Plasminogen Activator Inhibitor Type-1 Gene 4G/5G Polymorphism Is Associated with Hypertensive Patients in the Turkish Population

Abstract

This study has been performed on hypertensive patients in the Turkish population to determine the frequency of 4G/5G polymorphism genotypes of plasminogen activator inhibitor type-1 gene and with the aim of examining the role of this polymorphism in hypertension development. Genomic DNA obtained from 284 persons (176 patients with hypertension and 108 healthy controls) was used in the study. DNA was multiplied by polymerase chain reaction using 4G and 5G allele-specific primers. Polymerase chain reaction products were assessed by being exposed to 2% agarose gel electrophoresis. Results were evaluated with the chi-square test. The 4G allele frequency was 31.25% and the 5G allele frequency was 68.75% in patients, whereas it was 49/51% in a control group. 5G5G genotype was found statistically high (p < 0.001) in patients relative to controls. This study showed that the plasminogen activator inhibitor type-1 gene 4G/5G polymorphism and the 5G5G genotype appear to be associated with an elevated risk of developing hypertension in a representative sample of Turkish population.

Introduction

Hypertension, which is defined as arterial blood pressure rising above the acceptable limits, is one of the most important risk factors of terminal diseases such as stroke, myocardial infarct, and congestive cardiac failure (Ergün et al., 2002). According to the results of epidemiologic studies, hypertension comes forward as a health problem that is very common in the world (Yalçın and Fiahn, 2002; Gunes et al., 2004). Hypertension, whose etiopathogenesis is largely complex, is considered to stem from environmental factors within the existence of a genetic infrastructure. Gene mutations, excessive salt consumption, smoking, obesity, sedentary living, alcohol consumption, stress, and so forth may cause hypertension on their own or by showing a synergistic effect (Yalçın and Fiahn, 2002). It has been estimated that the genetic contribution to the regulation of blood pressure is around 30-50% (Aguayo and Fardella, 2009).

It has been observed that plasminogen activator inhibitor type-1 (PAI-1), which is one of the fibrinolytic system inhibitors, has an effect on hypertension. Plasma PAI-1 levels are found to be related to systolic blood pressure in healthy middle-aged men and women (Izzo and Black, 2003). In the studies carried out, it was determined that PAI-1 levels are related to a polymorphism created by the guanosine insertion/deletion variation (4G or 5G) on the 675 base upstream of transcriptional start area of the gene (accession number: P05121), which codes PAI-1 (serpin-1) protein (Gerning et al., 1997; Brown et al., 2001). Plasma PAI-1 levels of the 4G4G homozygous individuals are found to be higher compared with 4G5G heterozygous and 5G5G homozygous individuals (Lijnen, 2005; Wiklund et al., 2005; Ding et al., 2006; Su et al., 2006).

In the studies related to the occurrence of hypertension in our country, the occurrence has been determined as 31.8% for adult population (Altun et al., 2005). The aim of this study was to determine the frequency of PAI-1 4G/5G polymorphism genotypes and to present whether such polymorphism and hypertension is related.

Methods

Patients

This study included 284 subjects (173 women and 111 men) recruited from the Cardiology Department, Medical Faculty, Eskişehir Osmangazi University. Of them, 176 (106 women and 70 men; mean age ± standard error(SE) = 59.12 ± 0.70 years) have primary hypertension (systolic blood pressure ≥140 mmHg and diastolic blood pressure ≥90 mmHg) and 108 (67 females and 41 males; mean age ± SE = 54.44 ± 1.13 years) are healthy persons. In accordance with the study protocol approved by the ethics committee of the Medical Faculty, Eskişehir Osmangazi University, informed consent from each patient was obtained. The study population was a genetically homogeneous native Turkish population.

Gene analysis

DNA was extracted from 10 mL venous blood by the saline method and stored at +4°C. DNA was amplified by polymerase chain reaction (PCR) in a thermal cycler (Eppendorf Mastercycler Personal, Hamburg, Germany) with 0.5 μL of DNA sample and thermostable Taq polymerase (Sigma-Aldrich, St. Louis, MO). Allele-specific primers were used in the PCR. These primers (Integrated DNA Technologies, Leuven, Belgium) were as follows:

(a) For 5G allele, 5′-GTCTGGACACGTGGGGG-3′

(b) For 4G allele, 5′-GTCTGGACACGTGGGGA-3′

Each in combination with a common downstream primer 5′-TGCAGCCAGCCACGTGATTGTCTA-3′ gives rise to a 139-bp DNA fragment. A control upstream primer, 5′AAGCTTTTACCATGGTAACCCCTGGT-3′, was used as a positive control in the PCR. About 0.5 μL of DNA sample was amplified for 35 cycles with denaturation at 94°C for 60 s, annealing at 54°C for 30 s, and extension at 72°C for 40 s using a 25 μL PCR mixture containing a 50 pmol allele-specific primer, a 50 pmol common downstream primer, a 2.5 pmol control upstream primer, 1 × PCR buffer with magnesium chloride, 0.2 mM deoxyribonucleotide triphosphates (dNTPs), and 1.25 U Taq polymerase. The PCR products were separated by electrophoresis on 2% agarose gel containing 4 μL ethidium bromide (50 μg/mL) and were viewed by coupled charged device (CCD) camera. The results were evaluated using gel analysis software (Labwork, Cambridge, United Kingdom).

Statistical analysis

Statistical analysis was performed using Statistical Package for Social Sciences software package. The values were expressed as means ± SE. Alleles and genotype frequencies regarding PAI-1 gene 4G/5G polymorphism between patients and control subjects and sex were compared using the chi-square test. A p-value of less than 0.05 was considered statistically significant.

Results

Table 1 presents the distribution of PAI-1 gene 4G/5G genotypes in patients and controls. It is determined that, compared with the control group, the patient group had a significant statistical increase of 5G5G homozygosis (p < 0.001). Allele frequencies appear to be similar in the control group, whereas 5G allele frequency is found to be much higher compared with 4G allele frequency in the patient group.

Table 1.

Distribution of Plasminogen Activator Inhibitor Type-1 Gene 4G/5G Polymorphism Genotypes and Allele Frequencies in Patients and Controls

	Genotypes			Alleles
Groups	4G4G n (%)	4G5G n (%)	5G5G n (%)	4G n (%)	5G n (%)
Patient	23 (13.1)	64 (36.4)	89 (50.6)	110 (31.25)	242 (68.75)
Control	24 (22.2)	58 (53.7)	26 (24.1)	106 (49)	110 (51)
Statistics	p < 0.001; SD = 2; χ² = 19.676

Discussion

In this study, we have analyzed the distribution of PAI-1 gene 4G/5G polymorphism genotypes in Turkish hypertensive patients to assess its possible role in the pathogenesis of hypertension. This study is the first trial to specify the relationship between PAI-1 gene 4G/5G polymorphism and hypertension in adult patients in Turkey. The results of our study show that 5G5G genotype is statistically and significantly high among the PAI-1 gene 4G/5G polymorphism genotypes. On evaluating the allele frequencies, 5G allele frequency was found to be higher than 4G allele frequency in the patient group and such frequencies were found to be similar in the control group.

In the former studies related to the Turkish population, genotype percentages in the asthma group were found to be 26.5% for 4G4G, 43.9% for 4G5G, and 29.6% for 5G5G (Cosan et al., 2009) and for the stroke group, 45% for 4G4G, 12.3% for 4G5G, and 42.7% for 5G5G (Kucukarabaci et al., 2008). As no relation was found between such distribution and asthma, it was determined that 4G5G genotype has significantly decreased in the stroke group, compared with the control group. As the stroke groups were evaluated in terms of genotypes within themselves, 4G4G and 5G5G genotypes were found to significantly increase compared with 4G5G genotypes.

Likewise, a study carried out by Yılmaz et al. (2004) on Turkish patients with deep vein thrombosis reported that there is no significant difference between PAI-1 gene and 4G/5G genotypes. In a study carried out on hypertensive patients in a Chinese population, it was pointed out that there is a similarity in the genotype distribution of PAI-1 gene 4G/5G polymorphism and such gene polymorphism is not solely related to hypertension (Jeng, 2003).

In the study carried out by Martinez et al. in Spain, it was stated that PAI-1 gene 4G/5G polymorphism 4G4G genotype is a risk factor for the development of hypertension (Martinez et al., 2007).

As gene pools, life style, and gene-environment interactions vary between the populations, the risk cannot be supposed as identical in every population with respect to genotypes. Therefore, the differences will be manifested in the genetic relationship of PAI-1 gene 4G/5G polymorphism allele frequencies with diseases and populations (Cosan et al., 2009). The results of this study and former studies show that there are different genotype distributions within different disease groups of the same population. The results of this study show that the rate of 5G5G homozygosis of PAI-1 gene 4G/5G genotypes is high in Turkish patients and can be a marker in the development of hypertension.

Footnotes

Disclosure Statement

No competing financial interests exist.

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