Polymorphisms in MMP-2 and MMP-9 promoter regions are associated with endometriosis
2010; Elsevier BV; Volume: 94; Issue: 4 Linguagem: Inglês
10.1016/j.fertnstert.2009.12.024
ISSN1556-5653
AutoresMerli Saare, Merit Lamp, Tanel Kaart, Helle Karro, Ülle Kadastik, Andres Metspalu, Maire Peters, Andres Salumets,
Tópico(s)Apelin-related biomedical research
ResumoIn this case-control study, we investigated the potential associations of MMP-2 and MMP-9 gene promoter region polymorphisms as well as MMP-2 promoter haplotypes with susceptibility to endometriosis in women of caucasian origin. The results demonstrated that polymorphisms in MMP-2 (−735 C/T) and MMP-9 (−1562 C/T) were associated with elevated risk of endometriosis and that certain MMP-2 promoter haplotypes were more common in control group. In this case-control study, we investigated the potential associations of MMP-2 and MMP-9 gene promoter region polymorphisms as well as MMP-2 promoter haplotypes with susceptibility to endometriosis in women of caucasian origin. The results demonstrated that polymorphisms in MMP-2 (−735 C/T) and MMP-9 (−1562 C/T) were associated with elevated risk of endometriosis and that certain MMP-2 promoter haplotypes were more common in control group. Endometriosis is a multifactorial and polygenic gynecologic disorder that can have a negative impact on a woman's health and reproductive potential. Although endometriosis occurs in 6%–10% of women of reproductive age, the prevalence among infertile patients is 2–5 times higher (1Eskenazi B. Warner M. Epidemiology of endometriosis.Obstet Gynecol Clin North Am. 1997; 24: 235-258Abstract Full Text Full Text PDF PubMed Scopus (1130) Google Scholar). Sampson's implantation theory of the origin of endometriosis (2Sampson J. Peritoneal endometriosis due to menstrual dissemination of endometrial tissue into the peritoneal cavity.Am J Obstet Gynecol. 1927; 14: 422-469Abstract Full Text PDF Google Scholar) postulates that during menstruation, endometrial cells reflux into the abdomen and form ectopic endometriotic lesions. This process involves the physiologic processes of adhesion, proliferation, and angiogenesis. Additionally, complete remodeling of the extracellular matrix (ECM) is necessary for the ectopic growth of endometrial implants, which requires the presence of matrix metalloproteinases (MMPs). Endometrial stromal cells express several MMPs, including MMP-2 and MMP-9, which seem to play a key role in endometrial ECM breakdown. Altered expression of these proteinases may lead to the establishment and progression of endometriosis, because aberrant MMP-2 and MMP-9 gene expression has been reported in eutopic and ectopic endometrial tissue of endometriosis patients (3Hulboy D.L. Rudolph L.A. Matrisian L.M. Matrix metalloproteinases as mediators of reproductive function.Mol Hum Reprod. 1997; 3: 27-45Crossref PubMed Scopus (406) Google Scholar, 4Chung H.W. Wen Y. Chun S.H. Nezhat C. Woo B.H. Lake Polan M. Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-3 mRNA expression in ectopic and eutopic endometrium in women with endometriosis: a rationale for endometriotic invasiveness.Fertil Steril. 2001; 75: 152-159Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 5Shaco-Levy R. Sharabi S. Benharroch D. Piura B. Sion-Vardy N. Matrix metalloproteinases 2 and 9, E-cadherin, and [beta]-catenin expression in endometriosis, low-grade endometrial carcinoma and nonneoplastic eutopic endometrium.Eur J Obst Gynecol Reprod Biol. 2008; 139: 226-232Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 6DiCarlo C. Bonifacio M. Tommaselli G. Bifulco G. Guerra G. Nappi C. Metalloproteinases, vascular endothelial growth factor, and angiopoietin 1 and 2 in eutopic and ectopic endometrium.Fertil Steril. 2009; 91: 2315-2323Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Earlier studies have shown that some single-nucleotide polymorphisms (SNPs) in the promoter regions of MMP-2 and MMP-9 lead to changes in gene expression levels and thus could be associated with predisposition to a variety of diseases (7Yu C. Zhou Y. Miao X. Xiong P. Tan W. Lin D. Functional haplotypes in the promoter of matrix metalloproteinase-2 predict risk of the occurrence and metastasis of esophageal cancer.Cancer Res. 2004; 64: 7622-7628Crossref PubMed Scopus (180) Google Scholar, 8Kang S. Zhao X. Xing H. Wang N. Zhou R. Chen S. et al.Polymorphisms in the matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 and the risk of human adenomyosis.Environ Mol Mutagen. 2008; 49: 226-231Crossref PubMed Scopus (24) Google Scholar, 9Zhang B. Ye S. Herrmann S.M. Eriksson P. de Maat M. Evans A. et al.Functional polymorphism in the regulatory region of gelatinase B gene in relation to severity of coronary atherosclerosis.Circulation. 1999; 99: 1788-1794Crossref PubMed Scopus (534) Google Scholar). Four promoter SNPs (−735 C/T, −790 T/G, −1306 C/T, and −1575 G/A) have been described for MMP-2. The minor alleles of the −735 C/T and −1306 C/T SNPs were found to cause diminished promoter activity due to disruption of the transcription factor Sp1 binding element (7Yu C. Zhou Y. Miao X. Xiong P. Tan W. Lin D. Functional haplotypes in the promoter of matrix metalloproteinase-2 predict risk of the occurrence and metastasis of esophageal cancer.Cancer Res. 2004; 64: 7622-7628Crossref PubMed Scopus (180) Google Scholar, 10Price S. Greaves D. Watkins H. Identification of novel, functional genetic variants in the human matrix metalloproteinase-2 gene: role of Sp1 in allele-specific transcriptional regulation.J Biol Chem. 2001; 276: 7549-7558Crossref PubMed Scopus (359) Google Scholar). Also, the transcriptional activity of MMP-9 is reportedly influenced by the −1562 C/T SNP within the promoter region (9Zhang B. Ye S. Herrmann S.M. Eriksson P. de Maat M. Evans A. et al.Functional polymorphism in the regulatory region of gelatinase B gene in relation to severity of coronary atherosclerosis.Circulation. 1999; 99: 1788-1794Crossref PubMed Scopus (534) Google Scholar). Therefore, it is reasonable to assume that genetic variations in MMP-2 and MMP-9 promoter regions contribute to the predisposition to endometriosis. The aim of the present case-control study was to investigate associations between the risk of developing endometriosis and three SNPs (−735 C/T, −790 T/G, and −1575 G/A) in the MMP-2 promoter region and one SNP (−1562 C/T) in the MMP-9 promoter region, as well as to describe MMP-2 promoter haplotypes in women of caucasian origin. A total of 150 women (18–45 years old) with surgically and histologically confirmed endometriosis were recruited into the study from Tartu University Hospital's Women's Clinic and Nova Vita Clinic between February 2005 and February 2008. Disease stages according to the American Society for Reproductive Medicine revised classification system (11American Society for Reproductive MedicineRevised American Society for Reproductive Medicine classification of endometriosis.Fertil Steril. 1997; 67: 817-821Abstract Full Text PDF PubMed Scopus (2201) Google Scholar) were as follows: stage I-II: 92 patients; stage III-IV: 58 patients. Peripheral blood samples and a questionnaire of clinical data were collected from all participants. One hundred ninety-nine healthy women (30–50 years old) with proven fertility (at least two children) and no medical history of endometriosis were enrolled in the study as control subjects. Their DNA samples and medical records were obtained from the Estonian Genome Project. The study was approved by the Ethics Review Committee on Human Research of the University of Tartu (Tartu, Estonia), and informed consent was obtained from each participant. Three SNPs in the MMP-2 gene promoter region, rs2285053 (−735 C/T), rs243864 (−790 T/G), rs243866 (−1575 G/A), and one SNP in the MMP-9 gene promoter region, rs3918242 (−1562 C/T), were genotyped using polymerase chain reaction–based restriction fragment length polymorphism analysis. Primer sequences used are available upon request. All statistical tests were carried out using SPSS 17.0 software (SPSS, Chicago, IL). Student t test and χ2 test were used to compare the general characteristics of women and genotype frequencies between the study groups. Logistic regression analyses were used to evaluate the effect of genotype on risk for endometriosis. A P value of <.05 was considered to be statistically significant. Haplotypes for the MMP-2 promoter region were inferred using Phase software, version 2.1 (12Stephens M. Smith N. Donnelly P. A new statistical method for haplotype reconstruction from population data.Am J Hum Genet. 2001; 68: 978-989Abstract Full Text Full Text PDF PubMed Scopus (6338) Google Scholar, 13Stephens M. Scheet P. Accounting for decay of linkage disequilibrium in haplotype inference and missing data imputation.Am J Hum Genet. 2005; 76: 449-462Abstract Full Text Full Text PDF PubMed Scopus (1112) Google Scholar) and the Haploview program, version 4.0 (http://www.broad.mit.edu/mpg/haploview). The comparison of general characteristics revealed that the mean ± SD age was 32.1 ± 6.1 years for the endometriosis group and 39.8 ± 5.3 years for the control group (P<.0001). Women with endometriosis were taller than control subjects (167.2 ± 5.7 vs. 165.4 ± 5.4 cm, respectively; OR 1.06 for 1 cm increase, 95% CI 1.02–1.11; P=.003). They were also more likely to be nonsmokers compared with women in the control group (86.0% vs. 69.2%, respectively; active smoking vs. nonsmoking OR 0.37, 95% CI 0.21–0.63; P<.001). We determined the genotype frequencies for the MMP-2 −735 C/T, −790 T/G, and −1575 G/A and MMP-9 −1562 C/T polymorphisms. We found no significant differences in genotype frequencies of the MMP-2 −790 T/G and the −1575 G/A SNP between the patients and control subjects (data not shown). Logistic regression analysis showed that individuals with MMP-2 −735 CC genotype had almost twice the risk of endometriosis compared with women with TT or TC genotype (Table 1). By dividing patients according to the disease severity, we observed that women with MMP-2 −735 CC genotype had an elevated chance of developing stage I-II endometriosis compared with women with the TT or TC genotype (Table 1). The effect of the CC genotype on the risk of stage III-IV disease did not reach statistical significance.Table 1Frequency of MMP-2 and MMP-9 genotypes in control subjects and patients with endometriosis.Control subjectsAll patientsPatients with stage I-II endometriosisPatients with stage III-IV endometriosisGenotypen (%)n (%)OR (95% CI)P valuen (%)OR (95% CI)P valuen (%)OR (95% CI)P valueMMP-2 −735 CC142 (71.4)124 (82.7)1.95 (1.13–3.35).0278 (84.8)2.26 (1.17–4.38).0246 (79.3)1.57 (0.76–3.24)NS TT+TC57 (28.6)26 (17.3)Reference—14 (15.2)Reference—12 (20.7)Reference—MMP-9 −1562 TT3 (1.5)6 (4.0)3.61 (0.83–15.76)NS2 (2.2)1.75 (0.27–11.3)NS4 (6.9)7.73 (1.53–38.90).01 TC50 (25.1)44 (29.3)1.29 (0.79–2.13)NS24 (26.1)1.06 (0.59–1.89)NS20 (34.5)1.77 (0.91–3.42)NS TT+TC53 (26.6)50 (33.3)1.41 (0.87–2.28)NS26 (28.3)1.10 (0.62–1.93)NS24 (41.4)2.04 (1.09–3.83).03 CC146 (73.4)100 (66.7)Reference—66 (71.7)Reference—34 (58.6)Reference—Note: The number of patients was compared with the number of control subjects. Odds ratio was calculated using logistic regression analysis adjusted for height and smoking status (nonsmokers vs. smokers). CI = confidence interval; NS = not significant; OR = odds ratio. Open table in a new tab Note: The number of patients was compared with the number of control subjects. Odds ratio was calculated using logistic regression analysis adjusted for height and smoking status (nonsmokers vs. smokers). CI = confidence interval; NS = not significant; OR = odds ratio. All of the studied SNPs from the MMP-2 promoter region were in strong linkage disequilibrium (LD) and formed a single haploblock. Eight haplotypes were defined for the MMP-2 promoter region; three of them (HAP1–HAP3) occurred in 94.6% of subjects. The frequency of the most common haplotype, HAP1 (−735 C, −790 T, and −1575 G alleles), was significantly higher (P=.02) among women with endometriosis (65.8%) than control subjects (56.9%), but after a permutation test (n = 1000) it no longer reached statistical significance. The occurrences of HAP4 (−735 C, −790 G, and −1575 G alleles) and HAP5 (−735 C, −790 T, and −1575 A alleles) in the endometriosis group were 0.1% for both haplotypes and were significantly higher (3.4% and 3.3% for HAP4 and HAP5, respectively) in the control group (P<.01). The statistical significance of HAP4 and HAP5 remained relevant also after a permutation test (P=.01 for both haplotypes). The distribution of MMP-9 −1562 C/T genotypes was similar in endometriosis patients and control subjects (Table 1). However, after classifying patients into subgroups according to the disease severity, the MMP-9 −1562 TT genotype increased the odds ratio of stage III-IV endometriosis almost eightfold relative to the CC genotype (Table 1). When the TT and TC genotypes were combined, the risk of stage III-IV endometriosis was still two times higher than that in women with the major CC genotype. Only one earlier study has investigated the involvement of MMP-2 −735 C/T SNP in the development of endometriosis (14Kang S. Zhao X.W. Wang N. Chen S.C. Zhou R.M. Li Y. Association of polymorphisms of the MMP-2 and TIMP-2 genes with the risk of endometriosis in North Chinese women.Fertil Steril. 2008; 90: 2023-2029Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). In a population of women from North China, Kang et al. (14Kang S. Zhao X.W. Wang N. Chen S.C. Zhou R.M. Li Y. Association of polymorphisms of the MMP-2 and TIMP-2 genes with the risk of endometriosis in North Chinese women.Fertil Steril. 2008; 90: 2023-2029Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) did not observe any significant association between this variation and endometriosis. In contrast, we demonstrated that in a population of caucasian women, the CC genotype of the MMP-2 −735 C/T SNP results in almost twice the risk for endometriosis compared with women having either the TT or the TC genotype. This genotype was also clearly associated with an elevated risk of stage I-II endometriosis. Although the relationship between MMP-2 −735 C/T and endometriosis is only beginning to be defined, earlier in vivo and in vitro studies have associated the MMP-2 −735 C allele with increased promoter activity and susceptibility to certain cancers and heart failure (7Yu C. Zhou Y. Miao X. Xiong P. Tan W. Lin D. Functional haplotypes in the promoter of matrix metalloproteinase-2 predict risk of the occurrence and metastasis of esophageal cancer.Cancer Res. 2004; 64: 7622-7628Crossref PubMed Scopus (180) Google Scholar, 15Rollin J. Régina S. Vourc'h P. Iochmann S. Bléchet C. Reverdiau P. et al.Influence of MMP-2 and MMP-9 promoter polymorphisms on gene expression and clinical outcome of nonsmall cell lung cancer.Lung Cancer. 2007; 56: 273-280Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 16Vasku A. Goldbergova M. Holla L.I. Spinarova L. Spinar J. Vitovec J. et al.Two MMP-2 promoter polymorphisms (−790T/G and −735C/T) in chronic heart failure.Clin Chem Lab Med. 2003; 41: 1299-1303Crossref PubMed Scopus (31) Google Scholar). Those studies along with the present results suggest that the MMP-2 −735 CC genotype may increase the risk of endometriosis owing to locally higher MMP-2 transcriptional and enzymatic activities facilitating the invasion and survival of endometriotic implants in ectopic locations. The other two MMP-2 SNPs, −790 T/G and −1575 G/A, are neutral SNPs that have little to no effect on the promoter's activity (10Price S. Greaves D. Watkins H. Identification of novel, functional genetic variants in the human matrix metalloproteinase-2 gene: role of Sp1 in allele-specific transcriptional regulation.J Biol Chem. 2001; 276: 7549-7558Crossref PubMed Scopus (359) Google Scholar), and, based on our results, they are not relevant to endometriosis risk. Similar to three earlier studies (7Yu C. Zhou Y. Miao X. Xiong P. Tan W. Lin D. Functional haplotypes in the promoter of matrix metalloproteinase-2 predict risk of the occurrence and metastasis of esophageal cancer.Cancer Res. 2004; 64: 7622-7628Crossref PubMed Scopus (180) Google Scholar, 14Kang S. Zhao X.W. Wang N. Chen S.C. Zhou R.M. Li Y. Association of polymorphisms of the MMP-2 and TIMP-2 genes with the risk of endometriosis in North Chinese women.Fertil Steril. 2008; 90: 2023-2029Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 17Vasků A. Goldbergová M. Hollá L.I. Sisková L. Groch L. Beránek M. et al.A haplotype constituted of four MMP-2 promoter polymorphisms (−1575G/A, −1306C/T, −790T/G and −735C/T) is associated with coronary triple-vessel disease.Matrix Biol. 2004; 22: 585-591Crossref PubMed Scopus (66) Google Scholar), we also observed significant LD between all three SNPs studied in the MMP-2 promoter region. We identified three main MMP-2 haplotypes accounting for >90% of all haplotypes studied. The major haplotype, HAP1, was more common in patients, whereas haplotypes HAP4 and HAP5 were more frequent in control subjects. Similar findings were reported by Vasků et al. (17Vasků A. Goldbergová M. Hollá L.I. Sisková L. Groch L. Beránek M. et al.A haplotype constituted of four MMP-2 promoter polymorphisms (−1575G/A, −1306C/T, −790T/G and −735C/T) is associated with coronary triple-vessel disease.Matrix Biol. 2004; 22: 585-591Crossref PubMed Scopus (66) Google Scholar) in their study of four MMP-2 polymorphisms (−735 C/T, −790 T/G, −1306 C/T, and −1575 G/A), in which a significant association was found between the HAP1 haplotype and an elevated risk of coronary triple-vessel disease. Furthermore, Vasků et al. (17Vasků A. Goldbergová M. Hollá L.I. Sisková L. Groch L. Beránek M. et al.A haplotype constituted of four MMP-2 promoter polymorphisms (−1575G/A, −1306C/T, −790T/G and −735C/T) is associated with coronary triple-vessel disease.Matrix Biol. 2004; 22: 585-591Crossref PubMed Scopus (66) Google Scholar) identified two haplotypes similar to our HAP4 and HAP5 which were absent in patients but present in the control group. Because the HAP4 and HAP5 haplotypes were more prevalent among control subjects in the present study, we can assume that the presence of these haplotypes may protect women from developing endometriosis. In vitro expression studies on the MMP-9 −1562 C/T polymorphism have confirmed that the allelic change of C to T results in loss of a binding site for a transcription repressor protein, resulting in a doubling of promoter activity (9Zhang B. Ye S. Herrmann S.M. Eriksson P. de Maat M. Evans A. et al.Functional polymorphism in the regulatory region of gelatinase B gene in relation to severity of coronary atherosclerosis.Circulation. 1999; 99: 1788-1794Crossref PubMed Scopus (534) Google Scholar). In the present study, we found that the distribution of MMP-9 −1562 C/T genotypes were similar across both groups of women. However, when patients were divided into subgroups according to the disease severity, a significant association was observed between the occurance of the TT or TC genotype and stage III-IV endometriosis. Therefore we hypothesize that the presence of T allele in MMP-9 −1562 C/T polymorphism could indicate the development of only severe forms of endometriosis or create a precondition for progression of minimal-mild to moderate-severe form of endometriosis. However, this finding conflicts with two earlier studies that showed no significant relationship between the −1562 C/T SNP and stage III-IV endometriosis in Asian women (18Han Y.J. Kim H.N. Yoon J.K. Yi S.Y. Moon H.S. Ahn J.J. et al.Haplotype analysis of the matrix metalloproteinase-9 gene associated with advanced-stage endometriosis.Fertil Steril. 2009; 91: 2324-2330Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 19Shan K. Lian-Fu Z. Hui D. Wei G. Na W. Xia J. et al.Polymorphisms in the promoter regions of the matrix metalloproteinases-7, -9 and the risk of endometriosis and adenomyosis in China.Mol Hum Reprod. 2006; 12: 35-39Crossref PubMed Scopus (30) Google Scholar). Still, discrepancies between the studies could be due to population differences. The interpretation of the present study results is somewhat limited, because we included only fertile women with ≥2 children and no medical history of endometriosis in the control group. Therefore, we cannot entirely exclude the presence of undiagnosed asymptomatic endometriosis patients among the control women. However, we believe that the inclusion of undiagnosed cases could lead more likely to false-negative than false-positive associations, with a marginal dilution of the effects of the risk factors. In conclusion, the present case-control study demonstrated that individual variations in MMP-2 and MMP-9 promoter regions as well as certain MMP-2 promoter haplotypes may influence the susceptibility to endometriosis in a caucasian population.
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