Functional characterization of BRCA2 variants of uncertain significance identified in Korean breast cancer patients

Functional characterization of BRCA2 variants of uncertain significance identified in Korean breast cancer patients | 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 Research Article Functional characterization of BRCA2 variants of uncertain significance identified in Korean breast cancer patients Ah Reum Lim, Seung Pil Jung, Kyoungmi Kim, Hye Jin Choi, Kyong Hwa Park, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8617436/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Background Variants of uncertain significance (VUS) in BRCA2 remain a major challenge in the clinical management of breast cancer, particularly in Asian populations where population-specific reference data are limited. Functional assays may provide complementary biological evidence to support interpretation of these variants. Patients and methods: Three BRCA2 missense VUS—c.5969A > C (p.Asp1990Ala), c.3671G > A (p.Gly1224Asp), and c.5459G > A (p.Cys1820Tyr)—were selected based on their frequency in Korean cohorts and/or strong family history of breast cancer. Full-length BRCA2 constructs harboring each variant were introduced into BRCA2 -knockout DLD-1 cells. Functional consequences were evaluated using cellular sensitivity assays following cisplatin treatment and ionizing radiation, response to the PARP inhibitor olaparib, and homologous recombination (HR) efficiency assessed by a GFP-based reporter system. Results Cells expressing BRCA2 p.Asp1990Ala consistently demonstrated increased sensitivity to cisplatin, ionizing radiation, and olaparib, comparable to pathogenic controls. In addition, GFP-based HR assays revealed a marked reduction in HR repair efficiency in p.Asp1990Ala-expressing cells. In contrast, cells expressing BRCA2 p.Gly1224Asp or p.Cys1820Tyr exhibited DNA damage responses, drug sensitivity profiles, and HR activity similar to wild-type BRCA2 across all assays. Conclusions Among the three BRCA2 VUS analyzed, p.Asp1990Ala exhibited a pathogenic-like functional phenotype characterized by impaired homologous recombination repair, whereas p.Gly1224Asp and p.Cys1820Tyr appeared functionally neutral. These findings highlight the functional heterogeneity of BRCA2 VUS and support the role of functional assays as complementary tools for variant interpretation in breast cancer. BRCA2 Breast cancer Variants of uncertain significance Homologous recombination Functional assay Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Breast cancer is the most common malignancy among women worldwide, and hereditary predisposition plays a critical role in disease development and therapeutic decision-making [ 1 ]. Among hereditary breast cancer susceptibility genes, BRCA2 is a central tumor suppressor whose functional integrity is essential for maintaining genomic stability [ 2 ]. Pathogenic variants in BRCA2 are strongly associated with increased lifetime breast cancer risk and have direct implications for cancer surveillance, risk-reducing strategies, and systemic treatment selection [ 2 , 3 ]. The BRCA2 protein plays a pivotal role in homologous recombination (HR), a high-fidelity DNA repair pathway responsible for accurately repairing DNA double-strand breaks. Through direct interaction with RAD51, BRCA2 facilitates nucleoprotein filament formation and strand invasion, thereby ensuring error-free DNA repair [ 4 – 6 ]. Loss of BRCA2 function results in homologous recombination deficiency (HRD), leading to genomic instability and increased sensitivity to DNA-damaging agents, including platinum-based chemotherapy and poly(ADP-ribose) polymerase (PARP) inhibitors [ 7 – 9 ]. Accordingly, identification of pathogenic BRCA2 variants has become an integral component of precision treatment strategies in breast cancer [ 10 ]. With the widespread introduction of next-generation sequencing, particularly tumor-based genomic profiling, the clinical interpretation of BRCA2 variants has become increasingly complex. Tumor sequencing frequently identifies BRCA variants with variant allele frequencies close to 50%, a finding that strongly suggests a germline origin. In clinical practice, such results often prompt suspicion of hereditary cancer predisposition, especially when detected in patients with early-onset disease or a suggestive family history [ 11 , 12 ]. However, despite these features, a substantial proportion of these variants are ultimately classified as variants of uncertain significance (VUS). Unlike truncating variants with well-established clinical consequences, missense BRCA2 variants often lack sufficient evidence for definitive classification as pathogenic or benign [ 13 , 14 ]. This creates a clinically challenging gray zone in which variants that appear highly suggestive of germline inheritance based on tumor sequencing data cannot be used to guide genetic counseling, risk-reducing strategies, or treatment decisions [ 15 ]. The burden of this interpretive gap is particularly pronounced in non-Caucasian populations. Current variant classification frameworks, including the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines, rely heavily on population frequency data, previously reported clinical evidence, and reference databases that are largely derived from Caucasian populations [ 16 , 17 ]. Consequently, the application of these criteria is limited in Asian populations, where population-specific reference data remain underrepresented [ 18 ]. Multiple studies have reported higher rates of BRCA1/2 variants of uncertain significance in East Asian breast cancer cohorts, including Korean patients, reflecting both population-specific genetic architecture and insufficient representation in global databases [ 19 – 21 ]. Because of these limitations, efforts in Korea have increasingly focused on characterizing the distribution and frequency of BRCA1/2 VUS at the population level. In South Korea, a nationwide precision oncology initiative, K-MASTER, was conducted to perform large-scale tumor genome profiling in approximately 10,000 cancer patients. This program enabled us to systematically analyze the genome changes of various solid tumors including BRCA genes. In addition, we observed that a significant portion of BRCA2 mutations identified in the K-MASTER cohort were classified as uncertain significance mutations, and multiple missense mutations were repeated in individuals [ 22 ]. This makes sense to analyze BRCA in KMASTER data representing Korean solid cancer NGS results, and we thought it would be important to confirm the function of these VUSs through experiments if necessary. Functional assays have emerged as an important tool to address this unmet need. Current variant interpretation guidelines emphasize that well-validated functional studies can provide strong biological evidence by directly assessing the impact of a variant on protein function [ 16 , 17 ]. For BRCA2 , impairment of homologous recombination represents a particularly relevant functional endpoint, as HR deficiency provides a mechanistic link between molecular dysfunction and therapeutic vulnerability [ 9 , 23 ]. Assays evaluating cellular sensitivity to DNA-damaging agents, PARP inhibitors, and HR reporter systems therefore offer biologically meaningful insight into the functional consequences of individual BRCA2 variants [ 24 – 26 ]. In this study, motivated by observations from tumor-based genomic profiling and the clinical dilemma posed by germline-suspected BRCA2 VUS, we performed a comprehensive functional characterization of three BRCA2 missense variants of uncertain significance identified in Korean breast cancer patients. With the K-MASTER dataset, we evaluated the effects of each variant on the DNA damage response, including chemotherapeutic agent, ionizing radiation, and the PARP inhibitor olaparib, homologous recombination efficiency, and DNA damage factors. With this integrated functional approach, we wanted to clarify the variant-specific functional phenotype and provide complementary biological evidence related to the interpretation of BRCA2 VUS in breast cancer. Material and methods Study design and variant selection This study integrated population-scale tumor genomic data with functional assays to evaluate the biological significance of selected BRCA2 variants of uncertain significance (VUS) identified in Korean cancer patients. Candidate BRCA2 VUS were curated from the K-MASTER project database and from Korean breast cancer patients with strong suspicion of hereditary cancer based on family history. Among the BRCA2 VUS identified, three missense variants were selected for functional analysis based on recurrence in the K-MASTER cohort and/or association with suggestive family histories: c.5969A > C (p.Asp1990Ala) located in the BRC7 domain, c.3671G > A (p.Gly1224Asp) located in the BRC2 domain, and c.5459G > A (p.Cys1820Tyr) located in the spacer region between BRC5 and BRC6. These regions are known to play critical roles in RAD51 binding and homologous recombination repair. A known pathogenic BRCA2 variant, c.8167G > C (p.Asp2723His), was included as a pathogenic control, and wild-type BRCA2 served as a functional reference. K-MASTER cohort and BRCA variant analysis The K-MASTER project is a nationwide precision oncology initiative designed to perform large-scale tumor genomic profiling in Korean patients with advanced solid tumors. Tumor sequencing data from 9,677 patients were successfully analyzed between 2017 and 2021. Tumor specimens were sequenced using targeted gene panels, including the FIRST, CancerSCAN, K-MASTER 1.0, and K-MASTER 1.1 panels. For patients without available tumor tissue or with quality control failure, circulating tumor DNA from blood samples was analyzed using a targeted cancer panel. BRCA1 and BRCA2 variants detected in the K-MASTER cohort were annotated and classified using ClinVar and the Human Gene Mutation Database. Variants were categorized as pathogenic, likely pathogenic, benign, likely benign, variants of uncertain significance, or variants with conflicting interpretations. The distribution of BRCA2 variants by clinical significance, mutation type, and tumor origin was analyzed to identify recurrent VUS with potential biological relevance. Cell lines and culture conditions The human colorectal cancer cell line DLD-1 with homozygous deletion of BRCA2 ( BRCA2 knockout) was used as the experimental model. DLD-1 BRCA2 knockout cells were obtained from Horizon Discovery (Cambridge, UK). Cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin at 37°C in a humidified incubator with 5% CO₂. Wild-type DLD-1 cells were cultured under identical conditions and used as an additional reference where appropriate. Plasmid constructs and site-directed mutagenesis Full-length human BRCA2 cDNA constructs were used as templates for mutagenesis. Site-directed mutagenesis was performed to generate constructs harboring the selected VUS and the pathogenic control variant. Mutagenesis was conducted by a commercial service provider (BIOFACT, Daejeon, Korea) to ensure accuracy. All introduced variants were confirmed by Sanger sequencing. The resulting constructs included wild-type BRCA2 , one pathogenic variant, and three VUS constructs. These plasmids were used for transient transfection into DLD-1 BRCA2 knockout cells (Fig. 1 ). Transient transfection DLD-1 BRCA2 knockout cells were seeded at 70–80% confluence and transfected with plasmids encoding wild-type BRCA2 , pathogenic control, or VUS constructs using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. Cells were incubated for 48 hours following transfection before subsequent analyses. Western blot analysis Protein expression of BRCA2 constructs was confirmed by Western blot analysis. Cells were lysed using PRO-PREP protein extraction solution supplemented with protease and phosphatase inhibitor cocktails. Protein concentrations were quantified using the Bradford assay. Equal amounts of protein (30 µg) were separated by SDS–PAGE and transferred onto PVDF membranes. Membranes were blocked and incubated with a primary antibody against BRCA2 (Santa Cruz Biotechnology, sc-518154), followed by incubation with an appropriate secondary antibody. GAPDH was used as a loading control. Protein bands were visualized using enhanced chemiluminescence, and band intensity was assessed to confirm expression of full-length BRCA2 protein. DNA damage response assays using cisplatin To evaluate cellular responses to DNA damage, transfected cells were treated with cisplatin (Sigma-Aldrich). Cells were exposed to increasing concentrations of cisplatin for 24, 48, or 72 hours. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Japan) according to the manufacturer’s protocol. Absorbance was measured at 450 nm, and cell viability was calculated relative to untreated controls. Dose–response curves were generated, and IC50 values were calculated using GraphPad Prism software. Radiation-induced DNA damage assay To assess sensitivity to ionizing radiation, transfected cells were exposed to graded doses of radiation (1, 2, 4, and 8 Gy) using an X-ray irradiator (Xstrahl CIX3 Cabinet X-ray Irradiator). Following irradiation, cells were incubated for 24, 48, or 72 hours. Cell survival was measured using the CCK-8 assay and expressed as a percentage relative to non-irradiated controls. PARP inhibitor sensitivity assay To evaluate sensitivity to PARP inhibition, transfected cells were treated with olaparib (SelleckChem) at concentrations of 0, 0.1, 0.5, 1, 2, and 5 µM. After 48 hours of treatment, cell viability was assessed using the CCK-8 assay. Viability was calculated relative to untreated controls. Homologous recombination repair assay Homologous recombination repair efficiency was evaluated using a GFP-based reporter assay with I-SceI–induced double-strand breaks. DLD-1 BRCA2 knockout cells stably expressing the DR-GFP reporter were co-transfected with plasmids encoding I-SceI endonuclease and either wild-type BRCA2 , pathogenic control, or VUS constructs. Following transfection, cells were incubated to allow DNA repair. GFP-positive cells, indicative of successful HR-mediated repair, were visualized using fluorescence microscopy. HR efficiency was assessed by comparing the relative number of GFP-positive cells across experimental conditions (Fig. 2 ). Statistical analysis All experiments were performed in triplicate and repeated independently at least three times. Data are presented as mean ± standard deviation. Statistical analyses were conducted using GraphPad Prism version 10. Comparisons between groups were performed using one-way or two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test, as appropriate. A p-value < 0.05 was considered statistically significant. Results Identification and distribution of BRCA2 variants of uncertain significance in the K-MASTER cohort Tumor genomic sequencing data from 9,677 patients enrolled in the K-MASTER project were analyzed to characterize the distribution of BRCA1 and BRCA2 variants. Among these patients, BRCA1 variants were identified in 737 cases (7.6%), and BRCA2 variants were identified in 1,411 cases (14.6%) (Fig. 3 ). When classified according to clinical significance, a substantial proportion of detected variants were categorized as variants of uncertain significance. Specifically, BRCA2 VUS accounted for 7.04% of the total analyzed cohort. Missense variants represented the predominant mutation type among BRCA2 VUS, whereas pathogenic or likely pathogenic variants were more frequently associated with truncating mutations, including frameshift and nonsense variants ( Supplementary Fig. 1–2 ). This distribution indicated that missense substitutions constitute the major source of uncertainty in BRCA2 variant interpretation within the Korean cancer population (Table 1 ). Table 1 Frequent BRCA2 missense variants with uncertain or conflicting clinical significance identified in the K-MASTER cohort N DNA Change Protein Change VAF (%) Type Classification 56 c.6325G > A V2109I 41.51 Missense Conflicting interpretations of pathogenicity 28 c.4854T > A D1618E 46.22 Missense Uncertain significance 22 c.964A > C K322Q 45.4 Missense Conflicting interpretations of pathogenicity 19 c.5969A > C D1990A 46.41 Missense Conflicting interpretations of pathogenicity 14 c.623T > G V208G 43.58 Missense Uncertain significance 9 c.10131A > C E3377D 52.24 Missense Uncertain significance 7 c.7706G > A G2569D 42.55 Missense Uncertain significance 6 c.6496G > T V2166L 50.48 Missense Uncertain significance 4 c.3007C > G H1003D 50.2 Missense Uncertain significance Analysis of tumor types revealed that BRCA2 VUS were most frequently detected in breast cancer, followed by colorectal, gastric, lung, head and neck, and ovarian cancers ( Supplementary Fig. 3 ). Compared with the overall tumor distribution of the K-MASTER cohort, breast and ovarian cancers were relatively enriched among cases harboring BRCA2 VUS. Selection of BRCA2 VUS for functional analysis To prioritize candidates for functional evaluation, BRCA2 VUS were ranked based on frequency within the K-MASTER cohort and association with clinical features suggestive of hereditary cancer, including family history. Among recurrent variants, c.5969A > C (p.Asp1990Ala) was identified in 28 patients and exhibited a variant allele frequency close to 50%, suggesting a possible germline origin (Table 1 ). Several more frequently detected variants were excluded from further analysis because existing functional or clinical evidence supported a benign classification. Consequently, p.Asp1990Ala was selected as a representative recurrent BRCA2 VUS. In addition, two variants, c.3671G > A (p.Gly1224Asp) and c.5459G > A (p.Cys1820Tyr), were selected based on their presence in patients with strong family histories of breast or related cancers ( Supplementary Fig. 4 ). All three selected variants were located within or adjacent to BRC repeat regions implicated in RAD51 binding and homologous recombination repair (Fig. 4 ). Conservation and structural analysis of selected BRCA2 VUS Multiple sequence alignment of BRCA2 orthologs from six species demonstrated that the amino acid residues corresponding to p.Asp1990Ala, p.Gly1224Asp, and p.Cys1820Tyr were evolutionarily conserved ( Supplementary Fig. 5 ). Conservation across species suggested potential functional relevance of these residues. Three-dimensional structural modeling of the BRC repeats aligned to the RAD51–BRCA2 complex structure revealed distinct conformational features among the variants. Structural models predicted abnormal residue protrusion and altered local conformation for p.Asp1990Ala and p.Gly1224Asp, whereas p.Cys1820Tyr showed relatively preserved structural features ( Supplementary Fig. 6 ). Expression of BRCA2 wild-type, pathogenic, and VUS constructs in BRCA2 -deficient cells To assess the functional impact of each variant, full-length BRCA2 constructs encoding wild-type protein, a known pathogenic variant, or each VUS were transiently transfected into DLD-1 BRCA2 -knockout cells. Western blot analysis confirmed the expression of full-length BRCA2 protein in cells transfected with wild-type, pathogenic control, and all three VUS constructs. No BRCA2 signal was detected in untransfected BRCA2 -knockout cells ( Supplementary Fig. 7) . The expression levels of BRCA2 were comparable across wild-type, pathogenic, and VUS constructs, indicating that the observed functional differences were unlikely to be attributable to gross differences in protein expression or stability. Differential cellular response to cisplatin-induced DNA damage To evaluate the role of each BRCA2 variant in DNA damage response, transfected cells were treated with increasing concentrations of cisplatin, and cell viability was assessed at multiple time points. Wild-type BRCA2 -expressing cells exhibited a gradual, dose-dependent reduction in cell viability following cisplatin exposure, consistent with intact homologous recombination repair capacity. In contrast, BRCA2 -knockout cells showed marked sensitivity to cisplatin, with significantly reduced cell viability at lower drug concentrations ( Supplementary Fig. 8) . Cells expressing the pathogenic control variant demonstrated cisplatin sensitivity similar to BRCA2 -knockout cells. Notably, cells expressing BRCA2 p.Asp1990Ala also exhibited increased sensitivity to cisplatin, closely resembling the response observed in pathogenic control and knockout cells. The calculated IC50 values for p.Asp1990Ala were substantially lower than those for wild-type BRCA2 . In contrast, cells expressing BRCA2 p.Gly1224Asp or p.Cys1820Tyr maintained higher cell viability across the tested cisplatin concentrations, with dose–response curves comparable to wild-type BRCA2 -expressing cells. These findings indicate differential functional consequences among the three VUS with respect to cisplatin-induced DNA damage (Fig. 5 ). Increased sensitivity of BRCA2 p.Asp1990Ala to ionizing radiation To further assess DNA repair capacity, transfected cells were exposed to ionizing radiation, and cell survival was measured at 24, 48, and 72 hours following irradiation. Wild-type BRCA2 -expressing cells demonstrated relative resistance to low-dose radiation, with a progressive decline in survival at higher doses and later time points. In contrast, BRCA2 -knockout cells exhibited pronounced radiation sensitivity, with significantly reduced survival even at moderate radiation doses ( Supplementary Fig. 9) . Cells expressing the pathogenic control variant showed survival profiles similar to knockout cells. Importantly, cells expressing BRCA2 p.Asp1990Ala also displayed reduced survival following radiation exposure, closely paralleling the pathogenic control. At both 48 and 72 hours after irradiation, survival rates in p.Asp1990Ala-expressing cells were significantly lower than those observed in wild-type cells. Due to their wild-type–like behavior in cisplatin assays, p.Gly1224Asp- and p.Cys1820Tyr-expressing cells were not further evaluated in radiation assays. The radiation sensitivity observed in p.Asp1990Ala-expressing cells supports a functional defect in DNA damage repair pathways (Fig. 6 ) . Enhanced sensitivity of BRCA2 p.Asp1990Ala to PARP inhibition Given the established association between homologous recombination deficiency and PARP inhibitor sensitivity, the response of transfected cells to the PARP inhibitor olaparib was assessed. Wild-type BRCA2 -expressing cells demonstrated relative resistance to olaparib across a range of concentrations. In contrast, BRCA2 -knockout cells exhibited significantly reduced cell viability, consistent with HR deficiency. Cells expressing the pathogenic control variant showed olaparib sensitivity comparable to knockout cells. Similarly, cells expressing BRCA2 p.Asp1990Ala demonstrated increased sensitivity to olaparib, with IC50 values closer to those observed in pathogenic and knockout cells than to wild-type controls. These findings indicate that BRCA2 p.Asp1990Ala compromises cellular tolerance to PARP inhibition, consistent with impaired homologous recombination repair. In contrast, p.Gly1224Asp and p.Cys1820Tyr were not associated with increased olaparib sensitivity (Fig. 7 ). Impaired homologous recombination repair in BRCA2 p.Asp1990Ala assessed by GFP reporter assay To directly evaluate homologous recombination efficiency, a GFP-based reporter assay was performed using I-SceI–induced double-strand breaks. Cells expressing wild-type BRCA2 showed robust GFP signal, indicating efficient HR-mediated repair. In contrast, BRCA2 -knockout cells exhibited minimal GFP positivity, reflecting defective HR. Cells expressing the pathogenic control variant demonstrated markedly reduced GFP signal. Notably, cells expressing BRCA2 p.Asp1990Ala also showed a significant reduction in GFP-positive cells, comparable to the pathogenic control. This reduction indicates impaired HR repair capacity. In contrast, GFP signal intensity in cells expressing p.Gly1224Asp or p.Cys1820Tyr was comparable to wild-type BRCA2 , suggesting preserved HR function in these variants (Fig. 8 ). Discussion This study was initiated from a clinically relevant dilemma increasingly encountered in the era of tumor-based next-generation sequencing (NGS): the identification of BRCA2 variants that strongly suggest germline origin yet remain classified as variants of uncertain significance (VUS). Large-scale genomic screening efforts in Korea have demonstrated that a substantial proportion of BRCA2 variants detected through tumor sequencing are designated as VUS, often occurring at variant allele frequencies close to 50%, a pattern highly suggestive of germline inheritance [ 20 , 27 , 28 ]. Such findings frequently raise concerns regarding hereditary breast cancer; however, conventional classification frameworks provide limited guidance in these situations. Interpretation of BRCA1/2 VUS remains challenging under the current American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines, which rely on a multifactorial framework integrating population frequency, clinical data, segregation analysis, and functional evidence [ 29 , 30 ]. Because reference databases and population frequency data are largely derived from Caucasian populations, many variants identified in Asian cohorts fail to meet criteria for either pathogenic or benign classification and therefore remain designated as VUS [ 19 , 20 ]. This limitation has been consistently reported in Korean and other East Asian studies, underscoring the need for additional lines of evidence to contextualize variants encountered in real-world clinical practice. Based on this background, the present study focused on the functional characterization of selected BRCA2 missense VUS recurrently identified in the Korean cancer population and associated with clinical features suggestive of hereditary breast cancer. Given the essential role of the BRCA2 –RAD51 interaction in homologous recombination repair, variants located within or adjacent to BRC repeat regions were prioritized for experimental evaluation. Functional assessment was performed using a BRCA2 -deficient cellular model to directly evaluate DNA damage response, homologous recombination efficiency, and sensitivity to DNA-damaging agents and PARP inhibition. Among the three variants analyzed, BRCA2 p.Asp1990Ala consistently demonstrated impaired DNA repair function across multiple assays. Cells expressing this variant showed increased sensitivity to cisplatin and ionizing radiation, as well as heightened susceptibility to the PARP inhibitor olaparib, accompanied by reduced homologous recombination activity in a GFP-based reporter assay. The concordance of these findings across independent experimental platforms supports the biological relevance of this variant and suggests compromised BRCA2 -mediated DNA repair under genotoxic stress. Similar observations have been reported in prior functional studies demonstrating that a subset of BRCA VUS exhibit homologous recombination defects comparable to pathogenic variants [ 31 , 32 ]. In contrast, BRCA2 p.Gly1224Asp and p.Cys1820Tyr did not exhibit functional impairment in the experimental systems used. Despite their localization within regions implicated in RAD51 interaction and their identification in patients with suggestive family histories, these variants retained DNA repair capacity comparable to wild-type BRCA2 . This finding highlights the marked functional heterogeneity among BRCA2 missense variants and illustrates the limitations of predicting biological impact based solely on sequence conservation, domain location, or clinical suspicion. Such heterogeneity has been consistently described in previous functional complementation studies of BRCA missense variants [ 33 ]. The potential clinical relevance of functional heterogeneity among BRCA VUS has been increasingly recognized. Emerging clinical data suggest that some patients harboring BRCA VUS may demonstrate treatment responses closer to those observed in carriers of pathogenic variants than in patients with benign variants, particularly in the context of PARP inhibitor therapy [ 34 ]. While current clinical guidelines do not support therapeutic decision-making based on VUS status alone, functional data such as those presented here may provide important biological context for future integrative studies linking genotype, functional phenotype, and clinical outcome. Several limitations of this study should be acknowledged. First, all functional analyses were conducted in vitro using a single BRCA2 -knockout cell line, which may not fully recapitulate the complexity of tumor biology or tissue-specific contexts. Second, transient transfection was used to express BRCA2 constructs, potentially introducing variability in expression levels. Third, segregation analysis and longitudinal clinical outcome data were not incorporated, limiting direct correlation between functional findings and patient-level phenotypes. These limitations are consistent with those reported in prior functional studies of BRCA VUS and highlight the importance of integrative approaches combining functional assays with genetic and clinical data [ 29 , 35 ]. In conclusion, this study provides important insights into the functional impact of specific BRCA2 VUS, particularly within the Korean population. The results demonstrate that BRCA2 D1990A exhibits impaired DNA repair capabilities, aligning it closer to a pathogenic variant, whereas BRCA2 G1224D and BRCA2 C1820Y maintain DNA repair functionality similar to the wild-type BRCA2 . These findings support the importance of experimental validation for BRCA2 VUS to aid in reclassifying VUS. Further research is needed to confirm these findings in clinical settings and explore other VUS, ultimately enhancing personalized risk assessment and patient care. Declarations Ethics approval This study was approved by the Institutional Review Board of Korea University Anam Hospital (2017AN0401) and was conducted in accordance with the Declaration of Helsinki. Consent for publication Not applicable. The manuscript does not contain individual, identifiable patient data. Competing interests The authors declare no competing interests. Funding None. Authors’ contribution statements Conceptualization, Ah Reum Lim, Soohyeon Lee; Methodology, Ah Reum Lim, Kyoungmi Kim; Formal analysis, Ah Reum Lim; Investigation, Ah Reum Lim, Seung Pil Jung, Hye Jin Choi; Data curation, Ah Reum Lim, Kyong Hwa Park; Visualization, Ah Reum Lim; Resources, Seung Pil Jung, Hye Jin Choi; Supervision, Soohyeon Lee, Kyong Hwa Park; Writing—original draft, Ah Reum Lim; Writing—review and editing, Soohyeon Lee, Kyong Hwa Park, Kyoungmi Kim. 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Cancer Discov. 2022;12:938–48. https://doi.org/10.1158/2159-8290.Cd-21-1064 . Lord CJ, Ashworth A. PARP inhibitors: Synthetic lethality in the clinic. Science (1979). 2017;355:1152–8. https://doi.org/10.1126/science.aam7344 Olsen RK, Brøner S, Sabaratnam R, Doktor TK, Andersen HS, Bruun GH, et al. The ETFDH c.158A > G variation disrupts the balanced interplay of ESE- and ESS-binding proteins thereby causing missplicing and multiple Acyl-CoA dehydrogenation deficiency. Hum Mutat. 2014;35:86–95. https://doi.org/10.1002/humu.22455 . El-Seedy A, Girodon E, Norez C, Pajaud J, Pasquet MC, de Becdelièvre A, et al. CFTR mutation combinations producing frequent complex alleles with different clinical and functional outcomes. Hum Mutat. 2012;33:1557–65. https://doi.org/10.1002/humu.22129 . Moynahan ME, Jasin M. Loss of heterozygosity induced by a chromosomal double-strand break. Proc Natl Acad Sci U S A. 1997;94:8988–93. https://doi.org/10.1073/pnas.94.17.8988 . Kang E, Seong MW, Park SK, Lee JW, Lee J, Kim LS, et al. The prevalence and spectrum of BRCA1 and BRCA2 mutations in Korean population: recent update of the Korean Hereditary Breast Cancer (KOHBRA) study. Breast Cancer Res Treat. 2015;151:157–68. https://doi.org/10.1007/s10549-015-3377-4 . Han SA, Kim SW, Kang E, Park SK, Ahn SH, Lee MH, et al. The prevalence of BRCA mutations among familial breast cancer patients in Korea: results of the Korean Hereditary Breast Cancer study. Fam Cancer. 2013;12:75–81. https://doi.org/10.1007/s10689-012-9578-7 . Lindor NM, Guidugli L, Wang X, Vallée MP, Monteiro AN, Tavtigian S, et al. A review of a multifactorial probability-based model for classification of BRCA1 and BRCA2 variants of uncertain significance (VUS). Hum Mutat. 2012;33:8–21. https://doi.org/10.1002/humu.21627 . Lee JS, Oh S, Park SK, Lee MH, Lee JW, Kim SW, et al. Reclassification of BRCA1 and BRCA2 variants of uncertain significance: a multifactorial analysis of multicentre prospective cohort. J Med Genet. 2018;55:794–802. https://doi.org/10.1136/jmedgenet-2018-105565 . Guidugli L, Carreira A, Caputo SM, Ehlen A, Galli A, Monteiro AN, et al. Functional assays for analysis of variants of uncertain significance in BRCA2 . Hum Mutat. 2014;35:151–64. https://doi.org/10.1002/humu.22478 . Guidugli L, Pankratz VS, Singh N, Thompson J, Erding CA, Engel C, et al. A classification model for BRCA2 DNA binding domain missense variants based on homology-directed repair activity. Cancer Res. 2013;73:265–75. https://doi.org/10.1158/0008-5472.Can-12-2081 . Millot GA, Carvalho MA, Caputo SM, Vreeswijk MP, Brown MA, Webb M, et al. A guide for functional analysis of BRCA1 variants of uncertain significance. Hum Mutat. 2012;33:1526–37. https://doi.org/10.1002/humu.22150 . Chevrier S, Desmoulins I, Favier L, Ghiringhelli F, Bengrine L, Arnould L, et al. Prediction of olaparib sensitivity for variants of unknown significance in homologous repair genes. J Clin Oncol. 2019;37:3108. https://doi.org/10.1200/JCO.2019.37.15_suppl.3108 . De Paolis E, Paris I, Tilocca B, Roncada P, Foca L, Tiberi G, et al. Assessing the pathogenicity of BRCA1/2 variants of unknown significance: Relevance and challenges for breast cancer precision medicine. Front Oncol. 2022;12:1053035. https://doi.org/10.3389/fonc.2022.1053035 . Supplementary Files SupplementaryFigureBRCA2VUS.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major Revision 20 Apr, 2026 Reviewers agreed at journal 02 Mar, 2026 Reviewers invited by journal 08 Feb, 2026 Editor assigned by journal 16 Jan, 2026 First submitted to journal 15 Jan, 2026 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. 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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-8617436","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":587865904,"identity":"0ca21f03-ad08-484b-9fd7-92268b999ae7","order_by":0,"name":"Ah Reum Lim","email":"","orcid":"","institution":"Korea University Ansan Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ah","middleName":"Reum","lastName":"Lim","suffix":""},{"id":587865905,"identity":"5ecda59b-85e5-4ddc-9052-e990c3c1368b","order_by":1,"name":"Seung Pil Jung","email":"","orcid":"","institution":"Korea University Anam Hospital","correspondingAuthor":false,"prefix":"","firstName":"Seung","middleName":"Pil","lastName":"Jung","suffix":""},{"id":587865906,"identity":"8e80bd5c-4abd-4300-9d38-40e259bbc52a","order_by":2,"name":"Kyoungmi Kim","email":"","orcid":"","institution":"Korea University","correspondingAuthor":false,"prefix":"","firstName":"Kyoungmi","middleName":"","lastName":"Kim","suffix":""},{"id":587865907,"identity":"5f584bf0-bff3-4a05-8f9a-bff27c550bc5","order_by":3,"name":"Hye Jin Choi","email":"","orcid":"","institution":"Yonsei Cancer Center: Yonsei Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hye","middleName":"Jin","lastName":"Choi","suffix":""},{"id":587865908,"identity":"bbbdd17b-c9e6-46bf-9eb6-1796dd3c08ae","order_by":4,"name":"Kyong Hwa Park","email":"","orcid":"","institution":"Korea University Anam Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kyong","middleName":"Hwa","lastName":"Park","suffix":""},{"id":587865909,"identity":"4e0e9d0c-2133-4c6c-84ad-ec0bd20acc0b","order_by":5,"name":"Soohyeon Lee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYHACNgjFDsQfDsBFE4jQwszAwDiDZC3MPMRokW9vPvaYp8Imj4GZ+eBjmzM2+fzsBxg//GBIy8elxeDMsXRjnjNpxQzMbMnGOTfSLGf2JDBL9jDkWDbg0iKRYybN23Y4sYGZx0w658NhA4MDCQzSDAwVBjgdNgOk5R9IC//33xZALfbnHzD/xqeF4QZISwPYFjZmhhtAWyQS2IC25ODUAvRLmuScY2lALWzGkj1n0gwkbjxss+wxSMPtMGCISbypsUlsYG9++OHHMRsD/v7kwzd+VCTjdhgQMPEACfsDcD5jA9B2fBqASn7glx8Fo2AUjIKRDgANdlBCDMvUfQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-9665-062X","institution":"Korea University Anam Hospital","correspondingAuthor":true,"prefix":"","firstName":"Soohyeon","middleName":"","lastName":"Lee","suffix":""}],"badges":[],"createdAt":"2026-01-16 11:05:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8617436/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8617436/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102553478,"identity":"8713acf5-4b4f-46eb-8c77-0d0020783480","added_by":"auto","created_at":"2026-02-13 01:14:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":89611,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of the study design for functional characterization of BRCA2 variants\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8617436/v1/724924a1cd204ad563f4da18.png"},{"id":103503787,"identity":"2dd4b260-02d8-4e6d-addb-e20df39126d4","added_by":"auto","created_at":"2026-02-26 13:01:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":230250,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic overview of the GFP-based homologous recombination reporter assay\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8617436/v1/68d13f3753b35e99501725fa.png"},{"id":102747089,"identity":"c6b6080b-d00d-4664-a1da-9c8893686d53","added_by":"auto","created_at":"2026-02-16 09:03:48","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":50404,"visible":true,"origin":"","legend":"\u003cp\u003eAnalytical workflow for \u003cem\u003eBRCA1/2\u003c/em\u003e variant identification in the K-MASTER cohort\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8617436/v1/3d0cd40711ab83e03aade7b2.jpeg"},{"id":102553480,"identity":"ed88afc9-5825-4224-9622-fd9e6859fcef","added_by":"auto","created_at":"2026-02-13 01:14:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":125460,"visible":true,"origin":"","legend":"\u003cp\u003eGenomic locations of pathogenic and VUS \u003cem\u003eBRCA2\u003c/em\u003evariants analyzed in this study\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8617436/v1/773c9d258eb0d679e01efa17.png"},{"id":102553481,"identity":"62d54856-2bdc-4034-84b6-683fe8d9cadb","added_by":"auto","created_at":"2026-02-13 01:14:13","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":168854,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential sensitivity of \u003cem\u003eBRCA2 \u003c/em\u003ewild-type, knockout, pathogenic, and VUS-expressing cells to cisplatin Marks represent the mean cell viability (%) of triplicates, and error bars represent SD. **p \u0026lt; 0.01, *p \u0026lt; 0.001, two-way ANOVA.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8617436/v1/147518d706469622cf3d186a.jpeg"},{"id":102553485,"identity":"84675181-faaf-43c2-ab61-fd9ce4b4f309","added_by":"auto","created_at":"2026-02-13 01:14:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":41307,"visible":true,"origin":"","legend":"\u003cp\u003eRadiation-induced cell survival of BRCA2 wild-type, knockout, pathogenic, and VUS-expressing cells. Data are expressed as mean cell viability (%) relative to WT cells (set to 100%), with error bars representing standard deviations (SD) from triplicate experiments. Statistical significance was determined using two-way ANOVA (**p \u0026lt; 0.01, *p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8617436/v1/4911fb24ad4515f157ed0591.png"},{"id":102553479,"identity":"82b6874a-33b8-4cf3-b0b4-3c0daf1ad157","added_by":"auto","created_at":"2026-02-13 01:14:13","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":36678,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Olaparib on Cell Viability of \u003cem\u003eBRCA2\u003c/em\u003e WT, KO, VUS, and PV Cells. Data represent the mean cell viability (%) relative to untreated cells (set to 100%), with error bars indicating standard deviations (SD) from triplicate experiments. Statistical significance was determined using two-way ANOVA (**p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8617436/v1/0320f363f41f8fec5b31fc9c.png"},{"id":102553482,"identity":"f2020700-dbee-4414-a81e-4173fcb2851f","added_by":"auto","created_at":"2026-02-13 01:14:13","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":138468,"visible":true,"origin":"","legend":"\u003cp\u003eImmunofluorescence detection of homologous recombination activity using the GFP reporter assay (green).\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8617436/v1/9a090e0892019eab9c5fa47d.jpeg"},{"id":103508810,"identity":"98b6878b-bc6d-4411-a2b4-5bf841b5c9a8","added_by":"auto","created_at":"2026-02-26 13:54:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1927130,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8617436/v1/363e4b2e-b3e6-4c85-9166-1b53354e67d5.pdf"},{"id":102746829,"identity":"a7da32ec-3178-40a5-9c7d-bcb0c573295d","added_by":"auto","created_at":"2026-02-16 09:01:52","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1029631,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureBRCA2VUS.docx","url":"https://assets-eu.researchsquare.com/files/rs-8617436/v1/a715633b6f6eee45d80f6064.docx"}],"financialInterests":"","formattedTitle":"Functional characterization of BRCA2 variants of uncertain significance identified in Korean breast cancer patients","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer is the most common malignancy among women worldwide, and hereditary predisposition plays a critical role in disease development and therapeutic decision-making [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Among hereditary breast cancer susceptibility genes, \u003cem\u003eBRCA2\u003c/em\u003e is a central tumor suppressor whose functional integrity is essential for maintaining genomic stability [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Pathogenic variants in \u003cem\u003eBRCA2\u003c/em\u003e are strongly associated with increased lifetime breast cancer risk and have direct implications for cancer surveillance, risk-reducing strategies, and systemic treatment selection [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eBRCA2\u003c/em\u003e protein plays a pivotal role in homologous recombination (HR), a high-fidelity DNA repair pathway responsible for accurately repairing DNA double-strand breaks. Through direct interaction with RAD51, \u003cem\u003eBRCA2\u003c/em\u003e facilitates nucleoprotein filament formation and strand invasion, thereby ensuring error-free DNA repair [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Loss of \u003cem\u003eBRCA2\u003c/em\u003e function results in homologous recombination deficiency (HRD), leading to genomic instability and increased sensitivity to DNA-damaging agents, including platinum-based chemotherapy and poly(ADP-ribose) polymerase (PARP) inhibitors [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Accordingly, identification of pathogenic \u003cem\u003eBRCA2\u003c/em\u003e variants has become an integral component of precision treatment strategies in breast cancer [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWith the widespread introduction of next-generation sequencing, particularly tumor-based genomic profiling, the clinical interpretation of \u003cem\u003eBRCA2\u003c/em\u003e variants has become increasingly complex. Tumor sequencing frequently identifies \u003cem\u003eBRCA\u003c/em\u003e variants with variant allele frequencies close to 50%, a finding that strongly suggests a germline origin. In clinical practice, such results often prompt suspicion of hereditary cancer predisposition, especially when detected in patients with early-onset disease or a suggestive family history [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, despite these features, a substantial proportion of these variants are ultimately classified as variants of uncertain significance (VUS). Unlike truncating variants with well-established clinical consequences, missense \u003cem\u003eBRCA2\u003c/em\u003e variants often lack sufficient evidence for definitive classification as pathogenic or benign [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This creates a clinically challenging gray zone in which variants that appear highly suggestive of germline inheritance based on tumor sequencing data cannot be used to guide genetic counseling, risk-reducing strategies, or treatment decisions [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe burden of this interpretive gap is particularly pronounced in non-Caucasian populations. Current variant classification frameworks, including the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines, rely heavily on population frequency data, previously reported clinical evidence, and reference databases that are largely derived from Caucasian populations [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Consequently, the application of these criteria is limited in Asian populations, where population-specific reference data remain underrepresented [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Multiple studies have reported higher rates of \u003cem\u003eBRCA1/2\u003c/em\u003e variants of uncertain significance in East Asian breast cancer cohorts, including Korean patients, reflecting both population-specific genetic architecture and insufficient representation in global databases [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Because of these limitations, efforts in Korea have increasingly focused on characterizing the distribution and frequency of BRCA1/2 VUS at the population level.\u003c/p\u003e \u003cp\u003eIn South Korea, a nationwide precision oncology initiative, K-MASTER, was conducted to perform large-scale tumor genome profiling in approximately 10,000 cancer patients. This program enabled us to systematically analyze the genome changes of various solid tumors including \u003cem\u003eBRCA\u003c/em\u003e genes. In addition, we observed that a significant portion of \u003cem\u003eBRCA2\u003c/em\u003e mutations identified in the K-MASTER cohort were classified as uncertain significance mutations, and multiple missense mutations were repeated in individuals [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. This makes sense to analyze \u003cem\u003eBRCA\u003c/em\u003e in KMASTER data representing Korean solid cancer NGS results, and we thought it would be important to confirm the function of these VUSs through experiments if necessary.\u003c/p\u003e \u003cp\u003eFunctional assays have emerged as an important tool to address this unmet need. Current variant interpretation guidelines emphasize that well-validated functional studies can provide strong biological evidence by directly assessing the impact of a variant on protein function [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. For \u003cem\u003eBRCA2\u003c/em\u003e, impairment of homologous recombination represents a particularly relevant functional endpoint, as HR deficiency provides a mechanistic link between molecular dysfunction and therapeutic vulnerability [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Assays evaluating cellular sensitivity to DNA-damaging agents, PARP inhibitors, and HR reporter systems therefore offer biologically meaningful insight into the functional consequences of individual \u003cem\u003eBRCA2\u003c/em\u003e variants [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, motivated by observations from tumor-based genomic profiling and the clinical dilemma posed by germline-suspected \u003cem\u003eBRCA2\u003c/em\u003e VUS, we performed a comprehensive functional characterization of three \u003cem\u003eBRCA2\u003c/em\u003e missense variants of uncertain significance identified in Korean breast cancer patients. With the K-MASTER dataset, we evaluated the effects of each variant on the DNA damage response, including chemotherapeutic agent, ionizing radiation, and the PARP inhibitor olaparib, homologous recombination efficiency, and DNA damage factors. With this integrated functional approach, we wanted to clarify the variant-specific functional phenotype and provide complementary biological evidence related to the interpretation of \u003cem\u003eBRCA2\u003c/em\u003e VUS in breast cancer.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design and variant selection\u003c/h2\u003e \u003cp\u003eThis study integrated population-scale tumor genomic data with functional assays to evaluate the biological significance of selected \u003cem\u003eBRCA2\u003c/em\u003e variants of uncertain significance (VUS) identified in Korean cancer patients. Candidate \u003cem\u003eBRCA2\u003c/em\u003e VUS were curated from the K-MASTER project database and from Korean breast cancer patients with strong suspicion of hereditary cancer based on family history.\u003c/p\u003e \u003cp\u003eAmong the \u003cem\u003eBRCA2\u003c/em\u003e VUS identified, three missense variants were selected for functional analysis based on recurrence in the K-MASTER cohort and/or association with suggestive family histories: c.5969A\u0026thinsp;\u0026gt;\u0026thinsp;C (p.Asp1990Ala) located in the BRC7 domain, c.3671G\u0026thinsp;\u0026gt;\u0026thinsp;A (p.Gly1224Asp) located in the BRC2 domain, and c.5459G\u0026thinsp;\u0026gt;\u0026thinsp;A (p.Cys1820Tyr) located in the spacer region between BRC5 and BRC6. These regions are known to play critical roles in RAD51 binding and homologous recombination repair. A known pathogenic \u003cem\u003eBRCA2\u003c/em\u003e variant, c.8167G\u0026thinsp;\u0026gt;\u0026thinsp;C (p.Asp2723His), was included as a pathogenic control, and wild-type \u003cem\u003eBRCA2\u003c/em\u003e served as a functional reference.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eK-MASTER cohort and BRCA variant analysis\u003c/h3\u003e\n\u003cp\u003eThe K-MASTER project is a nationwide precision oncology initiative designed to perform large-scale tumor genomic profiling in Korean patients with advanced solid tumors. Tumor sequencing data from 9,677 patients were successfully analyzed between 2017 and 2021. Tumor specimens were sequenced using targeted gene panels, including the FIRST, CancerSCAN, K-MASTER 1.0, and K-MASTER 1.1 panels. For patients without available tumor tissue or with quality control failure, circulating tumor DNA from blood samples was analyzed using a targeted cancer panel.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e variants detected in the K-MASTER cohort were annotated and classified using ClinVar and the Human Gene Mutation Database. Variants were categorized as pathogenic, likely pathogenic, benign, likely benign, variants of uncertain significance, or variants with conflicting interpretations. The distribution of \u003cem\u003eBRCA2\u003c/em\u003e variants by clinical significance, mutation type, and tumor origin was analyzed to identify recurrent VUS with potential biological relevance.\u003c/p\u003e\n\u003ch3\u003eCell lines and culture conditions\u003c/h3\u003e\n\u003cp\u003eThe human colorectal cancer cell line DLD-1 with homozygous deletion of \u003cem\u003eBRCA2\u003c/em\u003e (\u003cem\u003eBRCA2\u003c/em\u003e knockout) was used as the experimental model. DLD-1 \u003cem\u003eBRCA2\u003c/em\u003e knockout cells were obtained from Horizon Discovery (Cambridge, UK). Cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin\u0026ndash;streptomycin at 37\u0026deg;C in a humidified incubator with 5% CO₂.\u003c/p\u003e \u003cp\u003eWild-type DLD-1 cells were cultured under identical conditions and used as an additional reference where appropriate.\u003c/p\u003e\n\u003ch3\u003ePlasmid constructs and site-directed mutagenesis\u003c/h3\u003e\n\u003cp\u003eFull-length human \u003cem\u003eBRCA2\u003c/em\u003e cDNA constructs were used as templates for mutagenesis. Site-directed mutagenesis was performed to generate constructs harboring the selected VUS and the pathogenic control variant. Mutagenesis was conducted by a commercial service provider (BIOFACT, Daejeon, Korea) to ensure accuracy. All introduced variants were confirmed by Sanger sequencing.\u003c/p\u003e \u003cp\u003eThe resulting constructs included wild-type \u003cem\u003eBRCA2\u003c/em\u003e, one pathogenic variant, and three VUS constructs. These plasmids were used for transient transfection into DLD-1 \u003cem\u003eBRCA2\u003c/em\u003e knockout cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eTransient transfection\u003c/h3\u003e\n\u003cp\u003eDLD-1 \u003cem\u003eBRCA2\u003c/em\u003e knockout cells were seeded at 70\u0026ndash;80% confluence and transfected with plasmids encoding wild-type \u003cem\u003eBRCA2\u003c/em\u003e, pathogenic control, or VUS constructs using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer\u0026rsquo;s instructions. Cells were incubated for 48 hours following transfection before subsequent analyses.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eProtein expression of \u003cem\u003eBRCA2\u003c/em\u003e constructs was confirmed by Western blot analysis. Cells were lysed using PRO-PREP protein extraction solution supplemented with protease and phosphatase inhibitor cocktails. Protein concentrations were quantified using the Bradford assay.\u003c/p\u003e \u003cp\u003eEqual amounts of protein (30 \u0026micro;g) were separated by SDS\u0026ndash;PAGE and transferred onto PVDF membranes. Membranes were blocked and incubated with a primary antibody against \u003cem\u003eBRCA2\u003c/em\u003e (Santa Cruz Biotechnology, sc-518154), followed by incubation with an appropriate secondary antibody. GAPDH was used as a loading control. Protein bands were visualized using enhanced chemiluminescence, and band intensity was assessed to confirm expression of full-length \u003cem\u003eBRCA2\u003c/em\u003e protein.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDNA damage response assays using cisplatin\u003c/h3\u003e\n\u003cp\u003eTo evaluate cellular responses to DNA damage, transfected cells were treated with cisplatin (Sigma-Aldrich). Cells were exposed to increasing concentrations of cisplatin for 24, 48, or 72 hours. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Japan) according to the manufacturer\u0026rsquo;s protocol. Absorbance was measured at 450 nm, and cell viability was calculated relative to untreated controls.\u003c/p\u003e \u003cp\u003eDose\u0026ndash;response curves were generated, and IC50 values were calculated using GraphPad Prism software.\u003c/p\u003e\n\u003ch3\u003eRadiation-induced DNA damage assay\u003c/h3\u003e\n\u003cp\u003eTo assess sensitivity to ionizing radiation, transfected cells were exposed to graded doses of radiation (1, 2, 4, and 8 Gy) using an X-ray irradiator (Xstrahl CIX3 Cabinet X-ray Irradiator). Following irradiation, cells were incubated for 24, 48, or 72 hours. Cell survival was measured using the CCK-8 assay and expressed as a percentage relative to non-irradiated controls.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePARP inhibitor sensitivity assay\u003c/h2\u003e \u003cp\u003eTo evaluate sensitivity to PARP inhibition, transfected cells were treated with olaparib (SelleckChem) at concentrations of 0, 0.1, 0.5, 1, 2, and 5 \u0026micro;M. After 48 hours of treatment, cell viability was assessed using the CCK-8 assay. Viability was calculated relative to untreated controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHomologous recombination repair assay\u003c/h2\u003e \u003cp\u003eHomologous recombination repair efficiency was evaluated using a GFP-based reporter assay with I-SceI\u0026ndash;induced double-strand breaks. DLD-1 \u003cem\u003eBRCA2\u003c/em\u003e knockout cells stably expressing the DR-GFP reporter were co-transfected with plasmids encoding I-SceI endonuclease and either wild-type \u003cem\u003eBRCA2\u003c/em\u003e, pathogenic control, or VUS constructs.\u003c/p\u003e \u003cp\u003eFollowing transfection, cells were incubated to allow DNA repair. GFP-positive cells, indicative of successful HR-mediated repair, were visualized using fluorescence microscopy. HR efficiency was assessed by comparing the relative number of GFP-positive cells across experimental conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were performed in triplicate and repeated independently at least three times. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical analyses were conducted using GraphPad Prism version 10. Comparisons between groups were performed using one-way or two-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post hoc test, as appropriate. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIdentification and distribution of\u003c/b\u003e \u003cb\u003eBRCA2\u003c/b\u003e \u003cb\u003evariants of uncertain significance in the K-MASTER cohort\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTumor genomic sequencing data from 9,677 patients enrolled in the K-MASTER project were analyzed to characterize the distribution of \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e variants. Among these patients, \u003cem\u003eBRCA1\u003c/em\u003e variants were identified in 737 cases (7.6%), and \u003cem\u003eBRCA2\u003c/em\u003e variants were identified in 1,411 cases (14.6%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). When classified according to clinical significance, a substantial proportion of detected variants were categorized as variants of uncertain significance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSpecifically, \u003cem\u003eBRCA2\u003c/em\u003e VUS accounted for 7.04% of the total analyzed cohort. Missense variants represented the predominant mutation type among \u003cem\u003eBRCA2\u003c/em\u003e VUS, whereas pathogenic or likely pathogenic variants were more frequently associated with truncating mutations, including frameshift and nonsense variants (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u0026ndash;2\u003c/b\u003e). This distribution indicated that missense substitutions constitute the major source of uncertainty in \u003cem\u003eBRCA2\u003c/em\u003e variant interpretation within the Korean cancer population (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFrequent \u003cem\u003eBRCA2\u003c/em\u003e missense variants with uncertain or conflicting clinical significance identified in the K-MASTER cohort\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDNA Change\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProtein Change\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVAF (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eType\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eClassification\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.6325G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eV2109I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e41.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMissense\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eConflicting interpretations of pathogenicity\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.4854T\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD1618E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e46.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMissense\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUncertain significance\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.964A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK322Q\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e45.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMissense\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eConflicting interpretations of pathogenicity\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.5969A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD1990A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e46.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMissense\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eConflicting interpretations of pathogenicity\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.623T\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eV208G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e43.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMissense\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUncertain significance\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.10131A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eE3377D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e52.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMissense\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUncertain significance\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.7706G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eG2569D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e42.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMissense\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUncertain significance\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.6496G\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eV2166L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMissense\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUncertain significance\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.3007C\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eH1003D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMissense\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUncertain significance\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAnalysis of tumor types revealed that \u003cem\u003eBRCA2\u003c/em\u003e VUS were most frequently detected in breast cancer, followed by colorectal, gastric, lung, head and neck, and ovarian cancers (\u003cb\u003eSupplementary Fig.\u0026nbsp;3\u003c/b\u003e). Compared with the overall tumor distribution of the K-MASTER cohort, breast and ovarian cancers were relatively enriched among cases harboring \u003cem\u003eBRCA2\u003c/em\u003e VUS.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSelection of\u003c/b\u003e \u003cb\u003eBRCA2\u003c/b\u003e \u003cb\u003eVUS for functional analysis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo prioritize candidates for functional evaluation, \u003cem\u003eBRCA2\u003c/em\u003e VUS were ranked based on frequency within the K-MASTER cohort and association with clinical features suggestive of hereditary cancer, including family history. Among recurrent variants, c.5969A\u0026thinsp;\u0026gt;\u0026thinsp;C (p.Asp1990Ala) was identified in 28 patients and exhibited a variant allele frequency close to 50%, suggesting a possible germline origin (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral more frequently detected variants were excluded from further analysis because existing functional or clinical evidence supported a benign classification. Consequently, p.Asp1990Ala was selected as a representative recurrent \u003cem\u003eBRCA2\u003c/em\u003e VUS. In addition, two variants, c.3671G\u0026thinsp;\u0026gt;\u0026thinsp;A (p.Gly1224Asp) and c.5459G\u0026thinsp;\u0026gt;\u0026thinsp;A (p.Cys1820Tyr), were selected based on their presence in patients with strong family histories of breast or related cancers (\u003cb\u003eSupplementary Fig.\u0026nbsp;4\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eAll three selected variants were located within or adjacent to BRC repeat regions implicated in RAD51 binding and homologous recombination repair (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eConservation and structural analysis of selected\u003c/b\u003e \u003cb\u003eBRCA2\u003c/b\u003e \u003cb\u003eVUS\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMultiple sequence alignment of \u003cem\u003eBRCA2\u003c/em\u003e orthologs from six species demonstrated that the amino acid residues corresponding to p.Asp1990Ala, p.Gly1224Asp, and p.Cys1820Tyr were evolutionarily conserved (\u003cb\u003eSupplementary Fig.\u0026nbsp;5\u003c/b\u003e). Conservation across species suggested potential functional relevance of these residues.\u003c/p\u003e \u003cp\u003eThree-dimensional structural modeling of the BRC repeats aligned to the RAD51\u0026ndash;BRCA2 complex structure revealed distinct conformational features among the variants. Structural models predicted abnormal residue protrusion and altered local conformation for p.Asp1990Ala and p.Gly1224Asp, whereas p.Cys1820Tyr showed relatively preserved structural features (\u003cb\u003eSupplementary Fig.\u0026nbsp;6\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression of\u003c/b\u003e \u003cb\u003eBRCA2\u003c/b\u003e \u003cb\u003ewild-type, pathogenic, and VUS constructs in\u003c/b\u003e \u003cb\u003eBRCA2\u003c/b\u003e\u003cb\u003e-deficient cells\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo assess the functional impact of each variant, full-length \u003cem\u003eBRCA2\u003c/em\u003e constructs encoding wild-type protein, a known pathogenic variant, or each VUS were transiently transfected into DLD-1 \u003cem\u003eBRCA2\u003c/em\u003e-knockout cells. Western blot analysis confirmed the expression of full-length \u003cem\u003eBRCA2\u003c/em\u003e protein in cells transfected with wild-type, pathogenic control, and all three VUS constructs. No \u003cem\u003eBRCA2\u003c/em\u003e signal was detected in untransfected \u003cem\u003eBRCA2\u003c/em\u003e-knockout cells (\u003cb\u003eSupplementary Fig.\u0026nbsp;7)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eThe expression levels of \u003cem\u003eBRCA2\u003c/em\u003e were comparable across wild-type, pathogenic, and VUS constructs, indicating that the observed functional differences were unlikely to be attributable to gross differences in protein expression or stability.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDifferential cellular response to cisplatin-induced DNA damage\u003c/h2\u003e \u003cp\u003eTo evaluate the role of each \u003cem\u003eBRCA2\u003c/em\u003e variant in DNA damage response, transfected cells were treated with increasing concentrations of cisplatin, and cell viability was assessed at multiple time points.\u003c/p\u003e \u003cp\u003eWild-type \u003cem\u003eBRCA2\u003c/em\u003e-expressing cells exhibited a gradual, dose-dependent reduction in cell viability following cisplatin exposure, consistent with intact homologous recombination repair capacity. In contrast, \u003cem\u003eBRCA2\u003c/em\u003e-knockout cells showed marked sensitivity to cisplatin, with significantly reduced cell viability at lower drug concentrations (\u003cb\u003eSupplementary Fig.\u0026nbsp;8)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eCells expressing the pathogenic control variant demonstrated cisplatin sensitivity similar to \u003cem\u003eBRCA2\u003c/em\u003e-knockout cells. Notably, cells expressing \u003cem\u003eBRCA2\u003c/em\u003e p.Asp1990Ala also exhibited increased sensitivity to cisplatin, closely resembling the response observed in pathogenic control and knockout cells. The calculated IC50 values for p.Asp1990Ala were substantially lower than those for wild-type \u003cem\u003eBRCA2\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn contrast, cells expressing \u003cem\u003eBRCA2\u003c/em\u003e p.Gly1224Asp or p.Cys1820Tyr maintained higher cell viability across the tested cisplatin concentrations, with dose\u0026ndash;response curves comparable to wild-type \u003cem\u003eBRCA2\u003c/em\u003e-expressing cells. These findings indicate differential functional consequences among the three VUS with respect to cisplatin-induced DNA damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIncreased sensitivity of\u003c/b\u003e \u003cb\u003eBRCA2\u003c/b\u003e \u003cb\u003ep.Asp1990Ala to ionizing radiation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further assess DNA repair capacity, transfected cells were exposed to ionizing radiation, and cell survival was measured at 24, 48, and 72 hours following irradiation.\u003c/p\u003e \u003cp\u003eWild-type \u003cem\u003eBRCA2\u003c/em\u003e-expressing cells demonstrated relative resistance to low-dose radiation, with a progressive decline in survival at higher doses and later time points. In contrast, \u003cem\u003eBRCA2\u003c/em\u003e-knockout cells exhibited pronounced radiation sensitivity, with significantly reduced survival even at moderate radiation doses (\u003cb\u003eSupplementary Fig.\u0026nbsp;9)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eCells expressing the pathogenic control variant showed survival profiles similar to knockout cells. Importantly, cells expressing \u003cem\u003eBRCA2\u003c/em\u003e p.Asp1990Ala also displayed reduced survival following radiation exposure, closely paralleling the pathogenic control. At both 48 and 72 hours after irradiation, survival rates in p.Asp1990Ala-expressing cells were significantly lower than those observed in wild-type cells.\u003c/p\u003e \u003cp\u003eDue to their wild-type\u0026ndash;like behavior in cisplatin assays, p.Gly1224Asp- and p.Cys1820Tyr-expressing cells were not further evaluated in radiation assays. The radiation sensitivity observed in p.Asp1990Ala-expressing cells supports a functional defect in DNA damage repair pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEnhanced sensitivity of\u003c/b\u003e \u003cb\u003eBRCA2\u003c/b\u003e \u003cb\u003ep.Asp1990Ala to PARP inhibition\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGiven the established association between homologous recombination deficiency and PARP inhibitor sensitivity, the response of transfected cells to the PARP inhibitor olaparib was assessed.\u003c/p\u003e \u003cp\u003eWild-type \u003cem\u003eBRCA2\u003c/em\u003e-expressing cells demonstrated relative resistance to olaparib across a range of concentrations. In contrast, \u003cem\u003eBRCA2\u003c/em\u003e-knockout cells exhibited significantly reduced cell viability, consistent with HR deficiency.\u003c/p\u003e \u003cp\u003eCells expressing the pathogenic control variant showed olaparib sensitivity comparable to knockout cells. Similarly, cells expressing \u003cem\u003eBRCA2\u003c/em\u003e p.Asp1990Ala demonstrated increased sensitivity to olaparib, with IC50 values closer to those observed in pathogenic and knockout cells than to wild-type controls.\u003c/p\u003e \u003cp\u003eThese findings indicate that \u003cem\u003eBRCA2\u003c/em\u003e p.Asp1990Ala compromises cellular tolerance to PARP inhibition, consistent with impaired homologous recombination repair. In contrast, p.Gly1224Asp and p.Cys1820Tyr were not associated with increased olaparib sensitivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eImpaired homologous recombination repair in\u003c/b\u003e \u003cb\u003eBRCA2\u003c/b\u003e \u003cb\u003ep.Asp1990Ala assessed by GFP reporter assay\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo directly evaluate homologous recombination efficiency, a GFP-based reporter assay was performed using I-SceI\u0026ndash;induced double-strand breaks.\u003c/p\u003e \u003cp\u003eCells expressing wild-type \u003cem\u003eBRCA2\u003c/em\u003e showed robust GFP signal, indicating efficient HR-mediated repair. In contrast, \u003cem\u003eBRCA2\u003c/em\u003e-knockout cells exhibited minimal GFP positivity, reflecting defective HR.\u003c/p\u003e \u003cp\u003eCells expressing the pathogenic control variant demonstrated markedly reduced GFP signal. Notably, cells expressing \u003cem\u003eBRCA2\u003c/em\u003e p.Asp1990Ala also showed a significant reduction in GFP-positive cells, comparable to the pathogenic control. This reduction indicates impaired HR repair capacity.\u003c/p\u003e \u003cp\u003eIn contrast, GFP signal intensity in cells expressing p.Gly1224Asp or p.Cys1820Tyr was comparable to wild-type \u003cem\u003eBRCA2\u003c/em\u003e, suggesting preserved HR function in these variants (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study was initiated from a clinically relevant dilemma increasingly encountered in the era of tumor-based next-generation sequencing (NGS): the identification of \u003cem\u003eBRCA2\u003c/em\u003e variants that strongly suggest germline origin yet remain classified as variants of uncertain significance (VUS). Large-scale genomic screening efforts in Korea have demonstrated that a substantial proportion of \u003cem\u003eBRCA2\u003c/em\u003e variants detected through tumor sequencing are designated as VUS, often occurring at variant allele frequencies close to 50%, a pattern highly suggestive of germline inheritance [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Such findings frequently raise concerns regarding hereditary breast cancer; however, conventional classification frameworks provide limited guidance in these situations.\u003c/p\u003e \u003cp\u003eInterpretation of \u003cem\u003eBRCA1/2\u003c/em\u003e VUS remains challenging under the current American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines, which rely on a multifactorial framework integrating population frequency, clinical data, segregation analysis, and functional evidence [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Because reference databases and population frequency data are largely derived from Caucasian populations, many variants identified in Asian cohorts fail to meet criteria for either pathogenic or benign classification and therefore remain designated as VUS [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This limitation has been consistently reported in Korean and other East Asian studies, underscoring the need for additional lines of evidence to contextualize variants encountered in real-world clinical practice.\u003c/p\u003e \u003cp\u003eBased on this background, the present study focused on the functional characterization of selected \u003cem\u003eBRCA2\u003c/em\u003e missense VUS recurrently identified in the Korean cancer population and associated with clinical features suggestive of hereditary breast cancer. Given the essential role of the \u003cem\u003eBRCA2\u003c/em\u003e\u0026ndash;RAD51 interaction in homologous recombination repair, variants located within or adjacent to BRC repeat regions were prioritized for experimental evaluation. Functional assessment was performed using a \u003cem\u003eBRCA2\u003c/em\u003e-deficient cellular model to directly evaluate DNA damage response, homologous recombination efficiency, and sensitivity to DNA-damaging agents and PARP inhibition.\u003c/p\u003e \u003cp\u003eAmong the three variants analyzed, \u003cem\u003eBRCA2\u003c/em\u003e p.Asp1990Ala consistently demonstrated impaired DNA repair function across multiple assays. Cells expressing this variant showed increased sensitivity to cisplatin and ionizing radiation, as well as heightened susceptibility to the PARP inhibitor olaparib, accompanied by reduced homologous recombination activity in a GFP-based reporter assay. The concordance of these findings across independent experimental platforms supports the biological relevance of this variant and suggests compromised \u003cem\u003eBRCA2\u003c/em\u003e-mediated DNA repair under genotoxic stress. Similar observations have been reported in prior functional studies demonstrating that a subset of BRCA VUS exhibit homologous recombination defects comparable to pathogenic variants [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn contrast, \u003cem\u003eBRCA2\u003c/em\u003e p.Gly1224Asp and p.Cys1820Tyr did not exhibit functional impairment in the experimental systems used. Despite their localization within regions implicated in RAD51 interaction and their identification in patients with suggestive family histories, these variants retained DNA repair capacity comparable to wild-type \u003cem\u003eBRCA2\u003c/em\u003e. This finding highlights the marked functional heterogeneity among \u003cem\u003eBRCA2\u003c/em\u003e missense variants and illustrates the limitations of predicting biological impact based solely on sequence conservation, domain location, or clinical suspicion. Such heterogeneity has been consistently described in previous functional complementation studies of \u003cem\u003eBRCA\u003c/em\u003e missense variants [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe potential clinical relevance of functional heterogeneity among \u003cem\u003eBRCA\u003c/em\u003e VUS has been increasingly recognized. Emerging clinical data suggest that some patients harboring \u003cem\u003eBRCA\u003c/em\u003e VUS may demonstrate treatment responses closer to those observed in carriers of pathogenic variants than in patients with benign variants, particularly in the context of PARP inhibitor therapy [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. While current clinical guidelines do not support therapeutic decision-making based on VUS status alone, functional data such as those presented here may provide important biological context for future integrative studies linking genotype, functional phenotype, and clinical outcome.\u003c/p\u003e \u003cp\u003eSeveral limitations of this study should be acknowledged. First, all functional analyses were conducted in vitro using a single \u003cem\u003eBRCA2\u003c/em\u003e-knockout cell line, which may not fully recapitulate the complexity of tumor biology or tissue-specific contexts. Second, transient transfection was used to express \u003cem\u003eBRCA2\u003c/em\u003e constructs, potentially introducing variability in expression levels. Third, segregation analysis and longitudinal clinical outcome data were not incorporated, limiting direct correlation between functional findings and patient-level phenotypes. These limitations are consistent with those reported in prior functional studies of \u003cem\u003eBRCA\u003c/em\u003e VUS and highlight the importance of integrative approaches combining functional assays with genetic and clinical data [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn conclusion, this study provides important insights into the functional impact of specific \u003cem\u003eBRCA2\u003c/em\u003e VUS, particularly within the Korean population. The results demonstrate that \u003cem\u003eBRCA2\u003c/em\u003e D1990A exhibits impaired DNA repair capabilities, aligning it closer to a pathogenic variant, whereas \u003cem\u003eBRCA2\u003c/em\u003e G1224D and \u003cem\u003eBRCA2\u003c/em\u003e C1820Y maintain DNA repair functionality similar to the wild-type \u003cem\u003eBRCA2\u003c/em\u003e. These findings support the importance of experimental validation for \u003cem\u003eBRCA2\u003c/em\u003e VUS to aid in reclassifying VUS. Further research is needed to confirm these findings in clinical settings and explore other VUS, ultimately enhancing personalized risk assessment and patient care.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Institutional Review Board of Korea University Anam Hospital (2017AN0401) and was conducted in accordance with the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable. The manuscript does not contain individual, identifiable patient data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contribution statements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, Ah Reum Lim, Soohyeon Lee; Methodology, Ah Reum Lim, Kyoungmi Kim; Formal analysis, Ah Reum Lim; Investigation, Ah Reum Lim, Seung Pil Jung, Hye Jin Choi; Data curation, Ah Reum Lim, Kyong Hwa Park; Visualization, Ah Reum Lim;\u003c/p\u003e\n\u003cp\u003eResources, Seung Pil Jung, Hye Jin Choi; Supervision, Soohyeon Lee, Kyong Hwa Park;\u003c/p\u003e\n\u003cp\u003eWriting—original draft, Ah Reum Lim; Writing—review and editing, Soohyeon Lee, Kyong Hwa Park, Kyoungmi Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. 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Assessing the pathogenicity of BRCA1/2 variants of unknown significance: Relevance and challenges for breast cancer precision medicine. Front Oncol. 2022;12:1053035. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fonc.2022.1053035\u003c/span\u003e\u003cspan address=\"10.3389/fonc.2022.1053035\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"breast-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"brca","sideBox":"Learn more about [Breast Cancer](http://link.springer.com/journal/12282)","snPcode":"12282","submissionUrl":"https://www.editorialmanager.com/brca/default2.aspx","title":"Breast Cancer","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"BRCA2, Breast cancer, Variants of uncertain significance, Homologous recombination, Functional assay","lastPublishedDoi":"10.21203/rs.3.rs-8617436/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8617436/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eVariants of uncertain significance (VUS) in \u003cem\u003eBRCA2\u003c/em\u003e remain a major challenge in the clinical management of breast cancer, particularly in Asian populations where population-specific reference data are limited. Functional assays may provide complementary biological evidence to support interpretation of these variants.\u003c/p\u003e\u003ch2\u003ePatients and methods:\u003c/h2\u003e \u003cp\u003eThree \u003cem\u003eBRCA2\u003c/em\u003e missense VUS\u0026mdash;c.5969A\u0026thinsp;\u0026gt;\u0026thinsp;C (p.Asp1990Ala), c.3671G\u0026thinsp;\u0026gt;\u0026thinsp;A (p.Gly1224Asp), and c.5459G\u0026thinsp;\u0026gt;\u0026thinsp;A (p.Cys1820Tyr)\u0026mdash;were selected based on their frequency in Korean cohorts and/or strong family history of breast cancer. Full-length \u003cem\u003eBRCA2\u003c/em\u003e constructs harboring each variant were introduced into \u003cem\u003eBRCA2\u003c/em\u003e-knockout DLD-1 cells. Functional consequences were evaluated using cellular sensitivity assays following cisplatin treatment and ionizing radiation, response to the PARP inhibitor olaparib, and homologous recombination (HR) efficiency assessed by a GFP-based reporter system.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCells expressing \u003cem\u003eBRCA2\u003c/em\u003e p.Asp1990Ala consistently demonstrated increased sensitivity to cisplatin, ionizing radiation, and olaparib, comparable to pathogenic controls. In addition, GFP-based HR assays revealed a marked reduction in HR repair efficiency in p.Asp1990Ala-expressing cells. In contrast, cells expressing \u003cem\u003eBRCA2\u003c/em\u003e p.Gly1224Asp or p.Cys1820Tyr exhibited DNA damage responses, drug sensitivity profiles, and HR activity similar to wild-type \u003cem\u003eBRCA2\u003c/em\u003e across all assays.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eAmong the three \u003cem\u003eBRCA2\u003c/em\u003e VUS analyzed, p.Asp1990Ala exhibited a pathogenic-like functional phenotype characterized by impaired homologous recombination repair, whereas p.Gly1224Asp and p.Cys1820Tyr appeared functionally neutral. These findings highlight the functional heterogeneity of \u003cem\u003eBRCA2\u003c/em\u003e VUS and support the role of functional assays as complementary tools for variant interpretation in breast cancer.\u003c/p\u003e","manuscriptTitle":"Functional characterization of BRCA2 variants of uncertain significance identified in Korean breast cancer patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-13 01:14:08","doi":"10.21203/rs.3.rs-8617436/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2026-04-20T22:02:12+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2026-03-02T23:26:16+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-09T00:08:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-16T12:57:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Breast Cancer","date":"2026-01-16T04:32:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"breast-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"brca","sideBox":"Learn more about [Breast Cancer](http://link.springer.com/journal/12282)","snPcode":"12282","submissionUrl":"https://www.editorialmanager.com/brca/default2.aspx","title":"Breast Cancer","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"632edc6d-f6fd-42ab-9c04-e95b6b8999cb","owner":[],"postedDate":"February 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T05:41:44+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-13 01:14:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8617436","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8617436","identity":"rs-8617436","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}