Role of tissue plasminogen activator
To determine the role of tissue plasminogen activator (tPA) Alu-repeat insertion/ deletion and plaminogen activator inhibitor PAI-1 4G/5G insertion/ deletion gene polymorphisms in myocardial infarction (MI) through a case-control study conducted among the Pakistani Population.
The genetic analysis of 211 patients suffering from MI and 165 healthy persons was carried out for the t-PA Alu-repeat insertion/ deletion and PAI-1 4G/5G insertion/ deletion gene polymorphisms. The target sequences of the two genes were amplified through Allele specific PCR. The amplified products were electrophoresed on 2% agarose gel. The allele frequencies were determined and chi-square analysis was performed in order to find out any significant difference among the patients and controls.
In MI patients the observed genotype distribution for 4G/4G, 4G/5G and 5G/5G alleles were 28.4%, 36.9% and 34.5% with no significant difference when compared to healthy controls with distribution values of 21.5%, 43.1 % and 35.4 %. The odd ratios (OR) for the risk allele (4G) is 1.17 (0.86-1.59) which showed that the 4G allele is not associated with MI. Similarly, for t-PA gene polymorphism, the observed genotype frequency for the Ins/Ins, Ins/Del and Del/Del were 26.3%, 30.6% and 41.3% among patients, the healthy controls also showed no significant difference with 28%, 24.4 % and 47.5% values. The risk allele Ins with OR value (0.85(0.63-1.15)) is also not associated with the MI. In case of the risk factors like smoking (OR= 3.45 (1.77-6.94)), diabetes (OR=9.75(6.89-13.77)) and family history (OR=1.72(1.08-2.73)) the significant association was observed with the disease.
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The t-PA and PAI-1 gene polymorphisms are not associated with MI in Pakistani population, whereas the environmental factors like cigarette smoking and diabetes are significantly associated with the disease progression. The family history of MI patients showed that some inherited genetic factor is involved in the disease onset in Pakistani population.
Key words: Myocardial Infarction (MI), tissue Plasminogen activator (tPA), Plasminogen Activator Inhibitor (PAI-1), Coronary Artery Disease (CAD), fibrinolysis.
Myocardial infarction (MI) is a coronary artery disease (CAD) caused by the degeneration of heart tissues due to obstruction of the blood supply to the heart muscles as a result of coronary thrombosis. It is one of the leading causes of morbidity and mortality worldwide [Joshi et al., 2007], estimates are that only 10% of all the patients of CAD are at risk of suffering from MI [Lewandrowski et al., 2002]. The major risk factors that are associated with MI include: cigarette smoking, high blood pressure, hypercholesterolemia and diabetes. In addition to these environmental factors and genetic factors like polymorphisms and family history also contribute towards disease progression [Margaglione et al., 1998; Crainich et al., 2003].
As MI is attributed to the development of blockage in the coronary arteries due to clot formation (thrombosis), it is therefore directly related to the thrombosis-fibrinolysis system. [Voestesch and Loscalzo, 2004]. The thrombosis-fibrinolytic system is regulated by the factors which are involved either in clot formation or clot degradation (fibrinolysis). To date a number of polymorphisms of the genes of the fibrinolytic proteins and their cross regulators have been studied to determine their role in the regulation and expression of these genes [de Lange et al., 2001]. The study of Knowles et al. 2007 showed that the polymorphisms in the genes encoding the components of thrombosis-fibrinolytic cascade might be a contributing factor in disease etiology by creating an imbalance between the two processes.
Tissue plasminogen activator (t-PA) and plasminogen activator nhibitor-1 (PAI-1) are the two important regulators of this pathway. The former is a serine protease found in endothelial cells and is involved in the breakdown of fibrin clot by converting plasminogen into plasmin, which then leads to fibrinolysis, whereas the latter is an inhibitor of t-PA and works by forming t-PA/PAI-1 complex thereby inducing the thrombosis [Robinson et al., 2006].
A number of polymorphisms occur in the genes encoding t-PA and PAI-1. In order to determine the role of such polymorphisms in gene expression, prospective and case-control studies have been conducted by different groups [Voestesch and Loscalzo, 2004].
The protein t-PA is encoded by PLAT (plasminogen activator tissue type isoform1) gene on chromosome 8p11.21 with 8 coding exons, and is the inducer of fibrinolysis. The increased enzyme activity causes hyper-fibrinolysis which might leads to excessive bleeding whereas it's decreased activity result in thrombosis. A common intronic insertion of an Alu-repeat of 311 bp is present in the intron 8 of the gene and is considered to be involved in plasma levels of tPA [Ridker et al., 1997; Nakazawa et al., 2001; Robinson et al., 2006]. A homozygous insertion in MI patient contributes a two fold increase in the risk for MI as compared to the heterozygous condition with Insertion/Deletion (Ins/Del), which imparts a 50% increased risk in MI [voestesch and Loscalzo, 2004].
The other important regulator is PAI-1, a glycoprotein of 50 KD, which belongs to a serine protease inhibitor super family (SERPINS). It is encoded by the PAI-1 gene on chromosome 7q22.1 with 13 coding exons. In PAI-1 a single nucleotide polymorphism (SNP) 4G/5G Insertion/Deletion at -675 bp in the promoter sequence is present. The homozygous 4G/4G polymorphism is associated with clot stabilization which leads to abnormal condition due to increased expression of PAI-1 gene product that inactivates the t-PA product from clot degradation and ultimately leads to MI [Voestesch and Loscalzo, 2004]. The higher level of PAI-1 has been observed in survivors of MI compared to normal healthy persons [Falk, 1995]. The higher transcription of the PAI-1 is responsible for the physiochemical stability of the t-PA-PAI-1 complex which results in low levels of free t-PA as a result of which fibrinolysis of the fibrin clot is inhibited thus the resultant thrombosis [Soysal et al., 2006]. The homozygous 5G/5G insertion has an additional repressor site for another transcriptional repressor, which makes 5G transcriptionally less active then 4G, and thus inhibits the expression of PAI-1 which then result in irregular fibrinolysis [Ericksson et al., 1995].
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In the present study the role of tPA Alu repeat I/D and PAI-1 4G/5G I/D polymorphisms as a genetic risk factor in MI was investigated through a case control association study involving the patients and unaffected individuals.
Materials and Methods
This study conforms to the Helsinki declaration and has the approval of the Shifa College of Medicine/Shifa International Hospital Ethics Committee/Institutional Review Board. All patients were informed in their local language about the purpose of the study and they gave their informed consent in writing before they were further assessed.
A total of 211 patients suffering from MI and 164 healthy individuals were selected for the study. The subjects were enlisted from the coronary care unit (CCU) of Rawalpindi General Hospital (RGH) and National Institute of Heart Diseases (NIHD) Rawalpindi. The patients were diagnosed using the standard WHO criteria, which is based on the complaint of typical chest pain for more than 20 min, higher than normal levels of the cardiac specific markers, and ST changes on the electrocardiography (ECG). The information about the risk factors including BMI, smoking habit, diabetes, hypertension, total cholesterol and family history were also obtained from the patients as well as healthy controls.
Blood Sampling and DNA extraction
The blood samples for DNA isolation of the patients and healthy controls were taken and collected in sterile 8.5ml vacutainer tubes containing Acid Citrate Dextrose (ACD) as an anticoagulant (Becton Dickinson product no 364606, New Jersey, USA, http://www.bd.com/). The blood samples were also collected in 5 ml BD vacutainer SST Advance Serum tubes (Becton Dickinson product no 367955, New Jersey, USA, http://www.bd.com/).to isolate the serum for lipid profile. The serum was separated by centrifugation at 4000 rpm for 10 min, just after the clotting of blood within the tube. The serum was stored on -20 C for further biochemical analysis to get the lipid profile. The lipid profile of both the patients and controls were obtained using Hitachi Automated Chemistry Analyzer 902 model [country]. For DNA isolation, the whole blood was used using standard organic phenol method of DNA isolation [Sambrooke and Russell, 2001].
The t-PA Alu repeat I/D and PAI-1 4G/5G gene polymorphisms were genotyped by allele specific Polymerase Chain Reaction (AS-PCR) as described by Sambrooke and Russell, 2001. The target DNA sequence was amplified by using primer sets for tissue-plasminogen activator (t-PA) gene polymorphism as described by Van der Bom et al. (1997). Similarly plasminogen activator inhibitor (PAI-1) target sequence was amplified by using primers from the study of Anderson et al. (1999).
The PCR reactions were performed with genomic DNA (50ng) in a total reaction mixture of 25 µl. Each reaction contained 0.2mM of each dNTPs, 1X Taq buffer, (75mM Tris- HCI (pH 8.8), 20mM (NH4) SO4 and 0.01 % Tween 20), 1.5 mM MgCl2, 0.4 µM of each forward and reverse primer, 0.12µM control primer for PAI-1 gene and 1 U/reaction of Taq polymerase. Similarly for the amplification of t-PA sequence, 0.6 µM of forward and reverse primers were used with the above mentioned conditions. PCR was performed in a Thermo Electron Corporation PCR system (PXE Thermal cycler Applied Biosystem Gene Amp PCR System 2700). Thermocycler profile for PAI-1 was as follows; initial denaturation at 95°C for 2 min followed by 45 cycles of denaturation at 94°C for 35 sec, primer annealing at 65°C 45 sec and primer extension at 72°C for 75 sec and a final extension at 72°C for 5 min.
The thermal cycling conditions for the t-PA were as follows; initial denaturation at 95°C for 5 min followed by 30 cycles of 94° C for 1 min, 58°C for 1 min, 72°C for 1 min and a final extension at 72°C for 5 min.
The amplified products were electrophoresed on 2% agarose gel for PAI-1and 1.5 % agarose gel for t-PA gene, and the bands were visualized by UV transilluminator. The images of the gels were then captured with a digital camera and documented using the software BioCap MW Version 11.01 software from Vilber Lourmat (France).
PAI-1 gene product showed a band of 139 bps for 4G or 140 bps for 5G gene polymorphism along with a control band of 256 bps long, whereas t-PA gene products showed a band of either 663 bps (deletion) or 964 bps (insertion) or both, after electrophoresis.
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The data were statistically analyzed with the SPSS software package version 14 (Statistical ver. 14 Packages for Social Sciences (SPSS) (Chicago, USA)). The genotypic frequencies were compared among the cases and control groups in order to determine the association of genotype with the disease.
A total of 375 individuals including 211 MI patients and 164 healthy controls were screened for the association of PAI-1 and t-PA gene polymorphism with mean age of 53.0 ± 11.0 (years) for MI patients and 42 ± 11.0 (years) among the healthy controls. Among the MI patients the mean age of men was 52.4 ± 10.2 (years) and 55.1 ± 13.4 (years) in female patients. Out of the 211 MI patients, 90 were male and were smokers. There was no significant difference in the Body Mass Index (kg/m2) of the cases and the healthy controls. The baseline characteristics of patients and controls are mentioned in table 1.
When the association analysis for the environmental factors among the cases and controls were carried out, three of the risk factors like smoking, diabetes and family history were also observed to be significantly different in MI patients compared to healthy controls (Table 1). A significant difference was observed regarding the smoking habit, with 45% of smokers suffering from MI as compared to healthy controls, 19.6% (p= 0.00008, OR=3.45, 95% CI=1.77-6.94). Similarly, a significant difference was also observed in diabetic condition in patients (27.8%) compared to the controls (3.8%) with OR=9.75 (6.89-13.77). Family history was also observed to be differed significantly in cases and controls with OR=1.72(1.08-2.73).
The genotype for PAI-1 4G/5G gene polymorphism among MI patients was as follows: 60 patients (28.4 %) were 4G/4G homozygous, 73 (34.5%) were 5G/5G homozygous whereas 78 (36.9%) were 4G/5G heterozygous. While in healthy controls, 34 individuals (21.5%) were 4G/4G homozygous, 56 (35.4 %) were homozygous for 5G/5G and 68 (43.1 %) were 4G/5G heterozygous (Table 2). The frequency of risk allele 4G was 46.9% in patients and 43.1% in the healthy individuals, which resulted in no significant difference (Table 3).
By applying the dominant model of inheritance for the PAI 4G/5G allele, the odds ratio (OR) for the 4G allele was observed to be OR=0.97(95% CI=0.62-1.52), which shows no significant difference among the cases and controls. While OR=1.45(CI 0.87-2.42) values observed in the recessive model for 4G allele also resulted in non-association of the allele with MI (Table 4 and 5).
The genotypic frequency of t-PA showed that 55 individuals (26.3%) were homozygous Ins/Ins, 64 (30.6%) were homozygous Del/Del and 90 (43.1%) were heterozygous Ins/Del in MI patients, whereas in controls the frequency was 46 (28%) Ins/Ins, 40 (24.4%) Del/Del and 76 (47.5%) Ins/Del respectively. The statistical analysis showed no significant difference in the genotype frequency among the two groups (Table 2). The frequency of Ins allele was 0.47 in cases compared to 0.51 in controls and no significant difference was observed through OR 0.85(0.63-1.15) (Table 3). The dominant and recessive models for t-PA genotype (OR=0.73(0.45-1.19) and OR=0.92(0.56-1.49) showed no significant association with MI (Table 4 and 5).
The case-control study was conducted to determine the association/ correlation of 4G/5G of PAI-1 gene and Ins/Del of t-PA gene polymorphisms as a genetic factor associated with MI. it was observed that neither of the polymorphism is responsible for the onset of MI in the Pakistani population. The study indicated the involvement of risk factors like smoking, diabetes and family history in the disease progression.
Impaired fibrinolytic activity has been associated with thrombus formation leading to MI and/or stroke. Several studies have been conducted to investigate the role of PAI-1 and t-PA in clot stabilization or degradation, respectively [Shoji et al., 2003].
T-PA, being a serine protease, activates the plasminogen to plasmin, which ultimately digests fibrin clots into fibrin degraded products. Several studies have shown the regulatory role of t-PA in fibrinolysis of the thrombus [Soysal et al. 2006]. The effect of genetic variation like Alu-repeat Ins/Del within the gene encoding t-PA have been reported and have been found to be associated with the increased or decreased expression level of t-PA in the serum. The Ins/Ins homozygous allele of t-PA has been found to be associated with an increased risk of MI [Jood et al. 2005].
PAI-1 is the main inhibitor of t-PA and it functions by forming t-PA/PAI-1 complex which then leads to thrombosis under normal conditions. Several SNPs or variations have been reported within this gene which can cause prolonged thrombosis that leads to MI [Voestesch and Loscalzo, 2004]. The 4G/5G SNP has been studied in different populations with the 4G allele reported as risk allele for MI. But this association has not been reported to be reproducible in different populations due to racial or ethnic variations [Petrovic et al., 2003].
Case control studies for CAD have been conducted in different populations, indicating a strong association of genetics with the disease [Hooper et al., 2000], but the findings are not consistent for different populations which might be due to racial or ethnic variations Castellano, 2006; Schoenhard et al., 2008]. In the present study we did not find any significant association of PAI-1 4G/5G gene polymorphism with MI, which is therefore in agreement with the work of Ridker et al. (1997), Doggen et al. (1999), Anderson et al. (1999), Hooper et al. (2000), Petrovic et al. (2003) and Crainich et al. (2003).
Similarly we also studied the Alu Ins/Del of t-PA gene in MI cases and healthy controls, our results show no significant difference between the allelic frequencies of the Ins/Del in MI cases and healthy controls (OR=0.85(0.63-1.15). Our finding is contradictory to the Rotterdam study, in which van der Bom et al. (1997) have indicated a two fold increase in risk in patients with an Ins/Ins genotype suffering from MI, but his finding is not consistent with the studies done by other groups[Ridker et al., 1997, Steed et al., 1998, Hooper et al., 2000]. Our findings are in agreement with Steed et al. (1998), who found Ins allele frequency of 0.57 in cases compared to 0.58 in healthy controls with no association of Ins/Del polymorphism of t-PA gene with MI (P >0.05). While, Hooper et al (2000) reported 0.37 Ins allele frequency in MI cases compared to 0.44 in healthy controls. In a recent study Nakazaw et al. (2001) found 55% of Ins allele among the Japanese population thus excluding the role of Alu repeat with disease association.
The 4G allele of PAI-1 gene is associated with it's over expression[Hooper et al., 2000], but we found no significant difference in the frequency of 4G allele in MI cases (0.47) and healthy persons (0.43) indicating the role of other factors in MI instead of PAI-1 gene polymorphism.
Different studies have shown that the expression of t-PA is effected by environmental factors like diabetes which leads to its lower expression decreased, whereas expression of PAI-1 higher due to elevated serum glucose, insulin resistance and triglycerides [Hooper et al., 2000, Hafer-Macko et al. 2007]. These studies support the view that complex mechanisms are involved in the impaired fibrinolysis despite having either 4G/5G or Ins/Del polymorphism among the patients.
The association of different environmental risk factors with MI was also determined (Table 1). Regarding the smoking habit of the patients (Current + Former smoker vs. Non smoker), 45% MI patients were found to be heavy smokers as opposed to 19.6% in the healthy controls (p<0.001). This finding is in agreement with the study of Jafary et al. (2007) that has shown a high prevalence of smoking (52%) among MI patients in Pakistan. It is therefore concluded that smoking is a major environmental risk factor that is associated with MI in Pakistan. Diabetes, being an important factor for impaired fibrinolysis (Hooper et al., 2000), was observed to be more prevalent among MI cases 27.8% compared to control individuals 3.8% with OR= 9.75(6.89-13.77). This finding is in agreement with the previous report of Ismail et al. (2004).
Family history was also observed to be one of the important risk factors for MI in Pakistani population (Soysal et al., 2006). In our study, we observed a significant association of positive parental family history with the onset of MI with OR=1.72 (1.08-2.73).
The total cholesterol level was lower in patients compared to the unaffected healthy individuals, but statistically no significant difference was observed between the two groups (p>0.05) (Table 1). This could be because most of the patients were on statins treatment at the time of their blood collection. This finding is in agreement with the observations of Juhan-Vague et al. (2003), who also found lower levels of cholesterol in MI patients in the HIFMECH study. HDL-cholesterol levels were observed significantly lower in the cases as compared to the healthy controls (p< 0.05), which is in agreement with the study of Salonen et al. (1991), who also found an inverse association of HDL-C and risk of MI.
From the studies conducted so far in different populations, it is established that cardiovascular disease like MI is not caused by single risk factor instead genetic and environmental factors contribute equally.
From this study it is therefore concluded that the Alu repeat Ins/Del polymorphism in t-PA gene and the 4G/5G polymorphism in the PAI-1 gene are not the genetic factors that are associated with the onset of MI in Pakistani population, the environmental factors like cigarette smoking and diabetes are significantly associated with the disease progression. The family history of MI patients showed that some inherited genetic factors are involved in the disease onset in Pakistani population.
We would like to thank all the patients and unaffected individuals for donating their blood. Funding for this work was provided to RQ by the Shifa College of Medicine, Islamabad, Pakistan.
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