Meta-analysis of the plasminogen activator inhibitor-1 (PAI-1) gene with insertion/deletion 4G/5G...

Asian Biomedicine Vol. 6 No. 2 April 2012; 203-217

Original article

Meta-analysis of the plasminogen activator inhibitor-1(PAI-1) gene with insertion/deletion 4G/5G polymorphismand its susceptibility to ischemic stroke in Thaipopulation

Anunchai Assawamakina, Narin Sriratanaviriyakulb,c, Yupaporn Lalerdb, Wanna Thongnoppakhunb,Oranud Praditsapb, Sissades Tongsimaa, Manop Pithukpakornb,d

aBiostatistics and Informatics Laboratory, Genome Institute, National Center for Genetic Engineeringand Biotechnology (BIOTEC), Pathumthani 12120, bDivision of Molecular Genetics, Department ofResearch and Development, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700Thailand, cInternal Medicine Residency Program, University of Hawaii, Hawaii 96822, USA, dDivisionof Medical Genetics, Department of Medicine, Faculty of Medicine, Siriraj Hospital, Mahidol University,Bangkok 10700, Thailand

Backgrounds: Plasminogen activator inhibitor-1 (PAI-1) gene was reported to associate with ischemic stroke,particularly the insertion/deletion 4G/5G promoter polymorphism. However, because of the complexity andmultifactorial nature of stroke, many previous studies showed inconsistent results.Objectives: We investigated whether PAI-1 gene polymorphism is associated with ischemic stroke by geneticassociation and meta-analysis in Thais.Methods: A case-control genetic association study on PAI-1 polymorphism was conducted. It contains 179patients with ischemic stroke and 229 healthy controls. We also conducted a meta-analysis by combining theresults from our work with other published association studies obtained between 1994 and 2010.Results: The genetic association studies on PAI-1 polymorphism on the Thai samples indicate marginal odds ratiobetween the PAI-1 polymorphisms and the risk of ischemic stroke. Combining our samples with all previouslyreported PAI-1 association studies, we did not find any genetic model significant by the lambda method. However,the allele-based method showed the recessive genetic model to be significant for the Asian-derived populationwhen analyzed separately from Caucasians.Conclusions: From the meta-analysis on our Thai data combined with other publications, it can be concluded thatthe homozygous-4G polymorphism is a risk factor for ischemic stroke in Asian, but not Caucasian populations.However, according to marginal odds ratio results, the cryptic genetic contribution, or other missing heritabilityshould be considered for the study of ischemic stroke for Thais.

Keywords: Genetic polymorphism, ischemic stroke, meta-analysis, Plasminogen activator inhibitor type 1 (PAI-1),SERPINE

DOI: 10.5372/1905-7415.0602.047

Despite the recent advancement in acute stroketherapy, stroke remains the leading cause of severedisability and mortality throughout the world [1, 2]. Inthe United States alone, the overall prevalence ofstroke is about 6.5 million people. Approximately795,000 individuals suffer from a new or recurrent

stroke. Furthermore, there were nearly 140,000 deathsfrom stroke-related causes in 2005 [2]. Similar largeoccurrences also appear in less developed countries[3]. One of the most effective measures in combatingstroke is deploying a primary prevention program inwhich identification of risk factors is the most importantaspect.

Stroke is a heterogeneous disease associatedwith many known conventional risk factorssuch as hypertension, diabetes, and dyslipidemia.Ischemic stroke caused by thrombosis is regarded as

Correspondence to: Manop Pithukpakorn, Division ofMolecular Genetics, Department of Research and Development,Faculty of Medicine, Siriraj Hospital, Mahidol University,Bangkok 10700 Thailand. E-mail: [email protected]

204 A. Assawamakin, et al.

a multifactorial disease. The thrombosis risk factorscan be generally classified into three groups, strong,moderate and weak based on the number of positiveassociations collected in HUGE Navigator [4]. Thestrong genetic risk factors include PAI-1, antithrombin,protein C and protein S, while the moderate groupcomprises factor V (F5) Leiden, prothrombin 20210A,non-O blood group, and fibrinogen 10034T. The weakgenetic risk factors were reported such as fibrinogen,factor XIII and factor XI variants. Identification ofstroke susceptibility genes and quantification ofassociated risks have been hampered by conflictingresults from underpowered case-control studies.

One leading predisposing factor of ischemicstroke is the plasminogen activator inhibitor-1(PAI-1) gene located on chromosome 7. Due to itsdirect involvement in thrombosis pathway, PAI-1 isan excellent candidate to investigate its role inischemic stroke. The product of PAI-1 acts as amajor inhibitor of tissue plasminogen activator (t-PA)by forming an irreversible one-to-one complex.This process leads to a decrease in fibrinolysis andthrombus degradation [5, 6].

Many polymorphisms have been identified inthis gene, one of which is “4/5” guanosine (4G/5G), asingle-base length polymorphism in the promoter regionof PAI-1. This particular polymorphism affects PAI-1levels [7-12]. Both 4G and 5G alleles have the samebinding site for the transcription activator. The 5G-allele, however, has an additional binding site for arepressor, resulting in lowering of the transcriptionrate and concomitant PAI-1 activity [8, 10]. Hence,homozygous 4G/4G individuals have the highestPAI-1 level, while 5G/5G homozygotes have thelowest.

Several studies have reported that the 4G/4Ggenotype increases the risk of myocardial infarctionand thromboembolism through impairment ofsecondary fibrinolytic function by elevated PAI-1activity [13, 14]. On the other hand, there is discordancebetween published studies regarding the associationof the PAI-1 gene 4G/5G polymorphism and stroke toincrease, neutral, or protective risk [7, 15-20]. Onlyone recent meta-analysis has found PAI-1 4G/5Gassociation with ischemic stroke risk in Han Chinese[3], whereas another meta-analysis [21] failed todemonstrate a significant association with otherpopulations.

The aims of this study were to investigate therole of 4G/5G PAI-1 polymorphism in the susceptibility

of stroke in Thai population and to characterize thedisease risks in different ethnic groups by gatheringall data from case-control studies, including data fromprevious two meta-analysis studies [3].

Materials and methodsStudy subjects and PAI-1 (SERPINE 1)genotypingCases and controls

Thai patients who had been diagnosed with acuteischemic stroke at Siriraj Hospital since October 2008and agreed to participate were invited in the study.Stroke was defined clinically using World HealthOrganization criteria. A diagnosis of ischemic strokewas made only if the patient has a clinical diagnosis ofstroke and if a computed tomography (CT) or magneticresonance imaging (MRI) scan of the brain done afteronset of the symptoms either was normal or showedthe relevant infarction. Patients with hemorrhagictransformation of an infarct were also eligible.Subtyping of the index ischemic stroke was donecentrally on the basis of the abstracted medical recordby a neurologist adjudicator blinded to genotypedata and personal identifiers. Because final subtypediagnosis could vary from initial diagnosis inapproximately one-third of the cases, the adjudicatorused all available and relevant information obtainedafter completion of the stroke work-up. The Trial ofORG10172 in Acute Stroke Treatment (TOAST)classification was mainly used in this study.

The control subjects were recruited from adultmen and women who must be at least 18 years old atthe time of enrollment. They must not have a strokeand must be genetically unrelated to the patients enrolledin this study. The Questionnaire for Verifying Stroke-free Status (QVSS) was used to confirm stroke-freestatus. Patients being treated for coronary or peripheralvascular disease were not included in the controls.This study protocol was reviewed and approved bythe Institutional Review Board (IRB) of the Facultyof Medicine, Siriraj Hospital. All participants signedwritten informed consent prior to the study. Clinicaldata as well as the DNA samples from peripheral bloodwere collected and compared with the control group.

Genotyping of plasminogen activator inhibitor(PAI)-1 promoter polymorphism, -675 ins/del (4G/5G)

Genomic DNAs were extracted from peripheralblood samples by the standard phenol/chloroform

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April 2012PAI-1 gene with insertion/deletion 4G/5G polymorphism

extraction method. For determination of PAI-1promoter polymorphism, -675 ins/del (4G/5G), SNPgenotyping was carried out using the PCR-DHPLCmethod. The 180 bp PCR products were generatedin 25-ml total volumes containing 10 pmoles of eachprimer: sense 5’-GCC AGA CAA GGT TGT TGACA-32 and anti-sense 52 -CAG CCA CGT GATTGT CTA GG-32 , 1.5 mM MgCl

2, 1x detergent-free

HF buffer (518), 200 μM dNTPs, 0.02 U Taq DNApolymerase (Phusion Hot-start High-fidelity DNApolymerase), and 100 ng of genomic DNA. A thermalcycle was performed for 30 seconds at 98°C todenature the DNA template, 40 cycles of 10 secondsat 98°C, 30 seconds at 64°C, and 30 seconds at 72°C,followed by 5 min at 72°C for the final extension.After PCR completion and heteroduplex induction,5-microliters of the amplified products were injectedinto the WAVE® Nucleic Acid Fragment AnalysisSystem (Transgenomic, Inc., Omaha, NE) operatedfor mutation detection in partially denaturing mode.The DNASep® HT column temperature was set at63°C to detect the 675 ins/del (4G/5G) SNP. The linearacetonitrile gradient at a flow rate of 0.9 ml/minutewas formed by mixing buffer A (0.1M TEAA) andbuffer B (0.1M TEAA, 25% acetonitrile) to create agradient of 49.1-59.1% buffer B over two minutes.After each analytical run, the column was cleanedand equilibrated. The eluted DNA fragments wereobserved by an ultraviolet (UV) detector. Thechromatographic elution profile of the polymorphismin a heterozygous state would show a unique peakpattern different from that of the homozygous wildtype, as confirmed by sequencing.

Identification of eligible studies and dataextraction

We examined all association studies betweenPAI-1 4G/5G polymorphisms and ischemic stroke.Articles in which PAI-1 4G/5G polymorphisms weredetermined in ischemic stroke patients and controls,during the years 1994 and 2008 were retrievedusing the PubMed, Embase, and HuGE navigatorsearch engines. The search keywords were “stroke”,“ischemic stroke”, “PAI-1”, “polymorphism”, and“genetic associations”. We also extensively reviewedthe references cited by these selected articles toidentify other publications that might have beenoverlooked by the keyword search. If multiplepublications from the same study group wereidentified, the latest and most complete results would

be used in the analysis. The search results wererestricted to English-language articles. The criteriaused to screen these articles are as follows;

1) The article must be a case-control studyregardless of the study sample size.

2) The article must be an association studybetween PAI-1 4G/5G genotypes and ischemic stroke.

3) The article must provide sufficient informationto calculate odds ratio (OR) and hence, the followingtypes of article must be excluded;

a. The article containing redundant dataoriginally from the same source(s)

b. The article that the number of wild-type/mutant genotypes cannot be ascertained

c. Linkage analysis and family-based studiesTwo investigators independently extracted the

following information: first author, a journal nameand year of publication, ethnicity of the studiedpopulation(s), the number of cases and controls, andthe genotype and allele frequency from the PAI-1 4G/5G polymorphisms. The same investigators alsoindependently assessed the quality of each study.Qualities of scoring criteria were modified from theprevious meta-analyses of the observational studies.Each paper was assigned with a score in the rangeof 0 (worst) to 12 (best). The third investigator wasappointed for adjudicating any disagreement betweenthe first two investigators.

Statistical analysisFor each eligible study, allele frequencies of

PAI-1 4G/5G from the control group were used tocompute Hardy–Weinberg equilibrium (HWE)using Fisher’s exact test. The studies that do notsatisfy HWE were removed from the analysis. Weexamined the different allelic effects of PAI-1 4G/5Gpolymorphism. For each study, the Odds Ratio (OR)and the corresponding 95% confidence interval (CI)were determined. We assessed the variation fromwithin the same study and among different studiesusing Cochran’s Q-statistic to evaluate the nullhypothesis that all studies should report the sameeffect resulting from PAI-1 4G/5G polymorphism.A significant Q-statistic value (p <0.10) indicatesheterogeneity of association results across the selectedstudies. Overall gene effect from the three groups ofgenotyping, namely 4G/4G, 4G/5G and 5G/5G, was thenanalyzed using logistic regression. When the calculationof gene effect is positive, the genetic model would beevaluated using lambda value (λ), which is the ratio


of log OR4G/5G

to log OR4G/4G

. The genetic model isevaluated as recessive, dominant and co-dominant ifλ = 0, 1, and 0.5 respectively. Pooled ORs wereestimated using both random and fixed effect models.Under the random effect model, weighting individualORs were carried out using the reciprocal of theirvariances. In addition, we also performed a subgroupanalysis to eliminate other factors such as ethnicityand age groups. Egger’s linear regression test andthe funnel plot were used to assess publication bias.All analyzed data were performed using the softwareMIX version 1.7. A p-value less than 0.05 is consideredstatistically significant, except for the test ofheterogeneity in which p <0.10 was used.

ResultsPatients and genotyping

One hundred and seventy nine patients with acuteischemic stroke and 229 healthy controls wererecruited to genotype 4G/5G PAI-1 polymorphism.No deviation from HWE was found (Exact tests;p = 0.596920). The PAI-1 4G-allele, which is themutant form, is more prevalent than the 5G-allele fromboth group (0.56 in cases and 0.53 in controls). Thegenotype frequencies of control group were 29.26%of 4G/4G, 48.03% of 4G/5G, and 22.70% of 5G/5G.Similar to control group, the genotype frequencies ofcases were 28.50% of 4G/4G, 54.19% of 4G/5G, and17.31% of 5G/5G. The 4G/5G genotype is the mostcommon in both groups followed by 4G/4G, and 5G/5G. Either genotype frequency or allele frequency wasnot significantly different between two groups,although a trend towards a higher prevalence ofthe heterozygous 4G/5G in the cases was observed[odds ratio (OR) 1.279; 95% confidence interval [CI]= 0.8638, 1.896]. In a regression analysis, the oddsratio (OR) for 4G/4G versus 4G/5G+5G/5G was1.038 (95% confidence interval [CI] = 0.674-1.598)(de Finetti); that is no clear association between4G/5G PAI-1 polymorphism and the acute ischemicstroke phenotype.

Meta-Analysis of PAI-1 4G/5GTwenty-seven studies were obtained using

keyword search from online publication databases.Three of these articles were the meta-analysis[3, 22]. Forty-nine case-control studies were reviewed[1, 3, 5-7, 9, 15-19, 22-50]. Twenty-five studies wereineligible, which can be characterized as 1) two studiesinvestigated stroke in the young 2) five studies were

irrelevant 3) seven studies had no genotypic data, and4) fourteen studies were duplicates [5, 7, 15-18, 28,31, 32, 36, 44, 46, 51]. Therefore, 26 studies includingpresent data from our study were considered inthis meta-analysis. There was, however, a diversityof ethnic groups studied from a wide geographicalrange. Thirteen studies were among Caucasians andthirteen studies were performed among Asians. Themeta-analysis experiments were performed on allstudies combined with no regard to ethnicity, andseparately for Caucasian and Asian populations(subgroup analysis). All individuals with stroke wererecruited only if an association exists between geneticsusceptibility and ischemic stroke in the studies. Thequality of these studies was determined by our meta-analysis ranged from 3 to 11.

The genotype frequencies of PAI-1 polymorphismwere classified as stroke and control. After addingthe results by combining with previous studies, meta-analysis was performed. The genotype distribution ofthe control group was in Hardy-Weinberg equilibrium(HWE). Heterogeneity between the studies wasobserved according to the calculation of bothI2 = 75.51% and Q statistic value <0.0001 respectively.Subgroup analysis was performed in order to handleethnic stratification. The heterogeneity factors weredescribed by I2 = 75.7 % and Q statistic value <0.0001in Asians and I2 = 69.08% and Q-statistic value<0.0004 in Caucasians. Therefore, the cause of theunderlying heterogeneity should be explored by furthersensitivity analysis.

A quality assessment scoring system was adoptedto review individual study bias so that the heterogeneitycan be re-calculated after excluding some studies.Similar heterogeneity results were obtained. We theninvestigated the most proper genetic model using thelambda method as described in the methodologysection. A non-significant genetic model was derivedfrom all analyses overall (λ = −1.65), and for Asian(λ = −0.53) and Caucasian (λ = −0.37) subgroupanalyses. Nonetheless, we could not find anappropriate genetic model for this polymorphism.Evidences from previous studies showed that therisk alleles of stroke affected only Asian population,while Caucasian results were inconclusive [3]. Thesubgroup analysis should be seriously considered. Weconducted the meta-analysis in all models to comparethe allele-based method (4G versus 5G), and thegenotype-based method (4G/4G vs. 5G/5G, 4G/4G vs.the remainders, and the remainders vs. 5G/5G).

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The 4G allele frequencies (pooled) from case(4,646) and control (6,214) individuals were 55.59%and 53.28% respectively. The 4G-allele frequencieswere not significantly different within Caucasian orAsian populations (57.69% and 53.97% among Asiancases and controls, and 53.53% and 54.41% amongCaucasian cases and controls). Overall, the per-alleleOR of the 4G allele was 1.0495 (95% confidenceintervals (CI) 0.9317 and 1.1822). The 4G per-alleleORs among Caucasian and Asian populations werenot significantly different (0.975 (0.8738-1.0879) and1.1752 (0.9087-1.5199), respectively). There wereno differences between random and fixed effectmodel calculations. The result showed no significantassociation between the alleles and ischemic stroke.

We then analyzed the genotypes from the 26eligible case–control studies with 4,646 cases and 6,214controls for the 4G/4G versus the remaining genotypes.The pooled ORs for the recessive, dominant, andcodominant models were not significantly differentfrom each other, and no significant associationswere observed as shown in Figure 1. Conversely,subgroup analysis revealed significant OR for therecessive model among the Asian population, but notCaucasians (OR=1.44, 95% CI 1.02–2.03) as shownin Figures 2 and 3. The 4G/4G polymorphism is thusa risk factor for ischemic stroke in the Asian, but notCaucasian population. Using Egger’s linear regressiontest, there was no evidence of publication bias or astudy-size effect from any conducted comparison.


Figure 1. Forest plots from meta-analysis of all 26 eligible case-control association studies (Caucasian and Asiancombined). The effect size of each study is represented by a closed square point (the size of each pointcorresponds inversely to its variance) where the horizontal rule shows the 95% confidence interval of eachpoint. The red diamond at the bottom of the plot indicates the summary of effect sizes from the meta-analysis.A: recessive model (4G/4G versus the remainder) B: codominant model (4G/4G versus 5G/5G) C: dominantmodel (5G/5G versus the remainder)

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Figure 2. Forest plots from meta-analysis of 13 Asian-derived case-control association studies. The effect size of eachstudy is represented by a closed square point (the size of each point corresponds inversely to its variance)whereas the horizontal rule shows the 95% confidence interval of each point. The red diamond at the bottom ofthe plot indicates the summary of effect sizes from the meta-analysis. A: recessive model (4G/4G versus theremainder) B: codominant model (4G/4G versus 5G/5G) C: dominant model (5G/5G versus the remainder)

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Figure 3. Forest plots from meta-analysis of 13 Caucasian case-control association studies. The effect size of each studyis represented by a closed square point (the size of each point corresponds inversely to its variance) where thehorizontal rule shows the 95% confidence interval of each point. The red diamond at the bottom of the plotindicates the summary of effect sizes from the meta-analysis. A: recessive model (4G/4G versus the remainder)B: codominant model (4G/4G versus 5G/5G) C: dominant model (5G/5G versus the remainder)


DiscussionOur findings reported no association between

the PAI-1 polymorphism and the risk of stroke inThai population. Due to the limitation of the samplesizes of our study, we then add our findings to previouspublished similar studies and performed meta-analysis.The meta-analysis of published studies demonstrateda significant association between the 4G/5Gpolymorphism in the promoter region of the PAI-1 geneand ischemic stroke in the Asian population. Theobserved association in Asians was robust underthe recessive model, which was consistent with theassumed role of this gene in the disease. On the otherhand, the 4G/5G polymorphism has no ischemicstroke association in Caucasians, which masks theassociation in Asians in the combined analysis.

Previous studies assumed that this polymorphismis likely to affect ischemic stroke in a recessive geneticmodel as the genotype reflects the expression of aprotein directly implicated in the disease. Surprisingly,the corresponding genetic model across all populationscould not be clearly established from these studies,which we showed using the lambda method.Nonetheless, comprehensive testing of all recessive,dominant and codominant models separately amongCaucasians and Asians did reveal that only therecessive model explains the association of 4G/4G inthe Asian population, which is in agreement with anearlier meta-analysis of the Han Chinese population[3]. Since stroke is regarded as a complex disease, itpotentially associates with many risk factors, bothenvironmental and genetic. Therefore, we might notbe able to unequivocally determine a genetic modelof the 4G/5G polymorphism among some populations,i.e. Caucasian.

Apart from the known effect of 4G/5Gpolymorphism on the expression of PAI-1 [7-12], otherfactors exist which control PAI-1 levels, such as verylow density lipoprotein [52], insulin and glucose [13].Moreover, these polymorphisms act as an acute-phasereactant [5, 8, 53], which means that difference inPAI-1 levels between 4G and 5G is more apparent inthe presence of stimulating factors [54]. It is worthnoting that although the 4G allele has been reportedto affect ischemic stroke, it is not yet establishedwhether it has a protective, neutral or harmful effect[13]. Therefore, all information pertaining to the 4Gallele and ischemic stroke was collated. The followingfindings might reveal the involvement of thispolymorphism in both being a risk and protective factor.

4G/4G genotype as a risk factor for ischemicstroke

1. 4G/4G polymorphism is related to higherPAI-1 levels in the circulation [7-12] and PAI-1 servesas a major physiological inhibitor of tissue plasminogenactivator (t-PA), thus impairing the fibrinolysis process[5, 6].

2. 4G/4G increases genetic susceptibility toelevated PAI-1 responses after an inflammatoryprocess. PAI-1 is considered as an acute-phasereactant which is regulated by inflammatory factorssuch as interleukin-1, TNF-α and TGF-β [5, 8, 53].It is believed that high levels of PAI-1 after a life-threatening condition impair fibrinolysis and increasesthe risk for ischemic stroke [39, 55].

3. Since ischemic stroke is one of the conditionsthat are caused by impaired circulation from thethromboembolic phenomenon, similar to coronaryartery disease and venous thromboembolism, theimpact of PAI-1 levels on the fibrinolysis pathway[5, 6] should be considered as a risk factor for thesediseases. For myocardial infarction, two meta-analysesshowed association of the 4G/4G genotype withincreased risk of myocardial infarction [14, 56]. Fordeep vein thrombosis, another meta-analysis partiallysupported the association of the 4G allele with therisk of venous thromboembolism [13, 21].

4. PAI-1 is associated with obesity, cholesteroland insulin resistance [27, 28, 57, 58], which areconsidered as risk factors for coronary artery diseaseand ischemic stroke. These might account for analternative hypothesis that this polymorphism alsoincreases the risk of myocardial infarction [59] as wellas ischemic stroke.

4G/4G genotype as a protective factor for ischemicstroke

Despite the impact of PAI-1 protein on thefibrinolysis pathway, many studies showed that the4G/4G genotype is associated with a decline in strokeincidence [16-20, 36, 38]. The current hypotheses areas follows:

1. Although tissue plasminogen activator (t-PA)has a major role in the fibrinolysis pathway, whichhelps clear clots, it also has a deleterious neurotoxiceffect. Neuronal death induced by t-PA can berescued by astrocytes and serpins, such as type-1plasminogen-activator inhibitor (PAI-1) andneuroserpin (NSP) [60].

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2. t-PA could also exacerbate the extracellularmatrix (ECM) degradation, which causesdestabilization of atherosclerotic plaques [61]. PAI-1serves as a protective role possibly involvingsuppression of matrix-degrading enzymes in theplaque, resulting in limited plaque growth andprevention of abnormal matrix remodeling [20, 62].

3. Animal studies have demonstrated that localcellular production of PAI-1 and t-PA might beinvolved in localized inflammatory processes, therebyplaying a major role in the process of atherosclerosisand plaque rupture [63, 64]. We hypothesize that atthe inflammatory sites, the putative elevation ofPAI-1 level is stronger for the 4G allele. This may, forinstance, result in plaque stabilization and increasedneutralization of t-PA, which is potentially neurotoxic[19].

4. 4G/4G polymorphism showed association withlower serum triglyceride levels in one study [38].

An alternative hypothesis is that the relationshipbetween elevated PAI-1 levels and myocardialinfarction is more straightforward than that of ischemicstroke. This hypothesis might explain the differencesin pathophysiological mechanisms betweenmyocardial infarction and stroke. In myocardialinfarction, rupture of atherosclerotic plaque is the mainmechanism and the plaques are associated withelevated PAI-1 level [65]. On the other hand, inthe case of ischemic stroke, small vessel diseaseand thromboembolism are more common causes ofcerebral ischemia than carotid and aortic archatherosclerosis [66].

With all the present information regarding theassociation between PAI-1 4G/5G polymorphism andrisk of ischemic stroke, it remains uncertain whethersuch a polymorphism has protective or harmful effect.Nevertheless, our meta-analysis revealed that ethnicitymight be an important factor in determining the effectof this polymorphism. Furthermore, there could bemany inherent variations across different studies thatinfluence the association result. First, ischemic strokeis a complex disease and genetic heterogeneity mayexist among different populations. Second, linkagedisequilibrium (LD) of the genetic markers fromdifferent studies may also contribute to the associationdiscrepancy. In other words, because differentpopulations sometimes have different LD patterns, thesame polymorphism could play a different role indisease susceptibility in different populations. Thisimplicates that the polymorphism might not be a causal

variant, i.e., it may be in the same LD block with anearby causal mutation in one population but not inanother [18, 67]. Third, the association differencesmight happen by chance, such as type I/II statisticalerrors, or due to lacking of multiple testing correctionwhich inflates the type I error [67]. Finally, theprevalence of the 4G allele varies among differentpopulations.

According to our comprehensive review results,the contribution of the 4G/5G polymorphism to ischemicstroke remains inconclusive. This may be because:

1. Stroke itself is a complex disease and hasmultiple risk factors.

2. PAI-1 protein may have variable influencesin different tissues, organs, conditions, or populationsas previously discussed in this paper.

3. The limitation from the individual studies’ biasincluding our meta-analysis, which exhibited highheterogeneity even in the subgroup analysis. Theheterogeneity could contribute to various factors, e.g.,ethnicity, particular sub-type of stroke, etc.

Meta-analysis, hence, should be interpreted verycarefully even when the meta-analysis was correctlycarried out.

ConclusionsAdding Thai samples to the pool of previously

published Asian data supports 4G/4G genotype as asignificant risk factor for Asians. Including data fromall populations resulted in non-significant association,which entails that PAI-1polymorphisms could be usedto predispose the risk of the disease only for specificpopulation. However, the low log OR

4G/4G from this

meta-analysis indicates that PAI-1 is not the only factorfor ischemic stroke, even though PAI-1 serves as amajor physiological inhibitor of tissue plasminogenactivator. Hence, the meta-analysis results from thisstudy support that the risk of ischemic stroke shouldnot be identified by only one major candidate PAI-1gene.

AcknowledgementsThe authors would like to thank Dr. Philip J. Shaw

for constructively commenting on the manuscript.Oranud Praditsap is supported by the ThailandResearch Fund (TRF) through the Royal GoldenJubilee Ph.D. Program (Grant No. PHD/4.I.MU.49/C.1). Sissades Tongsima is supported in part bythe National Center for Genetic Engineeringand Biotechnology (BIOTEC) and Anunchai


Assawamakin (BIOTEC Post-doctoral FellowshipsProgram). Finally, Manop Pithukpakorn and SissadesTongsima acknowledge Thailand Research Fund(TRF) and the National Research Council of Thailand(NRCT) for partially supporting this work.

The authors declare no conflict of interest inconducting this study. Anunchai Assawamakin andNarin Sriratanaviriyakul equally contributed to thework.

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Meta-analysis of the plasminogen activator inhibitor-1 (PAI-1) gene with insertion/deletion 4G/5G...

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