The importance of gene polymorphism in familial inheritance of endometriosis

Objective The study aimed to investigate familial transmission patterns in women with endometriosis by generating a customized single-nucleotide polymorphism (SNP) array.

Patients aged 18–45 who were diagnosed histopathologically with endometriosis were included in the study. Daughters and mothers of these patients were also included, regardless of whether they were diagnosed with endometriosis or not. The control group consisted of female patients of similar ages who were not diagnosed with endometriosis. The first stage of this investigation was the determination of the genes associated with the SNPs through meta-analyses in the field of endometriosis in the literature. The second stage was the creation of a unique SNP array by determining the SNPs in the selected target genes. We specifically evaluated whether SNPs in the WNT4 gene at locus 1p36.12 (rs7521902), the GREB1 gene at locus 2p25.1 (rs13391619), and the FN1 gene at locus 2q35 (rs1250248) were associated with endometriosis risk in the Turkish population.

The study included 91 participants, comprising 66 women diagnosed with endometriosis and 25 healthy controls. The analysis revealed statistically significant associations for the FN1 (rs1250248, G>A) and the GREB1 (rs13391619, T>C) variants among endometriosis patients and their mothers and daughters, indicating a possible familial genetic link.

These findings strengthen the evidence for a hereditary component in endometriosis and suggest that SNP-based genetic profiling may support earlier identification of at-risk individuals, enabling more timely surveillance and clinical intervention. 1 INTRODUCTION Endometriosis, characterized by the ectopic implantation of glandular and stromal cells normally located in the endometrium, is predominantly a disease of reproductive-age women, although it may be encountered from menarche to menopause. This chronic, inflammatory, and benign condition can localize to virtually any site in the body.1 Due to its potential for an asymptomatic clinical course, its overall prevalence in the general population is difficult to determine. However, it has been reported to affect approximately 5%–10% of women of reproductive age, and up to 35% of women experiencing infertility.2 Women with endometriosis often present with symptoms that significantly impact their quality of life, including dysmenorrhea (painful menstruation), dyspareunia (painful intercourse), chronic pelvic pain, and reproductive dysfunction. Although numerous pathophysiological mechanisms have been proposed to explain the development of endometriosis, the oldest and most widely accepted is “Sampson's theory.” This theory proposes that endometrial glands and stromal cells are shed into the peritoneal cavity during retrograde menstruation through the fallopian tubes. During menstruation, endometrial cells are shed into the peritoneal cavity in all women; however, in immunocompetent individuals, these cells, which can adhere, proliferate, differentiate, and invade the peritoneum, are typically eliminated by the immune system. Therefore, while retrograde menstruation occurs in the majority of women, the relatively low prevalence of endometriosis suggests that this theory alone is insufficient to fully explain its pathogenesis. This limitation has led to the development of alternative theories, particularly to account for cases involving extra-pelvic localization. These include the coelomic metaplasia theory, the induction theory, the implantation theory, and the lymphatic and hematogenous dissemination theories.3 Genetic transmission, now considered a fundamental mechanism underlying many diseases, is also one of the most recent hypotheses implicated in the development of endometriosis.4 Familial clustering, the diagnosis of endometriosis at similar ages in sisters, especially monozygotic twins, and associations with other immunological disorders such as systemic lupus erythematosus and Crohn's disease support a possible genetic basis. However, specific genes directly responsible for endometriosis have not yet been identified.5 Furthermore, mutations and polymorphisms, particularly single-nucleotide polymorphisms (SNPs) in genes responsible for hormone receptors and metabolism, have been implicated in the pathophysiology of the disease.6 The aim of the present study was to evaluate an SNP array containing genes with SNP variations identified in DNA obtained from patients with endometriosis to determine whether there is a familial transmission pattern of endometriosis within the Turkish population. Identifying genetic inheritance across generations within families could contribute to understanding the developmental causes of the disease, allow the classification of potential subtypes, and facilitate the development of non-invasive biomarkers for early diagnosis and timely treatment. Building on the findings of this study, future research involving larger cohorts using genotyping technologies and DNA sequencing methods may enable the diagnosis of endometriosis before clinical symptoms occur. Such early detection would provide the opportunity to initiate treatment earlier and potentially prevent the long-term adverse effects of the disease. 2 MATERIALS AND METHODS - Endometriosis group (cases): This group consisted of 22 women who underwent surgery and received a histopathologically confirmed diagnosis of endometriosis. - Familial extension: To investigate inheritance, the study also included the mothers and daughters of these 22 patients, regardless of whether they had an endometriosis diagnosis. This brought the total number of women associated with the “endometriosis lineage” to 66. - Control group: This group consisted of 25 age-matched healthy women who did not have a diagnosis or clinical suspicion of endometriosis. Patients were excluded from the study if they did not provide written informed consent, if their diagnosis of endometriosis was not histopathologically confirmed, or if they did not have an available daughter and/or mother to participate. The study was started after obtaining approval from the institutional ethics committee (Istanbul Faculty of Medicine Clinical Research Ethics Committee, approval no. 205612, dated 27/11/2020). All participants signed an informed consent form approved by the Ethics Committee, confirming their voluntary participation in the study. The study was supported by the Istanbul University Scientific Research Projects Unit (BAP) (project no. 37748). The collected peripheral blood samples were transported under cold chain conditions to the Department of Molecular Biology and Genetics at Istanbul University within 24 h. After the participants' blood was taken, the following stages were performed until the SNP analysis results were obtained. 2.1 Study design and genotyping A comprehensive literature review was conducted to determine which SNPs would be investigated in the three-generation study population from which serum samples were collected. Based on this review, three genomic regions were selected for SNP analysis. Due to the lack of existing studies on the genetic inheritance of endometriosis in Turkish women, SNP analyses performed in European populations were taken into consideration, taking into account ethnic differences. As a result, the most significant loci identified in previous studies were selected: WNT4 gene at locus 1p36.12 (rs7521902), GREB1 gene at locus 2p25.1 (rs133946619), and FN1 gene at locus 2q35 (rs1250248). The WNT4 gene encodes a protein essential for the development of the female genital tract. Its absence has been associated with Müllerian agenesis, a condition characterized by partial or complete failure of the female reproductive tract to develop. The GREB1 gene is involved in the early response within the estrogen signaling pathway. Given its role in estrogen metabolism, studies have shown that GREB1 expression is increased in endometriotic lesions, supporting its involvement in this estrogen-dependent disease. The FN1 gene plays a role in cellular adhesion and migration processes. Therefore, it has been implicated in embryogenesis, wound healing, blood coagulation, and metastasis.7 2.1.1 DNA extraction Peripheral blood samples were extracted using the GeneAll Exgene Blood SV DNA extraction kit. 2.1.2 Qubit fluorometric quantification A Qubit mix was prepared in a dark environment using 199 μL double-stranded DNA (dsDNA) Qubit buffer and 1 μL Qubit reagent per sample. Highly concentrated DNA samples were diluted to achieve uniform DNA concentrations across all samples. 2.1.3 Primer dilution This study specifically evaluated specific SNP loci for the WNT4, GREB1, and FN1 genes. The SNPs analyzed included rs7521902 (G>T) for WNT4 at locus 1p36.12, rs13391619 (G>A) for GREB1, and rs1250248 (A>G, T>C) for FN1. Specific forward and reverse primers were designed for each SNP. 2.1.4 Amplification—PCR Following optimization, polymerase chain reaction (PCR) amplifications for WNT4, GREB1, and FN1 were optimized using GC buffer under identical conditions (60°C for 1 min). After the amplification PCR, the resulting products were analyzed using agarose gel electrophoresis to confirm successful amplification. 2.1.5 Agarose gel preparation In agarose gel electrophoresis, all samples were selected with bands in the expected regions for the next step, ExoSAP treatment. ExoSAP is the purification method of choice for removing excess primers and nucleotides from amplification reactions. It is essential for successful sequencing, cloning, genotyping, and other DNA modification procedures. The reaction mixture contained exonuclease and alkaline phosphatase enzymes. Then, 5 μL of each PCR product were added to labeled tubes containing ExoSAP reagent and gently mixed. After vortexing and a brief centrifugation, the tubes were placed in a thermal cycler. After completing the ExoSAP reaction, the samples moved on to the BigDye Terminator Sequencing PCR step. During this stage, DNA sequences were randomly fragmented and terminated with fluorescently labeled ddNTPs. After completing the BigDye PCR reaction, the samples were prepared for purification. 2.1.6 Purification step Sephadex purification was used to separate nucleic acids from smaller molecules like primers, nucleotides, and dyes. Sephadex is a gel filtration resin composed of dextran cross-linked with epichlorohydrin. A prepared Sephadex solution was dispensed into individually filtered tubes for each sample at a volume of 700 μL per tube. All BigDye sequencing products were transferred into the appropriate columns and then spun at 2800 g for 3 min. After purifying the columns, 10 μL of each purified product was collected and transferred into a 96-well plate. The plate was loaded onto the 3130XL Genetic Analyzer Sanger sequencing system, and sequencing was initiated. Data analysis was conducted using Sequencing Analysis software. Each primer corresponded to a different genomic region, so care was taken to accurately identify the primer used and the specific target region. 2.2 Statistical analysis Statistical evaluations of SNP genotype analyses for endometriosis patients and their first-degree relatives (mother–child pairs) were performed using RGui (64-bit), R version 4.3.1 (2023) (R Computing Group, Vienna, Austria). Genotype and allele frequencies were calculated manually through direct counting. Comparisons of allele and genotype frequencies between patient and control groups were performed using Pearson's chi-square test (χ2). Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated using logistic regression analysis. Haplotype analyses evaluating the co-occurrence probabilities of rs1250248 (G/A) and rs13391619 (T/C) polymorphisms were performed using a two-tailed Fisher exact test. All P values were two-tailed, with a significance threshold of P less than 0.05. Hardy–Weinberg equilibrium (HWE) was used to assess genotype distribution consistency in patient and control groups. Observed and expected genotype frequencies and their compliance with HWE were calculated using the cog-genomics web tool. Expression quantitative trait loci (eQTL) data for SNPs in WNT4, FN1, and GREB1 genes were retrieved from the Genotype-Tissue Expression (GTEx) analysis portal (dbGaP accession phs000424.v8.p2). 3 RESULTS 3.1 The genotype and allele frequencies of rs7521902, rs1250248, and rs13391619 between endometriosis patients and healthy controls The rs7521902 (WNT4) C>A polymorphism showed comparable frequencies of the AA genotype in the endometriosis patient group (4.55%) and the control group (4%). The CC genotype was used as the reference. The AC genotype was observed twice as frequently in the patient group (50%) compared to the control group (24%) (Table 1). | Genotype/allele | Endometriosis patients (n = 22) | Frequency (%) | Healthy controls (n = 25) | Frequency (%) | OR | 95% CI | Chi-square (χ2) | P value | |---|---|---|---|---|---|---|---|---| | CC (Ref) | 12 | 54.55% | 18 | 72% | Ref | — | — | — | | AC | 8 | 36.36% | 6 | 24% | 2.000 | (1.021–4.575) | 1.25 | 0.134 | | AA | 2 | 9.09% | 1 | 4% | 3.000 | (1.040–17.443) | 0.87 | 0.351 | | C (Ref) | 32 | 72.73% | 42 | 84% | Ref | — | — | — | | A | 12 | 27.27% | 8 | 16% | 1.950 | (1.016–3.261) | 2.32 | 0.127 | - Abbreviations: CI, confidence interval; OR, odds ratio. The “A” allele of the rs7521902 polymorphism appeared at a higher frequency in endometriosis patients, but the difference between the patient and control groups was not statistically significant (P > 0.05). The rs1250248 (FN1) “G>A” polymorphism was statistically significant. When the GG genotype was used as the reference, the AG genotype was found to be considerably more prevalent in endometriosis patients (59.09%) than in healthy controls (12%) (P = 0.0011, OR = 12.380, 95% CI: 1.024–5.701, χ2 = 10.496). Furthermore, the presence of the “A” allele differed significantly between endometriosis patients and healthy controls (P = 0.0082, OR = 3.867, 95% CI: 1.016–3.149, χ2 = 6.982; Table 2). | Variant | Genotype/allele | Patient count (n = 22) | Control count (n = 25) | OR | 95% CI | Chi-square (χ2) | P value | |---|---|---|---|---|---|---|---| | Genotype | GG (Ref) | 7 (31.82%) | 20 (80%) | Ref | — | — | — | | AG | 13 (59.09%) | 3 (12%) | 12.380 | (1.024–5.701) | 10.496 | 0.0011 | | | AA | 2 (9.09%) | 2 (8%) | 2.857 | (1.034–11.565) | 0.923 | 0.336 | | | Allele | G (Ref) | 27 (61.36%) | 43 (86%) | Ref | — | — | — | | A | 17 (38.64%) | 7 (14%) | 3.867 | (1.016–3.149) | 6.982 | 0.0082 | - Abbreviations: CI, confidence interval; OR, odds ratio. The rs13391619 (GREB1) “T>C” polymorphism clearly demonstrated a significant difference in genotype frequency between endometriosis patients and healthy controls (P = 0.033, OR = 26.538, 95% CI: 1.049–31.614, χ2 = 4.527). Additionally, the “C” allele was more frequently observed in endometriosis patients (69.44%) than in controls (47.37%), although this difference did not reach statistical significance (P = 0.056; Table 3). | Variant | Genotype/allele | Patient count (n = 18) | Patient frequency (%) | Control count (n = 19) | Control frequency (%) | OR | 95% CI | Chi-square (χ2) | P value | |---|---|---|---|---|---|---|---|---|---| | Genotype | TT (Ref) | 0 | 0.00% | 7 | 36.84% | Ref | ||| | TC | 11 | 61.11% | 6 | 31.58% | 26.538 | (1.049–31.614) | 4.527 | 0.033 | | | CC | 7 | 38.89% | 6 | 31.58% | 17.307 | (1.049–32.693) | 3.358 | 0.066 | | | Allele | T (Ref) | 11 | 30.56% | 20 | 52.63% | Ref | ||| | C | 25 | 69.44% | 18 | 47.37% | 2.525 | (1.015–2.974) | 3.628 | 0.056 | - Abbreviations: CI, confidence interval; OR, odds ratio. 3.2 The genotype and allele frequencies of rs7521902, rs1250248, and rs13391619 polymorphisms in mothers and daughters of endometriosis patients compared with healthy controls DNA samples from mothers and daughters of endometriosis patients were analyzed using Sanger sequencing for the SNPs rs7521902, rs1250248, and rs13391619, located in the WNT4, FN1, and GREB1 genes, respectively. The resulting genotype data were compared with those of healthy controls. The analysis was divided into two comparison groups: “Endometriosis mothers versus controls” and “endometriosis daughters versus controls.” Individuals with homozygous deletions in the analyzed SNP regions were excluded from statistical evaluation. The rs7521902 (WNT4) “C>A” polymorphism showed a noticeable difference. Using the CC genotype as the reference, the AA genotype was observed more frequently in the mothers of endometriosis patients (9.09%) compared to the control group (4%). However, no statistically significant difference was found in the comparisons between either “endometriosis mothers versus healthy controls” or “endometriosis daughters versus healthy controls” (P > 0.05) (Table 4). | Genotype/allele | Endometriosis mothers (n = 22) | Frequency (%) | Healthy controls (n = 25) | Frequency (%) | Endometriosis daughters (n = 22) | Frequency (%) | OR (mothers vs. controls) | 95% CI | Chi-square (χ2) | P value | OR (daughters vs. controls) | 95% CI | Chi-square (χ2) | |---|---|---|---|---|---|---|---|---|---|---|---|---|---| | CC (Ref) | 14 | 63.64% | 18 | 72% | 15 | 68.18% | Ref | Ref | ||||| | AC | 6 | 27.27% | 6 | 24% | 7 | 31.82% | 1.285 | (1.021–4.575) | 0.136 | 0.712 | 1.4 | (1.020–4.169) | 0.262 | | AA | 2 | 9.09% | 1 | 4% | 0 | 0% | 2.571 | (1.040–17.443) | 0.548 | 0.459 | 0.397 | (1.053–42.126) | 0.306 | | C (Ref) | 34 | 77.27% | 42 | 84% | 37 | 84.09% | Ref | Ref | ||||| | A | 10 | 22.73% | 8 | 16% | 7 | 15.91% | 1.544 | (1.016–3.261) | 0.678 | 0.410 | 0.993 | (1.017–3.544) | 0.0003 | - Abbreviations: CI, confidence interval; OR, odds ratio. The rs1250248 (FN1) “G>A” polymorphism revealed a notable difference in the frequency of the AG genotype. Among daughters of endometriosis patients, the frequency reached 45.45%, significantly higher than the 12% observed in healthy controls (P = 0.009, OR = 7.407, 95% CI: 1.024–5.630, χ2 = 6.745; Table 5). Furthermore, the “A” allele of rs1250248 was also significantly more frequent in daughters of endometriosis patients than in healthy controls (P = 0.014, OR = 3.510, 95% CI: 1.016–3.165, χ2 = 5.964; Table 5). | Genotype/allele | Mothers (n = 22) | Frequency (%) | Controls (n = 25) | Frequency (%) | Daughters (n = 22) | Frequency (%) | OR (mothers vs. controls) | 95% CI | Chi-square (χ2) | P value | OR (daughters vs. controls) | 95% CI | Chi-square (χ2) | P value | |---|---|---|---|---|---|---|---|---|---|---|---|---|---|---| | GG (Ref) | 13 | 59.09% | 20 | 80% | 9 | 40.91% | Ref | Ref | |||||| | AG | 5 | 22.73% | 3 | 12% | 10 | 45.45% | 2.564 | (1.025–6.179) | 1.342 | 0.246 | 7.407 | (1.024–5.630) | 6.745 | 0.009 | | AA | 4 | 18.18% | 2 | 8% | 3 | 13.64% | 3.076 | (1.029–8.157) | 1.439 | 0.230 | 3.333 | (1.031–9.348) | 1.457 | 0.227 | | G (Ref) | 31 | 70.45% | 43 | 86% | 28 | 63.64% | Ref | Ref | |||||| | A | 13 | 29.55% | 7 | 14% | 16 | 36.36% | 2.576 | (1.016–3.241) | 3.252 | 0.071 | 3.510 | (1.016–3.165) | 5.964 | 0.014 | - Abbreviations: CI, confidence interval; OR, odds ratio. The rs13391619 (GREB1) “T>C” polymorphism, using the TT genotype as the reference, showed that both the TC and CC genotypes were more frequent in mothers of endometriosis patients (TC: 47.06%, CC: 41.18%) compared to healthy controls (TC: 31.58%, CC: 31.58%). However, these differences did not reach statistical significance (P > 0.05; Table 6). The “C” allele was notably the most prevalent in the “endometriosis patient mother” group, with a frequency of 64.71%, markedly higher than in any of the other groups (Table 6). | Genotype/allele | Mothers (n = 22) | Frequency (%) | Controls (n = 25) | Frequency (%) | Daughters (n = 22) | Frequency (%) | OR (mothers vs. controls) | 95% CI | Chi-square (χ2) | P value | OR (daughters vs. controls) | 95% CI | Chi-square (χ2) | |---|---|---|---|---|---|---|---|---|---|---|---|---|---| | TT (Ref) | 2 | 11.76% | 7 | 36.84% | 8 | 44.45% | Ref | Ref | ||||| | TC | 8 | 47.06% | 6 | 31.58% | 3 | 33.33% | 4.666 | (1.030–8.730) | 2.538 | 0.112 | 0.875 | (1.024–5.685) | 0.029 | | CC | 7 | 41.18% | 6 | 31.58% | 2 | 22.22% | 4.083 | (1.031–8.911) | 2.078 | 0.149 | 0.583 | (1.026–6.388) | 0.425 | | T (Ref) | 12 | 35.29% | 20 | 52.63% | 22 | 61.11% | Ref | Ref | ||||| | C | 22 | 64.71% | 18 | 47.37% | 14 | 38.89% | 2.037 | (1.015–2.959) | 2.160 | 0.707 | (1.014–2.878) | 0.540 | - Abbreviations: CI, confidence interval; OR, odds ratio. 3.3 Haplotype analysis of rs1250248 and rs13391619 polymorphisms between endometriosis patients and healthy controls The statistically significant associations observed for rs1250248 [G>A] (FN1) and rs13391619 [T>C] (GREB1) prompted a haplotype analysis to evaluate their combined distribution. Four haplotypes were assessed based on the respective alleles of rs1250248 and rs13391619. Haplotype frequencies were calculated, and comparisons between endometriosis patients and healthy controls were performed using a two-tailed Fisher exact test. As shown in Table 7, the G–T haplotype served as the reference. No significant associations were observed for the G–C and A–T haplotypes (P > 0.05). In contrast, the A–C haplotype showed a statistically significant difference between the groups (P = 0.0065) and was more frequently observed in endometriosis patients than in healthy controls (27.27% vs. 3.70%). | Haplotype (rs1250248-rs13391619) | Endometriosis patients (n = 22) | Frequency (%) | Healthy controls (n = 25) | Frequency (%) | Endometriosis versus healthy controls (P value)a | |---|---|---|---|---|---| | G–T (Reference) | 10 | 22.73% | 11 | 40.74% | Reference | | A–C | 12 | 27.27% | 1 | 3.70% | 0.0065 | | G–C | 16 | 36.36% | 11 | 40.74% | 0.476 | | A–T | 6 | 13.64% | 4 | 14.82% | 0.581 | - a Two-tailed Fisher exact test. 3.4 HWE analysis of rs7521902, rs1250248, and rs13391619 polymorphisms Table 8 presents the observed and expected genotype distributions for the rs7521902 (WNT4, C>A), rs1250248 (FN1, G>A), and rs13391619 (GREB1, T>C) polymorphisms, which were analyzed for compliance with Hardy–Weinberg equilibrium (HWE) in both endometriosis patients and healthy controls. For SNPs 1 (rs7521902), 2 (rs1250248), and 3 (rs13391619), the expected frequencies of major and minor alleles and the observed genotype frequencies were consistent with HWE (P > 0.05). However, SNP2 (rs1250248) in the healthy control group showed a significant deviation from HWE (P = 0.022). Allele and genotype frequency distributions based on HWE for both groups are illustrated in Figures 1-3. | SNP | Group | Major allele (A1) | Minor allele (A2) | Genotype counts | Observed heterozygote frequency | Expected heterozygote frequency | HWE P value | |---|---|---|---|---|---|---|---| | rs7521902 (WNT4) | Endometriosis patients | C | A | 10/11/1 | 0.50 | 0.417 | 0.464 | | rs7521902 (WNT4) | Healthy controls | C | A | 18/6/1 | 0.24 | 0.269 | 0.283 | | rs1250248 (FN1) | Endometriosis patients | G | A | 7/13/2 | 0.59 | 0.474 | 0.116 | | rs1250248 (FN1) | Healthy controls | G | A | 20/3/2 | 0.12 | 0.241 | 0.022 | | rs13391619 (GREB1) | Endometriosis patients | T | C | 0/11/7 | 0.611 | 0.424 | 0.069 | | rs13391619 (GREB1) | Healthy controls | T | C | 7/6/6 | 0.315 | 0.499 | 0.119 | - Abbreviations: HWE, Hardy Weinberg equilibrium; SNPs, single-nucleotide polymorphisms. 3.5 eQTL analyses of rs7521902, rs1250248, and rs13391619 single nucleotide polymorphisms Expression quantitative trait loci (eQTL) values associated with SNPs in the WNT4, FN1, and GREB1 genes were obtained from the GTEx analysis portal (dbGaP accession: phs000424.v8.p2). To assess eQTL associations for the rs7521902, rs1250248, and rs13391619 variants, each SNP was independently analyzed for tissue-specific expression in the uterus, ovary, and whole blood. A statistically significant association was observed between the rs1250248 variant and FN1 expression in ovarian tissue (eQTL P = 0.0087). Similarly, rs13391619 demonstrated a strong correlation with GREB1 expression in uterine tissue (eQTL P = 0.0015; Table 9). | Tissue | Gene | SNP | P value | NES (95% CI) | T-statistic | |---|---|---|---|---|---| | Uterus | WNT4 | rs7521902 | 0.44 | −0.073 | −0.78 | | FN1 | rs1250248 | 0.42 | −0.061 | −0.82 | | | GREB1 | rs13391619 | 0.0015 | 0.68 | 3.3 | | | Ovary | WNT4 | rs7521902 | 0.16 | −0.10 | −1.4 | | FN1 | rs1250248 | 0.0087 | −0.14 | −2.7 | | | GREB1 | rs13391619 | 0.58 | 0.084 | 0.55 | | | Whole blood | WNT4 | rs7521902 | 0.37 | −0.031 | −0.90 | | FN1 | rs1250248 | 0.54 | 0.026 | 0.61 | | | GREB1 | rs13391619 | N/A | N/A | N/A | - Note: Data obtained from the GTEx analysis portal. - Abbreviations: CI, confidence interval; eQTL, expression quantitative trait loci; NES, normalized effect size; SNP, single-nucleotide polymorphism. Figure 4 presents the normalized eQTL genotype distributions for WNT4, FN1, and GREB1 expression. 4 DISCUSSION The pathogenesis of endometriosis remains incompletely understood, but genetic predisposition is widely recognized as a significant contributing factor. The present study aimed to characterize SNPs in the WNT4 (rs7521902), FN1 (rs1250248), and GREB1 (rs13391619) genes across three generations (the patient, her mother, and her daughter) and to assess the potential influence of these polymorphisms on endometriosis susceptibility in the Turkish population. The study included 91 participants, comprising 66 women diagnosed with endometriosis and 25 healthy controls. Utilizing Sanger DNA sequencing, this research represents the first effort to compare candidate gene polymorphisms previously implicated in endometriosis development within the Turkish population. The analysis revealed statistically significant associations for the FN1 (rs1250248, G>A) and GREB1 (rs13391619, T>C) variants among endometriosis patients and their mothers and daughters, indicating a possible familial genetic link. All three variants (WNT4, rs7521902; FN1, rs1250248; GREB1 rs13391619) are located in non-coding regions that do not directly alter protein structure, rather than in the protein-coding exon sequences of the respective genes. The nucleotide substitutions rs1250248 G>A, rs13391619 T>C, and rs7521902 C>A do not alter the three-dimensional shape of FN1, GREB1, and WNT4 proteins, respectively; however, they do affect the expression levels of these proteins: Regarding the clinical significance of the findings, all patients found to carry the SNPs had histopathologically confirmed and clinically symptomatic endometriosis. In the maternal generation, although these women had not received a formal diagnosis in the past, clinical evaluations revealed symptoms strongly suggestive of endometriosis. Conversely, the daughters remained asymptomatic at the time of the study; however, this lack of clinical presentation is likely due to their younger age. Recent studies have increasingly focused on the genetic transmission of endometriosis, a complex condition shaped by hormonal, immunological, and environmental influences.8 A recent meta-analysis by Cardoso et al. highlighted the pivotal role of genome-wide association studies (GWAS) in identifying genetic variants associated with endometriosis risk. These analyses are crucial for unraveling the disease's multifactorial etiopathogenesis.9 Additionally, research into the genetic regulation of microRNAs, which are critical post-transcriptional gene regulators, has identified significant alterations in microRNA expression in women with endometriosis. These changes can impact intracellular signaling pathways and may drive epithelial-mesenchymal transition (EMT) in endometrial tissues, a key mechanism underlying the progression and invasiveness of endometriosis.10 Emerging evidence also suggests that microRNAs can modulate the WNT/β-catenin signaling pathway in endometriotic tissues, potentially affecting cell proliferation, differentiation, and invasive behavior within the endometrial microenvironment.11 Single-nucleotide polymorphisms are single-base variations in the DNA sequence that occur at specific nucleotide positions and differ among individuals. These genetic variations can significantly influence disease susceptibility and progression, making SNP studies an invaluable tool for uncovering the genetic basis of various medical conditions, including autoimmune disorders and cancers.12 In the context of endometriosis, a recent large-scale, multiancestry study involving over 900 000 women identified several critical genetic loci, including CDC42, SKAP1, and GREB1, that are significantly associated with endometriosis risk. This study underscored the importance of the WNT signaling pathway in the pathogenesis of endometriosis, reinforcing the role of this pathway in cellular differentiation, proliferation, and tissue remodeling processes relevant to endometriosis development.13 Furthermore, a recent meta-analysis integrating multiple independent datasets identified HLA-DQB1 as a critical gene associated with endometriosis, suggesting its potential utility as a diagnostic biomarker. This gene exhibited a central role within the broader genetic network of endometriosis, emphasizing its importance in the disease's underlying genetic architecture.14 Recent advances in non-invasive diagnostic techniques have also identified free circulating DNA (cfDNA) and differential methylation profiling as promising biomarkers for endometriosis. These approaches offer a non-invasive alternative to traditional diagnostic methods by detecting specific methylation patterns that distinguish endometriosis patients from healthy individuals.15 While smaller case–control studies on endometriosis have reported no significant differences in the polymorphisms and expression levels of genes believed to be involved in the disease's pathogenesis, such as vascular endothelial growth factor (VEGF),16-19 other studies have identified SNPs in proinflammatory cytokines, including interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α). These cytokines are closely associated with chronic inflammatory processes and may contribute to one of the major symptoms of endometriosis: pelvic pain. It is also well-established that women with relatively high estrogen levels are at an increased risk of developing endometriosis. Genetic studies focusing on estrogen receptors and related pathways have revealed the presence of polymorphisms at the receptor level in these women, further supporting the hormonal influence on disease susceptibility.20 Single-cell transcriptomic studies have revealed that endometriosis-associated mesothelial cells (EAMCs) interact with ectopic stromal cells, potentially contributing to progesterone resistance through intercellular communication.21 Emerging evidence has also highlighted the ERβ/QKI/circSMAD2 axis as a critical pathway in the progression of endometriosis, presenting a promising target for future therapeutic approaches.22 The WNT gene family plays a central role in fundamental biological processes, including differentiation, proliferation, cell motility, and apoptosis during embryogenesis. WNT proteins activate an intracellular signaling cascade, known as the WNT pathway, which regulates these critical cellular functions. Dysregulation or malfunction of this pathway can lead to abnormalities in cell growth, differentiation, and metabolism, contributing to the development of diseases such as cancer. The WNT signaling pathway plays a crucial role in epithelial-stromal cell communication, endometrial maturation, differentiation, and embryonic implantation within the endometrium.23 Recent studies have demonstrated that a common allele near the WNT4 gene significantly increases its expression in the endometrium, potentially enhancing uterine receptivity for embryo implantation while simultaneously elevating the risk of reproductive cancers and endometriosis. This variant has been shown to markedly upregulate WNT4 expression in the endometrial stroma, underscoring its dual role in both fertility and disease susceptibility.24 In the context of endometriosis, Liang et al. observed an overall downregulation of WNT expression in 30 endometriosis patients compared to 30 healthy controls, suggesting a potential role for WNT signaling disruption in the disease's pathogenesis.25 Additionally, extensive genetic mapping studies have established strong associations between biomarkers within or near the WNT4 gene and endometriosis susceptibility. For instance, Pagliardini et al. demonstrated that the rs7521902 polymorphism, located 21 kb from the WNT4 gene, may serve as a significant susceptibility locus for endometriosis.26 This SNP, situated at the 1p36.12 locus, has been associated with endometriosis susceptibility in women of various ethnic backgrounds, including British, Australian, Italian, and Japanese populations.23, 26, 27 In our study group, the rs7521902 SNP1 (C>A) variant of the WNT4 gene was found at similar frequencies among endometriosis patients with the homozygous AA genotype and healthy controls. However, the heterozygous AC genotype was significantly more frequent among endometriosis patients compared to the control group, suggesting a possible association between this SNP and an increased risk of endometriosis. Moreover, several candidate genes, such as GREB1 at the 2p25.1 locus and FN1 at the 2q35 locus, are highly expressed in cell types implicated in endometriosis pathogenesis, including neurons, immune cells, and epithelial cells.28 The GREB1 gene plays a critical role in hormone-responsive tissues and cancer. It is an estrogen-sensitive early response gene regulated by the estrogen receptor. A recent study identified a feedforward mechanism involving GREB1 and steroid receptors, demonstrating that GREB1 can modulate endometrial function through its interactions with progesterone-responsive genes, potentially influencing endometriosis development.29 However, in a study conducted on a Greek population, the rs11674184 polymorphism of the GREB1 gene was not found to be significantly associated with endometriosis, despite being one of the most consistently associated SNPs with endometriosis in European populations.30 The FN1 gene encodes fibronectin, a glycoprotein involved in cell adhesion and migration processes critical to embryogenesis, wound healing, and blood clotting. Although Matalliotaki et al. did not identify significant genetic differences in the GREB1 gene among endometriosis patients, their study provided evidence of a significant genotypic and allelic association between the FN1 (rs1250248) SNP and endometriosis.30 In our study group, endometriosis patients with the heterozygous AG genotype of the FN1 rs1250248 SNP2 variant were observed at a significantly higher frequency than healthy controls. Additionally, the A allele of this SNP was significantly more prevalent in the endometriosis patient group compared to the control group. Notably, the heterozygous AG genotype and A allele of the rs1250248 (FN1) SNP2 variant were also observed at significantly higher frequencies in the daughters of endometriosis patients compared to the healthy control group. Studies on the relationship between genetic polymorphisms and the risk of developing endometriosis in Japanese women have demonstrated that patients with endometriosis often carry the deletion (D)/deletion (D) genotype in insertion (I)/deletion (D) polymorphisms.31 Consistent with these findings, our study also observed this pattern with the rs13391619 (T>C) SNP3 variant of the GREB1 gene, where four endometriosis patients carried the homozygous D/D genotype. Notably, the same D/D genotype was present in the mothers and daughters of these patients, suggesting a potential familial inheritance pattern. No homozygous or heterozygous deletion genotypes were observed for the WNT4 and FN1 gene variants in our cohort. However, independent instances of patients, their mothers, and their daughters carrying the homozygous D/D genotype for the GREB1 SNP3 variant were identified. In our study group, endometriosis patients with the heterozygous TC genotype for the GREB1 SNP3 variant were observed at a significantly higher frequency compared to healthy controls. However, patients and control individuals with the homozygous TT (D/D) genotype were excluded from the genotype and allele frequency statistical analyses. Expression quantitative trait loci studies, which link genetic variants to cell-specific expression patterns in the endometrium and peritoneal fluid, remain critical for elucidating the molecular mechanisms underlying endometriosis.8 In our study, eQTL analyses of WNT4 (rs7521902), FN1 (rs1250248), and GREB1 (rs13391619) SNP variants in uterine, ovarian, and whole blood samples revealed significant associations. Specifically, the uterus eQTL P value for the GREB1 (rs13391619) variant was 0.0015, and the ovary eQTL P value for the FN1 (rs1250248) variant was 0.0087, both statistically significant, indicating notable expression differences. Our research represents the first study to evaluate candidate gene polymorphisms in the Turkish population. By focusing on genetic markers previously linked to endometriosis, this study fills the critical gap in regional genomic data. The primary limitation of this study is its relatively small sample size, which may constrain the statistical power and generalizability of the findings across the broader Turkish population. However, when considering the logistical challenges, technical complexities, and significant costs inherent in genetic research—particularly the use of Sanger DNA sequencing—the study population remained sufficient to identify statistically significant familial associations. Moreover, the unique three-generation design, including mothers and daughters, provides a depth of genetic insight that compensates for the cohort size and establishes a pioneering foundation for future large-scale genomic studies in this demographic. Additionally, the prospective cross-sectional design at a single tertiary center might not fully capture the long-term environmental or lifestyle factors that interact with these genetic polymorphisms. Finally, while Sanger sequencing provided reliable data, the study was limited to three specific genomic regions, suggesting that future research using NGS on larger cohorts is necessary for a more comprehensive genetic mapping. 5 CONCLUSION Our findings suggest that the FN1 rs1250248 (FN1, G>A) and the GREB1 rs13391619 (GREB1, T>C) polymorphisms, which have been reported in the literature as potentially involved in endometriosis pathogenesis using Sanger DNA sequencing, were statistically significant in the Turkish population. Future studies utilizing larger cohorts and advanced genetic techniques, such as targeted DNA fragment analysis and NGS, will be essential for validating these findings and further elucidating the role of these polymorphisms in the genetic architecture of endometriosis. The clinical application of this study lies in its potential for early intervention and personalized prevention strategies. By identifying familial genetic predispositions through specific SNP analysis, individuals at high risk can be diagnosed before the disease progresses to advanced stages or severely impacts their quality of life. This early detection allows for the implementation of preventative management plans, effectively preserving both fertility and long-term well-being by mitigating chronic pain and reproductive dysfunction before they manifest significantly. Ultimately, these findings facilitate a shift toward proactive care, ensuring that women with a hereditary link to endometriosis can maintain a higher quality of life through timely clinical monitoring and treatment. AUTHOR CONTRIBUTIONS Hale Goksever Celik: Conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, visualization, writing—original draft, writing—review and editing. Candan Eker: Formal analysis, methodology, software, validation, and writing—original draft. Berivan Guzelbag: Data curation and investigation. Ercan Bastu: Supervision, writing—review and editing. Tuba Gunel: Conceptualization, funding acquisition, investigation, methodology, visualization, supervision, writing—review and editing. ACKNOWLEDGMENTS The authors would like to thank the participants of this study. FUNDING INFORMATION The study was supported by the Istanbul University Scientific Research Projects Unit (BAP) under project number 37748. CONFLICT OF INTEREST STATEMENT The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. DATA AVAILABILITY STATEMENT None.