Multimodal analysis of rare BARD1 missense variant suggests its pathogenicity is conditional

Multimodal analysis of rare BARD1 missense variant suggests its pathogenicity is conditional | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Case Report Multimodal analysis of rare BARD1 missense variant suggests its pathogenicity is conditional Fiona Chan-Pak-Choon, Yuandi Gao, José Camacho-Valenzuela, Júlia-Jié Cabré-Romans, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7595877/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Hereditary breast cancer involves multiple risk genes, including BARD1 , which confers low to moderate risk and is associated with triple-negative breast cancer (TNBC). We report a proband with a primary ER+/PR+/HER2- breast cancer which recurred unilaterally as a TNBC and who later developed endometrial cancer. Clinical germline testing revealed a rare BARD1 missense variant of uncertain significance [c.2258G > T; p.(Gly753Val)]. Whole-exome sequencing of tumors and blood revealed BARD1 loss of heterozygosity and mutational signature 3 – indicative of homologous recombination (HR) repair deficiency – exclusively in the TNBC recurrence. Functional assays demonstrated impaired HR repair via increased PARP inhibitor sensitivity and reduced RAD51 foci formation. We hypothesize that the selective pressure exerted by tamoxifen resulted in breast cancer subtype switching and uncovered the pathogenic potential of the BARD1 p.(Gly753Val) variant. This case illustrates a previously unreported scenario where the pathogenicity of a germline BARD1 variant appears conditional on prior treatment. Figures Figure 1 Figure 2 Introduction Approximately 5–10% of all breast cancers are attributable to hereditary predisposition, with germline pathogenic variants (GPVs) in BRCA1 and BRCA2 accounting for 20–30% of such cases 1 , 2 . The genes associated with hereditary breast cancer can largely be grouped into three categories: high-risk genes - BRCA1 , BRCA2 , PALB2 , and TP53 ; moderate-risk genes – ATM and CHEK2 , which are primarily linked to estrogen receptor-positive (ER+) breast cancer; and a third group – BARD1 , RAD51C , and RAD51D , that confers low to moderate risk and is more strongly associated with triple-negative breast cancer (TNBC) 3 – 6 . These genes are typically included in multigene panel testing for individuals with suggestive personal or family histories, as identifying GPVs can inform clinical decision-making, including enhanced surveillance, risk-reducing strategies and future treatment options 3 , 7 . BARD1 (BRCA1-associated RING domain 1) encodes a protein that forms a heterodimer with BRCA1 in vivo via their respective Really Interesting New Gene (RING) domains 8 . The BRCA1–BARD1 complex plays a central role in homologous recombination (HR), the high-fidelity repair pathway for DNA double-strand breaks 9 , 10 . It modifies chromatin to enable access to damaged DNA, facilitates DNA end resection, and recruits and stabilizes RAD51 along with its mediators at damage sites to promote strand invasion and downstream HR repair processes 8 , 9 , 11 . Estimating the cancer risk associated with BARD1 GPVs remains challenging due to the rarity of carriers and the low to moderate penetrance of the variants 12 . BARD1 GPVs have been particularly associated with an elevated risk of triple-negative breast cancer (TNBC) 13 , 14 . Several case-control and population-based studies have reported odds ratios between 2.00 and 5.35 for overall breast cancer risk, with higher estimates for TNBC, ranging from 3.18 to 11.3 4,5,14–20 . However, ambiguity in the interpretation of many BARD1 variants limits their use in clinical risk assessment and management. To date, only truncating variants have been classified as likely pathogenic or pathogenic, while all missense variants continue to be classified as variants of uncertain significance (VUS) due to insufficient functional, epidemiologic, and segregation data 12 , 21 . We present the case of a proband with two primary malignancies (breast and endometrial cancers). Both the proband and her father, who was diagnosed with colon adenocarcinoma, were found to carry a rare missense variant in BARD1 (NM_000465.4): c.2258G > T; p.(Gly753Val). This variant is currently classified as a VUS (ClinVar ID: 649553). This study shows that detailed functional, molecular, and clinical data were required to resolve uncertainty regarding the pathogenicity of this variant. Results Case presentation The proband (Fig. 1 A, arrow) presented with breast carcinomas at age 50y (ER+/PR+/HER2-, pT1c pN1 pMx), 59y (TNBC, pTx pNx pMx – staging was not possible as the tumour was removed during diagnostic biopsy) and was later diagnosed with endometrial carcinoma at 62y (FIGO 1A). Her father was diagnosed with colon polyps at 67y and colon adenocarcinoma at 82y; two of her paternal aunts developed breast carcinomas at 56y and 81y, respectively (Fig. 1 A). Following the diagnosis of her second breast cancer, clinical germline testing in the proband revealed a rare BARD1 VUS [ BARD1 (NM_000465.4): c.2258 G > T; p.(Gly753Val)], occurring within the BRCA1 C-Terminus (BRCT) domain. For the ER+/PR + breast cancer, the proband received adjuvant chemotherapy with six cycles of docetaxel, doxorubicin, and cyclophosphamide (TAC), followed by three years of tamoxifen and an additional seven years of aromatase inhibitors until the diagnosis of her TNBC. For the TNBC, she underwent bilateral mastectomies and received four cycles of adjuvant docetaxel and cyclophosphamide. At age 62, she was treated for endometrial carcinoma with a total abdominal hysterectomy and bilateral salpingo-oophorectomy, without postoperative therapy. Genomic studies Whole-exome sequencing (WES) analysis performed on the proband’s blood, the two breast tumors, the endometrial tumor, as well as her father’s colon tumour and adjacent normal colon tissue confirmed the presence of the germline BARD1 VUS in all samples (Fig. 1 B). No likely causative variants were identified in other cancer predisposition genes (n = 144) in the samples based on our variant prioritization analysis. Copy number variation (CNV) analysis detected loss of heterozygosity (LOH) at the BARD1 locus in the proband’s TNBC only (Fig. 1 C). No second hits in BARD1 were identified in the other tumors. Clonal evolution analysis based on WES data was used to evaluate tumor relatedness across the proband’s tumors (Fig. 1 D). The first breast cancer (ER+/PR+) harbored 76 somatic variants (39 clonal, 38 subclonal), and the subsequent TNBC harbored 206 somatic variants (113 clonal, 98 subclonal), including 12 > 4bp deletions, indicative of increased genomic instability. These two tumors shared 23 clonal somatic variants, indicating that the second breast cancer was a recurrence rather than an independent primary tumor. In contrast, the endometrial tumor harbored 336 somatic variants (126 clonal, 210 subclonal) and showed no shared somatic variants with either breast cancer, supporting its origin as an independent primary tumor. Mutational signature analysis using the WES data revealed distinct mutational processes across the tumors (Fig. 1 E). The ER+/PR + tumor was dominated by Signature 1, associated with age-related mutagenesis. The TNBC displayed a more complex signature profile, with contributions from Signatures 1, 3 (homologous recombination (HR) repair deficiency), and 6 (DNA mismatch repair deficiency). The endometrial tumor exhibited Signatures 1, 2 (APOBEC activity), and 12 (unknown etiology). Functional studies To evaluate the functional impact of BARD1 VUS identified in the proband on HR repair, HCT116 cells engineered with both BARD1 alleles tagged with an auxin-inducible degron (AID) (HCT116- BARD1 AID/AID ) were used 22 . These cells carry a doxycycline-inducible E3 ubiquitin ligase, OsTIR1, which interacts with AID upon treatment with indole-3-acetic acid (IAA), leading to the rapid degradation of endogenous BARD1. Lentiviral transduction was used to introduce exogenous wild-type (WT) BARD1 (negative control), the patient-derived BARD1 variant [ BARD1 (NM_000465.4): c.2258 G > T; p.(G753V); hereafter G753V], the known functionally defective BARD1 missense variant [ BARD1 (NM_000465.4): c.159T > G; p.(Cys53Trp); hereafter C53W] 23 , 24 , or GFP (positive control lacking BARD1 expression). To assess the effective degradation of endogenous BARD1, immunoblotting was performed following IAA treatment. As expected, endogenous BARD1 was efficiently degraded in all conditions (Fig. 2 A). Lentiviral expression only achieved sub-physiological BARD1 levels, detectable at longer immunoblot exposures (Fig. 2 A, upper panel). The cell lines were then used to assess cell survival following treatment with olaparib, a poly(ADP-ribose) polymerase (PARP) inhibitor. Following auxin-induced degradation of endogenous BARD1, cells were chronically exposed to olaparib concentrations ranging from 0 to 500 nM for 10 days prior to staining with crystal violet (Fig. 2 B-C). In the absence of auxin, cell viability remained close to 100% across all cell lines. Upon auxin treatment, WT BARD1 maintained high viability across all olaparib concentrations (IC 50 = 825.8 nM), indicating effective functional complementation (Fig. 2 C). In contrast, cells expressing BARD1 G753V (IC 50 = 15.7 nM), BARD1 C53W (IC 50 = 6.2 nM), or GFP (IC 50 = 5.3 nM) showed decreasing viability with increasing olaparib concentrations. BARD1 C53W and GFP exhibited the greatest loss of viability, consistent with complete loss of BARD1 function. BARD1 G753V-expressing cells consistently showed intermediate sensitivity, with viability levels falling between those of BARD1 WT and BARD1 C53W or GFP, yet more closely resembling the viability of the latter. To further investigate the effect of the BARD1 variants on HR repair, the recruitment of the HR repair machinery to DNA damage sites was assessed by quantifying RAD51 foci in S-phase cells. Immunofluorescence staining was performed following auxin-induced degradation of endogenous BARD1 and exposure to ionizing radiation. Cells expressing the BARD1 G753V variant exhibited a mean number of RAD51 foci (5.71) similar to the known functionally impaired C53W variant (5.63). Both exhibited higher RAD51 foci counts than the control cells lacking exogenous BARD1 (4.88), but lower than those observed in cells expressing WT BARD1 (7.47) (Figs. 2 D-E). Discussion Here we report a rare BARD1 missense variant (ClinVar ID: 649553) identified in a woman with a recurrent primary breast cancer and endometrial cancer in the context of a family history of colon and breast cancers. Functional analysis of the variant revealed impaired HR repair. Molecular analyses of all four tumors – the proband’s breast cancers, endometrial carcinoma, and her father's colon adenocarcinoma – revealed that only the TNBC recurrence harbored a second hit (LOH) in BARD1 , suggesting biallelic inactivation. Notably, no second hit in BARD1 was detected in the proband’s primary breast cancer. Mutational Signature 3 was present only in the recurrent breast tumor, a TNBC. One could argue that the pathogenicity of the variant became evident only after analyzing the recurrent breast cancer. Moreover, we hypothesize that the pathogenic effect of the variant was conditional upon molecular switching, likely the result of the prior treatment of the primary breast cancer. Molecular subtype switching, also known as receptor conversion, from ER+/PR + primary breast cancers to metastatic TNBCs is a recognized phenomenon 25 – 28 . This shift is thought to arise from intratumor heterogeneity and the selective effects of chemotherapy and hormonal treatments, which can promote clonal selection of resistant tumor cells 28 , 29 . We hypothesize that chemotherapy and endocrine therapy following the primary diagnosis imposed selective pressure on the tumor, allowing a minor subpopulation of therapy-resistant clones – initially comprising ~ 5% ER- cells within a predominantly (~ 95%) ER + tumor – to survive and expand 28 , 29 . Over time, this subset acquired LOH of the wild-type BARD1 allele, resulting in loss of BARD1 functionality in the presence of G753V allele and thus HR repair deficiency, enabling further genomic instability which ultimately drove the emergence of the aggressive TNBC recurrence. Although receptor conversion is well documented, how such subtype changes might influence or reveal the pathogenic potential of an underlying germline variant remains unexplored. An alternative although not mutually exclusive hypothesis is that that the pre-existing germline BARD1 variant itself played a mechanistic role in this subtype transition, rather than the other way around. To our knowledge, the potential role of germline variants in influencing molecular subtype switching has not been studied. Finally, we acknowledge that the BARD1 variant could be a bystander in the process, that the LOH is incidental, and that the mutational signature 3 has other causes. The functional data, however, suggest otherwise. Compared with cells expressing WT BARD1, cells expressing G753V showed increased olaparib sensitivity, which along with reduced RAD51 foci formation indicated impaired HR repair. This raises the question of whether the recurrent TNBC might have responded to PARP inhibitor therapy such as olaparib. While BARD1 is not currently included in the eligibility criteria for PARP inhibitor therapy 31 , two reported cases involving BARD1 germline carriers – a patient with TNBC and another with neuroblastoma – showed marked responses to PARP inhibitor treatment 32 , 33 , highlighting the possibility that BARD1 inactivation may confer sensitivity to this therapy in certain settings. Classifying the BARD1 G753V variant remains inherently difficult. Like many rare missense variants in low-penetrance genes, it occupies a grey area under current ACMG guidelines, which rely heavily on recurrence in affected individuals, segregation data, and strong population evidence—criteria that are rarely met for genes like BARD1 34 . Additionally, functional validation is hindered by the lack of established pathogenic missense variants that could serve as positive controls, making it difficult to generate sufficiently robust experimental evidence to meet the ACMG guidelines 35 . Under the current ACMG criteria, this variant meets only PS3_supporting (functional data), PM2_supporting (absence from control population), and PP3 (in-silico predictions supporting a deleterious effect); it therefore remains a VUS. This challenge is further compounded in the present case, where we hypothesize that the pathogenicity of the variant is conditional, manifesting only after treatment-induced selective pressure in the TNBC. This points to the potential value of incorporating context-dependent factors such as tumour characteristics and prior treatment into variant interpretation frameworks. In conclusion, we identified a rare BARD1 germline VUS whose pathogenicity was only revealed after biallelic inactivation of BARD1 during molecular switching to a TNBC. This case illustrates the possibility of a potentially novel scenario whereby the pathogenicity of a cancer susceptibility allele is contingent upon therapeutic selective pressure. It underscores the importance of monitoring VUSs in HR repair genes, as these variants, despite not being initially causative, may drive therapy-resistant recurrences. We demonstrate that integrating genomic, functional, and clinical data can uncover these context-dependent effects that are not immediately apparent at diagnosis and, in the future, may enable more precise, personalized treatment strategies. Methods Patient samples Blood was collected from the proband, and formalin-fixed paraffin-embedded (FFPE) tumor blocks were obtained from both the proband and her father. The proband’s samples included primary breast cancer (ER+/PR+), TNBC recurrence, and endometrial cancer. The father’s samples comprised colon adenocarcinoma and adjacent normal colon tissue. Genomic DNA from the proband’s blood was extracted using the Gentra PureGene Blood Kit (Qiagen). DNA from FFPE tissue blocks was extracted using the QIAamp® DNA FFPE Tissue Kit (Qiagen), following the manufacturer’s protocols. Whole Exome Sequencing The DNA samples were analysed by WES. The Agilent SureSelect Human All Exon V7 kit (Agilent Technologies, Santa Clara, CA, USA) was used for exome capture, and the libraries were sequenced on the Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA) at the Centre d’expertise et de services Genome Quebec (Montreal, QC, Canada). The mean coverage was 260×. The Binary Alignment Map (BAM) files were realigned to the human reference genome hg19 with the Burrows-Wheeler Aligner (BWA) 36 . PCR duplicates were marked and removed using MarkDuplicates of The Genome Analysis Toolkit (GATK v.4.1.2.0) 37 . Germline variants were called with HaplotypeCaller and somatic variants were called with Mutect2 (GATK v.4.1.2.0) 38 . All the variants were annotated with ANNOVAR 39 . The variants were filtered to only retain those (1) with 4 reads supporting the variant, (2) 20× coverage at genomic locus, (3) with variant allele frequency (VAF) ³ 5%, (4) with frequency in normal population (ExAC) of < 0.001, and (v) predicted to be pathogenic by at least four out of six predictors (PROVEAN, FATHMM, MutationTaster, MutationAssessor, MetaSVM, MCAP and CADD). An additional filter consisting of two gene panels, a hereditary cancer gene panel and a tumour-specific gene panel, was included in our pipeline: (1) a panel made up of 144 cancer susceptibility genes aggregated from Rahman’s review and genes present in the Illumina TruSight’s (Illumina) and Invitae’s hereditary cancer panels 40 – 42 and (2) 637 genes from the Catalogue Of Somatic Mutations In Cancer (COSMIC)’s database version 98 43 . The resulting lists of variants were then manually inspected via the Integrative Genomics Viewer (IGV). Copy number variant analysis The matched normal/tumour BAM files were sorted by genomic coordinates with SortSam (GATK v.4.1.2.0) 37 . We used Sequenza 44 to perform a CNV analysis based on allele-specific segmentation on the sorted BAM files, which generated copy number integer values that were used to determine LOH status, along with a visual representation of the CNV detected across the genome. Clonal analysis and phylogenetic tree The phylogenetic tree of the three tumours from the proband were reconstructed using MesKit (version 1.18.0) 45 , which infers evolutionary relationships based on the presence or absence of somatic mutations. Clonal and subclonal mutations were classified using cancer cell fraction (CCF) estimates across multiple tumour regions: mutations with CCF consistent with presence in all cancer cells were considered clonal, while those present in subsets of cells were classified as subclonal. Mutational signatures Mutational signature analysis was performed using the MesKit (version 1.18.0) 45 . Somatic mutations were categorized into 96 trinucleotide contexts and grouped into truncal (shared across all tumor regions), and branch (private to individual regions) based on phylogenetic trees. Known COSMIC mutational signatures (COSMIC V2) 46 were then fitted to each profile. Similarity between original and reconstructed mutational profiles was measured using cosine similarity. Plasmid generation pDONR221-BARD1-WT was a gift from Dr. J.R. Chapman 22 , and variants present in the sequence were modified using site-directed mutagenesis (New England Biolabs) to match the RefSeq consensus sequence. Patient mutations were then inserted using site-directed mutagenesis (New England Biolabs) as per the manufacturer’s instructions. The BARD1 (WT, G753V, C53W) and GFP open reading frames were cloned into pLenti-PGK-NEO-DEST (a gift from Eric Campeau and Paul Kaufman, Addgene plasmid #19067 47 ) using LR clonase II (Life Technologies Inc.) following the manufacturer’s protocol. All plasmid sequences were verified using Oxford Nanopore Technologies long-read sequencing (Plasmidsaurus Inc). Cell line culture HCT116- BARD1 AID/AID cells, a gift from Dr. J.R. Chapman 22 , were maintained in Dulbecco’s modified Eagle medium (DMEM)–high glucose (Gibco, 11965092) supplemented with 10% FBS. Cultures were maintained at 37°C with 5% CO 2 . Stable expression of GFP or BARD1 (WT, G753V, C53W) was achieved using lentiviral transduction. Briefly, 5 x 10 5 HEK293T cells were seeded on a 6-well plate and co-transfected with 1.5 µg of the GFP or lentiviral vector GFP or BARD1 and third-generation lentiviral packaging vectors using 1.29 µg polyethylenimine per µg of DNA in Opti-MEM (Life Technologies Inc.). Viral supernatants were collected at 48 and 72 h post-transfection, syringe-filtered (0.45 µm), and immediately applied in a 1:1 (virus/medium) mixture over the HCT116- BARD1 AID/AID cells in the presence of 4 µg/ml polybrene (Millipore Sigma). Sixteen hours after transduction, viruses were removed, and cell populations were selected with 500 µg/ml G418 Sulfate (Life Technologies Inc.). Stably transduced cell populations were maintained in the presence of the antibiotic. Cell viability with crystal violet Cells were seeded at a density of 10 4 cells per well of a 12-well plate in the presence of 2 µg/ml doxycycline. After 24 h, 250 µM indole-3-acetic acid (IAA) or carrier (DMSO) was added. One hour after IAA or DMSO addition, olaparib was added to the indicated final concentrations. Ten days after plating, cells were washed and stained for 30 minutes with crystal violet (0.5% crystal violet in 25% methanol). Cells were then extensively washed with ddH 2 O and dried before scanning. For quantification, bound crystal violet was dissolved in 10% (v/v) acetic acid, and the absorbance of solubilized dye was measured at 595 nm. All experiments were performed in technical and biological triplicate. Data were analyzed using Prism 10 (GraphPad Software LLC), performing a 4-parameter non-linear fit for IC50 calculations. Representative wells were selected for display. Protein extraction and immunoblotting Cells were collected, washed and resuspended in 100 µl ice-cold benzonase cell lysis buffer (25 mM Tris-HCl pH 8.8, 40 mM NaCl, 0.05% SDS, 2 mM MgCl 2 , 20 U/ml benzonase (Millipore Sigma), and cOmplete mini EDTA-free protease inhibitor cocktail (Roche). Extracts were incubated at room temperature for 5 min, on ice for 90 min before centrifugation at 13,000g for 15 min at 4°C. The protein concentrations of clarified supernatants were measured by Bradford assay (Bio-Rad Laboratories). Subsequently, 4x NuPage LDS sample buffer (Life Technologies Inc.) was added, and samples were boiled at 95°C for 10 min. Equal amounts of protein were loaded on NuPAGE 4–12% 1.0 mm Bis-Tris polyacrylamide gels (Life Technologies Inc.) and transferred to 0.45-µm nitrocellulose membranes (GE Healthcare). Membranes were blocked with 5% milk in PBS–0.1% (v/v) Tween-20 (PBST) for 1 h and incubated overnight with primary antibody in a solution of 3% (w/v) bovine serum albumin (BSA) in PBST. Primary antibodies include: rabbit anti-BARD1 (1:1,000, Bethyl Laboratories, A300-265A-T), mouse anti-α-tubulin (1:2,000, Santa Cruz Biotechnology, sc-32293) and mouse anti-vinculin (1:25,000, Millipore Sigma, V9131). Following primary antibody incubation, membranes were incubated with either HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies (1:20,000, Jackson Laboratories). Membranes were developed with Pierce™ ECL Western Blotting Substrate (Life Technologies Inc.) and imaged using a ChemiDoc Imaging System (Bio-Rad Laboratories). Immunofluorescence staining Cells were seeded at a density of 1.5x10 4 cells per well of a 96-well plate in the presence of 2 µg/ml doxycycline. After 24 h, 250 µM indole-3-acetic acid (IAA) was added. Two hours after IAA addition, cells were irradiated at 5 Gy. Three hours post-irradiation, cells were fixed and permeabilized for 10 min using a solution of 2% (w/v) paraformaldehyde and 0.5% (v/v) Triton X-100 in PBS. After blocking with TBS–0.1% (v/v) Tween-20 (TBST) containing 1% (w/v) BSA for 30 min, cells were washed three times with PBS and incubated with primary antibodies for 1 h at room temperature, namely, anti-H2A.X (1:5,000, Biolegend, C613402) and anti-RAD51 (1:2000, Millipore Sigma, PC130). Secondary antibodies include anti-mouse and anti-rabbit Alexa Fluor 488- or 594-conjugated antibodies (Life Technologies Inc.). Cells were stained with 1 µg/ml 4, 6-diamidino-2-phenylindole (DAPI) for 5 min for nuclear staining and imaged using a Cytation C10 imaging reader with a 40× objective (BioTek). Foci quantification was performed using Cell Profiler, with cell cycle phases determined based on integrated nuclear DAPI intensities. All experiments were performed in technical and biological duplicates, with a minimum of 1,000 S-phase cells per experimental condition. Representative images were selected for display. Ethics statement Written informed consent was obtained from all patients involved, and the study protocol was approved by the McGill University Health Centre Research Ethics Board (Project No. MP-37-2019-4865). Declarations Conflicts of interest : The authors have no conflicts of interest to disclose. Author contribution FCPC collected samples, conducted the genomic analyses, and drafted the manuscript. YG, J-JC-R, and FCPC performed the functional analyses. JCV and PP assisted with the genomic analyses. LF performed the pathology review. BR, RC-M, and WDF supervised all aspects of the project and edited the manuscript. All authors reviewed and approved the final version of the manuscript for submission. Acknowledgements This study was funded by the Canadian Institutes of Health Research grant (FDN-148390) to WDF. BR is a Miguel Servet Fellow (CP21/00038) from the Instituto de Salud Carlos III (PI20/01721). RCM is a Junior 1 Scholar from the Fonds de Recherche du Québec–Santé (FRQS). FCPC received support from the George G Harris Fellowship, granted by the Faculty of Medicine and Health Sciences, McGill University. JCV was supported in part by an internal award from The Research Institute of the McGill University Centre (RI-MUHC). This research as well as biobanking of biological material and data was made possible in part through a collaboration with the Réseau de recherche sur le cancer (RRCancer) financially supported by the Oncopole, the FRQ cancer division, which receives funding from Merck Canada Inc., GSK, Pfizer and the Ministère de l'Économie, de l'Innovation et de l'Énergie du Québec. The RRCancer is affiliated to the Canadian Tumor Repository Network (CTRNet). Data availability The data supporting the findings of this study will be deposited in the European Genome-Phenome Archive and FigShare. Accession numbers will be provided in the final version of the manuscript. References Claus EB, Schildkraut JM, Thompson WD, Risch NJ (1996) The genetic attributable risk of breast and ovarian cancer. Cancer 77:2318–2324. https://doi.org/10.1002/(SICI)1097-0142(19960601)77:11%3C2318::AID-CNCR21%3E3.0.CO;2-Z De Brakeleer S et al (2010) Cancer predisposing missense and protein truncating BARD1 mutations in non-BRCA1 or BRCA2 breast cancer families. Hum Mutat 31:E1175–E1185. https://doi.org/10.1002/humu.21200 Turnbull C et al (2025) Breast cancer germline multigene panel testing in mainstream oncology based on clinical-public health utility: ESMO Precision Oncology Working Group recommendations. Ann Oncol 36:853–865. 10.1016/j.annonc.2025.04.012 Breast Cancer Risk Genes (2021) — Association Analysis in More than 113,000 Women. New England Journal of Medicine 384, 428–439, doi: 10.1056/NEJMoa1913948 Hu C et al (2021) A Population-Based Study of Genes Previously Implicated in Breast Cancer. N Engl J Med 384:440–451. 10.1056/NEJMoa2005936 Foulkes WD (2021) The ten genes for breast (and ovarian) cancer susceptibility. Nat Reviews Clin Oncol 18:259–260. 10.1038/s41571-021-00491-3 Sokolova A, Johnstone KJ, McCart Reed AE, Simpson PT, Lakhani SR (2023) Hereditary breast cancer: syndromes, tumour pathology and molecular testing. Histopathology 82:70–82. https://doi.org/10.1111/his.14808 Tarsounas M, Sung P (2020) The antitumorigenic roles of BRCA1–BARD1 in DNA repair and replication. Nat Rev Mol Cell Biol 21:284–299. 10.1038/s41580-020-0218-z Witus SR, Zhao W, Brzovic PS, Klevit RE (2022) BRCA1/BARD1 is a nucleosome reader and writer. Trends Biochem Sci 47:582–595. https://doi.org/10.1016/j.tibs.2022.03.001 Polak P et al (2017) A mutational signature reveals alterations underlying deficient homologous recombination repair in breast cancer. Nat Genet 49:1476–1486. 10.1038/ng.3934 Zhao W et al (2017) BRCA1–BARD1 promotes RAD51-mediated homologous DNA pairing. Nature 550:360–365. 10.1038/nature24060 Alenezi WM, Fierheller CT, Recio N, Tonin PN (2020) Literature Review of BARD1 as a Cancer Predisposing Gene with a Focus on Breast and Ovarian Cancers. Genes 11:856 De Brakeleer S et al (2016) Frequent incidence of BARD1-truncating mutations in germline DNA from triple-negative breast cancer patients. Clin Genet 89:336–340. https://doi.org/10.1111/cge.12620 Rofes P et al (2021) BARD1 Pathogenic Variants Are Associated with Triple-Negative Breast Cancer in a Spanish Hereditary Breast and Ovarian Cancer Cohort. Genes 12:150 Couch FJ et al (2017) Associations Between Cancer Predisposition Testing Panel Genes and Breast Cancer. JAMA Oncol 3:1190–1196. 10.1001/jamaoncol.2017.0424 Slavin TP et al (2017) The contribution of pathogenic variants in breast cancer susceptibility genes to familial breast cancer risk. NPJ Breast Cancer 3. 10.1038/s41523-017-0024-8 Castéra L et al (2018) Landscape of pathogenic variations in a panel of 34 genes and cancer risk estimation from 5131 HBOC families. Genet Med 20:1677–1686. 10.1038/s41436-018-0005-9 Weber-Lassalle N et al (2019) Germline loss-of-function variants in the BARD1 gene are associated with early-onset familial breast cancer but not ovarian cancer. Breast Cancer Res 21:55. 10.1186/s13058-019-1137-9 Suszynska M, Kozlowski P (2020) Summary of BARD1 Mutations and Precise Estimation of Breast and Ovarian Cancer Risks Associated with the Mutations. Genes (Basel) 11. 10.3390/genes11070798 Shimelis H et al (2018) Triple-Negative Breast Cancer Risk Genes Identified by Multigene Hereditary Cancer Panel Testing. J Natl Cancer Inst 110:855–862. 10.1093/jnci/djy106 Toh MR et al (2019) Functional analysis of clinical BARD1 germline variants. Cold Spring Harb Mol Case Stud 5. 10.1101/mcs.a004093 Becker JR et al (2021) BARD1 reads H2A lysine 15 ubiquitination to direct homologous recombination. Nature 596:433–437. 10.1038/s41586-021-03776-w Stewart MD et al (2018) BARD1 is necessary for ubiquitylation of nucleosomal histone H2A and for transcriptional regulation of estrogen metabolism genes. Proceedings of the National Academy of Sciences 115, 1316–1321. 10.1073/pnas.1715467115 Lee C et al (2015) Functional Analysis of BARD1 Missense Variants in Homology-Directed Repair of DNA Double Strand Breaks. Hum Mutat 36:1205–1214. 10.1002/humu.22902 Lindström LS et al (2012) Clinically Used Breast Cancer Markers Such As Estrogen Receptor, Progesterone Receptor, and Human Epidermal Growth Factor Receptor 2 Are Unstable Throughout Tumor Progression. J Clin Oncol 30:2601–2608. 10.1200/JCO.2011.37.2482 Schrijver WAME et al (2018) Receptor Conversion in Distant Breast Cancer Metastases: A Systematic Review and Meta-analysis. JNCI: J Natl Cancer Inst 110:568–580. 10.1093/jnci/djx273 Schwieger L et al (2024) Changes in expression of breast cancer tumor biomarkers between primary tumors and corresponding metastatic sites: common patterns and relationships with survival. Breast Cancer Res Treat 207:373–382. 10.1007/s10549-024-07368-w Dowling GP et al (2024) Receptor Discordance in Metastatic Breast Cancer; a review of clinical and genetic subtype alterations from primary to metastatic disease. Breast Cancer Res Treat 207:471–476. 10.1007/s10549-024-07431-6 Curtit E et al (2013) Discordances in Estrogen Receptor Status, Progesterone Receptor Status, and HER2 Status Between Primary Breast Cancer and Metastasis. Oncologist 18:667–674. 10.1634/theoncologist.2012-0350 Rivera B et al (2017) Functionally Null RAD51D Missense Mutation Associates Strongly with Ovarian Carcinoma. Cancer Res 77:4517–4529. 10.1158/0008-5472.Can-17-0190 Gradishar WJ et al (2023) NCCN Guidelines® Insights: Breast Cancer, Version 4.2023: Featured Updates to the NCCN Guidelines. J Natl Compr Canc Netw 21:594–608. 10.6004/jnccn.2023.0031 Zheng Y et al (2021) Functional consequences of a rare missense BARD1 c.403G > A germline mutation identified in a triple-negative breast cancer patient. Breast Cancer Res 23. 10.1186/s13058-021-01428-5 Cupit-Link M et al (2024) Response to PARP Inhibition in BARD1-Mutated Refractory Neuroblastoma. N Engl J Med 391:659–661. 10.1056/NEJMc2403316 Richards S et al (2015) Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Sci 17:405–423. 10.1038/gim.2015.30 Brnich SE et al (2019) Recommendations for application of the functional evidence PS3/BS3 criterion using the ACMG/AMP sequence variant interpretation framework. Genome Med 12:3. 10.1186/s13073-019-0690-2 Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754–1760. 10.1093/bioinformatics/btp324 McKenna A et al (2010) The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20:1297–1303. 10.1101/gr.107524.110 Poplin R et al (2018) Scaling accurate genetic variant discovery to tens of thousands of samples. bioRxiv 201178. 10.1101/201178 Wang K, Li M, Hakonarson H (2010) ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res 38:e164–e164. 10.1093/nar/gkq603 Rahman N (2014) Realizing the promise of cancer predisposition genes. Nature 505:302–308. 10.1038/nature12981 Illumina TruSight Cancer Gene List , https://www.illumina.com/content/dam/illumina-marketing/documents/products/gene_lists/gene_list_trusight_cancer.xlsx ( Invitae Invitae Multi-Cancer Panel , https://www.invitae.com/en/providers/test-catalog/test-01101 ( Tate JG et al (2018) COSMIC: the Catalogue Of Somatic Mutations In Cancer. Nucleic Acids Res 47:D941–D947. 10.1093/nar/gky1015 Favero F et al (2015) Sequenza: allele-specific copy number and mutation profiles from tumor sequencing data. Ann Oncol 26:64–70. 10.1093/annonc/mdu479 Liu M et al (2021) MesKit: a tool kit for dissecting cancer evolution of multi-region tumor biopsies through somatic alterations. GigaScience 10. 10.1093/gigascience/giab036 Alexandrov LB et al (2020) The repertoire of mutational signatures in human cancer. Nature 578:94–101. 10.1038/s41586-020-1943-3 Campeau E et al (2009) A Versatile Viral System for Expression and Depletion of Proteins in Mammalian Cells. PLoS ONE 4:e6529. 10.1371/journal.pone.0006529 Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7595877","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Case Report","associatedPublications":[],"authors":[{"id":513935996,"identity":"90e2e82e-4952-4835-8819-ab88c5b50952","order_by":0,"name":"Fiona Chan-Pak-Choon","email":"","orcid":"https://orcid.org/0000-0002-6317-1822","institution":"Department of Human Genetics, McGill University, Montreal, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"Fiona","middleName":"","lastName":"Chan-Pak-Choon","suffix":""},{"id":513935997,"identity":"31f61c4e-0588-4cb8-b19e-92054d3ae42c","order_by":1,"name":"Yuandi Gao","email":"","orcid":"https://orcid.org/0000-0002-1828-9776","institution":"Department of Human Genetics, McGill University, Montreal, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"Yuandi","middleName":"","lastName":"Gao","suffix":""},{"id":513935998,"identity":"93a3adf6-d96c-4be7-a487-37d856e76212","order_by":2,"name":"José Camacho-Valenzuela","email":"","orcid":"https://orcid.org/0000-0002-0081-7964","institution":"Department of Human Genetics, McGill University, Montreal, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"","lastName":"Camacho-Valenzuela","suffix":""},{"id":513935999,"identity":"26cbdd81-3adf-4852-81fa-d9c0e3c52323","order_by":3,"name":"Júlia-Jié Cabré-Romans","email":"","orcid":"","institution":"Department of Human Genetics, McGill University, Montreal, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"Júlia-Jié","middleName":"","lastName":"Cabré-Romans","suffix":""},{"id":513936000,"identity":"db8d6ff7-1568-4a84-9927-1a2e50688d48","order_by":4,"name":"Lili Fu","email":"","orcid":"https://orcid.org/0000-0003-2261-0902","institution":"Department of Pathology, McGill University, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"Lili","middleName":"","lastName":"Fu","suffix":""},{"id":513936001,"identity":"3b0c2c93-df49-4e18-a719-5ca152a735b0","order_by":5,"name":"Paz Polak","email":"","orcid":"https://orcid.org/0000-0002-2153-4488","institution":"Quest Diagnostics, Secaucus, NJ, USA","correspondingAuthor":false,"prefix":"","firstName":"Paz","middleName":"","lastName":"Polak","suffix":""},{"id":513936002,"identity":"9db9f81b-f56b-4348-b37d-a11fea6b04d6","order_by":6,"name":"Barbara Rivera","email":"","orcid":"https://orcid.org/0000-0001-9434-6288","institution":"Molecular Mechanisms and Experimental Therapy in Oncology Program, Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet de Llobregat, Barcelona, Spain","correspondingAuthor":false,"prefix":"","firstName":"Barbara","middleName":"","lastName":"Rivera","suffix":""},{"id":513936003,"identity":"a3df0c23-cc0f-4055-8464-108f4c46ae08","order_by":7,"name":"Raquel Cuella-Martin","email":"","orcid":"https://orcid.org/0000-0001-5682-5069","institution":"Department of Human Genetics, McGill University, Montreal, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"Raquel","middleName":"","lastName":"Cuella-Martin","suffix":""},{"id":513936004,"identity":"b2dd1f7a-c2b3-41f3-a958-0e8a83707dd5","order_by":8,"name":"William D. Foulkes","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIie3RPWrDMBTA8RcM0fKgq7vYV5ApeCrkKsqUJQGPGYJRKaRLaFZfIlAvniUM9uIlW8CLTS6QKXQKlUQDocSCbqXov/gD/XiWDOBy/cU870UwdSUcRpIDBPql6BIbGXFDUABo8mQIo7YxisANmXL9ZCO0VqRbFYBkQ2W2TGe7t3qvp4QPfICU+sOqFhAbKj+aclE080STKBP3SWzIuIWJP6eyX4tFcUCmCQMruagphlzSWXwloY1M198k5x6LD0QYQgfIxJD3FhGrRGZVGam96EP2o3yAPG5L2X+e2wDJa95vVmkY1/XxdFo+h8EAuYa39/qn+Pb1PyLdr5a7XC7Xv+8L5hRtPooFCJ8AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-7427-4651","institution":"Department of Human Genetics, McGill University, Montreal, QC, Canada","correspondingAuthor":true,"prefix":"","firstName":"William","middleName":"D.","lastName":"Foulkes","suffix":""}],"badges":[],"createdAt":"2025-09-12 02:57:31","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":true,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-7595877/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7595877/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":92068140,"identity":"bd8d3f65-aae8-4783-8fa1-1a1335260c65","added_by":"auto","created_at":"2025-09-24 09:22:56","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":126451,"visible":true,"origin":"","legend":"","description":"","filename":"BARD1finaldraft.docx","url":"https://assets-eu.researchsquare.com/files/rs-7595877/v1/9d34b8f12b5cd108fcc199af.docx"},{"id":92068138,"identity":"aa480748-90d1-4c46-9a6e-96d5f5eeb3ae","added_by":"auto","created_at":"2025-09-24 09:22:56","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":342,"visible":true,"origin":"","legend":"","description":"","filename":"rs7595877.json","url":"https://assets-eu.researchsquare.com/files/rs-7595877/v1/5147541b510cece2808436b4.json"},{"id":92068135,"identity":"13a4308d-8f99-46a3-a419-3998c5b74f56","added_by":"auto","created_at":"2025-09-24 09:22:56","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":104299,"visible":true,"origin":"","legend":"","description":"","filename":"rs75958770enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7595877/v1/db7b7aa9c1ebb7e2fb8f6fb3.xml"},{"id":92068141,"identity":"f891de46-e369-4619-a606-76377799d917","added_by":"auto","created_at":"2025-09-24 09:22:56","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":102174,"visible":true,"origin":"","legend":"","description":"","filename":"rs75958770structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7595877/v1/8dd5c8c1ce412c7b3813fa43.xml"},{"id":92068142,"identity":"8a5bd16d-1615-4e35-876e-a42c90f7fdfb","added_by":"auto","created_at":"2025-09-24 09:22:56","extension":"html","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":115147,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7595877/v1/d1e81389e82ab23aa5a51531.html"},{"id":92069408,"identity":"53a37cdb-c76c-43a5-b88c-ca685361563a","added_by":"auto","created_at":"2025-09-24 09:30:56","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":788891,"visible":true,"origin":"","legend":"\u003cp\u003eGenomic characterization of the \u003cem\u003eBARD1\u003c/em\u003e variant and tumor evolution in the proband. \u003cstrong\u003eA\u003c/strong\u003e Pedigree showing the proband (arrow) with two breast cancers (ER+/PR+ and TNBC) and endometrial cancer, her father with colon polyps and cancer, and her two paternal aunts with breast cancer. In addition, the family history was notable for reports of multiple relatives affected by various cancers, including breast, colon, cervical, and prostate cancers; however, these cases were excluded as they could not be confirmed by pathology report. \u003cstrong\u003eB\u003c/strong\u003e Schematic summary of whole-exome sequencing (WES) findings across blood and tumor samples from the proband and her father. The germline \u003cem\u003eBARD1\u003c/em\u003e variant (c.2258G\u0026gt;T; p.Gly753Val) was present in all tissues and loss of heterozygosity (LOH) was seen exclusively in the TNBC, suggesting biallelic inactivation. \u003cstrong\u003eC \u003c/strong\u003eCopy number variation (CNV) analysis confirming LOH at the BARD1 locus in the TNBC. \u003cstrong\u003eD\u003c/strong\u003e Clonal evolution analysis of the proband’s tumors. The ER+/PR+ and TNBC tumors shared a subset of clonal variants, supporting a recurrence. The endometrial carcinoma showed no shared variants, consistent with an independent primary tumor. \u003cstrong\u003eE\u003c/strong\u003e Mutational signature analysis of the three tumors. Signature 3, associated with homologous recombination (HR) deficiency, was detected only in the TNBC, alongside Signatures 1 and 6. The ER+/PR+ tumor exhibited only Signature 1, and the endometrial tumor displayed Signatures 1, 2, and 12\u003c/p\u003e","description":"","filename":"BARD1Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7595877/v1/227ab1260d9159f255e55804.jpg"},{"id":92068137,"identity":"d3e88741-ddc7-45af-b8bd-d128497828ed","added_by":"auto","created_at":"2025-09-24 09:22:56","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":768949,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional characterization of \u003cem\u003eBARD1\u003c/em\u003e G753V. \u003cstrong\u003eA \u003c/strong\u003eImmunoblot of whole-cell lysates collected 24 hours after auxin treatment. The upper panel shows expression of the indicated BARD1 transgenes (longer exposure), and the middle panel confirms degradation of endogenous BARD1 (BARD1-AID) (shorter exposure). Asterisks refer to non-specific bands (*). \u003cstrong\u003eB\u003c/strong\u003e Representative images of crystal violet–stained wells showing survival of HCT116-AID cells expressing wild-type (WT) BARD1, patient-derived G753V, functionally impaired C53W, or GFP control after 10-day exposure to increasing concentrations of olaparib, following auxin-induced degradation of endogenous BARD1. \u003cstrong\u003eC \u003c/strong\u003eQuantification of cell viability from \u003cstrong\u003eB\u003c/strong\u003e. Mean ± S.E.M. (N=3) and 4-parameter non-linear fit lines are depicted. IC₅₀ values were 825.8 nM for WT, 15.7 nM for G753V, 6.2 nM for C53W, and 5.3 nM for GFP \u003cstrong\u003eD\u003c/strong\u003e Representative immunofluorescence images of RAD51 foci in S-phase cells after exposure to ionizing radiation and depletion of endogenous BARD1. \u003cstrong\u003eE \u003c/strong\u003eQuantification of RAD51 foci per nucleus in S-phase cells. Mean (N=2), with a minimum of 1,000 cells per biological replicate, are depicted.\u003c/p\u003e","description":"","filename":"BARD1Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7595877/v1/d78e68ba21627a5cc754e429.jpg"},{"id":92071159,"identity":"ecfba08f-a583-4e41-9d1f-cee20552481a","added_by":"auto","created_at":"2025-09-24 09:46:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2189686,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7595877/v1/c882f4e9-22d2-42bb-81bb-49db3d03e798.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eMultimodal analysis of rare \u003cem\u003eBARD1\u003c/em\u003e missense variant suggests its pathogenicity is conditional\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eApproximately 5\u0026ndash;10% of all breast cancers are attributable to hereditary predisposition, with germline pathogenic variants (GPVs) in \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e accounting for 20\u0026ndash;30% of such cases \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The genes associated with hereditary breast cancer can largely be grouped into three categories: high-risk genes - \u003cem\u003eBRCA1\u003c/em\u003e, \u003cem\u003eBRCA2\u003c/em\u003e, \u003cem\u003ePALB2\u003c/em\u003e, and \u003cem\u003eTP53\u003c/em\u003e; moderate-risk genes \u0026ndash; \u003cem\u003eATM\u003c/em\u003e and \u003cem\u003eCHEK2\u003c/em\u003e, which are primarily linked to estrogen receptor-positive (ER+) breast cancer; and a third group \u0026ndash; \u003cem\u003eBARD1\u003c/em\u003e, \u003cem\u003eRAD51C\u003c/em\u003e, and \u003cem\u003eRAD51D\u003c/em\u003e, that confers low to moderate risk and is more strongly associated with triple-negative breast cancer (TNBC) \u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. These genes are typically included in multigene panel testing for individuals with suggestive personal or family histories, as identifying GPVs can inform clinical decision-making, including enhanced surveillance, risk-reducing strategies and future treatment options \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cem\u003eBARD1\u003c/em\u003e (BRCA1-associated RING domain 1) encodes a protein that forms a heterodimer with BRCA1 \u003cem\u003ein vivo via\u003c/em\u003e their respective Really Interesting New Gene (RING) domains \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The BRCA1\u0026ndash;BARD1 complex plays a central role in homologous recombination (HR), the high-fidelity repair pathway for DNA double-strand breaks \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. It modifies chromatin to enable access to damaged DNA, facilitates DNA end resection, and recruits and stabilizes RAD51 along with its mediators at damage sites to promote strand invasion and downstream HR repair processes \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eEstimating the cancer risk associated with \u003cem\u003eBARD1\u003c/em\u003e GPVs remains challenging due to the rarity of carriers and the low to moderate penetrance of the variants \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. BARD1 GPVs have been particularly associated with an elevated risk of triple-negative breast cancer (TNBC) \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Several case-control and population-based studies have reported odds ratios between 2.00 and 5.35 for overall breast cancer risk, with higher estimates for TNBC, ranging from 3.18 to 11.3 \u003csup\u003e4,5,14\u0026ndash;20\u003c/sup\u003e. However, ambiguity in the interpretation of many \u003cem\u003eBARD1\u003c/em\u003e variants limits their use in clinical risk assessment and management. To date, only truncating variants have been classified as likely pathogenic or pathogenic, while all missense variants continue to be classified as variants of uncertain significance (VUS) due to insufficient functional, epidemiologic, and segregation data \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWe present the case of a proband with two primary malignancies (breast and endometrial cancers). Both the proband and her father, who was diagnosed with colon adenocarcinoma, were found to carry a rare missense variant in \u003cem\u003eBARD1\u003c/em\u003e (NM_000465.4): c.2258G\u0026thinsp;\u0026gt;\u0026thinsp;T; p.(Gly753Val). This variant is currently classified as a VUS (ClinVar ID: 649553). This study shows that detailed functional, molecular, and clinical data were required to resolve uncertainty regarding the pathogenicity of this variant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCase presentation\u003c/h2\u003e\u003cp\u003eThe proband (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, arrow) presented with breast carcinomas at age 50y (ER+/PR+/HER2-, pT1c pN1 pMx), 59y (TNBC, pTx pNx pMx \u0026ndash; staging was not possible as the tumour was removed during diagnostic biopsy) and was later diagnosed with endometrial carcinoma at 62y (FIGO 1A). Her father was diagnosed with colon polyps at 67y and colon adenocarcinoma at 82y; two of her paternal aunts developed breast carcinomas at 56y and 81y, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Following the diagnosis of her second breast cancer, clinical germline testing in the proband revealed a rare \u003cem\u003eBARD1\u003c/em\u003e VUS [\u003cem\u003eBARD1\u003c/em\u003e(NM_000465.4): c.2258 G\u0026thinsp;\u0026gt;\u0026thinsp;T; p.(Gly753Val)], occurring within the BRCA1 C-Terminus (BRCT) domain. For the ER+/PR\u0026thinsp;+\u0026thinsp;breast cancer, the proband received adjuvant chemotherapy with six cycles of docetaxel, doxorubicin, and cyclophosphamide (TAC), followed by three years of tamoxifen and an additional seven years of aromatase inhibitors until the diagnosis of her TNBC. For the TNBC, she underwent bilateral mastectomies and received four cycles of adjuvant docetaxel and cyclophosphamide. At age 62, she was treated for endometrial carcinoma with a total abdominal hysterectomy and bilateral salpingo-oophorectomy, without postoperative therapy.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGenomic studies\u003c/h3\u003e\n\u003cp\u003eWhole-exome sequencing (WES) analysis performed on the proband\u0026rsquo;s blood, the two breast tumors, the endometrial tumor, as well as her father\u0026rsquo;s colon tumour and adjacent normal colon tissue confirmed the presence of the germline \u003cem\u003eBARD1\u003c/em\u003e VUS in all samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). No likely causative variants were identified in other cancer predisposition genes (n\u0026thinsp;=\u0026thinsp;144) in the samples based on our variant prioritization analysis. Copy number variation (CNV) analysis detected loss of heterozygosity (LOH) at the \u003cem\u003eBARD1\u003c/em\u003e locus in the proband\u0026rsquo;s TNBC only (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). No second hits in \u003cem\u003eBARD1\u003c/em\u003e were identified in the other tumors.\u003c/p\u003e\u003cp\u003eClonal evolution analysis based on WES data was used to evaluate tumor relatedness across the proband\u0026rsquo;s tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The first breast cancer (ER+/PR+) harbored 76 somatic variants (39 clonal, 38 subclonal), and the subsequent TNBC harbored 206 somatic variants (113 clonal, 98 subclonal), including 12\u0026thinsp;\u0026gt;\u0026thinsp;4bp deletions, indicative of increased genomic instability. These two tumors shared 23 clonal somatic variants, indicating that the second breast cancer was a recurrence rather than an independent primary tumor. In contrast, the endometrial tumor harbored 336 somatic variants (126 clonal, 210 subclonal) and showed no shared somatic variants with either breast cancer, supporting its origin as an independent primary tumor.\u003c/p\u003e\u003cp\u003eMutational signature analysis using the WES data revealed distinct mutational processes across the tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). The ER+/PR\u0026thinsp;+\u0026thinsp;tumor was dominated by Signature 1, associated with age-related mutagenesis. The TNBC displayed a more complex signature profile, with contributions from Signatures 1, 3 (homologous recombination (HR) repair deficiency), and 6 (DNA mismatch repair deficiency). The endometrial tumor exhibited Signatures 1, 2 (APOBEC activity), and 12 (unknown etiology).\u003c/p\u003e\n\u003ch3\u003eFunctional studies\u003c/h3\u003e\n\u003cp\u003eTo evaluate the functional impact of \u003cem\u003eBARD1\u003c/em\u003e VUS identified in the proband on HR repair, HCT116 cells engineered with both \u003cem\u003eBARD1\u003c/em\u003e alleles tagged with an auxin-inducible degron (AID) (HCT116-\u003cem\u003eBARD1\u003c/em\u003e\u003csup\u003eAID/AID\u003c/sup\u003e) were used \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. These cells carry a doxycycline-inducible E3 ubiquitin ligase, OsTIR1, which interacts with AID upon treatment with indole-3-acetic acid (IAA), leading to the rapid degradation of endogenous BARD1. Lentiviral transduction was used to introduce exogenous wild-type (WT) BARD1 (negative control), the patient-derived \u003cem\u003eBARD1\u003c/em\u003e variant [\u003cem\u003eBARD1\u003c/em\u003e(NM_000465.4): c.2258 G\u0026thinsp;\u0026gt;\u0026thinsp;T; p.(G753V); hereafter G753V], the known functionally defective \u003cem\u003eBARD1\u003c/em\u003e missense variant [\u003cem\u003eBARD1\u003c/em\u003e(NM_000465.4): c.159T\u0026thinsp;\u0026gt;\u0026thinsp;G; p.(Cys53Trp); hereafter C53W] \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, or GFP (positive control lacking BARD1 expression).\u003c/p\u003e\u003cp\u003eTo assess the effective degradation of endogenous BARD1, immunoblotting was performed following IAA treatment. As expected, endogenous BARD1 was efficiently degraded in all conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Lentiviral expression only achieved sub-physiological BARD1 levels, detectable at longer immunoblot exposures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, upper panel).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe cell lines were then used to assess cell survival following treatment with olaparib, a poly(ADP-ribose) polymerase (PARP) inhibitor. Following auxin-induced degradation of endogenous BARD1, cells were chronically exposed to olaparib concentrations ranging from 0 to 500 nM for 10 days prior to staining with crystal violet (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C). In the absence of auxin, cell viability remained close to 100% across all cell lines. Upon auxin treatment, WT BARD1 maintained high viability across all olaparib concentrations (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;825.8 nM), indicating effective functional complementation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In contrast, cells expressing BARD1 G753V (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;15.7 nM), BARD1 C53W (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.2 nM), or GFP (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;5.3 nM) showed decreasing viability with increasing olaparib concentrations. BARD1 C53W and GFP exhibited the greatest loss of viability, consistent with complete loss of BARD1 function. BARD1 G753V-expressing cells consistently showed intermediate sensitivity, with viability levels falling between those of BARD1 WT and BARD1 C53W or GFP, yet more closely resembling the viability of the latter.\u003c/p\u003e\u003cp\u003eTo further investigate the effect of the \u003cem\u003eBARD1\u003c/em\u003e variants on HR repair, the recruitment of the HR repair machinery to DNA damage sites was assessed by quantifying RAD51 foci in S-phase cells. Immunofluorescence staining was performed following auxin-induced degradation of endogenous BARD1 and exposure to ionizing radiation. Cells expressing the BARD1 G753V variant exhibited a mean number of RAD51 foci (5.71) similar to the known functionally impaired C53W variant (5.63). Both exhibited higher RAD51 foci counts than the control cells lacking exogenous BARD1 (4.88), but lower than those observed in cells expressing WT BARD1 (7.47) (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-E).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere we report a rare \u003cem\u003eBARD1\u003c/em\u003e missense variant (ClinVar ID: 649553) identified in a woman with a recurrent primary breast cancer and endometrial cancer in the context of a family history of colon and breast cancers. Functional analysis of the variant revealed impaired HR repair. Molecular analyses of all four tumors \u0026ndash; the proband\u0026rsquo;s breast cancers, endometrial carcinoma, and her father's colon adenocarcinoma \u0026ndash; revealed that only the TNBC recurrence harbored a second hit (LOH) in \u003cem\u003eBARD1\u003c/em\u003e, suggesting biallelic inactivation. Notably, no second hit in \u003cem\u003eBARD1\u003c/em\u003e was detected in the proband\u0026rsquo;s primary breast cancer. Mutational Signature 3 was present only in the recurrent breast tumor, a TNBC. One could argue that the pathogenicity of the variant became evident only after analyzing the recurrent breast cancer. Moreover, we hypothesize that the pathogenic effect of the variant was conditional upon molecular switching, likely the result of the prior treatment of the primary breast cancer.\u003c/p\u003e\u003cp\u003eMolecular subtype switching, also known as receptor conversion, from ER+/PR\u0026thinsp;+\u0026thinsp;primary breast cancers to metastatic TNBCs is a recognized phenomenon \u003csup\u003e\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. This shift is thought to arise from intratumor heterogeneity and the selective effects of chemotherapy and hormonal treatments, which can promote clonal selection of resistant tumor cells \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. We hypothesize that chemotherapy and endocrine therapy following the primary diagnosis imposed selective pressure on the tumor, allowing a minor subpopulation of therapy-resistant clones \u0026ndash; initially comprising\u0026thinsp;~\u0026thinsp;5% ER- cells within a predominantly (~\u0026thinsp;95%) ER\u0026thinsp;+\u0026thinsp;tumor \u0026ndash; to survive and expand \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Over time, this subset acquired LOH of the wild-type \u003cem\u003eBARD1\u003c/em\u003e allele, resulting in loss of BARD1 functionality in the presence of G753V allele and thus HR repair deficiency, enabling further genomic instability which ultimately drove the emergence of the aggressive TNBC recurrence. Although receptor conversion is well documented, how such subtype changes might influence or reveal the pathogenic potential of an underlying germline variant remains unexplored. An alternative although not mutually exclusive hypothesis is that that the pre-existing germline \u003cem\u003eBARD1\u003c/em\u003e variant itself played a mechanistic role in this subtype transition, rather than the other way around. To our knowledge, the potential role of germline variants in influencing molecular subtype switching has not been studied. Finally, we acknowledge that the \u003cem\u003eBARD1\u003c/em\u003e variant could be a bystander in the process, that the LOH is incidental, and that the mutational signature 3 has other causes. The functional data, however, suggest otherwise.\u003c/p\u003e\u003cp\u003eCompared with cells expressing WT BARD1, cells expressing G753V showed increased olaparib sensitivity, which along with reduced RAD51 foci formation indicated impaired HR repair. This raises the question of whether the recurrent TNBC might have responded to PARP inhibitor therapy such as olaparib. While \u003cem\u003eBARD1\u003c/em\u003e is not currently included in the eligibility criteria for PARP inhibitor therapy \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, two reported cases involving \u003cem\u003eBARD1\u003c/em\u003e germline carriers \u0026ndash; a patient with TNBC and another with neuroblastoma \u0026ndash; showed marked responses to PARP inhibitor treatment \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, highlighting the possibility that \u003cem\u003eBARD1\u003c/em\u003e inactivation may confer sensitivity to this therapy in certain settings.\u003c/p\u003e\u003cp\u003eClassifying the \u003cem\u003eBARD1\u003c/em\u003e G753V variant remains inherently difficult. Like many rare missense variants in low-penetrance genes, it occupies a grey area under current ACMG guidelines, which rely heavily on recurrence in affected individuals, segregation data, and strong population evidence\u0026mdash;criteria that are rarely met for genes like \u003cem\u003eBARD1\u003c/em\u003e \u003csup\u003e34\u003c/sup\u003e. Additionally, functional validation is hindered by the lack of established pathogenic missense variants that could serve as positive controls, making it difficult to generate sufficiently robust experimental evidence to meet the ACMG guidelines \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eUnder the current ACMG criteria, this variant meets only PS3_supporting (functional data), PM2_supporting (absence from control population), and PP3 (in-silico predictions supporting a deleterious effect); it therefore remains a VUS. This challenge is further compounded in the present case, where we hypothesize that the pathogenicity of the variant is conditional, manifesting only after treatment-induced selective pressure in the TNBC. This points to the potential value of incorporating context-dependent factors such as tumour characteristics and prior treatment into variant interpretation frameworks.\u003c/p\u003e\u003cp\u003eIn conclusion, we identified a rare \u003cem\u003eBARD1\u003c/em\u003e germline VUS whose pathogenicity was only revealed after biallelic inactivation of \u003cem\u003eBARD1\u003c/em\u003e during molecular switching to a TNBC. This case illustrates the possibility of a potentially novel scenario whereby the pathogenicity of a cancer susceptibility allele is contingent upon therapeutic selective pressure. It underscores the importance of monitoring VUSs in HR repair genes, as these variants, despite not being initially causative, may drive therapy-resistant recurrences. We demonstrate that integrating genomic, functional, and clinical data can uncover these context-dependent effects that are not immediately apparent at diagnosis and, in the future, may enable more precise, personalized treatment strategies.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePatient samples\u003c/h2\u003e\u003cp\u003eBlood was collected from the proband, and formalin-fixed paraffin-embedded (FFPE) tumor blocks were obtained from both the proband and her father. The proband\u0026rsquo;s samples included primary breast cancer (ER+/PR+), TNBC recurrence, and endometrial cancer. The father\u0026rsquo;s samples comprised colon adenocarcinoma and adjacent normal colon tissue.\u003c/p\u003e\u003cp\u003eGenomic DNA from the proband\u0026rsquo;s blood was extracted using the Gentra PureGene Blood Kit (Qiagen). DNA from FFPE tissue blocks was extracted using the QIAamp\u0026reg; DNA FFPE Tissue Kit (Qiagen), following the manufacturer\u0026rsquo;s protocols.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eWhole Exome Sequencing\u003c/h3\u003e\n\u003cp\u003eThe DNA samples were analysed by WES. The Agilent SureSelect Human All Exon V7 kit (Agilent Technologies, Santa Clara, CA, USA) was used for exome capture, and the libraries were sequenced on the Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA) at the Centre d\u0026rsquo;expertise et de services Genome Quebec (Montreal, QC, Canada). The mean coverage was 260\u0026times;. The Binary Alignment Map (BAM) files were realigned to the human reference genome hg19 with the Burrows-Wheeler Aligner (BWA) \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. PCR duplicates were marked and removed using MarkDuplicates of The Genome Analysis Toolkit (GATK v.4.1.2.0) \u003csup\u003e37\u003c/sup\u003e. Germline variants were called with HaplotypeCaller and somatic variants were called with Mutect2 (GATK v.4.1.2.0) \u003csup\u003e38\u003c/sup\u003e. All the variants were annotated with ANNOVAR \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The variants were filtered to only retain those (1) with 4 reads supporting the variant, (2) 20\u0026times; coverage at genomic locus, (3) with variant allele frequency (VAF) \u0026sup3; 5%, (4) with frequency in normal population (ExAC) of \u0026lt;\u0026thinsp;0.001, and (v) predicted to be pathogenic by at least four out of six predictors (PROVEAN, FATHMM, MutationTaster, MutationAssessor, MetaSVM, MCAP and CADD).\u003c/p\u003e\u003cp\u003eAn additional filter consisting of two gene panels, a hereditary cancer gene panel and a tumour-specific gene panel, was included in our pipeline: (1) a panel made up of 144 cancer susceptibility genes aggregated from Rahman\u0026rsquo;s review and genes present in the Illumina TruSight\u0026rsquo;s (Illumina) and Invitae\u0026rsquo;s hereditary cancer panels \u003csup\u003e\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e and (2) 637 genes from the Catalogue Of Somatic Mutations In Cancer (COSMIC)\u0026rsquo;s database version 98 \u003csup\u003e43\u003c/sup\u003e. The resulting lists of variants were then manually inspected via the Integrative Genomics Viewer (IGV).\u003c/p\u003e\n\u003ch3\u003eCopy number variant analysis\u003c/h3\u003e\n\u003cp\u003eThe matched normal/tumour BAM files were sorted by genomic coordinates with SortSam (GATK v.4.1.2.0) \u003csup\u003e37\u003c/sup\u003e. We used Sequenza \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e to perform a CNV analysis based on allele-specific segmentation on the sorted BAM files, which generated copy number integer values that were used to determine LOH status, along with a visual representation of the CNV detected across the genome.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eClonal analysis and phylogenetic tree\u003c/h2\u003e\u003cp\u003eThe phylogenetic tree of the three tumours from the proband were reconstructed using MesKit (version 1.18.0) \u003csup\u003e45\u003c/sup\u003e, which infers evolutionary relationships based on the presence or absence of somatic mutations. Clonal and subclonal mutations were classified using cancer cell fraction (CCF) estimates across multiple tumour regions: mutations with CCF consistent with presence in all cancer cells were considered clonal, while those present in subsets of cells were classified as subclonal.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eMutational signatures\u003c/h2\u003e\u003cp\u003eMutational signature analysis was performed using the MesKit (version 1.18.0) \u003csup\u003e45\u003c/sup\u003e. Somatic mutations were categorized into 96 trinucleotide contexts and grouped into truncal (shared across all tumor regions), and branch (private to individual regions) based on phylogenetic trees. Known COSMIC mutational signatures (COSMIC V2) \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e were then fitted to each profile. Similarity between original and reconstructed mutational profiles was measured using cosine similarity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003ePlasmid generation\u003c/h2\u003e\u003cp\u003epDONR221-BARD1-WT was a gift from Dr. J.R. Chapman \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, and variants present in the sequence were modified using site-directed mutagenesis (New England Biolabs) to match the RefSeq consensus sequence. Patient mutations were then inserted using site-directed mutagenesis (New England Biolabs) as per the manufacturer\u0026rsquo;s instructions. The BARD1 (WT, G753V, C53W) and GFP open reading frames were cloned into pLenti-PGK-NEO-DEST (a gift from Eric Campeau and Paul Kaufman, Addgene plasmid #19067 \u003csup\u003e47\u003c/sup\u003e) using LR clonase II (Life Technologies Inc.) following the manufacturer\u0026rsquo;s protocol. All plasmid sequences were verified using Oxford Nanopore Technologies long-read sequencing (Plasmidsaurus Inc).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eCell line culture\u003c/h2\u003e\u003cp\u003eHCT116-\u003cem\u003eBARD1\u003c/em\u003e\u003csup\u003eAID/AID\u003c/sup\u003e cells, a gift from Dr. J.R. Chapman \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, were maintained in Dulbecco\u0026rsquo;s modified Eagle medium (DMEM)\u0026ndash;high glucose (Gibco, 11965092) supplemented with 10% FBS. Cultures were maintained at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Stable expression of GFP or BARD1 (WT, G753V, C53W) was achieved using lentiviral transduction. Briefly, 5 x 10\u003csup\u003e5\u003c/sup\u003e HEK293T cells were seeded on a 6-well plate and co-transfected with 1.5 \u0026micro;g of the GFP or lentiviral vector GFP or BARD1 and third-generation lentiviral packaging vectors using 1.29 \u0026micro;g polyethylenimine per \u0026micro;g of DNA in Opti-MEM (Life Technologies Inc.). Viral supernatants were collected at 48 and 72 h post-transfection, syringe-filtered (0.45 \u0026micro;m), and immediately applied in a 1:1 (virus/medium) mixture over the HCT116-\u003cem\u003eBARD1\u003c/em\u003e\u003csup\u003eAID/AID\u003c/sup\u003e cells in the presence of 4 \u0026micro;g/ml polybrene (Millipore Sigma). Sixteen hours after transduction, viruses were removed, and cell populations were selected with 500 \u0026micro;g/ml G418 Sulfate (Life Technologies Inc.). Stably transduced cell populations were maintained in the presence of the antibiotic.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eCell viability with crystal violet\u003c/h2\u003e\u003cp\u003eCells were seeded at a density of 10\u003csup\u003e4\u003c/sup\u003e cells per well of a 12-well plate in the presence of 2 \u0026micro;g/ml doxycycline. After 24 h, 250 \u0026micro;M indole-3-acetic acid (IAA) or carrier (DMSO) was added. One hour after IAA or DMSO addition, olaparib was added to the indicated final concentrations. Ten days after plating, cells were washed and stained for 30 minutes with crystal violet (0.5% crystal violet in 25% methanol). Cells were then extensively washed with ddH\u003csub\u003e2\u003c/sub\u003eO and dried before scanning. For quantification, bound crystal violet was dissolved in 10% (v/v) acetic acid, and the absorbance of solubilized dye was measured at 595 nm. All experiments were performed in technical and biological triplicate. Data were analyzed using Prism 10 (GraphPad Software LLC), performing a 4-parameter non-linear fit for IC50 calculations. Representative wells were selected for display.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eProtein extraction and immunoblotting\u003c/h2\u003e\u003cp\u003eCells were collected, washed and resuspended in 100 \u0026micro;l ice-cold benzonase cell lysis buffer (25 mM Tris-HCl pH 8.8, 40 mM NaCl, 0.05% SDS, 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 20 U/ml benzonase (Millipore Sigma), and cOmplete mini EDTA-free protease inhibitor cocktail (Roche). Extracts were incubated at room temperature for 5 min, on ice for 90 min before centrifugation at 13,000g for 15 min at 4\u0026deg;C. The protein concentrations of clarified supernatants were measured by Bradford assay (Bio-Rad Laboratories). Subsequently, 4x NuPage LDS sample buffer (Life Technologies Inc.) was added, and samples were boiled at 95\u0026deg;C for 10 min. Equal amounts of protein were loaded on NuPAGE 4\u0026ndash;12% 1.0 mm Bis-Tris polyacrylamide gels (Life Technologies Inc.) and transferred to 0.45-\u0026micro;m nitrocellulose membranes (GE Healthcare). Membranes were blocked with 5% milk in PBS\u0026ndash;0.1% (v/v) Tween-20 (PBST) for 1 h and incubated overnight with primary antibody in a solution of 3% (w/v) bovine serum albumin (BSA) in PBST. Primary antibodies include: rabbit anti-BARD1 (1:1,000, Bethyl Laboratories, A300-265A-T), mouse anti-α-tubulin (1:2,000, Santa Cruz Biotechnology, sc-32293) and mouse anti-vinculin (1:25,000, Millipore Sigma, V9131). Following primary antibody incubation, membranes were incubated with either HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies (1:20,000, Jackson Laboratories). Membranes were developed with Pierce\u0026trade; ECL Western Blotting Substrate (Life Technologies Inc.) and imaged using a ChemiDoc Imaging System (Bio-Rad Laboratories).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e\u003cp\u003eCells were seeded at a density of 1.5x10\u003csup\u003e4\u003c/sup\u003e cells per well of a 96-well plate in the presence of 2 \u0026micro;g/ml doxycycline. After 24 h, 250 \u0026micro;M indole-3-acetic acid (IAA) was added. Two hours after IAA addition, cells were irradiated at 5 Gy. Three hours post-irradiation, cells were fixed and permeabilized for 10 min using a solution of 2% (w/v) paraformaldehyde and 0.5% (v/v) Triton X-100 in PBS. After blocking with TBS\u0026ndash;0.1% (v/v) Tween-20 (TBST) containing 1% (w/v) BSA for 30 min, cells were washed three times with PBS and incubated with primary antibodies for 1 h at room temperature, namely, anti-H2A.X (1:5,000, Biolegend, C613402) and anti-RAD51 (1:2000, Millipore Sigma, PC130). Secondary antibodies include anti-mouse and anti-rabbit Alexa Fluor 488- or 594-conjugated antibodies (Life Technologies Inc.). Cells were stained with 1 \u0026micro;g/ml 4, 6-diamidino-2-phenylindole (DAPI) for 5 min for nuclear staining and imaged using a Cytation C10 imaging reader with a 40\u0026times; objective (BioTek). Foci quantification was performed using Cell Profiler, with cell cycle phases determined based on integrated nuclear DAPI intensities. All experiments were performed in technical and biological duplicates, with a minimum of 1,000 S-phase cells per experimental condition. Representative images were selected for display.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eEthics statement\u003c/h2\u003e\u003cp\u003eWritten informed consent was obtained from all patients involved, and the study protocol was approved by the McGill University Health Centre Research Ethics Board (Project No. MP-37-2019-4865).\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003e\u003cb\u003eConflicts of interest\u003c/b\u003e:\u003c/h2\u003e\u003cp\u003eThe authors have no conflicts of interest to disclose.\u003c/p\u003e\u003ch2\u003eAuthor contribution\u003c/h2\u003e\u003cp\u003eFCPC collected samples, conducted the genomic analyses, and drafted the manuscript. YG, J-JC-R, and FCPC performed the functional analyses. JCV and PP assisted with the genomic analyses. LF performed the pathology review. BR, RC-M, and WDF supervised all aspects of the project and edited the manuscript. All authors reviewed and approved the final version of the manuscript for submission.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eThis study was funded by the Canadian Institutes of Health Research grant (FDN-148390) to WDF. BR is a Miguel Servet Fellow (CP21/00038) from the Instituto de Salud Carlos III (PI20/01721). RCM is a Junior 1 Scholar from the Fonds de Recherche du Qu\u0026eacute;bec\u0026ndash;Sant\u0026eacute; (FRQS). FCPC received support from the George G Harris Fellowship, granted by the Faculty of Medicine and Health Sciences, McGill University. JCV was supported in part by an internal award from The Research Institute of the McGill University Centre (RI-MUHC).\u003c/p\u003e\u003cp\u003eThis research as well as biobanking of biological material and data was made possible in part through a collaboration with the R\u0026eacute;seau de recherche sur le cancer (RRCancer) financially supported by the Oncopole, the FRQ cancer division, which receives funding from Merck Canada Inc., GSK, Pfizer and the Minist\u0026egrave;re de l'\u0026Eacute;conomie, de l'Innovation et de l'\u0026Eacute;nergie du Qu\u0026eacute;bec. The RRCancer is affiliated to the Canadian Tumor Repository Network (CTRNet).\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eThe data supporting the findings of this study will be deposited in the European Genome-Phenome Archive and FigShare. Accession numbers will be provided in the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eClaus EB, Schildkraut JM, Thompson WD, Risch NJ (1996) The genetic attributable risk of breast and ovarian cancer. Cancer 77:2318\u0026ndash;2324. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/(SICI)1097-0142(19960601)77:11%3C2318::AID-CNCR21%3E3.0.CO;2-Z\u003c/span\u003e\u003cspan address=\"10.1002/(SICI)1097-0142(19960601)77:11%3C2318::AID-CNCR21%3E3.0.CO;2-Z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDe Brakeleer S et al (2010) Cancer predisposing missense and protein truncating BARD1 mutations in non-BRCA1 or BRCA2 breast cancer families. Hum Mutat 31:E1175\u0026ndash;E1185. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/humu.21200\u003c/span\u003e\u003cspan address=\"10.1002/humu.21200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTurnbull C et al (2025) Breast cancer germline multigene panel testing in mainstream oncology based on clinical-public health utility: ESMO Precision Oncology Working Group recommendations. Ann Oncol 36:853\u0026ndash;865. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.annonc.2025.04.012\u003c/span\u003e\u003cspan address=\"10.1016/j.annonc.2025.04.012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBreast Cancer Risk Genes (2021) \u0026mdash; Association Analysis in More than 113,000 Women. \u003cem\u003eNew England Journal of Medicine\u003c/em\u003e 384, 428\u0026ndash;439, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1056/NEJMoa1913948\u003c/span\u003e\u003cspan address=\"10.1056/NEJMoa1913948\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHu C et al (2021) A Population-Based Study of Genes Previously Implicated in Breast Cancer. N Engl J Med 384:440\u0026ndash;451. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1056/NEJMoa2005936\u003c/span\u003e\u003cspan address=\"10.1056/NEJMoa2005936\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFoulkes WD (2021) The ten genes for breast (and ovarian) cancer susceptibility. Nat Reviews Clin Oncol 18:259\u0026ndash;260. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41571-021-00491-3\u003c/span\u003e\u003cspan address=\"10.1038/s41571-021-00491-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSokolova A, Johnstone KJ, McCart Reed AE, Simpson PT, Lakhani SR (2023) Hereditary breast cancer: syndromes, tumour pathology and molecular testing. Histopathology 82:70\u0026ndash;82. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/his.14808\u003c/span\u003e\u003cspan address=\"10.1111/his.14808\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTarsounas M, Sung P (2020) The antitumorigenic roles of BRCA1\u0026ndash;BARD1 in DNA repair and replication. Nat Rev Mol Cell Biol 21:284\u0026ndash;299. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41580-020-0218-z\u003c/span\u003e\u003cspan address=\"10.1038/s41580-020-0218-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWitus SR, Zhao W, Brzovic PS, Klevit RE (2022) BRCA1/BARD1 is a nucleosome reader and writer. Trends Biochem Sci 47:582\u0026ndash;595. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tibs.2022.03.001\u003c/span\u003e\u003cspan address=\"10.1016/j.tibs.2022.03.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePolak P et al (2017) A mutational signature reveals alterations underlying deficient homologous recombination repair in breast cancer. Nat Genet 49:1476\u0026ndash;1486. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/ng.3934\u003c/span\u003e\u003cspan address=\"10.1038/ng.3934\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao W et al (2017) BRCA1\u0026ndash;BARD1 promotes RAD51-mediated homologous DNA pairing. Nature 550:360\u0026ndash;365. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nature24060\u003c/span\u003e\u003cspan address=\"10.1038/nature24060\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlenezi WM, Fierheller CT, Recio N, Tonin PN (2020) Literature Review of BARD1 as a Cancer Predisposing Gene with a Focus on Breast and Ovarian Cancers. Genes 11:856\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDe Brakeleer S et al (2016) Frequent incidence of BARD1-truncating mutations in germline DNA from triple-negative breast cancer patients. Clin Genet 89:336\u0026ndash;340. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/cge.12620\u003c/span\u003e\u003cspan address=\"10.1111/cge.12620\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRofes P et al (2021) BARD1 Pathogenic Variants Are Associated with Triple-Negative Breast Cancer in a Spanish Hereditary Breast and Ovarian Cancer Cohort. Genes 12:150\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCouch FJ et al (2017) Associations Between Cancer Predisposition Testing Panel Genes and Breast Cancer. JAMA Oncol 3:1190\u0026ndash;1196. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1001/jamaoncol.2017.0424\u003c/span\u003e\u003cspan address=\"10.1001/jamaoncol.2017.0424\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSlavin TP et al (2017) The contribution of pathogenic variants in breast cancer susceptibility genes to familial breast cancer risk. NPJ Breast Cancer 3. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41523-017-0024-8\u003c/span\u003e\u003cspan address=\"10.1038/s41523-017-0024-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCast\u0026eacute;ra L et al (2018) Landscape of pathogenic variations in a panel of 34 genes and cancer risk estimation from 5131 HBOC families. Genet Med 20:1677\u0026ndash;1686. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41436-018-0005-9\u003c/span\u003e\u003cspan address=\"10.1038/s41436-018-0005-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWeber-Lassalle N et al (2019) Germline loss-of-function variants in the BARD1 gene are associated with early-onset familial breast cancer but not ovarian cancer. Breast Cancer Res 21:55. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13058-019-1137-9\u003c/span\u003e\u003cspan address=\"10.1186/s13058-019-1137-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSuszynska M, Kozlowski P (2020) Summary of BARD1 Mutations and Precise Estimation of Breast and Ovarian Cancer Risks Associated with the Mutations. Genes (Basel) 11. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/genes11070798\u003c/span\u003e\u003cspan address=\"10.3390/genes11070798\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShimelis H et al (2018) Triple-Negative Breast Cancer Risk Genes Identified by Multigene Hereditary Cancer Panel Testing. J Natl Cancer Inst 110:855\u0026ndash;862. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/jnci/djy106\u003c/span\u003e\u003cspan address=\"10.1093/jnci/djy106\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eToh MR et al (2019) Functional analysis of clinical BARD1 germline variants. Cold Spring Harb Mol Case Stud 5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1101/mcs.a004093\u003c/span\u003e\u003cspan address=\"10.1101/mcs.a004093\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBecker JR et al (2021) BARD1 reads H2A lysine 15 ubiquitination to direct homologous recombination. Nature 596:433\u0026ndash;437. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41586-021-03776-w\u003c/span\u003e\u003cspan address=\"10.1038/s41586-021-03776-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStewart MD et al (2018) BARD1 is necessary for ubiquitylation of nucleosomal histone H2A and for transcriptional regulation of estrogen metabolism genes. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e 115, 1316\u0026ndash;1321. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.1715467115\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1715467115\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee C et al (2015) Functional Analysis of BARD1 Missense Variants in Homology-Directed Repair of DNA Double Strand Breaks. Hum Mutat 36:1205\u0026ndash;1214. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/humu.22902\u003c/span\u003e\u003cspan address=\"10.1002/humu.22902\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLindstr\u0026ouml;m LS et al (2012) Clinically Used Breast Cancer Markers Such As Estrogen Receptor, Progesterone Receptor, and Human Epidermal Growth Factor Receptor 2 Are Unstable Throughout Tumor Progression. J Clin Oncol 30:2601\u0026ndash;2608. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1200/JCO.2011.37.2482\u003c/span\u003e\u003cspan address=\"10.1200/JCO.2011.37.2482\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchrijver WAME et al (2018) Receptor Conversion in Distant Breast Cancer Metastases: A Systematic Review and Meta-analysis. JNCI: J Natl Cancer Inst 110:568\u0026ndash;580. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/jnci/djx273\u003c/span\u003e\u003cspan address=\"10.1093/jnci/djx273\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchwieger L et al (2024) Changes in expression of breast cancer tumor biomarkers between primary tumors and corresponding metastatic sites: common patterns and relationships with survival. Breast Cancer Res Treat 207:373\u0026ndash;382. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10549-024-07368-w\u003c/span\u003e\u003cspan address=\"10.1007/s10549-024-07368-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDowling GP et al (2024) Receptor Discordance in Metastatic Breast Cancer; a review of clinical and genetic subtype alterations from primary to metastatic disease. Breast Cancer Res Treat 207:471\u0026ndash;476. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10549-024-07431-6\u003c/span\u003e\u003cspan address=\"10.1007/s10549-024-07431-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCurtit E et al (2013) Discordances in Estrogen Receptor Status, Progesterone Receptor Status, and HER2 Status Between Primary Breast Cancer and Metastasis. Oncologist 18:667\u0026ndash;674. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1634/theoncologist.2012-0350\u003c/span\u003e\u003cspan address=\"10.1634/theoncologist.2012-0350\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRivera B et al (2017) Functionally Null RAD51D Missense Mutation Associates Strongly with Ovarian Carcinoma. Cancer Res 77:4517\u0026ndash;4529. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1158/0008-5472.Can-17-0190\u003c/span\u003e\u003cspan address=\"10.1158/0008-5472.Can-17-0190\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGradishar WJ et al (2023) NCCN Guidelines\u0026reg; Insights: Breast Cancer, Version 4.2023: Featured Updates to the NCCN Guidelines. J Natl Compr Canc Netw 21:594\u0026ndash;608. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.6004/jnccn.2023.0031\u003c/span\u003e\u003cspan address=\"10.6004/jnccn.2023.0031\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZheng Y et al (2021) Functional consequences of a rare missense BARD1 c.403G\u0026thinsp;\u0026gt;\u0026thinsp;A germline mutation identified in a triple-negative breast cancer patient. Breast Cancer Res 23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13058-021-01428-5\u003c/span\u003e\u003cspan address=\"10.1186/s13058-021-01428-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCupit-Link M et al (2024) Response to PARP Inhibition in BARD1-Mutated Refractory Neuroblastoma. N Engl J Med 391:659\u0026ndash;661. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1056/NEJMc2403316\u003c/span\u003e\u003cspan address=\"10.1056/NEJMc2403316\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRichards S et al (2015) Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Sci 17:405\u0026ndash;423. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/gim.2015.30\u003c/span\u003e\u003cspan address=\"10.1038/gim.2015.30\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBrnich SE et al (2019) Recommendations for application of the functional evidence PS3/BS3 criterion using the ACMG/AMP sequence variant interpretation framework. Genome Med 12:3. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13073-019-0690-2\u003c/span\u003e\u003cspan address=\"10.1186/s13073-019-0690-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754\u0026ndash;1760. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/bioinformatics/btp324\u003c/span\u003e\u003cspan address=\"10.1093/bioinformatics/btp324\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcKenna A et al (2010) The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20:1297\u0026ndash;1303. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1101/gr.107524.110\u003c/span\u003e\u003cspan address=\"10.1101/gr.107524.110\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePoplin R et al (2018) Scaling accurate genetic variant discovery to tens of thousands of samples. bioRxiv 201178. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1101/201178\u003c/span\u003e\u003cspan address=\"10.1101/201178\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang K, Li M, Hakonarson H (2010) ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res 38:e164\u0026ndash;e164. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/nar/gkq603\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkq603\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRahman N (2014) Realizing the promise of cancer predisposition genes. Nature 505:302\u0026ndash;308. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nature12981\u003c/span\u003e\u003cspan address=\"10.1038/nature12981\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIllumina \u003cem\u003eTruSight Cancer Gene List\u003c/em\u003e, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.illumina.com/content/dam/illumina-marketing/documents/products/gene_lists/gene_list_trusight_cancer.xlsx\u003c/span\u003e\u003cspan address=\"https://www.illumina.com/content/dam/illumina-marketing/documents/products/gene_lists/gene_list_trusight_cancer.xlsx\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eInvitae \u003cem\u003eInvitae Multi-Cancer Panel\u003c/em\u003e, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.invitae.com/en/providers/test-catalog/test-01101\u003c/span\u003e\u003cspan address=\"https://www.invitae.com/en/providers/test-catalog/test-01101\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTate JG et al (2018) COSMIC: the Catalogue Of Somatic Mutations In Cancer. Nucleic Acids Res 47:D941\u0026ndash;D947. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/nar/gky1015\u003c/span\u003e\u003cspan address=\"10.1093/nar/gky1015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFavero F et al (2015) Sequenza: allele-specific copy number and mutation profiles from tumor sequencing data. Ann Oncol 26:64\u0026ndash;70. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/annonc/mdu479\u003c/span\u003e\u003cspan address=\"10.1093/annonc/mdu479\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu M et al (2021) MesKit: a tool kit for dissecting cancer evolution of multi-region tumor biopsies through somatic alterations. \u003cem\u003eGigaScience\u003c/em\u003e 10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/gigascience/giab036\u003c/span\u003e\u003cspan address=\"10.1093/gigascience/giab036\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlexandrov LB et al (2020) The repertoire of mutational signatures in human cancer. Nature 578:94\u0026ndash;101. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41586-020-1943-3\u003c/span\u003e\u003cspan address=\"10.1038/s41586-020-1943-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCampeau E et al (2009) A Versatile Viral System for Expression and Depletion of Proteins in Mammalian Cells. PLoS ONE 4:e6529. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0006529\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0006529\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"McGill University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7595877/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7595877/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHereditary breast cancer involves multiple risk genes, including \u003cem\u003eBARD1\u003c/em\u003e, which confers low to moderate risk and is associated with triple-negative breast cancer (TNBC). We report a proband with a primary ER+/PR+/HER2- breast cancer which recurred unilaterally as a TNBC and who later developed endometrial cancer. Clinical germline testing revealed a rare \u003cem\u003eBARD1\u003c/em\u003e missense variant of uncertain significance [c.2258G\u0026thinsp;\u0026gt;\u0026thinsp;T; p.(Gly753Val)]. Whole-exome sequencing of tumors and blood revealed \u003cem\u003eBARD1\u003c/em\u003e loss of heterozygosity and mutational signature 3 \u0026ndash; indicative of homologous recombination (HR) repair deficiency \u0026ndash; exclusively in the TNBC recurrence. Functional assays demonstrated impaired HR repair \u003cem\u003evia\u003c/em\u003e increased PARP inhibitor sensitivity and reduced RAD51 foci formation. We hypothesize that the selective pressure exerted by tamoxifen resulted in breast cancer subtype switching and uncovered the pathogenic potential of the \u003cem\u003eBARD1\u003c/em\u003e p.(Gly753Val) variant. This case illustrates a previously unreported scenario where the pathogenicity of a germline \u003cem\u003eBARD1\u003c/em\u003e variant appears conditional on prior treatment.\u003c/p\u003e","manuscriptTitle":"Multimodal analysis of rare BARD1 missense variant suggests its pathogenicity is conditional","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-24 09:22:51","doi":"10.21203/rs.3.rs-7595877/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2b849279-7b1d-4558-9d19-cdd412f502c0","owner":[],"postedDate":"September 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-24T09:22:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-24 09:22:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7595877","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7595877","identity":"rs-7595877","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}