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Association of CDKN2B-AS and WNT4 genetic polymorphisms in Korean patients with endometriosis

      Objective

      To investigate whether specific genetic polymorphisms in the cyclin-dependent kinase inhibitor 2B antisense RNA (CDKN2B-AS) gene and near the wingless-type MMTV integration site family member 4 (WNT4) gene are associated with endometriosis in a Korean population.

      Design

      Case-control genetic association study.

      Setting

      University.

      Patient(s)

      Surgically or histologically diagnosed cases of endometriosis (n = 673) and controls (n = 500) among a population of ethnic Koreans.

      Intervention(s)

      None.

      Main Outcome Measure(s)

      Genotype distribution and synergistic interaction.

      Result(s)

      Significant differences were found in the allele distributions of the CC genotype of the rs10965235 single-nucleotide polymorphism (SNP) of the CDKN2B-AS gene and the GG genotype of the rs16826658 SNP on chromosome 1p36 between the endometriosis cases and the controls (rs10965235: 69.7% CC, 26.9% CA, and 3.4% AA vs. 59.2% CC, 35.2% CA, and 5.6% AA; rs16826658: 33.7% GG, 48.4% GT, and 17.8% TT vs. 25.6% GG, 49.8% GT, and 24.6% TT, respectively). A significant interaction was not found between the CC genotype of the rs10965235 SNP and the GG genotype of the rs16826658 SNP after Bonferroni correction (32.8% of CC + GG and 67.2% of CC + non-GG in the endometriosis cases vs. 25.0% of CC + GG and 75.0% of CC + non-GG in the controls).

      Conclusion(s)

      Our results suggest that the rs10965235 SNP in the CDKN2B-AS gene and the rs16826658 SNP near the WNT4 gene were significantly associated with endometriosis in this Korean population.

      Key Words

      Discuss: You can discuss this article with its authors and with other ASRM members at http://fertstertforum.com/leegh-cdkn2bas-wnt4-polymorphisms-korean-endometriosis/
      Endometriosis is defined as the presence of endometrial tissue outside the uterus, and this condition affects 6%–10% of women of reproductive age. Endometriosis causes diverse clinical manifestations including subfertility, pelvic pain, and dysmenorrhea, and has a significant detrimental impact on quality of life and work productivity (
      • Giudice L.C.
      • Kao L.C.
      ,
      • Kim S.H.
      • Chae H.D.
      • Kim C.H.
      • Kang B.M.
      Update on the treatment of endometriosis.
      ).
      The etiology of endometriosis remains elusive; however, many hypothesis-driven candidate gene studies and several hypothesis-generating genome-wide association studies (GWAS) have reported that several specific single-nucleotide polymorphism (SNP) loci may be associated with endometriosis (
      • Lee G.H.
      • Choi Y.M.
      • Kim S.H.
      • Hong M.A.
      • Oh S.T.
      • Lim Y.T.
      • et al.
      Association of tumor necrosis factor-{alpha} gene polymorphisms with advanced stage endometriosis.
      ,
      • Uno S.
      • Zembutsu H.
      • Hirasawa A.
      • Takahashi A.
      • Kubo M.
      • Akahane T.
      • et al.
      A genome-wide association study identifies genetic variants in the CDKN2BAS locus associated with endometriosis in Japanese.
      ,
      • Painter J.N.
      • Anderson C.A.
      • Nyholt D.R.
      • Macgregor S.
      • Lin J.
      • Lee S.H.
      • et al.
      Genome-wide association study identifies a locus at 7p15.2 associated with endometriosis.
      ). Several studies have demonstrated an association between endometriosis and candidate genes in terms of hormone action, steroid biosynthesis, inflammatory cytokines, and autoimmune factors; however, these findings have not been replicated or corroborated in different populations (
      • Lee G.H.
      • Kim S.H.
      • Choi Y.M.
      • Suh C.S.
      • Kim J.G.
      • Moon S.Y.
      Estrogen receptor beta gene +1730 G/A polymorphism in women with endometriosis.
      ,
      • Lee G.H.
      • Choi Y.M.
      • Kim J.M.
      • Shin J.J.
      • Kim J.G.
      • Moon S.Y.
      Association of epidermal growth factor receptor gene polymorphisms with advanced endometriosis in a Korean population.
      ). This lack of reproducibility in SNP studies has led to the recent development of GWAS, which have identified significant genetic associations by mapping 100,000 common and very densely spaced SNPs that were selected to capture the genetic architecture.
      Two recently published GWAS, including a study by the BioBank Japan (BBJ) project (
      • Uno S.
      • Zembutsu H.
      • Hirasawa A.
      • Takahashi A.
      • Kubo M.
      • Akahane T.
      • et al.
      A genome-wide association study identifies genetic variants in the CDKN2BAS locus associated with endometriosis in Japanese.
      ) and a study by the International Endogene Consortium (
      • Painter J.N.
      • Anderson C.A.
      • Nyholt D.R.
      • Macgregor S.
      • Lin J.
      • Lee S.H.
      • et al.
      Genome-wide association study identifies a locus at 7p15.2 associated with endometriosis.
      ) in a European population, have identified several SNPs associated with endometriosis. However, the top loci in these two studies were inconsistent, especially SNPs rs10965235 and rs16826658. The sentinel SNP rs10965235 is located in intron 6 of the cyclin-dependent kinase inhibitor 2B antisense RNA (CDKN2B-AS) gene, and this SNP was strongly associated with endometriosis in the BBJ study (
      • Uno S.
      • Zembutsu H.
      • Hirasawa A.
      • Takahashi A.
      • Kubo M.
      • Akahane T.
      • et al.
      A genome-wide association study identifies genetic variants in the CDKN2BAS locus associated with endometriosis in Japanese.
      ). However, this SNP was monomorphic in the populations in the International Endogene Consortium study (
      • Painter J.N.
      • Anderson C.A.
      • Nyholt D.R.
      • Macgregor S.
      • Lin J.
      • Lee S.H.
      • et al.
      Genome-wide association study identifies a locus at 7p15.2 associated with endometriosis.
      ). In addition, the rs16826658 SNP on chromosome 1p36, which was associated with endometriosis in the BBJ study, was not significant (P>.001) in the International Endogene Consortium study (
      • Painter J.N.
      • Anderson C.A.
      • Nyholt D.R.
      • Macgregor S.
      • Lin J.
      • Lee S.H.
      • et al.
      Genome-wide association study identifies a locus at 7p15.2 associated with endometriosis.
      ). Furthermore, Nyholt et al. (
      • Nyholt D.R.
      • Low S.K.
      • Anderson C.A.
      • Painter J.N.
      • Uno S.
      • Morris A.P.
      • et al.
      Genome-wide association meta-analysis identifies new endometriosis risk loci.
      ) conducted an extended meta-analysis using the combined data from GWAS in Australia, the United Kingdom, and Japan. They found that the rs1537377 SNP, which is located in the CDKN2B-AS gene, was in linkage disequilibrium with the rs10965235 SNP. On the basis of the reliability and reproducibility of the data produced in these GWAS, we hypothesized that the two loci, which were identified in Asian populations, may confer risk for developing endometriosis in a Korean population and investigated whether an epistatic interaction occurs between the rs10965235 and rs16826658 SNPs.

      Materials and methods

       Subjects

      Peripheral blood was obtained from a total of 1,173 patients who underwent laparoscopic surgery, exploratory laparotomy, or transabdominal hysterectomy in the gynecologic clinic at Seoul National University Hospital. All of the subjects were of Korean origin, which is a single ethnic trait. After a pathologist reviewed the specimens to confirm the histologic diagnosis of endometriosis, 673 patients with endometriosis were recruited, and 500 patients without evidence of endometriosis served as controls.
      Endometriosis was staged by clinicians according to the revised classification of the American Society for Reproductive Medicine (ASRM) (
      Revised American Society for Reproductive Medicine classification of endometriosis: 1996.
      ). A total of 124 and 549 patients were diagnosed with stage I/II and stage III/IV endometriosis, respectively. The indications for surgery among the endometriosis group included dysmenorrhea, pelvic pain, infertility, and adnexal mass. Patients with leiomyoma, adenomyosis, and invasive carcinoma of the uterine cervix or ovary were excluded from the control group. The indications for surgery in the control group included benign ovarian cyst, tubal ligation, and tubal reanastomosis. None of the subjects received hormone therapy (HT) during the previous 12 months.
      The institutional review board for human research of Seoul National University Hospital approved this study, and written informed consent was obtained from each study participant. The mean age of the patients with endometriosis was significantly lower than that of the controls (31.3 ± 5.5 years vs. 46.3 ± 5.3 years; P<.001).

       SNP Genotyping

      Genomic DNA was extracted from peripheral blood using the Wizard DNA Purification Kit (Promega). The rs10965235 SNP in the CDKN2B-AS gene and the rs16826658 SNP on chromosome 1p36 were genotyped using a minor groove binder primer/probe TaqMan assay and the ABI 7500 real-time polymerase chain reaction (PCR) system (Applied Biosystems). Primer pairs and probes were used for rs10965235 C > A (Assays-on-Demand, C__31288946_10; Applied Biosystems) and rs16826658 G > T (Assays-on-Demand, C___2454433_10; Applied Biosystems). The PCR mixture consisted of 10 μL of TaqMan Universal PCR Master Mix 2x (Applied Biosystems) and 25 ng of DNA. The PCR cycling conditions consisted of one 2-minute cycle at 50°C and one 10-minute cycle at 95°C, followed by 40 cycles at 95°C for 15 seconds and at 60°C for 1 minute. Distilled water was used as a negative PCR control in each amplification.

       Statistical Analysis

      The ages of each group of patients were normally distributed according to the Kolmogorov-Smirnov test and thus were compared using Student's t test.
      Distributions of the genotypes were examined for significant deviation from Hardy-Weinberg equilibrium using a goodness-of-fit χ2 test. The χ2 test was used to examine the differences in the proportions of genotypes between the endometriosis cases and controls. The patients were divided into groups that consisted of women with stages I/II and III/IV disease, and the distribution of the three polymorphisms in these groups was analyzed separately.
      For the analysis of epistasis, first, we extracted from all SNP samples carrying four genotype categories (CC, non-CC, GG, and non-GG). Second, we conducted logistic regression analysis on the combination of each genotype patterns in accordance with another genotype. The statistical significance was specified using the Bonferroni correction to ensure consistency in the probability of type I error caused by four independent hypotheses (Pc value=.05/4).
      All of the data analyses were performed using the Statistical Package for the Social Science software package (version 18.0, SPSS Inc.).

      Results

      Genotyping of the rs10965235 SNP in the CDKN2B-AS gene and rs16826658 near the wingless-type MMTV integration site family member 4 (WNT4) gene was successfully performed in all subjects, and the genotype distributions of both groups were in Hardy-Weinberg equilibrium. The distribution of the rs10965235 SNP in the CDKN2B-AS gene varied significantly between the endometriosis cases and the healthy controls (CC, CA, and AA genotypes; 69.7%, 26.9%, and 3.4% vs. 59.2%, 35.3% and 5.6%, respectively; P=7.16E−04). For the rs10965235 SNP in the CDKN2B-AS gene, which is monoallelic in nature in white populations but not in Japanese populations, the risk allele frequency (RAF) in the patients with endometriosis was higher than that in the controls (C allele frequency: 83.1% vs. 76.8%, respectively; P=1.30E−04, odds ratio [OR] = 1.489, 95% confidence interval [CI] 1.213–1.827).
      For further analysis, the endometriosis group was divided into subgroups according to the revised ASRM classification. We found differences in the frequency of the rs10965235 polymorphisms between patients with stage III/IV disease and the controls (P=3.40E−05), but we did not find any differences between the stage I/II patients and the controls (P=.521) (Table 1). Furthermore, a dose-response relationship was observed between the severity of endometriosis and the C RAF (stage I/II, P=.375; stage III/IV, P=3.40E−05).
      Table 1The genotypes and allele frequencies of the rs10965235 SNP in the CDKN2B-AS gene in endometriosis patients and healthy controls.
      10965235 CANo.GenotypeAllele frequency
      CC (%)CA (%)AA (%)P value
      Evaluated using the χ2 test to compare the case group with the control group.
      C (%)A (%)P value
      Evaluated using the χ2 test to compare the case group with the control group.
      OR (95% CI)
      Endometriosis cases673469 (69.7)181 (26.9)23 (3.4)7.16E−041,119 (83.1)227 (16.9)1.30E−041.489 (1.213–1.827)
       Stage I/II12480 (64.5)37 (29.8)7 (5.6).521197 (79.4)51 (20.6).375
       Stage III/IV549389 (70.9)144 (26.2)16 (2.9)2.19E−04922 (84.0)176 (16.0)3.40E−051.583 (1.272–1.968)
      Control group500296 (59.2)176 (35.2)28 (5.6)768 (76.8)232 (23.2)
      Note: CDKN2B-AS = cyclin-dependent kinase inhibitor 2B antisense RNA; CI = confidence interval; OR = odds ratio; SNP = single-nucleotide polymorphism.
      a Evaluated using the χ2 test to compare the case group with the control group.
      For the rs16826658 SNP, the GG genotype was more common in the patients with endometriosis compared with the controls (33.7% GG, 48.4% GT, and 17.8% TT vs. 25.6% GG, 49.8% GT, and 24.6% TT, respectively, P=1.73E−03). For the rs16826658 SNP, which is located approximately 16 kb upstream of the WNT4 gene, the RAF was higher in the patients with endometriosis than in the controls (G allele frequency: OR = 1.351, 95% CI 1.146–1.592, P=3.36E−04). When the patients were divided into stage I/II and stage III/IV groups, according to the revised ASRM classification, significant differences in the genotype distribution were detected between the stage III/IV patients with endometriosis and the controls (P=5.56E−04) but not between the stage I/II patients with endometriosis and the controls (P=.123). An increased risk of disease was associated with the G RAF in patients with stage III/IV endometriosis but not in patients with stage I/II endometriosis (Table 2). The combined effect of the two SNPs was borderline statistically significant after Bonferroni correction (32.8% for the patients with both genotypes vs. 25.0% for the controls, P=.021, Pc value=.0125) (Table 3).
      Table 2The genotypes and allele frequencies of the rs16826658 SNP near the WNT4 gene in endometriosis patients and healthy controls.
      16826658 GTNo.GenotypeAllele frequency
      GG (%)GT (%)TT (%)P value
      Evaluated using the χ2 test to compare the case group with the control group.
      G (%)T (%)P value
      Evaluated using the χ2 test to compare the case group with the control group.
      OR (95% CI)
      Endometriosis cases673227 (33.7)326 (48.4)120 (17.8)1.73E−03780 (57.9)566 (42.1)3.36E−041.351 (1.146–1.592)
       Stage I/II12440 (32.3)63 (50.8)21 (16.9).043143 (57.7)105 (42.3).043
       Stage III/IV549187 (34.1)263 (47.9)99 (18.0)2.78E−03637 (58.0)461 (42.0)5.56E−041.354 (1.140–1.609)
      Control group500128 (25.6)249 (49.8)123 (24.6)505 (50.5)495 (49.5)
      Note: CI = confidence interval; OR = odds ratio; SNP = single-nucleotide polymorphism; WNT4 = wingless-type MMTV integration site family member 4.
      a Evaluated using the χ2 test to compare the case group with the control group.
      Table 3The interaction between the rs10965235 and rs16826658 single-nucleotide polymorphisms in a case-control study in Korea.
      rs10965235/rs16826658No.Case (%)Controls (%)P value
      P<.0125 was considered statistically significant after the Bonferroni correction.
      CC/GG228154 (67.5)74 (32.4).021
      CC/non-GG537315 (58.7)222 (41.3)
      Non-CC/GG12773 (57.5)54 (42.5).042
      Non-CC/non-GG281131 (46.6)150 (53.4)
      a P<.0125 was considered statistically significant after the Bonferroni correction.

      Discussion

      In the present study, we observed the genotypes and RAFs of two polymorphic sites and found significant associations between the rs10965235 SNP in the CDKN2B-AS gene, the rs16826658 SNP near the WNT4 gene, and the development of endometriosis in a Korean population. A significantly higher incidence of the CC genotype of the rs10965235 SNP and the GG genotype of the rs16826658 SNP was detected in patients with endometriosis compared with the controls. Furthermore, we observed an epistatic interaction between these two SNPs that may provide valuable information about susceptibility to complex diseases (
      • Julià A.
      • Moore J.
      • Miquel L.
      • Alegre C.
      • Barceló P.
      • Ritchie M.
      • et al.
      Identification of a two-loci epistatic interaction associated with susceptibility to rheumatoid arthritis through reverse engineering and multifactor dimensionality reduction.
      ).
      In the BBJ study, Uno et al. (
      • Uno S.
      • Zembutsu H.
      • Hirasawa A.
      • Takahashi A.
      • Kubo M.
      • Akahane T.
      • et al.
      A genome-wide association study identifies genetic variants in the CDKN2BAS locus associated with endometriosis in Japanese.
      ) investigated the rs10965235 SNP, which is located in the CDKN2B-AS gene on chromosome 9P21, to determine the association between this SNP and endometriosis. The risk allele C has an allele frequency of 0.85 in the Japanese population but not in the European population (
      • Painter J.N.
      • Anderson C.A.
      • Nyholt D.R.
      • Macgregor S.
      • Lin J.
      • Lee S.H.
      • et al.
      Genome-wide association study identifies a locus at 7p15.2 associated with endometriosis.
      ). Our replication study of 673 patients and 500 controls found a significant relationship between this SNP and endometriosis, especially in patients with stage III/IV endometriosis. An increased risk of disease was associated with the CC genotype frequency in patients with stage I/II endometriosis (P=.521) and stage III/IV endometriosis (P=2.19E−04). Pagliardini et al. (
      • Pagliardini L.
      • Gentilini D.
      • Vigano P.
      • Panina-Bordignon P.
      • Busacca M.
      • Candiani M.
      • et al.
      An Italian association study and meta-analysis with previous GWAS confirm WNT4, CDKN2BAS and FN1 as the first identified susceptibility loci for endometriosis.
      ) demonstrated that the rs1333049 SNP was significantly associated with an increased OR for more severe stages of the disease compared with the general population (P=1.66E−03, OR = 1.32, 95% CI 1.11–1.57). However, this association was not observed for the rs10965235 SNP because this SNP was previously demonstrated to be monomorphic in individuals of European descent and is located in the same linkage disequilibrium block as the rs1333049 SNP.
      In a combined analysis of several SNPs near the WNT4 gene, the BBJ study revealed a possible association between endometriosis and the rs16826658 SNP on chromosome 1p36 (P=1.66E−06, OR = 1.20, 95% CI 1.11–1.29). The rs16826658 SNP is located approximately 16 kb upstream of the WNT4 gene, which is important for steroidogenesis, the development of ovarian follicles, and the development of the female reproductive tract (
      • Franco H.L.
      • Dai D.
      • Lee K.Y.
      • Rubel C.A.
      • Roop D.
      • Boerboom D.
      • et al.
      WNT4 is a key regulator of normal postnatal uterine development and progesterone signaling during embryo implantation and decidualization in the mouse.
      ). Therefore, the rs16826658 SNP may be a plausible candidate SNP for the development of endometriosis based on these biological functions. However, Painter et al. (
      • Painter J.N.
      • Anderson C.A.
      • Nyholt D.R.
      • Macgregor S.
      • Lin J.
      • Lee S.H.
      • et al.
      Genome-wide association study identifies a locus at 7p15.2 associated with endometriosis.
      ) reported that the SNP rs75210902, which is located 22 kb upstream of the WNT4 gene, was more highly associated with endometriosis in a European cohort (P=9.0E−05, OR = 1.17, 95% CI 1.08–1.25) than the rs16826658 SNP (P=3.4E−03, OR = 1.10, 95% CI 1.03–1.18). In addition, Albertsen et al. (
      • Albertsen H.M.
      • Chettier R.
      • Farrington P.
      • Ward K.
      Genome-wide association study link novel loci to endometriosis.
      ) found weak evidence of an allelic association in the rs16826658 SNP (P=.05, OR = 1.07). In contrast to these weak associations that have been reported for European groups, our results indicated a significant association between the rs16826658 SNP and endometriosis (P=1.73E−03), especially in patients with stage III/IV disease who are known to have substantially greater genetic loading (P=2.78E−03). In addition, we found a statistically significant association between the RAF distribution in the patients (RAF = 0.579) and the controls (RAF = 0.505), despite the common occurrence of the minor allele (P=3.36E−04, OR = 1.351, 95% CI 1.146–1.592). The previous Japanese GWAS data demonstrated that the rs16826658 locus had G RAFs of 0.568 and 0.522 in patients with endometriosis and controls, respectively. These allelic frequencies were associated with an increased risk of endometriosis in a Japanese population (P=1.66E−06, OR = 1.20, 95% CI 1.11–1.29) (Table 4).
      Table 4Differences in the genotype frequencies of the rs10965235 and rs16826658 single-nucleotide polymorphisms between three studies.
      10965235 CAStudy (year)Case CC (%)RAF (%)Control CC (%)RAF (%)P value
      Uno et al. (2010)1,031 (72.5)2,422 (85.1)860 (65.4)2,137 (81.3)1.52E−04
      Present study (2014)469 (69.7)1,119 (83.1)296 (59.2)768 (76.8)1.30E−04
      Painter et al. (2010)Monomorphic
      16826658 GTCase GGRAFControl GGRAFP value
      Uno et al. (2010)465 (32.7)1,632 (57.3)360 (27.3)1,364 (51.7)3.46E−05
      Albertsen et al.
      Genotype frequencies were calculated from the RAF.
      (2013)
      343 (17.0)1,664 (41.2)2,269 (15.7)11,460 (39.6).05
      Present study (2014)227 (33.7)780 (57.9)128 (25.6)505 (50.5)3.36E−04
      Note: RAF = risk allele frequency.
      a Genotype frequencies were calculated from the RAF.
      Due to the high frequency of stage III/IV disease in our case buildup because the patients were recruited in a tertiary referral center, a significant association was found between the two SNPs and stage III/IV endometriosis. However, Painter et al. (
      • Painter J.N.
      • Anderson C.A.
      • Nyholt D.R.
      • Macgregor S.
      • Lin J.
      • Lee S.H.
      • et al.
      Genome-wide association study identifies a locus at 7p15.2 associated with endometriosis.
      ) described a stronger association between these SNPs and stage III/IV patients with endometriosis compared with all of the disease stages, thus providing empirical evidence that stage III/IV cases could provide more meaningful data. It remains unclear whether minimal and mild cases of endometriosis in asymptomatic women progress through stage III/IV and whether stage I/II endometriosis represents an epiphenomenon rather than a disease entity (
      • Koninckx P.R.
      • Oosterlynck D.
      • D'Hooghe T.
      • Meuleman C.
      Deeply infiltrating endometriosis is a disease whereas mild endometriosis could be considered a non-disease.
      ).
      Assuming that our control group was completely free of endometriosis due to their older age, this case-control series may have led to a systematic overestimation of the true ORs, which may not accurately reflect the prevalence of endometriosis in the general population (
      • Hadfield R.M.
      • Manek S.
      • Weeks D.E.
      • Mardon H.J.
      • Barlow D.H.
      • Kennedy S.H.
      • et al.
      Linkage and association studies of the relationship between endometriosis and genes encoding the detoxification enzymes GSTM1, GSTT1 and CYP1A1.
      ,
      • Lee G.H.
      • Choi Y.M.
      • Kim S.H.
      • Hong M.A.
      • Ku S.Y.
      • Kim S.H.
      • et al.
      Interleukin-2 receptor beta gene C627T polymorphism in Korean women with endometriosis: a case-control study.
      ). To overcome this potential problem, we used a larger population than any of the previous study groups.
      In addition to the genotype and allele frequencies, we investigated the presence of epistasis between the two SNPs because increasing amounts of evidence have shown that gene-to-gene interactions may be relevant in determining an individual's susceptibility to complex diseases (
      • Pagliardini L.
      • Gentilini D.
      • Vigano P.
      • Panina-Bordignon P.
      • Busacca M.
      • Candiani M.
      • et al.
      An Italian association study and meta-analysis with previous GWAS confirm WNT4, CDKN2BAS and FN1 as the first identified susceptibility loci for endometriosis.
      ,
      • Cordell H.J.
      Detecting gene-gene interactions that underlie human diseases.
      ). Although patients who were carrying both the CC genotype of the rs10965235 SNP and the GG genotype of the rs16826658 SNP were more frequently found in the cases compared with the controls, only a borderline significant association was observed after the Bonferroni correction because testing of four independent hypotheses is assumed (Pc=.0125).
      In conclusion, the hypothesis that two polymorphisms (rs10965235 and rs16826658) may affect the risk of endometriosis in the Korean population was confirmed in the present study. Similar to the positive association that was found in the GWAS data from a Japanese population, the rs10965235 and rs16826658 sites were associated with endometriosis in Korean women in the present study. Further studies should be conducted on a larger population to clarify the association between these two polymorphic markers and the risk of endometriosis because genetic polymorphisms often vary among different ethnic groups.

      References

        • Giudice L.C.
        • Kao L.C.
        Endometriosis Lancet. 2004; 364: 1789-1799
        • Kim S.H.
        • Chae H.D.
        • Kim C.H.
        • Kang B.M.
        Update on the treatment of endometriosis.
        Clin Exp Reprod Med. 2013; 40: 55-59
        • Lee G.H.
        • Choi Y.M.
        • Kim S.H.
        • Hong M.A.
        • Oh S.T.
        • Lim Y.T.
        • et al.
        Association of tumor necrosis factor-{alpha} gene polymorphisms with advanced stage endometriosis.
        Hum Reprod. 2008; 23: 977-981
        • Uno S.
        • Zembutsu H.
        • Hirasawa A.
        • Takahashi A.
        • Kubo M.
        • Akahane T.
        • et al.
        A genome-wide association study identifies genetic variants in the CDKN2BAS locus associated with endometriosis in Japanese.
        Nat Genet. 2010; 42: 707-710
        • Painter J.N.
        • Anderson C.A.
        • Nyholt D.R.
        • Macgregor S.
        • Lin J.
        • Lee S.H.
        • et al.
        Genome-wide association study identifies a locus at 7p15.2 associated with endometriosis.
        Nat Genet. 2011; 43: 51-54
        • Lee G.H.
        • Kim S.H.
        • Choi Y.M.
        • Suh C.S.
        • Kim J.G.
        • Moon S.Y.
        Estrogen receptor beta gene +1730 G/A polymorphism in women with endometriosis.
        Fertil Steril. 2007; 88: 785-788
        • Lee G.H.
        • Choi Y.M.
        • Kim J.M.
        • Shin J.J.
        • Kim J.G.
        • Moon S.Y.
        Association of epidermal growth factor receptor gene polymorphisms with advanced endometriosis in a Korean population.
        Eur J Obstet Gynecol Reprod Biol. 2012; 164: 196-199
        • Nyholt D.R.
        • Low S.K.
        • Anderson C.A.
        • Painter J.N.
        • Uno S.
        • Morris A.P.
        • et al.
        Genome-wide association meta-analysis identifies new endometriosis risk loci.
        Nat Genet. 2012; 44: 1355-1359
      1. Revised American Society for Reproductive Medicine classification of endometriosis: 1996.
        Fertil Steril. 1997; 67: 817-821
        • Julià A.
        • Moore J.
        • Miquel L.
        • Alegre C.
        • Barceló P.
        • Ritchie M.
        • et al.
        Identification of a two-loci epistatic interaction associated with susceptibility to rheumatoid arthritis through reverse engineering and multifactor dimensionality reduction.
        Genomics. 2007; 90: 6-13
        • Pagliardini L.
        • Gentilini D.
        • Vigano P.
        • Panina-Bordignon P.
        • Busacca M.
        • Candiani M.
        • et al.
        An Italian association study and meta-analysis with previous GWAS confirm WNT4, CDKN2BAS and FN1 as the first identified susceptibility loci for endometriosis.
        J Med Genet. 2013; 50: 43-46
        • Franco H.L.
        • Dai D.
        • Lee K.Y.
        • Rubel C.A.
        • Roop D.
        • Boerboom D.
        • et al.
        WNT4 is a key regulator of normal postnatal uterine development and progesterone signaling during embryo implantation and decidualization in the mouse.
        FASEB J. 2011; 25: 1176-1187
        • Albertsen H.M.
        • Chettier R.
        • Farrington P.
        • Ward K.
        Genome-wide association study link novel loci to endometriosis.
        PLoS One. 2013; 8: e58257
        • Koninckx P.R.
        • Oosterlynck D.
        • D'Hooghe T.
        • Meuleman C.
        Deeply infiltrating endometriosis is a disease whereas mild endometriosis could be considered a non-disease.
        Ann N Y Acad Sci. 1994; 734: 333-341
        • Hadfield R.M.
        • Manek S.
        • Weeks D.E.
        • Mardon H.J.
        • Barlow D.H.
        • Kennedy S.H.
        • et al.
        Linkage and association studies of the relationship between endometriosis and genes encoding the detoxification enzymes GSTM1, GSTT1 and CYP1A1.
        Mol Hum Reprod. 2001; 7: 1073-1078
        • Lee G.H.
        • Choi Y.M.
        • Kim S.H.
        • Hong M.A.
        • Ku S.Y.
        • Kim S.H.
        • et al.
        Interleukin-2 receptor beta gene C627T polymorphism in Korean women with endometriosis: a case-control study.
        Hum Reprod. 2009; 24: 2596-2599
        • Cordell H.J.
        Detecting gene-gene interactions that underlie human diseases.
        Nat Rev Genet. 2009; 10: 392-404