-treated cells exhibited higher levels of the BRCA1-BARD1-RAD51 complex during irradiation, even in the presence of compound C, an AMP-activated protein kinase inhibitor. BARD1 knockdown confirmed its critical role in metformin-mediated inhibition of endothelial senescence. Metformin increased blood vessel sprouting and decreased SA β-gal activity in mouse aortas. Collectively, our findings provide new insights into how metformin can prevent endothelial cell senescence by promoting BARD1-related DNA damage repair, suggesting that metformin may be an effective anti-aging agent and a promising therapeutic for protecting against radiation-induced cardiotoxicity.

Mutations in exon region of BRCA1-related RING domain 1 gene and risk of breast cancer.

BackgroundBRCA1‐associated RING Domain 1 (BARD1) is an important gene related to breast cancer development. However, the role of BARD1 mutations in breast cancer remains inconclusive. This study is to investigate the relationship between exon mutations of BARD1 gene and the risk of early‐onset breast cancer.MethodsTotally, 60 cases of early‐onset breast cancer patients (age 30–40 years) and 240 healthy women (age 30–40 years) were enrolled. Exon mutations of BARD1 were detected and analyzed by direct sequencing and SNaPshot.ResultsThe risk of breast cancer was increased by 3.475 times in carriers with deletion mutation at rs28997575 site of BARD1 (aOR1 = 3.475, 95%CI = 1.302–9.276) (p = 0.013). The risk of breast cancer in carriers with GC genotype at rs2229571 site of BARD1 was reduced by 72.6% (aOR1 = 0.274, 95%CI = 0.134–0.562) (p = 0.001), and that in carriers with CC genotype was reduced by 82.8% (aOR1 = 0.172, 95%CI = 0.076–0.392) (p = 0.001). After stratification with family history, the difference of rs2229571 site mutation genotype was statistically significant (OR = −2.169, 95%CI = 0.016–0.828, p = 0.032). Additionally, the frequency distribution of breast cancer family history in the case group (15%) was significantly more than that in the control group (6.7%) (p = 0.037).ConclusionThe deletion mutation at rs28997575 locus of the BARD1 gene can significantly increase the risk of breast cancer. The mutation genotype of rs2229571 locus can significantly reduce the risk of breast cancer. Family history is associated with BARD1 gene polymorphism. A family history of breast cancer may be a risk factor for breast cancer.

Computational structural assessment of BReast CAncer type 1 susceptibility protein (BRCA1) and BRCA1-Associated Ring Domain protein 1 (BARD1) mutations on the protein-protein interface.

Breast cancer type 1 susceptibility protein (BRCA1) is closely related to the BRCA2 (breast cancer type 2 susceptibility protein) and BARD1 (BRCA1-associated RING domain-1) proteins. The homodimers were formed through their RING fingers; however they form more compact heterodimers preferentially, influencing BRCA1 residues 1-109 and BARD1 residues 26-119. We implemented an integrative computational pipeline to screen all the mutations in BRCA1 and identify the most significant mutations influencing the Protein-Protein Interactions (PPI) in the BRCA1-BARD1 protein complex. The amino acids involved in the PPI regions were identified from the PDBsum database with the PDB ID: 1JM7. We screened 2118 missense mutations in BRCA1 and none in BARD1 for pathogenicity and stability and analyzed the amino acid sequences for conserved residues. We identified the most significant mutations from these screenings as V11G, M18K, L22S, and T97R positioned in the PPI regions of the BRCA1-BARD1 protein complex. We further performed protein-protein docking using the ZDOCK server. The native protein-protein complex showed the highest binding score of 2118.613, and the V11G mutant protein complex showed the least binding score of 1992.949. The other three mutation protein complexes had binding scores between the native and V11G protein complexes. Finally, a molecular dynamics simulation study using GROMACS was performed to comprehend changes in the BRCA1-BARD1 complex's binding pattern due to the mutation. From the analysis, we observed the highest deviation with lowest compactness and a decrease in the intramolecular h-bonds in the BRCA1-BARD1 protein complex with the V11G mutation compared to the native complex or the complexes with other mutations.