ACE and MTHFR gene polymorphisms in unexplained recurrent pregnancy loss

Transcription

1 doi: /j x J. Obstet. Gynaecol. Res. Vol. 34, No. 3: , June 2008 ACE and MTHFR gene polymorphisms in unexplained recurrent pregnancy loss Venkatesan Vettriselvi, Krishnaswami Vijayalakshmi, Solomon F. D. Paul and Perumal Venkatachalam Department of Human Genetics, Sri Ramachandra University, Porur, Chennai, Tamil Nadu, India Abstract Aim: To assess the association between polymorphisms in angiotensin converting enzyme and methylene tetrahydrofolate reductase genes and recurrent pregnancy loss by a case-control study in South Indian women. Methods: DNA was extracted from peripheral blood leukocytes of 104 women with Recurrent Pregnancy Loss (RPL) and 120 controls. Genotyping of ACE Insertion Deletion and MTHFR C677T polymorphism were carried out by PCR and PCR-RFLP, respectively. Results: No statistically significant difference was observed in the distribution of genotypes between cases and controls for ACE and MTHFR polymorphisms. Further, the combination of MTHFR and ACE genotypes failed to reveal an association. Conclusion: In conclusion, the present study reveals lack of association of MTHFR C677T and ACE I/D polymorphisms in RPL in South Indian women. However, we cannot exclude the possibility that other polymorphisms of ACE and MTHFR genes could be associated with the disease and might be clinically useful as a marker to assess risk for RPL. Key words: ACE, MTHFR, polymorphism, recurrent pregnancy loss. Introduction Idiopathic recurrent pregnancy loss (RPL) represents a clinically challenging problem in obstetric practice and affects upto 2% of women in the reproductive age group. Though several etiological factors have been proposed, in approximately 50% of these cases, the underlying pathophysiological mechanisms remain undetermined. 1 Pregnancies require an even balance of coagulation and fibrinolysis in order to avoid excess fibrin accumulation in placental vessels and intervillous spaces as well as to secure fibrin polymerization and stabilization of the placental basal plate. 2,3 Adequate fine-tuning of fibrinolysis is mandatory in order to prevent hemorrhage and hence minimal alterations in the fibrinolysis cascade leading to either hypo- or hyperfibrinolysis are suspected to interfere with placentation and early pregnancy. 4 Hence genes regulating the vascular tone may serve as vital etiological factors. The physiological remodeling of spiral arteries throughout pregnancy is mediated by the reninangiotensin system (RAS), which is one of the main factors regulating blood pressure, fluid and electrolyte balance. 5 Angiotensin converting enzyme (ACE) encoded by the ACE gene, is a key component of the Renin Angiotensin system mediating the conversion of angiotensin I to angiotensin II. In vitro and in vivo studies indicate that ACE interferes with hemostasis through different mechanisms, including an influence on fibrinolysis, platelet aggregation and blood clotting. 6 Though the human ACE gene contains a number of variable polymorphic regions that can be of potential use in genetic analysis of populations, 7 the insertion/ deletion (I/D) polymorphism in intron 16, has been extensively investigated. During pregnancy, marked Received: August Accepted: October Reprint request to: Dr Perumal Venkatachalam, Department of Human Genetics, Sri Ramachandra University, Porur, Chennai , Tamil Nadu, India. venkip@yahoo.com 2008 The Authors 301

2 V. Vettriselvi et al. changes in hemostasis take place and ACE D/D genotype has been proposed as a new thrombophilic factor influencing pregnancy negative events. 8 In addition to the well-known vasomotor functions, reninangiotensin system is also involved in key events of the inflammatory process by increasing vascular permeability 9 and contributing to the recruitment of inflammatory cells. 10 Regarding hemostasis, several reactions are modulated by the renin-angiotensin system, and evidence exists for an association between the ACE D/D genotype and increased risk of thrombotic events. 11 Moreover, ACE by bradykinin degradation reduces nitric oxide levels, therefore contributing to endothelial dysfunction. Lately there has been a renewed interest in abnormal homocysteine metabolism; mild to moderate hyperhomocysteinemia is now recognized as an independent risk factor for occlusive arterial and venous disease. 12,13 Because RPL has been associated with diverse prothrombotic conditions, such as the presence of antiphospholipid antibodies or activated protein C resistance (factor V Leiden mutation); 14,15 it is assumed that hyperhomocysteinemia and its resulting vascular damage and procoagulant effect could play a significant role in the pathogenesis of recurrent unexplained spontaneous abortion. Despite the high number of publications describing methionine homocysteine metabolism in the recent years, the pathogenic role of increased homocysteine concentrations is poorly understood. As hyper homocysteinaemia may reflect reduced remethylation, it has been postulated that elevated total homocysteine is associated with impaired DNA methylation and gene expression, 16 leading to disturbed chorionic vasculogen. Methylenetetrahydrofolate reductase (MTHFR) is involved in the synthesis of 5- methylenetetrahydrofolate, which is a cofactor in the enzymatic formation of methionine from homocysteine. The gene encoding MTHFR has been mapped to chromosomal region 1p36.3. Mutations in MTHFR gene are associated with hyperhomocysteinemia and increased thrombotic tendency. 12,13 A polymorphism of MTHFR (C677T) leads to an alanine to valine amino acid substitution within the predicted catalytic domain of MTHFR. Based on their biological functions, ACE and MTHFR can be seen as candidate genes for recurrent pregnancy loss. In this study we attempted to establish an association between insertion deletion polymorphism in ACE gene and the C677T polymorphism in MTHFR gene and the occurrence of RPL in a south Indian population. We hypothesized that the incidence Table 1 Characteristics of cases with recurrent pregnancy loss (RPL) and controls in the South Indian population Cases (n = 104) Controls (n = 120) n % n % Age (years) Pregnancy loss of the polymorphisms would be increased among the women with RPL. Materials and Methods Subjects This case control study was performed in the city of Chennai, Tamil Nadu, during the year s RPL was defined as having a history of two or more spontaneous consecutive miscarriages less than 20 weeks of gestation, with the same partner. The medical history of women with recurrent pregnancy loss was examined and those with anatomic, hormonal, chromosomal, infectious or autoimmune causes were excluded from the study. All the females included are primary aborters with no live child. Routine diagnostic procedures such as karyotyping of the partners, torch test, identification of anti-phospholipid antibodies and hysteroscopic examination were used to rule out known causes of pregnancy loss. None of them had any pregnancy-related complications such as hypertension, thyroid abnormalities, diabetes, etc. The study comprised of 104 RPL women aged years and 120 controls aged years. The control women were volunteers with atleast two live births, without any history of abortion, endometriosis or infertility. The study was approved by the Institutional Medical ethics committee. The population characteristics of both cases and controls and the number of RPLs are presented in Table 1. DNA extraction and genotyping Blood samples were collected from the subjects with informed written consent. Genomic DNA was isolated from whole blood by the salting out method. 17 ACE genotypes were determined by PCR as previously described by Rigat et al. 7 The PCR products were The Authors

3 ACE and MTHFR polymorphisms in RPL visualized by 2% agarose gel electrophoresis and the genotype was determined by the presence or absence of 490 and 190 bp PCR amplicons. Presence of 490 bp amplicon indicates the insertion genotype and 190 bp indicates the deletion genotype, both 490 and 190 bp indicates the heterozygous insertion deletion genotype. The presence of C677T mutation in the MTHFR gene was determined by PCR followed by restriction fragment length polymorphism as described by Frosst et al. 18 The PCR product of 198 bp was digested by Hinf I (New England Biolabs), separated on a 3.5% agarose gel. The T substitution at nucleotide 677 creates a Hinf I restriction site with subsequent cleavage of the original 198 bp PCR fragment into 175 bp and a 23 bp fragments. In the absence of the mutation, no cleavage is observed. Statistical analysis The allele and genotype frequency of ACE and MTHFR genes were calculated for cases and controls. The relative risk of the variant genotypes was determined by calculating the odds ratio (OR) at 95% confidence interval (CI). Logistic regression analysis was used to calculate the age adjusted odds ratio. The risk associated with the various double genotype combinations was also assessed by binary logistic regression. Further, the genotype distribution based on age and number of abortions was determined and risk was estimated by OR at 95%CI. All the statistical analyzes were done with SPSS version 13. Results The distribution of MTHFR and ACE allele and genotype frequency among the RPL patients and controls are shown in Table 2. For the ACE polymorphisms of the 104 patients analyzed 40% had I/I genotype, 38% were I/D and 22% were D/D. Among the 120 controls 46% had I/I genotype, 31% were I/D and 23% had D/D genotype. The frequency of D allele was 0.41 in patients and 0.25 in controls. There was no significant difference in the frequency of the deletion allele between the patients and controls. The expected genotype frequencies were calculated and the genotypes of the patients as well as the controls were in Hardy Weinberg equilibrium. Of the 104 RPL patients analyzed for the MTHFR polymorphism, 83% were C/C, 14% were C/T and 3% were T/T genotype. Among the 120 controls 82% were C/C, 16% were C/T and 2% were T/T (Table 2). The frequency of C allele was 0.90 in patients and controls. Thus there was no significant difference in the frequency of mutant T allele between the patients and controls. The expected genotype frequencies were calculated and the genotypes of the patients as well as the controls were found to follow Hardy Weinberg equilibrium. To evaluate the interaction between the genotypes, we examined the combined effect of MTHFR and ACE genotypes (Table 3). Taking the risk of the combined wild type genotypes as a baseline reference category, the odds ratios were calculated for the combination of genotypes. No double genotypes revealed statistically significant association with the risk of RPL. To determine the association of MTHFR and ACE genes with age, we performed stratified analysis between cases and controls. There was no significant difference between the cases and controls based on age (Table 4). Further, the stratified analysis of ACE and MTHFR genotypes based on the number of abortions; 2 and 3 or more revealed no statistically significant difference (Table 5). Discussion Idiopathic recurrent pregnancy loss appears to be a multifactorial process and increases in frequency when Table 2 Association MTHFR and ACE genotypes with recurrent pregnancy loss Gene Genotype frequencies Age adjusted OR 95% CI Controls (n = 120) Cases (n = 104) ACE I/I 55 (46%) 42 (40%) 1.0 I/D 38 (31%) 39 (38%) D/D 27 (23%) 23 (22%) MTHFR C/C 98 (82%) 86 (83%) 1.0 C/T 19 (16%) 15 (14%) T/T 3 (2%) 3 (3%) The Authors 303

4 V. Vettriselvi et al. Table 3 Distribution of the various double genotype combinations of MTHFR and ACE genes and risk of RPL MTHFR ACE Controls (n = 120) Cases (n = 104) OR 95% CI CC I/I 46 (38%) 35 (35%) I/D 33 (28%) 31 (31%) D/D 19 (16%) 20 (19%) CT I/I 8 (7%) 6 (6%) I/D 4 (3%) 6 (6%) D/D 7 (5%) 3 (3%) TT I/I 1 (1%) 1 (1%) I/D 1 (1%) 2 (2%) D/D 1 (1%) 0 Table 4 Distribution of MTHFR and ACE genotypes based on age Gene Genotype Age OR 95% CI Controls (%) Cases (%) MTHFR 30 n = 48 n = 83 C/C 40 (83%) 71 (85%) 1.0 C/T 5 (11%) 10 (12%) T/T 3 (6%) 2 (3%) >30 n = 72 n = 21 C/C 58 (81%) 15 (72%) 1.0 C/T 14 (19%) 5 (23%) T/T 0 1 (5%) ACE 30 n = 48 n = 83 I/I 20 (42%) 36 (43%) 1.0 I/D 19 (40%) 29 (35%) D/D 9 (18%) 18 (22%) >30 n = 72 n = 21 I/I 18 (25%) 5 (24%) 1.0 I/D 35 (49%) 6 (29%) D/D 19 (26%) 10 (47%) Table 5 Association of MTHFR and ACE genotypes and number of abortions Gene Genotype Number of abortions OR 95% CI 2(n = 30) 3 (n = 74) MTHFR C/C C/T T/T ACE I/I I/D D/D there are two or more concomitant thrombophilic defects. This is the first report on the investigation of frequency and interaction of the MTHFR C677T and ACE I/D polymorphism among the women with RPL in a South Indian population. Our data, however, fall short of showing any association between the examined polymorphism and the occurrence of RPL. The transformation of spiral arteries to large vessels of low resistance has been reported as a crucial event for the viability of the embryo in early pregnancy. In the present study we did not find any significant association between the ACE genotypes and RPL. This is in conflict with the earlier studies. Data by Fatini et al. shows the ACE I/D polymorphism to be a stronger risk The Authors

5 ACE and MTHFR polymorphisms in RPL factor for RPL. 19 Buchholz et al. reported that the homozygosity for the D allele of ACE results in elevated PAI-1 concentration and hypofibrinolysis and thus associated with an elevated risk of RPL in Caucasians. 3 Concerning the MTHFR C677T polymorphism, we found no significant difference in genotype distribution for the MTHFR C677T mutation between patients with recurrent unexplained pregnancy loss and healthy controls. Earlier studies have provided inconsistent results regarding the association of RPL and MTHFR C677T genotypes. Nelen et al. reported an increased prevalence of the homozygous MTHFR C677T genotype among Dutch women with 2 consecutive spontaneous miscarriages. 20 Quere et al. in a retrospective case-control study found a similar prevalence (20%) of homozygosity for MTHFR C677T among French patients with recurrent unexplained spontaneous abortion. 21 In contrast, in a recent case-control study that compared 94 Italian patients with 150 controls, Grandone et al. did not find a difference between the groups in the distribution of genotype frequencies for the MTHFR mutation. 22 In a recent meta-analysis on six studies, only one found MTHFR T/T genotype to be a significant risk factor for idiopathic recurrent miscarriage. The remaining five studies gave non-significant ORs. However, the pooled estimate for the MTHFR T/T genotype was significant with an OR of 1.4 (95% CI ). 23 This could be explained by the fact that the role MTHFR C677T homozygosity in the pathogenesis of venous or arterial thrombosis, although related to high homocysteine levels, is controversial. 12 On the other hand, hyperhomocysteinemia is a situation that is easily corrected by the use of folic acid during pregnancy, thus confounding the results. It is reasonable to speculate that disturbances in the folate metabolism act as cofactors in a multifactorial disease process. In summary our study falls short of proving our hypothesis, however, it is the limitation of our study that we cannot specify at what level an increase or decrease in protein expression regulated by the investigated genes could affect the risk of developing embryo. Based on their biological functions of the two genes investigated and the negative results obtained, we doubt that investigating these polymorphisms in the embryonic/trophoblast tissue would provide any additional information. However, we cannot exclude the possibility that other polymorphisms of Angiotensinogen and MTHFR genes and the combination of these polymorphisms could be associated with the disease and might be clinically useful as a marker to assess women s risk for RPL. References 1. Stephenson MD. Frequency of factors associated with habitual abortion in 197 couples. Fertil Steril 1996; 66: Preston FE, Rosendaal FR, Walker ID. Increased fetal loss in women with heritable thrombophilia. Lancet 1996; 348: Buchholz T, Thaler CJ. Inherited thrombophilia: impact on human reproduction. Am J Reprod Immunol 2003; 49: Hickey M, Fraser IS. Clinical implications of disturbances of uterine vascular morphology and function. 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