Association Between Plasminogen Activator Inhibitor-1-675 4G/5G Insertion/Deletion Polymorphism and Chronic Obstructive Pulmonary Disease

ABSTRACT

Molecular pathology of chronic obstructive pulmonary disease (COPD) is still being investigated to discover relationships with disease pathogenesis. Evidence of plasminogen activator inhibitor-1 (PAI-1) overexpression in the sputum and the blood of COPD patients is growing. We aimed to investigate the potential relation between PAI-1 promoter 4G/5G insertion/deletion polymorphism and COPD development. In a case-control study, we genotyped 117 COPD patients and 160 control subjects for PAI-1 promoter 4G/5G polymorphism by an allele-specific polymerase chain reaction analysis. All subjects were male smokers. In the co-dominant model, there was a significant difference in the distribution of 5G/5G, 4G/5G and 4G/4G genotypes between COPD patients and controls (p = 0.002). In the recessive model, carriers of 4G/4G genotype were significantly higher in COPD patients than controls (p = 0.01). Carriers of 4G/4G genotype were at higher risk to develop COPD than those carrying 5G/5G or 4G/5G genotypes (crude odds ratio (OR) = 2.10, 95% confidence interval (CI) = 1.19–3.73, adjusted OR = 2.5, 95% CI = 1.22–3.99). In conclusion, PAI-1 4G/5G genetic variations are associated with COPD development in males.

KEYWORDS:

• Association
• COPD
• PAI-1 4G/5G

Introduction

Chronic obstructive pulmonary disease (COPD) prevalence is increasing and it is expected to be one of the highest five chronic diseases in terms of international mortality and morbidity by 2030 (1). Even though cigarette smoking is the most important risk factor for COPD, only 20–30% of chronic smokers develop COPD (2). In addition to smoking, other environmental and genetic factors and gene environment relationships affect COPD development (3). Genomics provide a chance to understand the pathogenesis of COPD and implicate this to improved clinical management (4). Current concept of COPD pathogenesis is that lung injury by inhaled tobacco leads to small and large airway inflammation. Chronic inflammation may induce destruction of parenchymal tissue leading to emphysema and disruption of normal tissue repair mechanisms and defense pathways causing small airway fibrosis (5).

Plasminogen activator inhibitor 1 (PAI-1), also known as SERPINE1 (Serpin Peptidase Inhibitor, Clade E (Nexin, Plasminogen Activator Inhibitor Type 1), Member 1), is a protease inhibitor, and inhibitor of fibrinolysis. PAI-1 is reported to play a significant role in the development and progression to fibrosis (pathological formation of connective tissue) in inflammatory conditions where fibrin is deposited in tissues. The genetic and environmental determinants of PAI-1 expression are not fully understood (6, 7). Gene variability may also contribute to the level of PAI-1 biosynthesis (8). The human PAI-1 gene is located on chromosome 7. A guanosine insertion/deletion polymorphism in the promoter region of PAI-1 gene at the −675 bp position, named 4G/5G (rs1799889), has been reported (9).

To our knowledge the potential associations between COPD risk and PAI-1 4G/5G polymorphism are not previously investigated, the aim of our study was to investigate the distribution of genotypes and the frequency of alleles of the 4G/5G polymorphism in patients with COPD and analyze the relation between development of COPD and PAI-1 4G/5G polymorphism.

Materials and methods

Study population

The procedures followed were in accordance with the ethical standards of the responsible institutional committee on human experimentation and with the Declaration of Helsinki 1975, as revised in 2008.

The study comprised 117 COPD patients and 160 apparently healthy controls. They were all Egyptians, an ethnic group in North Africa. They were all males. Patients with COPD were aged 54.8 ± 4.4 years. Control subjects were aged 53.1 ± 14.3 years. All subjects were smokers. COPD was diagnosed on the basis of history, and clinical and radiological examinations; and confirmed by spirometry (post-bronchodilator forced expiratory volume in one second (FEV¹)/forced vital capacity (FVC) ratio % <70%). According to Global initiative for Chronic Obstructive Lung disease (GOLD) (5), 79 COPD patients were GOLD 3 and 38 patients were GOLD 4. Any patient with a history of bronchial asthma, diabetes, hypertension and any other associated primary neoplasm, or^{Figure 1} its history was excluded from the study. Patients gave a written consent prior to blood and data collection.

DNA isolation

High molecular weight DNA from peripheral blood of COPD cases and controls was isolated using DNA purification kit (QIAamp®DNA Blood Mini Kit, Qiagen, 28159 Avenue Stanford, Valencia, USA). DNA was quantified spectrophotometrically at 260 nm and 280 nm and stored at −20°C.

Genotyping assays

DNA was amplified for molecular detection of PAI-1 4G/5G insertion/deletion polymorphism located within the promoter region-675 base pairs (bp) upstream of the transcription start site by an allele-specific polymerase chain reaction analysis using specific primers as previously described (10). PAI-1: 5′ AAG CTT TTA CCA TGG TAA CCC CTG GT 3′, PAI-2: 5′ TGC AGC CAG CCA CGT GAT TGT CTA 3′, PAI-4G: 5′ GTC TGG ACA CGT GGG GA, and 3′ PAI-5G: 5′ GTC TGG ACA CGT GGG GG 3′. Both PAI-1 and PAI-2 amplify a 257-bp product from either allele. The PAI-4G specifically amplifies the 4G allele, and PAI-5G specifically amplifies the 5G allele. Amplification reactions were performed in volume of 25 µl containing 1 µg DNA, both the PAI-1 and PAI-2 primers and either the PAI-4GA or the PAI-5G primer in GoTaq® Green Master Mix, 2X (Promega corporation, USA) consisted of GoTaq® DNA Polymerase supplied in 2X Green GoTaq® Reaction Buffer (pH 8.5), 400 µM dNTPs and 3 mM MgCl₂. The protocol begins with a 94°C hold for 5 minutes followed by 40 cycles, each consists of 1-minute denaturation at 94°C, 45-seconds annealing at 58°C and 1-minute extension at 72°C. An additional extension for 15 minutes at 72°C was added after the last cycle, followed by a 4°C incubation. The products were visualized after ethidium bromide containing agarose gel electrophoresis. Each successful reaction produced the expected 257-bp control band and an additional band in either the PAI-4G or PAI-5G-containing reaction. Non-template controls were included in every PCR run. Representative gel for the three genotypes is shown in Figure 1.

Figure 1. 2% Agarose gel electrophoresis of PCR products. Lanes 1 and 2 represent 4G/5G genotype. Lanes 3 and 4 represent 4G/4G genotype. Lanes 5 and 6 represent 5G/5G genotype. Lane 7 is a molecular weight marker.

Statistical analysis

Demographic data and spirometric parameters are presented as mean ± standard deviation (SD). χ2 test was used to compare the proportions of categorical variables in cases and controls. The distributions of genotypes for PAI-1 gene were tested for the Hardy–Weinberg heredity equilibrium by exact test. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to examine the association between the PAI-1 4G/5G genotypes and COPD development. Binary logistic regression was done to confirm the association between PAI-1 4G/5G gene polymorphisms and COPD by adjustment for smoking index. The co-dominant, the dominant and the recessive models were used. p < 0.05 was considered significant.

Results

The study included 117 COPD patients (GOLD 3 and 4) in addition to 160 control subjects. All subjects were males. They were all smokers. Age did not differ significantly between patients and controls (p = 0.21). Age, smoking index and pulmonary function tests data of our subjects are presented in Table 1.

Table 1. Age, smoking index and pulmonary function data of study subjects (n = 277).

Parameters	Controls (n = 160) (mean ± SD)	Patients (n = 117) (mean ± SD)
Age (years)	53.1 ± 14.3	54.8 ± 4.4
Smoking index (Pack-years)	14.4 ± 11.8	38.3 ± 14.6
FEV^1% predicted	88.3 ± 10.4	37.9 ± 8.5
FEV^1/FVC ratio %	77.9 ± 4.2	46.8 ± 8.8

FEV¹ = forced expiratory volume in one second; FVC = forced vital capacity.

Genotype distribution in the control population did not deviate from Hardy–Weinberg equilibrium (p = 0.75). There was Hardy–Weinberg disequilibrium among cases (p < 0.0001) (Table 2).

Table 2. PAI-1 exact test for Hardy–Weinberg equilibrium (n = 277).

	N11	N12	N22	N1	N2	p
All subjects	98	117	62	313	241	0.02
Controls	52	81	27	185	135	0.75
Patients	46	36	35	128	106	<0.0001

^{Table 3Table 4}

The frequency distribution of PAI-1 4G/5G polymorphism is presented in Table 3. In the co-dominant model, there was a significant difference in the distribution of 5G/5G, 4G/5G and 4G/4G genotypes between COPD patients and controls (p = 0.002). In crude analysis, carriers of 4G/5G genotype were at lower risk to develop COPD than those carrying 5G/5G genotype (crude OR = 0.50, 95% CI = 0.29–0.88). After adjustment for smoking index, 4G/5G and 4G/4G genotypes were not related to COPD development (adjusted OR = 0.77, 95% CI = 0.48–1.25 and adjusted OR = 1.66, 95% CI = 0.96–2.55, respectively). In the dominant model, carriers of 4G/5G or 4G/4G genotypes did not differ significantly between COPD patients and controls compared to carriers of 5G/5G genotype (p = 0.24). In the recessive model, carriers of 4G/4G genotype were significantly higher in COPD patients than controls (p = 0.01). Carriers of 4G/4G genotype were at higher risk for COPD development than those carrying 5G/5G or 4G/5G genotypes (crude OR = 2.10, 95% CI = 1.19–3.73, adjusted OR = 2.5, 95% CI = 1.22–3.99) (Table 3).

Table 3. PAI-1 4G/5G polymorphism association with COPD.

Model	Genotype	Controls (n = 160)	Patients (n = 117)	p	Crude OR (95% CI)	Adjusted OR (95% CI)
Co-dominant	5G/5G	52 (32.5%)	46 (39.3%)	0.002	1.00	1.00
	4G/5G	81 (50.6%)	36 (30.8%)		0.50 (0.29–0.88)	0.77 (0.48–1.25)
	4G/4G	27 (16.9%)	35 (29.9%)		1.47 (0.77–2.78)	1.66 (0.96–2.55)
Dominant	5G/5G	52 (32.5%)	46 (39.3%)	0.24	1.00	1.00
	4G/5G-4G/4G	108 (67.5%)	71 (60.7%)		0.74 (0.45–1.22)	0.87 (0.52–1.43)
Recessive	5G/5G-4G/5G	133 (83.1%)	82 (70.1%)	0.01	1.00	1.00
	4G/4G	27 (16.9%)	35 (29.9%)		2.10 (1.19–3.73)	2.5 (1.22–3.99)

The frequency distribution of PAI-1 5G and 4G alleles did not differ significantly between COPD patients and controls (p = 0.46) (Table 4).

Table 4. PAI-1 4G and 5G allele frequencies and association with COPD.

	All subjects (n = 277)		Controls (n = 160)		Patients (n = 117)
Allele	Count	Proportion	Count	Proportion	Count	Proportion	p	Crude OR (95% CI)	Adjusted OR (95% CI)
5G	313	0.56	185	0.58	128	0.55	0.46	1.00	1.00
4G	241	0.44	135	0.42	106	0.45		1.13 (0.81–1.59)	1.44 (0.85–2.12)

Discussion

The rare α-1-antitrypsin deficiency is the only documented genetic cause of COPD (11). A number of studies have been performed to find other susceptibility genes of COPD. To our knowledge, this is the first report investigating the potential influence of PAI-1 4G/4G polymorphism on COPD development. Our results showed that PAI-1 4G/4G genotype was significantly more associated with COPD development compared to 5G/5G or 4G/5G genotypes (p = 0.01) and carriers of PAI-1 4G/4G genotype were at higher risk to develop COPD than carriers of 5G/5G or 4G/5G genotypes (crude OR = 2.10, adjusted OR = 2.5).

PAI-1 is one of antiproteases, and it has been shown to be upregulated in the lung (12) and increased in the blood (13) during hypoxia. It has been overexpressed in the lung with exposure to lipopolysaccharide, cigarette smoke (14, 15), or oxidative stress via nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) (16). Elevated levels of PAI-1 in sputum have been reported in two different case–control studies on COPD patients (16, 17). Moreover, Xiao et al. (18) reported that PAI-1 in induced sputum of COPD patients showed significant negative correlation with FEV¹%. Furthermore, van den Borst et al. (19) reported higher plasma PAI-1 in COPD patients.

Studies indicate that the protein encoded by the 4G-allele possesses higher activity than that encoded by the 5G-allele. This is because the 5G-allele contains an additional binding site for a DNA-binding protein that acts as a transcriptional repressor (20, 21). Studies on different populations have shown that individuals with 4G/4G genotype have significantly higher plasma PAI-1 levels than those with 5G/5G genotype (22, 23).

The link between PAI-1 polymorphism and COPD risk is observably important not only for understanding COPD pathogenesis but also for selecting patients for potential PAI-1 target therapy. One can assume that PAI-1 may be a potential therapeutic target for COPD patients with 4G/4G genotype. Perhaps, lower PAI-1 levels could lead to less inhibition of fibrinolysis and more rapid degradation of the fibrin; part of COPD pathogenesis. Schuliga et al. (24) review highlighted the potential of fibrinolytic agents, including small molecule inhibitors of PAI-1, as treatments for chronic respiratory disease. PAI-1 genotype–phenotype association studies in COPD patients are needed.

In conclusion, our findings indicate PAI-1 4G/5G genetic variants association with COPD development in males. Further independent studies using a larger population, studying genotypic phenotypic associations, including both sexes and applied on different GOLD stages are necessary in order to clarify the relation between PAI-1 4G/5G gene polymorphism and pathogenesis of COPD.

Author contributions

Both authors shared in study design, implementation, and data analysis. Essa ES drafted the manuscript and both authors edited it for intellectual content. All authors have approved the final article.