European Journal of Gynaecological Oncology,2025,46(10):21-30 DOI:10.22514/ejgo.2025.128
Original Research
PRDM6 promoter methylation as a potential epigenetic biomarker in BRCA-associated ovarian cancer
Numan Comert1, Ozge Sukruoglu Erdogan2, Betul Celik Demirbas2, Seda Kilic Erciyas2, Ahmet Dinc1, Özge Pasin3, Hülya Yazıcı4, Seref Bugra Tuncer2,*,

1Institute of Graduate Studies in Health Sciences, Istanbul University, 34126 Istanbul, Türkiye

2Department of Cancer Genetics, Oncology Institute, Istanbul University, 34093 Istanbul, Türkiye

3Department of Biostatistics and Medical Informatics, Hamidiye Medical Faculty, Health Sciences University, 34668 Istanbul, Türkiye

4Department of Medical Biology, Faculty of Medicine, Istanbul Health and Technology University, 34275 Istanbul, Türkiye

*Corresponding Author(s):seref.tuncer@istanbul.edu.tr (Seref Bugra Tuncer)

History Submitted: 05 June 2025 | Accepted: 21 July 2025 | Published: 15 October 2025
Copyright:  ©2025  The Author(s). Published by MRE Press.
This is an open access article under the CC BY 4.0 license (https://creativecommons.org/licenses/by/4.0/).

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Abstract

Background: Ovarian cancer (OC) remains one of the most lethal gynecologic malignancies, primarily due to late-stage diagnosis. Aberrant DNA methylation is critical in tumorigenesis and represents a promising avenue for biomarker development. We hypothesized that PR/SET domain 6 (PRDM6) methylation is associated with breast cancer gene (BRCA) mutation status in OC. Methods: Peripheral blood samples were collected from 387 patients with high-grade serous ovarian cancer, 50 individuals with benign ovarian conditions, and 100 healthy controls. DNA methylation was evaluated using methylation-sensitive restriction enzymes (MSREs) and subsequently analyzed by real-time polymerase chain reaction (PCR). Descriptive statistics were employed to summarize categorical and continuous variables. Associations between PRDM6 methylation and clinical parameters, including BRCA mutation status, cancer antigen 125 (CA-125) levels, and age, were statistically analyzed. Results: PRDM6 methylation was detected in 53.9% of OC patients, 60.0% of individuals with benign ovarian disease, and 37.0% of healthy controls. The methylation frequency in OC patients was significantly higher than in healthy controls (p = 0.005). PRDM6 promoter methylation was detected in 64.8% of BRCA-mutated OC patients compared to 50.3% of BRCA-negative patients, indicating a significant association between BRCA mutation status and PRDM6 methylation (p = 0.016). No significant associations were found between PRDM6 methylation and age, menopausal status, or CA-125 levels. Conclusions: PRDM6 methylation may serve as a non-invasive biomarker for early detection in high-risk populations, particularly in BRCA mutation carriers.

Keywords:Ovarian cancer;DNA methylation;Epigenetic biomarker;PRDM6 methylation;BRCA mutation
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Cite this article

Numan Comert, Ozge Sukruoglu Erdogan, Betul Celik Demirbas, Seda Kilic Erciyas, Ahmet Dinc, Özge Pasin, et al.PRDM6 promoter methylation as a potential epigenetic biomarker in BRCA-associated ovarian cancer.European Journal of Gynaecological Oncology,2025,46(10):21-30 DOI:10.22514/ejgo.2025.128

1. Introduction

Ovarian cancer (OC) is the seventh most common cancer among women in Türkiye, with an estimated 3855 new cases diagnosed annually, representing approximately 2.6% of all female cancers, according to the Global Cancer Observatory (GLOBOCAN) 2022 data [1]. Globally and nationally, cancer remains a significant public health concern. To improve survival rates, there is an urgent need to develop new strategies focused on early detection and diagnosis [2]. OC arises from the uncontrolled proliferation of cells originating from the ovarian tissue [3].

Extensive research has revealed a variety of genetic and epigenetic alterations in both oncogenes and tumor suppressor genes in OC. In addition, loss of heterozygosity (LOH) has been observed in several chromosomal regions implicated in ovarian tumorigenesis [4]. Similar to other malignancies, the initiation and progression of OC are driven not only by somatic genetic mutations but also by epigenetic mechanisms, including DNA methylation, histone modification, and regulation of non-coding RNAs. These epigenetic processes contribute to dysregulated gene expression without altering the underlying DNA sequence [5, 6].

While earlier studies provided limited insight into the role of DNA methylation in OC, recent advances have highlighted the importance of epigenetic regulation in ovarian carcinogenesis. Promoter hypermethylation of tumor suppressor genes such as BRCA1, Homeobox A9 (HOXA9), Ras association domain family member 1 (RASSF1A), Secreted protein acidic and rich in cysteine (SPARC), and Hypermethylated In Cancer 1 (HIC1) has been implicated in OC development [7, 8]. Furthermore, the inactivation of DNA damage response (DDR) genes through promoter methylation has been linked to defective DNA repair mechanisms and increased genomic instability in OC. Notably, hypermethylation-induced silencing of key DDR genes has also been correlated with unfavorable clinical outcomes, thus underscoring the potential prognostic value of epigenetic dysregulation [5]. Our previous research, involving monozygotic twins—one healthy and one diagnosed with OC—identified promoter hypermethylation of the PRDM6 gene exclusively in the affected twin, suggesting a possible role for PRDM6 in ovarian tumorigenesis [9].

PRDM6 belongs to the protein (PR) domain-containing (PRDM) gene family, which is known for its roles in transcriptional regulation and chromatin remodeling. PRDM6 encodes a histone methyltransferase that modulates gene expression through epigenetic modifications [10]. Its oncogenic potential has been demonstrated in various malignancies. For instance, Schmidt et al. [11] reported that PRDM6 localizes to the nuclei of neural epithelial stem cells and contributes to medulloblastoma by repressing chromatin accessibility and altering gene expression patterns. Although this finding was specific to brain tumors, it highlights the broader oncogenic potential of PRDM6 through epigenetic pathways, suggesting a similar role in the pathogenesis of other cancers, including OC. Given the critical role of BRCA genes in DNA repair and PRDM6’s potential role in epigenetic instability, we hypothesized that alterations in PRDM6 methylation might be particularly relevant in the context of BRCA-associated ovarian carcinogenesis. The role of PRDM6 in OC, particularly in high-grade serous ovarian cancer (HGSOC), remains inadequately characterized. However, existing studies provide insights into its potential involvement in cancer biology through its functions in cell cycle regulation and the epithelial-to-mesenchymal transition (EMT) [10, 12, 13]. While the connection between PRDM6 and OC remains largely unexplored, the existing body of literature emphasizes a need for more focused investigations. The exploration of PRDM6 in the context of OC holds promise, particularly in understanding the dynamic regulatory networks that govern tumor biology and patient outcomes.

In our previous research, we conducted comprehensive genomic profiling, including healthy and affected individuals with serous OC within a family. Building on the preliminary findings, the present study investigates the methylation status of PRDM6 in peripheral blood samples from 387 patients with serous OC, 50 individuals with benign ovarian conditions, and 100 healthy controls. We aimed to investigate whether PRDM6 methylation is associated with BRCA mutations, with a particular focus on its potential role in prognosis and risk stratification [9, 14] and whether it could serve as a diagnostic biomarker.

2. Materials and methods

2.1 Study cohort

This study included a total of 537 female participants, comprising 387 patients diagnosed with high-grade serous ovarian carcinoma (HGSOC), 50 individuals with benign ovarian diseases, and 100 healthy controls. All ovarian cancer cases included in this study were histopathologically confirmed as HGSOC by board-certified gynecologic pathologists, under the diagnostic criteria established by the World Health Organization (WHO). To ensure diagnostic precision and subtype verification, histological assessment was supported by immunohistochemical staining for key markers, including p53, Antigen Kiel 67 (Ki-67), Wilms Tumor 1 (WT-1), and p16. These markers were evaluated to distinguish HGSOC from other histological subtypes and to validate the inclusion of cases in the molecular analyses. No borderline ovarian tumors were identified or included in the cohort. Somatic BRCA testing was not performed, as the study focused on germline mutations in patients selected according to the National Comprehensive Cancer Network (NCCN) criteria for hereditary breast and ovarian cancer (HBOC) risk.

Peripheral blood samples were collected from all participants at the Istanbul University Oncology Institute between 2021 and 2023. All individuals provided written informed consent before inclusion in the study. The study was approved by the Ethics Committee of the Istanbul Faculty of Medicine (Ethics Committee Approval: Meeting No. 2021/1122; No. 2019/1161).

2.2 DNA extraction and methylation analysis

Genomic DNA was extracted from peripheral blood lymphocytes using the Quick-DNA™ Miniprep Plus Kit (D4069, Zymo Research, Irvine, CA, USA), following the manufacturer’s protocol. The combination of Methylation-Sensitive Restriction Enzyme (MSRE) and quantitative PCR (qPCR) is frequently preferred over bisulfite sequencing due to its practical advantages, including cost-efficiency, operational simplicity, and high sensitivity. Notably, MSRE + qPCR enables accurate quantification of DNA methylation from minimal input material, making it particularly well-suited for clinical and research applications where sample availability is limited [15]. DNA methylation analysis of the PRDM6 gene was performed using the OneStep qMethyl™ Kit (D5310, Zymo Research, Irvine, CA, USA), which enables the direct quantification of DNA methylation without the need for bisulfite conversion. Real-time PCR amplification was performed using an Applied Biosystems Real-Time PCR System, with SYTO 9 fluorescent dye and gene-specific primers targeting the PRDM6 promoter region. The methylation status was quantified by comparing threshold cycle (Ct) values between methylation-sensitive and control reactions. The sequences of the primers used for PRDM6 methylation analysis were as follows:

• Forward primer (PRDM6METF1): 5′-GGA GTT GGT GCC TTC TCT AAC-3′

• Reverse primer (PRDM6METR1): 5′-CTC GAC TGC CTC CCA AAC-3′

All reactions were performed in duplicate, and negative controls were included to ensure the absence of contamination. BRCA1/2 mutation analysis was conducted at the Istanbul University Cancer Genetics Laboratory using a targeted next-generation sequencing (NGS) approach on the Illumina MiSeq® platform (Benchtop Sequencer, Illumina, San Diego, CA, USA) in combination with SOPHiA DDM® software (version7.5.1, SOPHiA GENETICS, Saint-Sulpice, Switzerland) for variant calling and interpretation. Genomic DNA was isolated from peripheral blood samples using the QIAamp DNA Micro Kit (56304, Qiagen, Hilden, NRW, Germany) or GeneAll® Exgene™ Kit (1051013, GeneAll Biotechnology, Siheung, South Korea), and DNA quality was assessed by NanoDrop spectrophotometry. Library preparation was performed using the BRCA MASTR Plus Dx kit (8031360, SOPHiA GENETICS, Saint-Sulpice, VD, Switzerland), following an amplicon-based enrichment strategy. The libraries were purified using AMPure XP beads and quantified prior to sequencing. For quality control, each run was spiked with 6% PhiX control. Sequencing results were analyzed using the human genome reference GRCh37/hg19. Variants were classified according to the American College of Medical Genetics and Genomics (ACMG) guidelines, using population and functional databases including ClinVar, single-nucleotide polymorphism database (dbSNP), genome aggregation database (GnomAD), and human gene mutation database (HGMD). Pathogenic and likely pathogenic variants were confirmed by Sanger sequencing, and large genomic rearrangements were assessed using Multiplex Ligation-dependent Probe Amplification (MLPA; MRC-Holland, Amsterdam, Netherlands).

Samples exhibiting methylation levels ≤6% were classified as unmethylated, while those with values exceeding 6% were considered methylated. The 6% cutoff was selected based on the manufacturer’s specifications provided in the OneStep qMethyl™ Kit manual (D5310, Zymo Research Corp., Irvine, CA, USA), which recommends this threshold as a standard reference for methylation quantification. All measurements and classifications in the current study were performed in accordance with this validated guideline [16, 17, 18].

2.3 Statistical analysis

Statistical analyses were conducted using IBM SPSS Statistics version 26.0 (IBM Corp., Armonk, NY, USA). Descriptive statistics were used to summarize demographic and clinical characteristics. Comparisons between groups were made using Pearson’s Chi-square test, Fisher’s Exact test, and the Kruskal-Wallis test, as appropriate. A p-value < 0.05 was considered statistically significant.

3. Results

3.1 Descriptive statistics and cohort characteristics

Descriptive statistics were reported as counts and percentages for categorical variables (e.g., BRCA mutation status, diagnosis, clinical stage, histological grade, histological subtype, ethnicity, and menopausal status) and as means, standard deviations, medians, minimum and maximum values for continuous variables (e.g., age and CA-125 levels before and after treatment).

A total of 387 serous OC patients, 50 individuals with benign ovarian disease, and 100 healthy controls were included in the final analysis. The number of individuals included in each analysis may vary slightly due to missing data. Among individuals with benign ovarian disease, the histological subtypes were as follows: 3 hemorrhagic cysts, 22 simple anechoic cysts, 19 endometriomas, 3 cases with both endometriomas and simple cysts, one teratoma, and one dermoid cyst. Ethnically, the cohort was predominantly Turkish (68.5%), followed by individuals of Balkan (18.6%) and Eastern Anatolian (other ethnicities 9.0%) origin.

3.2 Patient demographics

The majority of patients were over 45 years old (67.2%), with a predominant late-stage diagnosis (Stage 3: 52.7%, Stage 4: 13.4%). Among OC patients, 23.8% (n = 92) were found to carry a BRCA mutation (either BRCA1 or BRCA2), while 76.2% (n = 295) were BRCA mutation-negative. All individuals in the benign ovarian disease group and the healthy control group were BRCA mutation-negative. The cohort included individuals from Turkish (68.5%), Balkan (18.6%), and Eastern Anatolian Regions (9.0%), with other ethnicities.

3.3 PRDM6 methylation analysis

PRDM6 methylation status was assessed in peripheral blood samples from all three study groups. A methylation threshold of 6% was applied; samples with methylation levels ≤6% were considered unmethylated, while those >6% were categorized as methylated. Consistent with Feng et al. [19] (2021), who demonstrated that methylation thresholds of 5–8% have clinical relevance in HGSOC, our 6% cutoff aligns with established epigenetic biomarker standards. The methylation frequency of the PRDM6 gene was: 52.0% in OC patients (n = 375, the total number of OC patients was 387; however, 12 individuals were excluded from the study due to unavailable expression data), 60.0% in the individuals with benign ovarian disease group (n = 50), and 37.0% in healthy controls (n = 100) (Fig. 1). A significant difference in PRDM6 promoter methylation was observed across the study groups (p = 0.005). While 37.0% of healthy controls exhibited PRDM6 methylation, the frequency was markedly higher among individuals with benign ovarian disease (60.0%) and OC patients (53.9%) (Table 1). These findings suggest that PRDM6 methylation may be associated with ovarian pathology in general, rather than being specific to malignant transformation.

Methylation distribution of the PRDM6 gene among study groups. PRDM6: PR/SET domain 6.

Fig. 1.Methylation distribution of the PRDM6 gene among study groups. PRDM6: PR/SET domain 6.

Table 1.Comparison of PRDM6 methylation status across study groups.
Healthy controlsIndividuals with benign ovarian diseaseOvarian cancerp-value
PRDM6 methylation
Unmethylated63 (63.0%)20 (40.0%)173 (46.1%)0.005
Methylated37 (37.0%)30 (60.0%)202 (53.9%)

PRDM6: PR/SET domain 6.

3.4 Association between BRCA status and PRDM6 methylation

Among OC patients with BRCA1/2 mutations (n = 91), 64.8% (59/91) exhibited PRDM6 promoter methylation, whereas 35.2% (32/91) had unmethylated PRDM6 status. This indicates that PRDM6 methylation is significantly enriched in BRCA mutation carriers, suggesting a potential link between inherited BRCA alterations and epigenetic changes in the PRDM6 gene. The statistically significant p-value (p = 0.016) (Table 2) supports the hypothesis that BRCA-associated ovarian tumors may follow distinct epigenetic regulatory pathways compared to BRCA-negative cases, potentially influencing tumor biology or therapeutic responses.

Table 2.Relationship between BRCA mutation status and PRDM6 methylation in ovarian cancer patients.
MethylatedUnmethylatedTotalp-value
BRCA status
BRCA+59 (64.8%)32 (35.2%)910.016
BRCA−143 (50.3%)141 (49.7%)284

BRCA: Breast Cancer Gene.

3.5 Association of PRDM6 methylation with clinical parameters

Further analyses were conducted to investigate whether PRDM6 methylation was associated with selected clinical variables, including age at diagnosis, age at menopause, and pre- and post-treatment CA-125 levels. No statistically significant differences were observed between methylated and unmethylated groups for any of the variables examined (Table 3).

Table 3.Evaluation of PRDM6 methylation in relation to quantitative variables.
VariableMethylation statusnMeanMedianSDMinMaxp-value
First CA-125 (individuals with benign ovarian disease)
Unmethylated205.830.0015.990.0064.000.661
Methylated306.030.0019.230.0084.00
First CA-125 (ovarian cancer)
Unmethylated52791.72364.501236.287.976600.000.491
Methylated65710.81221.001406.092.2010,389.50
Last CA-125 (ovarian cancer)
Unmethylated45388.8245.00943.573.004284.500.092
Methylated54269.7219.501076.092.207709.00
Diagnosis age in years (healthy control)
Unmethylated6342.2441.007.4130650.584
Methylated3743.0842.007.463065
Diagnosis age in years (individuals with benign ovarian disease)
Unmethylated2038.3540.008.7123570.079
Methylated3033.4731.509.051850
Diagnosis age in years (ovarian cancer)
Unmethylated15051.9450.0012.4417840.769
Methylated18250.7650.009.392384
Menopause age in years (ovarian cancer)
Unmethylated8646.7348.005.3128560.507
Methylated11346.1746.006.152660

SD: Standard deviation; Min: Minimum; Max: Maximum; CA-125: Cancer antigen 125; PRDM6: PR/SET domain 6.

4. Discussion

OC remains one of the leading causes of cancer-related mortality among women worldwide [20]. In this study, we investigated the potential of the PRDM6 gene as a non-invasive epigenetic biomarker by evaluating its methylation status in peripheral blood DNA. We aimed to assess the diagnostic utility of PRDM6 methylation and to contribute to the understanding of molecular mechanisms underlying ovarian tumorigenesis. Survival in OC is strongly influenced by the stage at diagnosis; earlier detection significantly improves prognosis. While CA-125 and human epididymis protein 4 (HE4) are currently the most commonly used biomarkers for diagnosis [21], they have several limitations [22], including low sensitivity in early-stage disease and lack of specificity [23, 24, 25]. One of the key challenges in OC is the lack of early, reliable biomarkers. Given that DNA hypermethylation often occurs in the early stages of tumorigenesis [26, 27, 28], the identification of methylation-based biomarkers, such as PRDM6, may offer a promising strategy for early detection. Our analysis revealed that PRDM6 promoter methylation was present in 53.9% of OC patients, 60% of individuals with benign ovarian disease, and 37% of healthy controls. Notably, methylation frequency in the healthy control group was significantly lower than that observed in the OC group (p = 0.005), suggesting a potential association between PRDM6 methylation and malignant transformation.

Given the role of PRDM6 in chromatin remodeling and gene silencing through histone methylation, alterations in its methylation status may influence key regulatory pathways involved in ovarian tumorigenesis. Methylation of histones can lead to either gene activation or repression, depending on the specific site of methylation [29, 30, 31]. Since BRCA1 and BRCA2 are essential for DNA damage repair via homologous recombination [32, 33], aberrant PRDM6 methylation might further disrupt genomic stability, particularly in BRCA-mutated tumors. Therefore, we investigated the potential association between PRDM6 promoter methylation and BRCA mutation status to explore whether epigenetic regulation of PRDM6 contributes to the distinct molecular landscape of BRCA-related OC. A significant association was also found between PRDM6 methylation and BRCA mutation status among OC patients. BRCA mutation-positive individuals exhibited a higher frequency of PRDM6 methylation compared to BRCA-negative individuals (p = 0.016). This suggests that PRDM6 methylation may be more prevalent in genetically predisposed OC cases and could potentially contribute to tumor development in this subgroup. It is important to note that this study was designed to assess the germline contribution of BRCA mutations to epigenetic regulation, and somatic testing was not within the scope of the current analysis. However, all tumor samples underwent detailed histopathological and immunohistochemical evaluation to confirm HGSOC subtype.

Interestingly, PRDM6 promoter methylation was also observed at a higher frequency in individuals with benign ovarian disease (60.0%) compared to healthy controls (37.0%), and was even slightly higher than in OC patients (53.9%). This finding suggests that epigenetic alterations in PRDM6 may not be limited to malignant transformation but may also occur during benign ovarian pathologies, possibly reflecting chronic inflammation, hormonal fluctuations, or increased cellular turnover [34]. Indeed, the association between DNA methylation changes and benign ovarian conditions is an area of growing interest, particularly in efforts to improve the molecular understanding of early events in ovarian tumorigenesis [35]. Prior studies have demonstrated that specific DNA methylation patterns can help differentiate between benign and malignant ovarian lesions [36]. For example, the methylation status of the Growth Hormone Secretagogue Receptor (GHSR) gene is significantly higher in malignant ovarian tumors compared to benign lesions, underscoring the discriminatory potential of gene-specific epigenetic changes [37]. Although PRDM6 methylation does not show a clear-cut distinction between benign and malignant tissues, the elevated levels observed in benign conditions may still reflect an active epigenetic landscape influenced by inflammation and hormonal signaling.

Furthermore, studies profiling normal ovarian tissues, benign lesions, and malignant tumors have revealed variable methylation signatures that are thought to be shaped by the immune microenvironment, particularly the presence of cytokines and inflammatory cells [38]. Chronic inflammation is a known contributor to neoplastic progression through mechanisms that promote cellular proliferation and resistance to apoptosis. These inflammatory processes may also drive DNA methylation changes in benign ovarian tissues [39]. In addition to inflammatory signaling, hormonal regulation plays a pivotal role in shaping the epigenetic profile of ovarian tissue. Hormonal cycles influence gene expression via methylation modifications, and disruptions in hormonal homeostasis may increase susceptibility to neoplastic transformation, even in benign neoplasms [39]. For instance, in benign mucinous cystadenomas, the complexity of methylation regulation is compounded by histopathological heterogeneity and microenvironmental influences such as localized inflammation [40]. A deeper understanding of the biological mechanisms linking DNA methylation to the tissue microenvironment in benign ovarian conditions may thus open new avenues for early detection and clinical stratification. Importantly, the integration of methylation profiling using minimally invasive methods, such as liquid biopsies, holds promise for differentiating between benign and malignant ovarian lesions in real time [39]. Therefore, while PRDM6 methylation alone may not provide sufficient diagnostic specificity, it could contribute to multi-marker panels aimed at improving diagnostic accuracy and guiding patient management. The observation that PRDM6 methylation levels were higher in individuals with benign ovarian conditions than in malignant cases suggests that its standalone diagnostic utility may be limited. However, elevated PRDM6 methylation may serve as an additional risk indicator, particularly in BRCA mutation carriers.

Although PRDM6 methylation was also detected in individuals with benign ovarian conditions, its higher frequency was observed among OC patients, particularly those with BRCA mutations. The increased methylation of PRDM6 observed in BRCA-mutated patients can be interpreted within the framework of the “second hit” hypothesis, where the initial BRCA1/2 mutation compromises homologous recombination repair (HRR), thereby creating genomic instability [41]. BRCA1 and BRCA2 play critical roles in maintaining genomic stability through HRR of DNA double-strand breaks. Defects in HRR caused by BRCA mutations impair HRR, leading to genomic instability [42, 43, 44]. This genomic instability may facilitate epigenetic alterations, such as the hypermethylation of genes involved in cell proliferation and differentiation, including PRDM6 [45]. Hypermethylation of PRDM6 could act as a secondary hit that synergizes with HRR deficiency, enhancing tumor progression by modulating gene expression profiles critical to tumor biology [46].

The increased methylation of PRDM6 observed in BRCA-mutant tumors may critically influence key oncogenic pathways, particularly the wingless-related integration site (Wnt)/β-catenin signaling cascade and epithelial-to-mesenchymal transition (EMT), both of which are fundamental in cancer progression. PRDM6 has been shown to regulate transcriptional programs associated with cell fate, and its methylation-induced silencing could disrupt standard control over the Wnt/β-catenin pathway, which is known to sustain stemness and promote chemoresistance in cancers, including breast tumors [47, 48, 49]. In BRCA-deficient contexts, where DNA repair is compromised, epigenetic alterations such as PRDM6 hypermethylation may amplify β-catenin activity, thereby driving aggressive tumor phenotypes linked to enhanced proliferation and metastatic potential through EMT activation [50, 51]. Furthermore, the loss of PRDM6 function through methylation may derepress EMT-promoting transcription factors, such as snail family transcriptional repressor 1 (SNAI1) and twist family BHLH transcription factor 1 (TWIST1), thereby facilitating cellular plasticity and invasiveness [29]. These molecular events could represent adaptive tumor strategies to overcome therapeutic pressures, supported by compensatory activation of Wnt and EMT pathways in BRCA-mutant malignancies [29, 52]. Collectively, this suggests a mechanistic model in which homologous recombination deficiency in BRCA-mutant tumors leads to PRDM6 hypermethylation, disrupting Wnt/β-catenin signaling, and thereby enhancing EMT processes. Targeting PRDM6-mediated epigenetic dysregulation, particularly in BRCA-mutant tumors, may overcome the therapeutic resistance associated with Wnt/EMT activation. While our data suggest Wnt/EMT involvement, future studies should validate PRDM6’s direct role in these pathways in OC models. A hypothetical model summarizing these interactions—including PRDM6 hypermethylation, BRCA-associated homologous recombination deficiency, and downstream activation of Wnt/β-catenin signaling and EMT—is presented in Fig. 2.

Proposed regulatory mechanism by which PRDM6 methylation may contribute to ovarian cancer development and progression. PRDM6: PR/SET domain 6; BRCA: Breast Cancer Gene; HR: homologous recombination; Wnt: wingless-related integration site.

Fig. 2.Proposed regulatory mechanism by which PRDM6 methylation may contribute to ovarian cancer development and progression. PRDM6: PR/SET domain 6; BRCA: Breast Cancer Gene; HR: homologous recombination; Wnt: wingless-related integration site.

However, no significant associations were identified between PRDM6 methylation and other clinical parameters such as CA-125 levels, age at diagnosis, or menopausal age. The lack of association between PRDM6 methylation and CA-125 levels suggests that this epigenetic alteration may occur independently of tumor burden, positioning it as an early event in carcinogenesis. PRDM6 encodes a transcriptional repressor involved in chromatin remodeling and has known interactions with key epigenetic regulators, such as histone deacetylases (HDACs) and histone methyltransferases [53, 54]. It plays a role in maintaining cellular proliferative capacity and inhibiting differentiation, particularly in smooth muscle and neural tissues. While the role of PRDM6 in developmental biology has been well described, studies on its epigenetic regulation in cancer, especially OC, remain scarce.

Our findings provide one of the first large-scale assessments of PRDM6 methylation in this context, suggesting that its epigenetic dysregulation may contribute to ovarian tumorigenesis. In this study, DNA methylation was evaluated using MSREs and subsequently analyzed by real-time PCR. The selection of a methylation threshold, such as 6%, is a crucial step in analyzing DNA methylation, particularly in cancer studies like those on OC. By setting a threshold of 6%, researchers ensure that low levels of methylation, which may lack biological relevance, do not confound insights into the association between methylation status and tumor behavior. This is particularly important as low or negligible methylation levels may occur due to variability within both normal and cancerous cell populations [55, 56, 57]. Moreover, clinical studies have indicated that significant methylation changes (i.e., above 6%) can serve as biomarkers for prognosis and response to treatment in ovarian cancer. For instance, high-grade serous OC patients exhibiting elevated levels of methylated genes have been correlated with poor prognostic outcomes and chemoresistance [19].

Despite these promising results, our study has some limitations. While peripheral blood is a minimally invasive and clinically feasible source for methylation analysis, it may not fully capture tumor-specific epigenetic alterations. The limitations of using peripheral blood to reflect tumor epigenetics involve several key factors that affect the accuracy, sensitivity, and specificity of the detected DNA methylation alterations. While peripheral blood has been explored as a non-invasive source for monitoring cancer biomarkers, including epigenetic modifications, it presents various challenges in accurately mirroring the tumor environment. In addition, detecting hypermethylation in peripheral blood represents a feasible and scientifically supported strategy, consistent with well-characterized methylation alterations observed in cancer, underscoring its utility as a potential biomarker for ovarian cancer. However, adopting a fixed methylation threshold (6%) may not fully capture the nuanced and heterogeneous nature of epigenetic regulation. Additionally, a key limitation of our study is the absence of an analysis exploring how PRDM6 methylation may interact with other genetic predispositions or environmental exposures that could modulate ovarian cancer susceptibility. Lastly, although our OC cohort was relatively large, further validation in independent and prospective cohorts, along with mechanistic studies, is necessary to confirm the biological and clinical significance of PRDM6 methylation. The extent to which PRDM6 methylation in peripheral blood reflects epigenetic alterations in tumor tissue warrants further investigation through matched blood–tumor sample analyses in future studies.

5. Conclusions

Our findings indicate that PRDM6 promoter methylation is significantly associated with ovarian cancer and BRCA mutation status, supporting its potential as a promising non-invasive blood-based epigenetic biomarker. This association highlights the possible role of PRDM6 in ovarian tumorigenesis, particularly in genetically predisposed individuals. To fully establish the clinical relevance of these results, further studies are needed to validate them in larger, independent cohorts and to elucidate the molecular mechanisms by which PRDM6 contributes to ovarian cancer development. Future research should also explore its utility in early detection and risk stratification, as well as its potential as a target for epigenetic-based therapeutic strategies.

Availability of data and materials

The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Author contributions

NC and OSE—designed the research study. SKE and BCD—provided help and advice on technical aspects of the research. AD—helped with the experiments. ÖP—analyzed the data. HY—selected the patients to be included in this study. SBT—was the principal investigator of the study.

Ethics approval and consent to participate

Approvals were received for the study from the Istanbul Faculty of Medicine Ethics Committee (Ethics Committee Approval No. 2021/1122; No. 2019/1161). All individuals provided written informed consent before inclusion in the study.

Acknowledgment

We gratefully acknowledge the support of the Istanbul University Scientific Research Projects Coordination Unit, whose contributions played a vital role in the successful execution of this study.

Funding

This study was funded by the Scientific Research Projects Coordination Unit of Istanbul University (Project number: TYL-2021-38160 and TOA-2020-35780).

Conflict of interest

The authors declare no conflict of interest.

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