We investigated polymorphisms of plasminogen activator inhibitor-1 (PAI-1), angiotensin converting enzyme (ACE ) and coagulation factor XIII (FXIII) genes and their association with recurrent spontaneous abortion (RSA) in Iranian patients and normal healthy controls. Ten (18.5%) patients were homozygote (4G/4G) for PAI-1 polymorphism, in contrast with two (2%) controls (p = 0.001). Patients with homozygote 4G mutation were significantly more prone to RSA in contrast to others (odds ratio: 11.0, 95% CI: 2.3-52.4). Nineteen (30.2%) patients and 25 (26.6%) controls were homozygote (DD) for ACE polymorphism. We observed only two patients and one control with homozygosity (34leu) for FXIII polymorphism. 4G/4G polymorphism for PAI-1 gene could be a thrombophilic mutation leading to abortion in Iranian population

Aarabi, M.

Akhondi, M.A.

Arefi, S.

Memariani, T.

Modarressi, M.H.

Zadeh, S.H.

English

Golestan University of Medical Sciences Repository

SHORT REPORTPolymorphisms of plasminogen activator inhibitor-1, angiotensin convertingenzyme and coagulation factor XIII genes in patients with recurrent spontaneousabortionMAHMOUD AARABI1,2, TOKTAM MEMARIANI1, SOHEILA AREFI3, MOHSEN AARABI4,SEDIGHEH HANTOOSH ZADEH5, MEHDI A. AKHONDI1, & MOHAMMAD H. MODARRESSI61Department of Reproductive Genetics, Reproductive Biotechnology Research Center, Avicenna Research Institute, Tehran, Iran,2Department of Anatomy and Cell Biology, Queen’s University, Kingston, ON, Canada, 3Department of ReproductiveEndocrinology, Avicenna Research Institute, Tehran, Iran, 4Department of Social Medicine, Golestan University of Medical Sciences,Gorgan, Iran, 5Department of Obstetrics & Gynecology, Tehran University of Medical Sciences, Tehran, Iran, and 6Department ofMedical Genetics, Tehran University of Medical Sciences, Tehran, Iran(Received 4 January 2010; revised 4 July 2010; accepted 22 July 2010)AbstractWe investigated polymorphisms of plasminogen activator inhibitor-1 (PAI-1), angiotensin converting enzyme (ACE ) andcoagulation factor XIII (FXIII) genes and their association with recurrent spontaneous abortion (RSA) in Iranian patientsand normal healthy controls. Ten (18.5%) patients were homozygote (4G/4G) for PAI-1 polymorphism, in contrast with two(2%) controls (p¼ 0.001). Patients with homozygote 4G mutation were significantly more prone to RSA in contrast toothers (odds ratio: 11.0, 95% CI: 2.3–52.4). Nineteen (30.2%) patients and 25 (26.6%) controls were homozygote (DD) forACE polymorphism. We observed only two patients and one control with homozygosity (34leu) for FXIII polymorphism.4G/4G polymorphism for PAI-1 gene could be a thrombophilic mutation leading to abortion in Iranian population.Keywords: Recurrent spontaneous abortion, thrombophilia, plasminogen activator inhibitor-1, angiotensin converting enzyme,coagulation factor XIIIIntroductionRecurrent spontaneous abortion (RSA) is among the mostcommon complications of pregnancy, affecting more than5% of women. It might cause considerable emotionaldistress in couples who desire to have children. Theetiology of RSA remains unexplained in many patients, butabout 55% of patients are suspected to be associated withthrombophilic defects [1].Thrombophilia can be characterized as an increasedtendency towards thrombosis through enhanced coagula-tion. It includes several rare inherited abnormalities, oftenleading to thrombosis in young people [2]. Thrombophiliais shown to be associated with venous thromboembolism,deep vein thrombosis, pulmonary embolism, myocardialinfarction and cerebral vein thrombosis [1,2].Pregnancy is stable when there is a balance betweenmaternal coagulation and fibrinolysis. This balance pre-vents excess fibrin deposition in placental vessels andintravillous spaces, secures fibrin polymerization andstabilizes the placental basal plate [3]. Women withthrombophilic defects are at increased risk, not only forpregnancy associated thromboembolism, but also for othervascular complications, such as pre-eclampsia and fetalloss [2].Homeostasis abnormalities could be due to heritable oracquired defects which lead to clinical thrombophilia.Inherited abnormalities include mutations of either thegenes encoding natural anticoagulants like antithrombinIII, proteins C and S [1], or clotting factors likeprothrombin, factor V Leiden (FVL) and methylenetetra-hydrofolate reductase (MTHFR) [4].In addition to above genetic defects, polymorphisms ofplasminogen activator inhibitor-1 (PAI-1), coagulationfactor XIII (FXIII) and angiotensin converting enzyme(ACE) genes have been mentioned. These genes appear tobe a cause of impaired fibrinolysis which could promotethe development of RSA [5].PAI-1 has a vital role in hypofibrinolysis and thromboticcomplications. The gene expression is modulated by a4G/5G polymorphism in the promoter region, which islocated 675 bp upstream from the start site of transcription[6]. The 4G allele binds to transcription-regulating factorsand produces more PAI-1 protein than 5G allele. Highlevels of PAI-1 concentration could result in increasedthrombosis through the defect in fibrinolysis and coagula-tion. This can contribute to initiation of placental damageand thrombotic complication [7].ACE regulates thrombosis through generating angioten-sin II from angiotensin I [2]. In addition, ACE is a keycomponent of the rennin–angiotensin system and affectshomeostasis [3].The level of enzyme is related to poly-morphism of ACE gene consisting of the insertion/deletion (I/D) of a 287-bp fragment in intron 16 [8].Correspondence: M. H. Modarressi, MD, PhD. Department of Medical Genetics, Tehran University of Medical Sciences, Tehran, Iran PO Box: 14155-6447.Tel: þ98-21-8895-3005. Fax: þ98-21-8895-3005. E-mail: modaresi@tums.ac.irMahmoud Aarabi and Toktam Memariani contributed equally to this work.The Journal of Maternal-Fetal and Neonatal Medicine, March 2011; 24(3): 545–548ISSN 1476-7058 print/ISSN 1476-4954 online  2011 Informa UK, Ltd.DOI: 10.3109/14767058.2010.511331J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by University of Glasgow on 05/06/13For personal use only.Blood FXIII plays an important role in fibrin stabiliza-tion and protection of fibrin from proteolytic degradation.It is a pro-transglutaminase of tetrameric structure (A2B2)consisting of two potentially active A subunits (FXIII-A)and two inhibitory/protective B subunits (FXIII-B) [7].The Val34Leu polymorphism in exon 2 of the FXIII-Agene could have an antifibronolytic effect through the earlycross-linking of fibrin fibers [9].In this study, we have investigated the association ofRSA and PAI-1 4G/5G, ACE D/I and FXIII Val 34 Leupolymorphisms in Iranian patients and healthy controls.MethodsPatientsPatients with a history of spontaneous abortion and healthywomen without history of abortion as control group wereregistered in Avicenna Infertility Clinic and Department ofObstetrics & Gynecology of Imam Hospital, Tehran, Iran.The inclusion criterion was having the experience of atleast three unexplained consecutive spontaneous abortionsbefore 25 weeks of gestation. We also studied the patientswith two unexplained consecutive spontaneous abortionsbefore 25 weeks of gestation. For all patients, we collectedmedical history as well as results of physical examination,routine laboratory, endocrinologic and immunologicaltests for autoantibodies from the database of above centers.We excluded patients with known risk factors for sponta-neous abortion: anatomical abnormalities, endocrinologicdysfunctions, autoimmune diseases, urogenital infectionand inflammatory pelvic disease. All patients and normalhealthy controls were Iranian.Approval from the Avicenna Research Institute’s ethics &human rights committee was obtained for the use of bloodsamples and study protocol. Before study, all participantssigned the informed consent form.Determination of genotypeTo analyze the D/I polymorphism in intron 16 of the ACEgene and the 7675 4G/5G polymorphism in the promoterregion of the PAI-1 gene and Val 34 Leu FXIIIpolymorphisms, 5 ml of blood samples were obtained.Genomic DNA was then extracted from leukocytes usingsalting out standard protocol. Genomic DNA was ampli-fied by polymerase chain reaction (PCR) using gene-specific primers (Figure 1).Each 25 ml of reaction contained 2.5 ml of 106PCRreaction buffer (100 mM Tris-HCl, 500 mM KCl pH:8.3), 1.5 ml MgCl2 (25 mM), 1 ml of forward and reverseprimers (10 mM), 1U DNA polymerase (5U, ROCHE,Mannheim, Germany) and 150 ng of DNA. After dena-turation at 948C for 5 min, DNA fragments were eitheramplified for 35 cycles for FXIII (948C for 30 s, 588C for30 s and 728C for 30 s), 35 cycles for ACE (948C for 30 s,708C for 40 s and 728C for 1 min), and 32 cycles for PAI-1polymorphisms (948C for 30 s, 608C for 30 s and 728C for1 min). PCR cycles were followed by final extension step at728C for 7 min.For the amplification of PAI-1 gene, we designed aforward primer different from the native sequence in orderto introduce a site for BseR I restriction enzyme.Originally, the 148 bp PCR product has no restriction sitefor enzyme, but combination of nucleotide substitutionand genetic abnormality creates the enzyme cleavage siteand cleaves the amplicon into two fragments (110 bp and38 bp). The D/I genotype of the ACE gene was describedby 190 bp PCR product length in deletion, as well as490 bp in insertion. Finally, PCR product of FXIII Val 34Leu polymorphism had a length of 192 bp with norestriction site for DdeI enzyme. The mutation in FXIIIcreated a restriction site and produced two fragments(161 bp and 31 bp) after enzyme digestion.PCR products were separated on 1.5% agarose gel andvisualized by ethidium bromide staining under UV light.Digested and undigested PCR products were separated bypolyacrylamide gel electrophoresis (PAGE) and visualizedusing silver staining method.Statistical methodsPatients were categorized according to their genotypes.Statistical analysis was performed with the StatisticalPackage for Social Sciences (SPSS, version 16 forWindows, Chicago, IL). Results of the two groups werecompared using Fisher’s exact test or w2 test, as well ast-test for quantitative variables. An a level of 0.05 was usedto indicate statistical significance. We firstly comparedFigure 1. Genotyping assays for PAI-1, ACE and factor XIII polymorphisms. Primer sequences are listed from 50 to 30, and the uppersequence represents the forward primer; the lower sequence, the reverse primer. The underlined bases were changed from the nativesequence to introduce a restriction enzyme recognition sequence that would result in digestion of the PCR product to the fragments shownin parentheses.546 M. Aarabi et al.J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by University of Glasgow on 05/06/13For personal use only.patients with RSA (3 abortions) and controls. In the nextstep, the comparison was done for patients with two ormore abortions and controls.ResultsAfter excluding the patients with known risk factors ofspontaneous abortion, we investigated 63 patients and 114healthy women of the control group for the PAI-1, ACE,and FXIII gene polymorphisms. Mean age at registrationwas 32.5 (95% CI: 31.1–34.0) years for patients and32.9(95% CI: 31.4–34.5) years for controls. The differencebetween ages in patient population versus the control groupwas not statistically significant (T-Test, p¼ 0.01). Meanfrequency of abortion among 63 patients was 4.6 (95% CI:4.0–5.3) with a range of 3–15. Neither physical examina-tion nor laboratory tests were significantly different amongpatient and control groups.The incidence of the PAI-1 4G/5G polymorphism inpatients was 10 (18.5%) for homozygous 4G, 23 (42.6%)for heterozygous 4G/5G and 21 (38.9%) for homozygous5G versus 2 (2.0%), 66 (66.7%) and 31 (31.3%) incontrols, respectively. The prevalence of polymorphism(4G/4G) in patients was significantly higher than controls(w2 test: p¼ 0.001). Patients with homozygote 4G/4Gpolymorphism were significantly more prone to RSA incontrast to controls (OR: 11.0, 95% CI: 2.3–52.4)(Table I). Interestingly, the frequency of 4G polymorphismwas higher among the participants with two or moreabortions rather than controls (w2 test: p¼ 00.1). We foundthat this polymorphism could result in the higher risk ofhaving two or more abortions in our study population (OR:8.2, 95% CI: 1.8–36.5) (Table II).The frequency of ACE D/I polymorphism in patientswas 14 (22.2%) for homozygous II, 30 (47.6%) forheterozygous DI and 19 (30.2%) for homozygous DD;whereas the frequency in the control group was 22(23.4%), 47 (50%) and 25 (26.6%), respectively. The rateof mutation in patients was not statistically higher thancontrol group.FXIII 34Val, Val 34Leu, and 34Leu genotypes wereobserved in 71.9%, 24.6% and 3.5% of patients, respec-tively. There was no significant difference between patientsand controls (Figure 2).We also combined the results of polymorphisms of abovethree genes to find any probable differences betweenpatients and controls. However, we were unable to find anysignificant associations. More details of results are shownin Tables I and II.DiscussionPolymorphism analysis of genes involved in thrombophiliashould be investigated in different ethnics. Detection ofthose polymorphisms with RSA can help clinicians todesign better therapeutic strategies.In this study, we observed the significant increase ofPAI-1 (4G/4G) polymorphism in patients with RSA.Table I. Frequency of homozygous mutations in patient group andcontrols.Patients ControlsORNo. Fr. No. Fr. p value (CI 95%)PAI-1 10 18.5 2 2.0 0.001 11.0 (2.3–52.4)ACE 19 30.2 25 26.6 0.626 1.2 (0.6–2.4)FXIII 2 3.5 1 0.9 0.277 3.9 (0.3–43.5)PAI-1&ACE 2 3.3 0 0 0.125 NSPAI-1&FXIII 0 0 0 0 NS NSACE&FXIII 0 0 0 0 NS NSPAI-1&ACE&FXIII 0 0 0 0 NS NSOR, odds ratio; CI, confidence interval; Fr., frequency; NS, notsignificant.Table II. Frequency of homozygous mutations in participants withtwo or more spontaneous abortions and controls.Patients ControlsORNo. Fr. No. Fr. p value (CI 95%)PAI-1 16 14.4 2 2.06 0.001 8.2 (1.8–36.5)ACE 38 29.5 25 26.6 0.639 1.2 (0.7–2.1)FXIII 2 1.7 1 0.9 1 1.8 (0.2–20.1)PAI-1&ACE 3 2.4 0 0 0.25 NSPAI-1&FXIII 0 0 0 0 NS NSACE&FXIII 0 0 0 0 NS NSPAI-1&ACE&FXIII 0 0 0 0 NS NSOR, odds ratio; CI, confidence interval; Fr. Frequency; NS, notsignificant.Figure 2. PCR products of thrombophilic mutations among patients with RSA. M: marker, 1: homozygote patient, 2: normal heterozygote,3: normal homozygote (A: PAI-1, B: ACE, C: FXIII).PAI-1 4G/4G genotype is common among Iranian women with recurrent spontaneous abortion 547J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by University of Glasgow on 05/06/13For personal use only.We also found this polymorphism as a risk factor forspontaneous abortion. Three-fold elevations in the level offunctionally active plasminogen have been reported as arisk factor for pregnancy disorders and fetal damage [6]. Inaddition, Kohler has described higher concentrations ofPAI-1 as well as increased frequency of PAI-1 4G/4Ggenotype in patients with myocardial infarction [10]. Theirdata suggested that the PAI-1 4G and FXIII 34Leuvariants synergistically contribute to impaired fibrinolysisbecause of reduced activity of the fibrinolytic system andincreased resistance of the fibrin network to fibrinolysis[7,10]. Our data support parts of their suggestion that PAI-1 4G variant might be a risk factor for recurrent abortion,but does not support the role of FXIII 34Leu. On the otherhand, our findings are supported by other studies whichreported the elevated plasminogen activator inhibitor type1 (PAI-1) and PAI-1 polymorphisms [7] in patients withRSA and thrombotic homeostasis defects. This maypromote the development of early pregnancy loss inaffected women by insufficient trophoblast invasionand unbalanced fibrin deposition in the early placentalcirculation.We considered three or more spontaneous abortions asthe definition of RSA in this study. However, we found thatthe PAI-1 4G polymorphism is even more frequent amongparticipants with two or more abortions over the controls.This finding highlights the important role of 4G poly-morphism as a potential risk factor of spontaneousabortion. We recommend the screening of this polymorph-ism not only for women with RSA, but for women with thehistory of two or more spontaneous abortions.We demonstrated that homozygosity for the ACE allelesis not a risk factor for RSA in our population. Thedifference of patterns of I/D polymorphism in ACE gene issuggested to be an important risk factor for cardiovascularmorbidity and mortality in Western and Asian individuals.This might be relevant in explaining differences ofthromboembolic diseases in different populations. ACED allele is demonstrated to be in association with increasedlevel of PAI-1 and susceptibility to fibrinolysis [8]. Wecould not find any significant association between ACEpolymorphism as well as combination of polymorphismsand RSA in our study. This finding could be due to thedifferences between ethnicity of participating women instudies. The strong association between the ethnicity of thestudy population and the polymorphism frequencies ispossible and should be considered [11].We previously analyzed the frequency of FVL(G1691A), MTHFR (C677T), and FII (G20210A) muta-tions in Iranian patients who suffered from unexplainedinfertility or RSA and compared the results with healthycontrols. Those data showed a skew towards highermutation frequencies of FVL and MTHFR in the patients[4]. Investigation of the cumulative risk associated with acombination of those three mutations and sequencevariants of the PAI-1, ACE and FXIII genes in ourpatients revealed no association between above six poly-morphisms as a risk factor for pregnancy loss.This study along with our previous published dataemphasize that analysis of PAI-1 (4G/4G) polymorphism,FVL (G1691A) and MTHFR (C677T) mutations shouldbe included in the routine work-up of patients with RSA.The clinical implications of these data need to beaddressed in a prospective study to answer the questionwhether or not homozygous individuals should be treatedwith heparin to prevent RSA.AcknowledgementsThe authors thank Dr. Mohammad Reza Sadeghi PhD,Dr. Reza Behjati MD and Ms. Safoora Soleimani Fakhr,Avicenna Research Institute for their kind and helpfulcontribution in performing the study.References1. Middeldorp S. Pregnancy failure and heritable thrombophilia.Semin Hematol 2007;44:93–97.2. Kempf Haber M, Klimek M. Thrombophilia in pregnancyand its influence on venous thromboembolism and recurrentmiscarriages. Przegl Lek 2005;62:164–168.3. Buchholz T, Thaler CJ. Inherited thrombophilia: impact onhuman reproduction. Am J Reprod Immunol 2003;49:1–13.4. Behjati R, Modarressi MH, Jeddi-Tehrani M, Dokoohaki P,Ghasemi J, Zarnani AH, et al. Thrombophilic mutations inIranian patients with infertility and recurrent spontaneousabortion. Ann Hematol 2006;85:268–271.5. Glueck CJ, Wang P, Goldenberg N, Sieve L. Pregnancy loss,polycystic ovary syndrome, thrombophilia, hypofibrinolysis,enoxaparin, metformin. Clin Appl Thromb Hemost 2004;10:323–334.6. Balta G, Altay C, Gurgey A. PAI-1 gene 4G/5G genotype: arisk factor for thrombosis in vessels of internal organs. Am JHematol 2002;71:89–93.7. Coulam CB, Wallis D, Weinstein J, DasGupta DS,Jeyendran RS. Comparison of thrombophilic gene mutationsamong patients experiencing recurrent miscarriage and deepvein thrombosis. Am J Reprod Immunol 2008;60:426–431.8. Wiwanitkit V. Angiotensin-converting enzyme gene poly-morphism: I and D alleles from some different countries.Clin Appl Thromb Hemost 2004;10:179–182.9. Bereczky Z, Katona E, Muszbek L. Fibrin stabilization (FactorXIII), fibrin structure and thrombosis. Pathophysiol HaemostThromb 2003/2004;33:430–437.10. Kohler HP. Role of blood coagulation factor XIII in vasculardiseases. Swiss Med Wkly 2001;131:31–34.11. Gohil R, Peck G, Sharma P. The genetics of venousthromboembolism. A meta-analysis involving approximately120,000 cases and 180,000 controls. Thromb Haemost 2009;102:360–370.548 M. Aarabi et al.J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by University of Glasgow on 05/06/13For personal use only.

Polymorphisms of plasminogen activator inhibitor-1, angiotensin converting enzyme and coagulation factor XIII genes in patients with recurrent spontaneous abortion

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Polymorphisms of plasminogen activator inhibitor-1, angiotensin converting enzyme and coagulation factor XIII genes in patients with recurrent spontaneous abortion

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