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Genetic variant in glutathione peroxidase 1 gene is associated with an increased risk of coronary artery disease in a Chinese population
Clinica Chimica Acta 395 (2008) 89–93
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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Genetic variant in glutathione peroxidase 1 gene is associated with an increased risk of coronary artery disease in a Chinese population Na-Ping Tang a,1, Lian-Sheng Wang a,b,1, Li Yang c,1, Hai-Juan Gu a, Qing-Min Sun a, Ri-Hong Cong a, Bo Zhou a, Huai-Jun Zhu a, Bin Wang a,⁎ a b c
Key Laboratory of Reproductive Medicine, Department of Pharmacology, Nanjing Medical University, Nanjing, Jiangsu Province 210029, China Department of Cardiology, First Affiliated Hospital of Nanjing Medical University, Nanjing, China Department of General Surgery, First Affiliated Hospital of Nanjing Medical University, Nanjing, China
A R T I C L E
I N F O
Article history: Received 3 November 2007 Received in revised form 28 February 2008 Accepted 16 May 2008 Available online 25 May 2008 Keywords: Coronary artery disease Genetic variant Glutathione peroxidase 1 Oxidative stress Polymorphism
A B S T R A C T Background: Glutathione peroxidase 1 (GPX1), the key antioxidant enzyme in vascular endothelial cells, has been shown to exert a protective effect against the presence of coronary artery disease (CAD). The 198Pro/leu variant, located at codon 198 of GPX1 gene, has recently been linked to cardiovascular disease, but data were inconsistent. We investigated the association between the occurrence of CAD and the 198Pro/leu variant in a Chinese population. Methods: A total of 265 unrelated CAD patients and 265 age- and sex-matched control subjects were recruited in this study. The GPX1 198Pro/leu genotype was determined using polymerase chain reaction– restriction fragment length polymorphism. Results: Compared to the 198Pro/Pro carriers, subjects with the variant genotypes (198Pro/leu and 198Leu/ leu) had a significantly higher risk of CAD (adjusted OR = 2.02, 95%CI = 1.27–3.22). In stratified analyses, the variant genotypes were significantly associated with increased CAD risk in subjects b 64 y (adjusted OR = 2.41, 95%CI = 1.16–4.98), males (adjusted OR = 1.86, 95%CI = 1.09–3.18) and non-smokers (adjusted OR = 2.40, 95% CI = 1.15–5.01). However, no significant association was observed between this variant and the severity of CAD. Conclusion: These data provide evidence that GPX1 198Pro/leu variant genotypes are significantly associated with CAD risk in this Chinese population. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Oxidative stress has recently been suggested to play a central role in the pathogenesis of atherosclerosis [1–3]. Antioxidant enzymes, including superoxide dismutase, catalase, and glutathione peroxidase (GPX), are essential for defense against oxidative stress [4–6]. One of these enzymes, GPX, is a soluble selenoprotein [7,8]. To date, 4 different GPX forms have been identified, all of which contain selenocysteine at their active sites [7]. GPX1, the ubiquitous intracellular form and key antioxidant enzyme within many cells, including the endothelium, converts hydrogen peroxide to water and lipid peroxides to their respective alcohols using reduced glutathione as an essential co-substrate [8]. Multiple lines of evidence supported the involvement of GPX1 in protecting vessels against oxidative stress and atherogenesis [9–15]. Recent studies show that, in vascular endothe-
⁎ Correspondence author. Department of Pharmacology, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, Jiangsu Province, China. Tel./fax: +86 25 86862884. E-mail address: [email protected] (B. Wang). 1 Contributed equally to this work. 0009-8981/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2008.05.013
lial cells, shear stress can upregulate the expression and enzymatic activity of GPX1 [9]. In mouse model, deficiency of GPX1 leads to endothelial dysfunction [10] and accelerates the progression of atherosclerosis [11]. In addition, GPX1 deficiency is accompanied by increased periadvential inflammation, neointima formation, and collagen deposition surrounding the coronary arteries [12]. Furthermore, decreased GPX1 activity was observed in patients with coronary artery disease (CAD) and those with acute myocardial infarction, and associated with the extent of atherosclerosis [13–15]. GPX1, the gene coding for glutathione peroxidase 1, is located on chromosome 3p21.3 [16] and it is composed of 2 exons with a 1.42 kb region [17]. A genetic variant (198Pro/leu, rs1050450) at codon 198 of GPX1 gene, resulting in the substitution of leucine (CTC) for proline (CCC), was identified by Moscow et al. [18]. Recent studies have confirmed that the GPX1 198Leu allele was associated with a significantly decreased GPX1 enzymatic activity compared with the 198Pro allele [19–21]. The role of this variant has been extensively studied in cancer [18–20,22–26]. However, studies investigating the association between this variant and vascular disease are rare [21,27– 29]. Additionally, data on the effect of this variant on risk of cardiovascular disease are inconsistent [21,28,29]. Thus, we conducted a
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Table 1 Baseline characteristics of cases and controls Characteristics
Cases (n = 265)
Controls (n = 265)
P
Sex (male), n (%) Age (years) BMI (kg/m2) Hypertension, n (%) Diabetes, n (%) Dyslipidemia, n (%) Smoking, n (%) TC (mmol/l) TG (mmol/l) HDL-C (mmol/l) LDL-C (mmol/l) Glucose (mmol/l) Number of diseased vessels Single vessel, n (%) Double vessels, n (%) Triple vessels, n (%)
194 (73.2) 64 (56–71) 25.1 ± 3.3 194 (73.2) 60 (22.6) 21 (7.9) 127 (47.9) 4.11 (3.48–4.71) 1.46 (1.05–2.14) 0.98 (0.84–1.14) 2.36 (1.83–2.82) 5.19 (4.68–5.95)
194 (73.2) 64 (55–71) 23.8 ± 3.6 117 (44.2) 44 (16.6) 8 (3.0) 83 (31.3) 3.95 (3.31–4.54) 1.14 (0.79–1.62) 1.08 (0.90–1.29) 2.14 (1.66–2.63) 4.88 (4.41–5.60)
NS NS b0.001 b0.001 NS 0.013 b0.001 0.036 b0.001 b0.001 0.005 b0.001
97 (36.6) 82 (30.9) 86 (32.5)
– – –
– – –
BMI (normal distributed) was expressed as mean ± SD and compared by student's Student's t-test. Age, TC, TG, HDL-C, LDL-C and glucose (abnormal distributed) were expressed as median (25th–75th percentiles) and analyzed by Mann–Whitney U-test. Other data were expressed as frequencies and percentages and evaluated by χ2-test. BMI, body mass index (normal reference range 18.5–23 kg/m2); HDL-C, high density lipoprotein cholesterol (0.83–1.97 mmol/l); LDL-C, low density lipoprotein cholesterol (1.85–3.65 mmol/l); TC, total cholesterol (3.15–6.25 mmol/l); TG, triglyceride (0.48–1.88 mmol/l).
hospital-based case-control study with CAD patients confirmed by coronary angiography and age- and sex-matched controls to evaluate the association between the risk of CAD and the GPX1 198Pro/leu variant in a Chinese population. 2. Materials and methods 2.1. Study population Consecutive 265 CAD patients were recruited from the inpatients who were admitted to Nanjing Medical University Affiliated Hospital because of angina pectoris or other symptoms or signs of cardiovascular diseases. An additional 265 patients also admitted to this hospital served as the control group. They were selected during the same time as cases and matched by age (±5 years) and sex. Considering that it was unethical to perform coronary angiography to rule out the presence of asymptomatic CAD, the following inclusion criteria were used: no history of angina pectoris, without any symptoms or signs of other atherosclerotic vascular diseases. All subjects enrolled in this study were Han Chinese and residing in or nearby Jiangsu province. They had no family history of CAD and no history of significant concomitant diseases including cardiomyopathy, bleeding disorders, renal failure, previous thoracic irradiation therapy and malignant diseases. This study was approved by the Nanjing Medical University Affiliated Hospital Ethics Committee and informed consent was obtained from each participant. The definition of CAD was based on angiographic criteria; the coronary angiograms were reviewed by experienced cardiologists who were unaware that the patients were to be included in this study. Coronary artery disease was defined as angiographic evidence of at least one segment of a major coronary artery including the left anterior descending, left circumflex or right coronary artery with N50% organic stenosis. The severity of CAD was expressed as single vessel disease, double vessel disease and triple vessel disease depending on the number of the main vessels with stenosis. Hypertension, diabetes mellitus, and dyslipidemia were defined as those reported in our previous study [3]. Briefly, hypertension was defined as resting systolic blood pressure N140 mm Hg and/or diastolic blood pressure N90 mm Hg or in the presence of active treatment with antihypertensive agents. Diabetes was defined as fasting blood glucose N 7.8 mmol/l or a diagnosis of diabetes needing diet or antidiabetic drug therapy. Dyslipidemia was defined as total cholesterol level of N6.2 mmol/l or on drugs. Smoking was defined as ≥ 10 cigarettes/d. 2.2. Laboratory measurements Fasting blood samples were obtained by venipuncture in the early morning. The levels of plasma total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) were measured enzymatically (First Chemical Co. Japan) on a chemistry analyzer (Olympus Au2700). Glucose levels were measured by a glucose oxidase method (Reagent kit, Diagnostic Chemicals Ltd., Oxford, CT). 2.3. Genotyping Genomic DNA was isolated from leukocyte pellet using standard phenol-chloroform extraction. The GPX1 198Pro/leu (C to T variant) variant was determined using
polymerase chain reaction (PCR)-restriction fragment length polymorphism employed primers 5′- TCC AGA CCA TTG ACA TCG AG -3′ (forward) and 5′- ACT GGG ATC AAC AGG ACC AG -3′ (reverse). A 222-base pair (bp) DNA fragment containing the polymorphic site was amplified by PCR in the T1 Thermocycler (Biometra, Goettingen, Germany) with a total volume of 20 μl, containing 2 μl 10 × PCR buffer, 1.125 mmol/l MgCl2, 0.15 mmol/l dNTPs, 0.25 μmol/l each primer, 100 ng of genomic DNA and 1.5 U of Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania). The PCR amplification was performed with the following conditions: initial denaturation at 94 °C for 8 min, amplification for 35 cycles at 94 °C for 30 s, 59 °C for 30 s and 72 °C for 30 s, followed by a final elongation step at 72 °C for 7 min. An aliquot of 5 μl of the PCR product was digested with 10 U of ApaI (GGGCCC, New England BioLabs, Waltham, MA) in 2 μl of 10 × NEB buffer 4 (50 mmol/l potassium acetate, 20 mmol/l Tris-acetate, 10 mmol/l magnesium acetate and 1 mmol/l dithiothreitol), 0.2 μl of 100 μg/ml bovine serum albumin and 12.3 μl dH2O at 25 °C for 16 h. After restriction enzyme digestion, the products were separated on a 2% agarose gel, and visualized by staining with 0.5 μg/ml of ethidium bromide under ultraviolet light. The 222-bp PCR product was cleaved into 2 fragments of 170 and 52 bp in the presence of the 198Pro (C) homozygous, 3 fragments of 222, 170 and 52 bp for the 198Pro/leu (CT) heterozygote, while the 198Leu (T) homozygous remained uncleaved showing the 222 bp PCR product. About 10% of the samples were randomly selected to perform the repeated assays, and the results were 100% concordant. Two researchers, blinded to the clinical data, scored the genotypes independently. 2.4. Statistical analysis The normality of the distribution was analyzed using the Kolmogorov–Smirnov test. Differences of continuous variables without skewness (presented as mean ± SD) between 2 groups were calculated by the Student's t-test. Differences of continuous variables departing from the normal distribution even after transformation (presented as median and interquartile range) between 2 groups were analyzed by Mann–Whitney U-test. Categorical variables were presented using frequency counts and compared by χ2-test. The χ2 goodness-of-fit test was used to identify significant departures from the Hardy–Weinberg equilibrium. A binary logistic regression analysis was used for the evaluation of the independent effect of GPX1 genotypes on the development of CAD, adjusted for the presence of risk factors including age, sex, body mass index (BMI), smoking status, hypertension, diabetes and dyslipidemia. Odds ratio (OR) and 95% confidence interval (CI) were calculated. The linear trend in the association of GPX1 198Pro/leu polymorphism with CAD severity was evaluated by χ2-test for trend. A value of P b 0.05 was considered statistically significant. Statistical analyses were performed with the SPSS 13.0 (SPSS Inc., Chicago, IL, USA).
3. Results 3.1. Baseline characteristics A total of 265 CAD patients and 265 age- and sex-matched controls were included in this study. The baseline characteristics are shown in Table 1. No significant difference was found between the cases and controls in age [64 (56–71) vs. 64 (55–71) y, P = 0.504] and sex (male: 73.2% vs. 73.2%, P = 1.000). As expected, compared to the control group, the CAD group had higher BMI, numbers of hypertension, dyslipidemia and rate of smoking, as well as higher levels of TC, TG, LDL-C and glucose but lower HDL-C, all of which are established CAD risk factors. By coronary angiography, 97 (36.6%) CAD cases had single vessel disease, 82 (30.9%) had double vessel disease and 86 (32.5%) had triple vessel disease. In CAD patients with diabetes, 15 (25.0%) had single vessel disease, 24 (40.0%) had double vessel disease and 21 (35.0%) had triple vessel disease.
Table 2 Genotype and allele distribution of the 198Pro/leu (C to T variant) polymorphism in cases and controls Group
Genotype, n (%) CC
Cases (n = 265) Controls (n = 265)
CT
197 (74.3) 65 (24.5) 222 (83.8) 43 (16.2) χ2 = 8.973, df = 2 P = 0.011
Allele, n (%) TT
C-allele
3 (1.1) 0 (0)
459 (86.6) 71 (13.4) 487 (91.9) 43 (8.1) χ2 = 7.706, df = 1 P = 0.006
T-allele
Distributions of the 198Pro/leu genotypes in both cases and controls were in Hardy– Weinberg equilibrium (χ2 = 0.864, P = NS and χ2 = 2.066, P = NS, respectively, calculated by χ2 goodness-of-fit test).
N.-P. Tang et al. / Clinica Chimica Acta 395 (2008) 89–93
3.2. GPX1 198Pro/leu genotypes and allele frequencies Table 2 shows the distribution of GPX1 198Pro/leu genotypes and alleles of the two groups. In both cases and controls, the genotype distributions were consistent with those predicted by the Hardy– Weinberg equilibrium (χ2 = 0.864, P = 0.353 and χ2 = 2.066, P = 0.151, respectively). In the case group, the frequencies of the 198Pro/Pro, 198Pro/leu, and 198Leu/leu genotypes were 74.3%, 24.5% and 1.1%; while in the control group, the frequencies were 83.8% and 16.2% for the 198Pro/Pro and 198Pro/leu genotypes, respectively, and no 198Leu/leu individuals were found. The 198Leu allele frequency was significantly higher in the case group than those observed in the control group (13.4% vs. 8.1%, P = 0.006). In addition, among CAD patients with diabetes, 11 (18.3%) carried the variant genotypes (198Pro/leu, and 198Leu/leu).
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Table 4 Stratified analysis for the variant 198Pro/leu (C to T variant) genotypes in cases and controls Smoking status
CT + TT/CC Cases controls
Unadjusted model
P
OR(95% CI) Ageb b 64 y ≥ 64 y Sex Male Female Smoking status Yes No
Adjusted modela
P
OR(95% CI)
36/93 18/109 32/104 25/113
2.34 (1.25–4.40) 1.39 (0.77–2.50)
0.008 NS
3.41 (1.62–7.19) 1.41 (0.75–2.67)
0.001 NS
53/141 32/162 15/56 11/60
1.90 (1.16–3.12) 1.46 (0.62–3.45)
0.011 NS
2.30 (1.34–3.95) 1.51 (0.59–3.87)
0.003 NS
26/101 14/69 42/96 29/153
1.27 (0.62–2.60) 2.31 (1.35–3.95)
NS 0.002
1.38 (0.63–3.01) 2.70 (1.50–4.84)
NS 0.001
a
Adjusted for age, sex, BMI, smoking status, hypertension, diabetes and dyslipidemia (logistic regression analysis). Stratified by the median age (64 years) of controls.
3.3. GPX1 198Pro/leu variant and CAD risk
b
Table 3 shows the risk estimates for the variant GPX1 genotypes among CAD patients compared with controls. Since three 198Leu/leu homozygotes were found in case group and none in the control group, the variant genotypes including 198Pro/leu and 198Leu/leu were ascribed to a single group. Overall, the unadjusted OR for subjects with variant genotypes was 1.78 (95% CI = 1.16–2.73, P = 0.008) relative to wild-type 198Pro/Pro carriers. After adjustment for age, sex, BMI, smoking status, hypertension, diabetes and dyslipidemia, the association persisted (adjusted OR = 2.02, 95% CI = 1.27–3.22, P = 0.003). 3.4. Stratified analysis The results of stratified analyses by the median age of controls (64 years), sex and smoking status with the GPX1 variant genotypes are presented in Table 4. In the stratified analysis by age, the effect of variant genotypes (198Pro/leu and 198Leu/leu) on the increased risk of CAD in subjects with age b64 y were significant (adjusted OR = 3.41, 95% CI = 1.62–7.19, P = 0.001), while in subjects with age ≥64 y, no statistical significance was found (P = NS). The increased risk associated with the variant genotypes tended to be more evident in male subjects (adjusted OR = 2.30, 95% CI = 1.34–3.95, P = 0.003). But in female subjects, the association between the GPX1 polymorphism and CAD risk was not statistically significant (P = NS). Compared with the 198Pro/Pro genotype, the adjusted OR for the variant genotypes was 2.70 (95% CI = 1.50–4.84, P = 0.001) among non-smokers and 1.38 (95% CI = 0.63–3.01, P = NS) among smokers. Patients with CAD were also subclassified into 3 subgroups (single, double and triple vessel disease) according to the number of affected coronary arteries. However, no statistically significant association was noted between the GPX1 198Pro/leu variant and the severity of CAD (data not shown). 4. Discussion In this hospital-based case-control study, we identified the role of GPX1 198Pro/leu variant in CAD susceptibility in a Chinese population. Our results indicate that the variant 198Leu allele is significantly associated with the increased risk of CAD in this Chinese population. 198Pro/leu variant is located in exon 2 of GPX1 gene with proline to
Table 3 Risk estimates for the variant 198Pro/leu (C to T variant) genotypes Genotype
Cases/controls
Unadjusted model
P
OR(95% CI) Overall CC CT + TT a
265/265 197/222 68/43
1.00 1.78 (1.16–2.73)
Adjusted modela
P
OR(95% CI)
0.008
1.00 2.02 (1.27–3.22)
0.003
Adjusted for age, sex, BMI, smoking status, hypertension, diabetes and dyslipidemia (logistic regression analysis).
leucine transition at codon 198 [18]. To date, very few studies have linked the GPX1 198Pro/leu variant to cardiovascular disease [21,28,29]. Hamanishi et al. [21] showed that, in Japanese type 2 diabetic patients, those with 198Pro/leu genotype had significantly higher mean intimamedia thickness of common carotid arteries (1.04± 0.23 vs. 0.91 ± 0.18 mm, P = 0.0028) and prevalence of cardiovascular disease (24.2% vs. 10.6%, P = 0.035) and peripheral vascular disease (15.2% vs. 7.9%, P = 0.027) than those with 198Pro/Pro genotype [21]. Nemoto et al. [29] also reported that the 198Pro/leu genotype is associated with significantly higher coronary artery calcium score among type 2 diabetic patients in Japan [29]. However, Sergeeva et al. [28] reported that there was no significant association between the GPX1 198Pro/leu polymorphism and either myocardial infarction or stroke in hypertensive non-insulin-dependent diabetes mellitus patients in Russian [28]. In the present study, a significant difference of GPX1 198Pro/leu genotype distribution was found between CAD cases and controls. In order to eliminate the influence caused by CAD risk factors, we performed a binary logistic regression analysis when assessing the impact of the GPX1 198Pro/leu polymorphism on the susceptibility to CAD. Compared to the 198Pro/Pro genotype carriers, subjects with the variant genotypes (198Pro/leu and 198Leu/leu) had a 78% increased risk of CAD. After adjustment for age, sex, BMI, smoking status, hypertension, diabetes and dyslipidemia, the difference still existed, confirming that this variant was truly associated with CAD risk in this Chinese population. The effect of the GPX1 198Pro/leu variant on the function of GPX1 enzyme is considerable. Ravn-Haren et al. [19] reported that the variant allele of GPX1 198Pro/leu polymorphism was correlated with lower GPX1 activity [19]. Hu and Diamond [20] also demonstrated that the 198Leu allele was less responsive to the stimulation of GPX1 enzyme activity than 198Pro allele [20]. Furthermore, Hamanishi et al. [21] confirmed that the 198Leu allele combined with Ala6 allele had significantly lower GPX1 activity than the combination of 198Pro and Ala5 (20.1 ± 10.6 vs. 32.9 ± 9.5 units/mg protein, P = 0.0230) [21]. GPX1 is a cytosolic isoform of GPX and is a key antioxidant enzyme within many cells, including vascular endothelial cells, converts hydrogen peroxide to water and lipid peroxides to their respective alcohols [7,8]. Previous studies have indicated biological relevance for GPX1 in atherogenesis. Forgione et al. [12] demonstrated that GPX1 deficiency was accompanied by increased periadvential inflammation, neointima formation, and collagen deposition surrounding the coronary arteries [12]. Studies also showed that deficiency of GPX1 was associated with the endothelial dysfunction and the progression of atherosclerosis in mice [10,11]. Furthermore, decreased GPX1 activity was observed in patients with CAD and those with acute myocardial infarction [13,14], suggesting the protective role of GPX1 against the development of CAD. Thus, it was reasonable to suppose that our observation of an increased risk of CAD in subjects with GPX1 198Pro/leu variant genotypes might be due to the decreased GPX1 enzyme activity.
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In this study, we also investigated the association of GPX1 198Pro/ leu variant with the presence of CAD in the 2 groups that were subdivided according to the median age (64 y) of the control subjects. For the subjects with age b64 y, the variant genotypes showed significantly higher risk of CAD than wild-type 198Pro/Pro. However, it was without significance among subjects with age ≥64 y. Our findings are in agreement with the conclusions of the Swedish twin study that when people die of CAD at younger ages, genetic mechanisms play a greater role than in deaths at an older age [30]. In addition, a hypothesis could also be suggested that, as subjects having CAD before 64 y showed higher prevalence of 198Pro/leu or 198Leu/leu genotypes, this polymorphism could be associated with the premature atherosclerosis in humans. Although this is just a hypothesis to interpret the results of our study, these results could be a clue to solve the mystery of the GPX1 gene in human aging and atherosclerosis. And further studies are needed to confirm this hypothesis. Our data also showed that the association between the increased CAD risk and the variant genotypes was more evident in male subjects than in female subjects. Several investigators have reported the sexspecific effects of genotypes [23,25]. However, the results were inconsistent [26]. Zhao et al. [25] reported that a significant decreased risk of recurrence in patients with superficial bladder cancer was found in female subjects with the variant genotypes (adjusted hazard ratio = 0.28, 95%CI = 0.09–0.90) while not in male subjects (adjusted hazard ratio = 0.77, 95%CI = 0.49–1.23) [25]. In addition, RaaschouNielsen et al. [23] showed that the homozygote 198Leu/leu was significantly associated with decreased risk of lung cancer only in female subjects (adjusted incidence rate ratio = 0.35, 95%CI = 0.15– 0.85) but not in male subjects (adjusted incidence rate ratio = 0.96, 95% CI = 0.47–1.94) [23]. However, previous study performed in 630 male subjects by Ratnasinghe et al. [26] showed that the variant genotypes were significantly associated with the increased risk of lung cancer (198Leu/leu vs. 198Pro/Pro: adjusted OR = 2.3, 95%CI = 1.3–3.8; 198Pro/ leu vs. 198Pro/Pro: adjusted OR = 1.8, 95%CI = 1.2–2.8) [26]. The stratified analysis by smoking status revealed that, among non-smokers, those with the variant genotypes had significantly higher risk of CAD, while among smokers, no statistical significance was noted. Smoking has been strongly implicated as a risk factor for atherosclerosis [31]. Multiple evidences support the major pathological mechanism of oxidative stress linking smoking to atherosclerosis [32–35]. Smoking is known to increase lipid peroxidation, which contributes to the atherogenic potential tobacco use [32–34]. Studies have also reported that smokers have an increased susceptibility of LDL to oxidation and that, in smokers there are higher levels of oxidized LDL which participates in early stages of the atherosclerosis [32,34,35]. Importantly, recent study by Ravn-Haren et al. [19] has shown that GPX1 activity was tended to be lower in smokers compared with non-smokers [19]. Thus, we speculated that, the decreased GPX1 activity caused by smoking might mask the effect of the GPX1 198Pro/leu variant among smokers and, with the variant genotypes, the decreased GPX1 activity and enhanced production of reactive oxygen radicals might be more evident in non-smokers. Several limitations in the present study should be acknowledged. Firstly, the relatively small sample size of our study, especially after stratifying by age, sex and smoking status, may limit the statistical power. Nevertheless, the present study provides valuable insights and interesting information and may serve to guide future studies in this area. Secondly, although we selected control subjects who had no history of angina pectoris and no symptoms or signs of other atherosclerotic vascular diseases, we could not exclude the possibility that some of them were affected by CAD without having performed coronary angiography. Finally, the GPX1 198Pro/leu polymorphism associated with CAD in this study may be in linkage disequilibrium with polymorphisms of other nearby genes that actually contributed to the development of CAD.
In conclusion, we conducted a pilot study to reveal the association of GPX1 198Pro/leu polymorphism with susceptibility to CAD. Our data provide evidence that the GPX1 198Pro/leu variant genotypes are associated with the increased risk of CAD in the Chinese population, supporting that the GPX1 198Pro/leu variant is involved in the cardiovascular disease. The potential interactions between the GPX1 198Pro/leu genotype and age, sex or smoking also have been demonstrated in this study. Acknowledgements The project was supported by grants from the National Natural Science Foundation of China (No 30672486), the Natural Science Foundation of Jiangsu Province (No BK2006525), “333 Project” and “Qinglan Project” Funding for the Young Academic Leader of Jiangsu Province to B Wang. References [1] Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med 1999;340:115–26. [2] Harrison D, Griendling KK, Landmesser U, Hornig B, Drexler H. Role of oxidative stress in atherosclerosis. 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[24] Yang P, Bamlet WR, Ebbert JO, Taylor WR, de Andrade M. Glutathione pathway genes and lung cancer risk in young and old populations. Carcinogenesis 2004;25:1935–44. [25] Zhao H, Liang D, Grossman HB, Wu X. Glutathione peroxidase 1 gene polymorphism and risk of recurrence in patients with superficial bladder cancer. Urology 2005;66:769–74.
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Contents lists available at ScienceDirect
Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Genetic variant in glutathione peroxidase 1 gene is associated with an increased risk of coronary artery disease in a Chinese population Na-Ping Tang a,1, Lian-Sheng Wang a,b,1, Li Yang c,1, Hai-Juan Gu a, Qing-Min Sun a, Ri-Hong Cong a, Bo Zhou a, Huai-Jun Zhu a, Bin Wang a,⁎ a b c
Key Laboratory of Reproductive Medicine, Department of Pharmacology, Nanjing Medical University, Nanjing, Jiangsu Province 210029, China Department of Cardiology, First Affiliated Hospital of Nanjing Medical University, Nanjing, China Department of General Surgery, First Affiliated Hospital of Nanjing Medical University, Nanjing, China
A R T I C L E
I N F O
Article history: Received 3 November 2007 Received in revised form 28 February 2008 Accepted 16 May 2008 Available online 25 May 2008 Keywords: Coronary artery disease Genetic variant Glutathione peroxidase 1 Oxidative stress Polymorphism
A B S T R A C T Background: Glutathione peroxidase 1 (GPX1), the key antioxidant enzyme in vascular endothelial cells, has been shown to exert a protective effect against the presence of coronary artery disease (CAD). The 198Pro/leu variant, located at codon 198 of GPX1 gene, has recently been linked to cardiovascular disease, but data were inconsistent. We investigated the association between the occurrence of CAD and the 198Pro/leu variant in a Chinese population. Methods: A total of 265 unrelated CAD patients and 265 age- and sex-matched control subjects were recruited in this study. The GPX1 198Pro/leu genotype was determined using polymerase chain reaction– restriction fragment length polymorphism. Results: Compared to the 198Pro/Pro carriers, subjects with the variant genotypes (198Pro/leu and 198Leu/ leu) had a significantly higher risk of CAD (adjusted OR = 2.02, 95%CI = 1.27–3.22). In stratified analyses, the variant genotypes were significantly associated with increased CAD risk in subjects b 64 y (adjusted OR = 2.41, 95%CI = 1.16–4.98), males (adjusted OR = 1.86, 95%CI = 1.09–3.18) and non-smokers (adjusted OR = 2.40, 95% CI = 1.15–5.01). However, no significant association was observed between this variant and the severity of CAD. Conclusion: These data provide evidence that GPX1 198Pro/leu variant genotypes are significantly associated with CAD risk in this Chinese population. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Oxidative stress has recently been suggested to play a central role in the pathogenesis of atherosclerosis [1–3]. Antioxidant enzymes, including superoxide dismutase, catalase, and glutathione peroxidase (GPX), are essential for defense against oxidative stress [4–6]. One of these enzymes, GPX, is a soluble selenoprotein [7,8]. To date, 4 different GPX forms have been identified, all of which contain selenocysteine at their active sites [7]. GPX1, the ubiquitous intracellular form and key antioxidant enzyme within many cells, including the endothelium, converts hydrogen peroxide to water and lipid peroxides to their respective alcohols using reduced glutathione as an essential co-substrate [8]. Multiple lines of evidence supported the involvement of GPX1 in protecting vessels against oxidative stress and atherogenesis [9–15]. Recent studies show that, in vascular endothe-
⁎ Correspondence author. Department of Pharmacology, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, Jiangsu Province, China. Tel./fax: +86 25 86862884. E-mail address: [email protected] (B. Wang). 1 Contributed equally to this work. 0009-8981/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2008.05.013
lial cells, shear stress can upregulate the expression and enzymatic activity of GPX1 [9]. In mouse model, deficiency of GPX1 leads to endothelial dysfunction [10] and accelerates the progression of atherosclerosis [11]. In addition, GPX1 deficiency is accompanied by increased periadvential inflammation, neointima formation, and collagen deposition surrounding the coronary arteries [12]. Furthermore, decreased GPX1 activity was observed in patients with coronary artery disease (CAD) and those with acute myocardial infarction, and associated with the extent of atherosclerosis [13–15]. GPX1, the gene coding for glutathione peroxidase 1, is located on chromosome 3p21.3 [16] and it is composed of 2 exons with a 1.42 kb region [17]. A genetic variant (198Pro/leu, rs1050450) at codon 198 of GPX1 gene, resulting in the substitution of leucine (CTC) for proline (CCC), was identified by Moscow et al. [18]. Recent studies have confirmed that the GPX1 198Leu allele was associated with a significantly decreased GPX1 enzymatic activity compared with the 198Pro allele [19–21]. The role of this variant has been extensively studied in cancer [18–20,22–26]. However, studies investigating the association between this variant and vascular disease are rare [21,27– 29]. Additionally, data on the effect of this variant on risk of cardiovascular disease are inconsistent [21,28,29]. Thus, we conducted a
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Table 1 Baseline characteristics of cases and controls Characteristics
Cases (n = 265)
Controls (n = 265)
P
Sex (male), n (%) Age (years) BMI (kg/m2) Hypertension, n (%) Diabetes, n (%) Dyslipidemia, n (%) Smoking, n (%) TC (mmol/l) TG (mmol/l) HDL-C (mmol/l) LDL-C (mmol/l) Glucose (mmol/l) Number of diseased vessels Single vessel, n (%) Double vessels, n (%) Triple vessels, n (%)
194 (73.2) 64 (56–71) 25.1 ± 3.3 194 (73.2) 60 (22.6) 21 (7.9) 127 (47.9) 4.11 (3.48–4.71) 1.46 (1.05–2.14) 0.98 (0.84–1.14) 2.36 (1.83–2.82) 5.19 (4.68–5.95)
194 (73.2) 64 (55–71) 23.8 ± 3.6 117 (44.2) 44 (16.6) 8 (3.0) 83 (31.3) 3.95 (3.31–4.54) 1.14 (0.79–1.62) 1.08 (0.90–1.29) 2.14 (1.66–2.63) 4.88 (4.41–5.60)
NS NS b0.001 b0.001 NS 0.013 b0.001 0.036 b0.001 b0.001 0.005 b0.001
97 (36.6) 82 (30.9) 86 (32.5)
– – –
– – –
BMI (normal distributed) was expressed as mean ± SD and compared by student's Student's t-test. Age, TC, TG, HDL-C, LDL-C and glucose (abnormal distributed) were expressed as median (25th–75th percentiles) and analyzed by Mann–Whitney U-test. Other data were expressed as frequencies and percentages and evaluated by χ2-test. BMI, body mass index (normal reference range 18.5–23 kg/m2); HDL-C, high density lipoprotein cholesterol (0.83–1.97 mmol/l); LDL-C, low density lipoprotein cholesterol (1.85–3.65 mmol/l); TC, total cholesterol (3.15–6.25 mmol/l); TG, triglyceride (0.48–1.88 mmol/l).
hospital-based case-control study with CAD patients confirmed by coronary angiography and age- and sex-matched controls to evaluate the association between the risk of CAD and the GPX1 198Pro/leu variant in a Chinese population. 2. Materials and methods 2.1. Study population Consecutive 265 CAD patients were recruited from the inpatients who were admitted to Nanjing Medical University Affiliated Hospital because of angina pectoris or other symptoms or signs of cardiovascular diseases. An additional 265 patients also admitted to this hospital served as the control group. They were selected during the same time as cases and matched by age (±5 years) and sex. Considering that it was unethical to perform coronary angiography to rule out the presence of asymptomatic CAD, the following inclusion criteria were used: no history of angina pectoris, without any symptoms or signs of other atherosclerotic vascular diseases. All subjects enrolled in this study were Han Chinese and residing in or nearby Jiangsu province. They had no family history of CAD and no history of significant concomitant diseases including cardiomyopathy, bleeding disorders, renal failure, previous thoracic irradiation therapy and malignant diseases. This study was approved by the Nanjing Medical University Affiliated Hospital Ethics Committee and informed consent was obtained from each participant. The definition of CAD was based on angiographic criteria; the coronary angiograms were reviewed by experienced cardiologists who were unaware that the patients were to be included in this study. Coronary artery disease was defined as angiographic evidence of at least one segment of a major coronary artery including the left anterior descending, left circumflex or right coronary artery with N50% organic stenosis. The severity of CAD was expressed as single vessel disease, double vessel disease and triple vessel disease depending on the number of the main vessels with stenosis. Hypertension, diabetes mellitus, and dyslipidemia were defined as those reported in our previous study [3]. Briefly, hypertension was defined as resting systolic blood pressure N140 mm Hg and/or diastolic blood pressure N90 mm Hg or in the presence of active treatment with antihypertensive agents. Diabetes was defined as fasting blood glucose N 7.8 mmol/l or a diagnosis of diabetes needing diet or antidiabetic drug therapy. Dyslipidemia was defined as total cholesterol level of N6.2 mmol/l or on drugs. Smoking was defined as ≥ 10 cigarettes/d. 2.2. Laboratory measurements Fasting blood samples were obtained by venipuncture in the early morning. The levels of plasma total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) were measured enzymatically (First Chemical Co. Japan) on a chemistry analyzer (Olympus Au2700). Glucose levels were measured by a glucose oxidase method (Reagent kit, Diagnostic Chemicals Ltd., Oxford, CT). 2.3. Genotyping Genomic DNA was isolated from leukocyte pellet using standard phenol-chloroform extraction. The GPX1 198Pro/leu (C to T variant) variant was determined using
polymerase chain reaction (PCR)-restriction fragment length polymorphism employed primers 5′- TCC AGA CCA TTG ACA TCG AG -3′ (forward) and 5′- ACT GGG ATC AAC AGG ACC AG -3′ (reverse). A 222-base pair (bp) DNA fragment containing the polymorphic site was amplified by PCR in the T1 Thermocycler (Biometra, Goettingen, Germany) with a total volume of 20 μl, containing 2 μl 10 × PCR buffer, 1.125 mmol/l MgCl2, 0.15 mmol/l dNTPs, 0.25 μmol/l each primer, 100 ng of genomic DNA and 1.5 U of Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania). The PCR amplification was performed with the following conditions: initial denaturation at 94 °C for 8 min, amplification for 35 cycles at 94 °C for 30 s, 59 °C for 30 s and 72 °C for 30 s, followed by a final elongation step at 72 °C for 7 min. An aliquot of 5 μl of the PCR product was digested with 10 U of ApaI (GGGCCC, New England BioLabs, Waltham, MA) in 2 μl of 10 × NEB buffer 4 (50 mmol/l potassium acetate, 20 mmol/l Tris-acetate, 10 mmol/l magnesium acetate and 1 mmol/l dithiothreitol), 0.2 μl of 100 μg/ml bovine serum albumin and 12.3 μl dH2O at 25 °C for 16 h. After restriction enzyme digestion, the products were separated on a 2% agarose gel, and visualized by staining with 0.5 μg/ml of ethidium bromide under ultraviolet light. The 222-bp PCR product was cleaved into 2 fragments of 170 and 52 bp in the presence of the 198Pro (C) homozygous, 3 fragments of 222, 170 and 52 bp for the 198Pro/leu (CT) heterozygote, while the 198Leu (T) homozygous remained uncleaved showing the 222 bp PCR product. About 10% of the samples were randomly selected to perform the repeated assays, and the results were 100% concordant. Two researchers, blinded to the clinical data, scored the genotypes independently. 2.4. Statistical analysis The normality of the distribution was analyzed using the Kolmogorov–Smirnov test. Differences of continuous variables without skewness (presented as mean ± SD) between 2 groups were calculated by the Student's t-test. Differences of continuous variables departing from the normal distribution even after transformation (presented as median and interquartile range) between 2 groups were analyzed by Mann–Whitney U-test. Categorical variables were presented using frequency counts and compared by χ2-test. The χ2 goodness-of-fit test was used to identify significant departures from the Hardy–Weinberg equilibrium. A binary logistic regression analysis was used for the evaluation of the independent effect of GPX1 genotypes on the development of CAD, adjusted for the presence of risk factors including age, sex, body mass index (BMI), smoking status, hypertension, diabetes and dyslipidemia. Odds ratio (OR) and 95% confidence interval (CI) were calculated. The linear trend in the association of GPX1 198Pro/leu polymorphism with CAD severity was evaluated by χ2-test for trend. A value of P b 0.05 was considered statistically significant. Statistical analyses were performed with the SPSS 13.0 (SPSS Inc., Chicago, IL, USA).
3. Results 3.1. Baseline characteristics A total of 265 CAD patients and 265 age- and sex-matched controls were included in this study. The baseline characteristics are shown in Table 1. No significant difference was found between the cases and controls in age [64 (56–71) vs. 64 (55–71) y, P = 0.504] and sex (male: 73.2% vs. 73.2%, P = 1.000). As expected, compared to the control group, the CAD group had higher BMI, numbers of hypertension, dyslipidemia and rate of smoking, as well as higher levels of TC, TG, LDL-C and glucose but lower HDL-C, all of which are established CAD risk factors. By coronary angiography, 97 (36.6%) CAD cases had single vessel disease, 82 (30.9%) had double vessel disease and 86 (32.5%) had triple vessel disease. In CAD patients with diabetes, 15 (25.0%) had single vessel disease, 24 (40.0%) had double vessel disease and 21 (35.0%) had triple vessel disease.
Table 2 Genotype and allele distribution of the 198Pro/leu (C to T variant) polymorphism in cases and controls Group
Genotype, n (%) CC
Cases (n = 265) Controls (n = 265)
CT
197 (74.3) 65 (24.5) 222 (83.8) 43 (16.2) χ2 = 8.973, df = 2 P = 0.011
Allele, n (%) TT
C-allele
3 (1.1) 0 (0)
459 (86.6) 71 (13.4) 487 (91.9) 43 (8.1) χ2 = 7.706, df = 1 P = 0.006
T-allele
Distributions of the 198Pro/leu genotypes in both cases and controls were in Hardy– Weinberg equilibrium (χ2 = 0.864, P = NS and χ2 = 2.066, P = NS, respectively, calculated by χ2 goodness-of-fit test).
N.-P. Tang et al. / Clinica Chimica Acta 395 (2008) 89–93
3.2. GPX1 198Pro/leu genotypes and allele frequencies Table 2 shows the distribution of GPX1 198Pro/leu genotypes and alleles of the two groups. In both cases and controls, the genotype distributions were consistent with those predicted by the Hardy– Weinberg equilibrium (χ2 = 0.864, P = 0.353 and χ2 = 2.066, P = 0.151, respectively). In the case group, the frequencies of the 198Pro/Pro, 198Pro/leu, and 198Leu/leu genotypes were 74.3%, 24.5% and 1.1%; while in the control group, the frequencies were 83.8% and 16.2% for the 198Pro/Pro and 198Pro/leu genotypes, respectively, and no 198Leu/leu individuals were found. The 198Leu allele frequency was significantly higher in the case group than those observed in the control group (13.4% vs. 8.1%, P = 0.006). In addition, among CAD patients with diabetes, 11 (18.3%) carried the variant genotypes (198Pro/leu, and 198Leu/leu).
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Table 4 Stratified analysis for the variant 198Pro/leu (C to T variant) genotypes in cases and controls Smoking status
CT + TT/CC Cases controls
Unadjusted model
P
OR(95% CI) Ageb b 64 y ≥ 64 y Sex Male Female Smoking status Yes No
Adjusted modela
P
OR(95% CI)
36/93 18/109 32/104 25/113
2.34 (1.25–4.40) 1.39 (0.77–2.50)
0.008 NS
3.41 (1.62–7.19) 1.41 (0.75–2.67)
0.001 NS
53/141 32/162 15/56 11/60
1.90 (1.16–3.12) 1.46 (0.62–3.45)
0.011 NS
2.30 (1.34–3.95) 1.51 (0.59–3.87)
0.003 NS
26/101 14/69 42/96 29/153
1.27 (0.62–2.60) 2.31 (1.35–3.95)
NS 0.002
1.38 (0.63–3.01) 2.70 (1.50–4.84)
NS 0.001
a
Adjusted for age, sex, BMI, smoking status, hypertension, diabetes and dyslipidemia (logistic regression analysis). Stratified by the median age (64 years) of controls.
3.3. GPX1 198Pro/leu variant and CAD risk
b
Table 3 shows the risk estimates for the variant GPX1 genotypes among CAD patients compared with controls. Since three 198Leu/leu homozygotes were found in case group and none in the control group, the variant genotypes including 198Pro/leu and 198Leu/leu were ascribed to a single group. Overall, the unadjusted OR for subjects with variant genotypes was 1.78 (95% CI = 1.16–2.73, P = 0.008) relative to wild-type 198Pro/Pro carriers. After adjustment for age, sex, BMI, smoking status, hypertension, diabetes and dyslipidemia, the association persisted (adjusted OR = 2.02, 95% CI = 1.27–3.22, P = 0.003). 3.4. Stratified analysis The results of stratified analyses by the median age of controls (64 years), sex and smoking status with the GPX1 variant genotypes are presented in Table 4. In the stratified analysis by age, the effect of variant genotypes (198Pro/leu and 198Leu/leu) on the increased risk of CAD in subjects with age b64 y were significant (adjusted OR = 3.41, 95% CI = 1.62–7.19, P = 0.001), while in subjects with age ≥64 y, no statistical significance was found (P = NS). The increased risk associated with the variant genotypes tended to be more evident in male subjects (adjusted OR = 2.30, 95% CI = 1.34–3.95, P = 0.003). But in female subjects, the association between the GPX1 polymorphism and CAD risk was not statistically significant (P = NS). Compared with the 198Pro/Pro genotype, the adjusted OR for the variant genotypes was 2.70 (95% CI = 1.50–4.84, P = 0.001) among non-smokers and 1.38 (95% CI = 0.63–3.01, P = NS) among smokers. Patients with CAD were also subclassified into 3 subgroups (single, double and triple vessel disease) according to the number of affected coronary arteries. However, no statistically significant association was noted between the GPX1 198Pro/leu variant and the severity of CAD (data not shown). 4. Discussion In this hospital-based case-control study, we identified the role of GPX1 198Pro/leu variant in CAD susceptibility in a Chinese population. Our results indicate that the variant 198Leu allele is significantly associated with the increased risk of CAD in this Chinese population. 198Pro/leu variant is located in exon 2 of GPX1 gene with proline to
Table 3 Risk estimates for the variant 198Pro/leu (C to T variant) genotypes Genotype
Cases/controls
Unadjusted model
P
OR(95% CI) Overall CC CT + TT a
265/265 197/222 68/43
1.00 1.78 (1.16–2.73)
Adjusted modela
P
OR(95% CI)
0.008
1.00 2.02 (1.27–3.22)
0.003
Adjusted for age, sex, BMI, smoking status, hypertension, diabetes and dyslipidemia (logistic regression analysis).
leucine transition at codon 198 [18]. To date, very few studies have linked the GPX1 198Pro/leu variant to cardiovascular disease [21,28,29]. Hamanishi et al. [21] showed that, in Japanese type 2 diabetic patients, those with 198Pro/leu genotype had significantly higher mean intimamedia thickness of common carotid arteries (1.04± 0.23 vs. 0.91 ± 0.18 mm, P = 0.0028) and prevalence of cardiovascular disease (24.2% vs. 10.6%, P = 0.035) and peripheral vascular disease (15.2% vs. 7.9%, P = 0.027) than those with 198Pro/Pro genotype [21]. Nemoto et al. [29] also reported that the 198Pro/leu genotype is associated with significantly higher coronary artery calcium score among type 2 diabetic patients in Japan [29]. However, Sergeeva et al. [28] reported that there was no significant association between the GPX1 198Pro/leu polymorphism and either myocardial infarction or stroke in hypertensive non-insulin-dependent diabetes mellitus patients in Russian [28]. In the present study, a significant difference of GPX1 198Pro/leu genotype distribution was found between CAD cases and controls. In order to eliminate the influence caused by CAD risk factors, we performed a binary logistic regression analysis when assessing the impact of the GPX1 198Pro/leu polymorphism on the susceptibility to CAD. Compared to the 198Pro/Pro genotype carriers, subjects with the variant genotypes (198Pro/leu and 198Leu/leu) had a 78% increased risk of CAD. After adjustment for age, sex, BMI, smoking status, hypertension, diabetes and dyslipidemia, the difference still existed, confirming that this variant was truly associated with CAD risk in this Chinese population. The effect of the GPX1 198Pro/leu variant on the function of GPX1 enzyme is considerable. Ravn-Haren et al. [19] reported that the variant allele of GPX1 198Pro/leu polymorphism was correlated with lower GPX1 activity [19]. Hu and Diamond [20] also demonstrated that the 198Leu allele was less responsive to the stimulation of GPX1 enzyme activity than 198Pro allele [20]. Furthermore, Hamanishi et al. [21] confirmed that the 198Leu allele combined with Ala6 allele had significantly lower GPX1 activity than the combination of 198Pro and Ala5 (20.1 ± 10.6 vs. 32.9 ± 9.5 units/mg protein, P = 0.0230) [21]. GPX1 is a cytosolic isoform of GPX and is a key antioxidant enzyme within many cells, including vascular endothelial cells, converts hydrogen peroxide to water and lipid peroxides to their respective alcohols [7,8]. Previous studies have indicated biological relevance for GPX1 in atherogenesis. Forgione et al. [12] demonstrated that GPX1 deficiency was accompanied by increased periadvential inflammation, neointima formation, and collagen deposition surrounding the coronary arteries [12]. Studies also showed that deficiency of GPX1 was associated with the endothelial dysfunction and the progression of atherosclerosis in mice [10,11]. Furthermore, decreased GPX1 activity was observed in patients with CAD and those with acute myocardial infarction [13,14], suggesting the protective role of GPX1 against the development of CAD. Thus, it was reasonable to suppose that our observation of an increased risk of CAD in subjects with GPX1 198Pro/leu variant genotypes might be due to the decreased GPX1 enzyme activity.
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In this study, we also investigated the association of GPX1 198Pro/ leu variant with the presence of CAD in the 2 groups that were subdivided according to the median age (64 y) of the control subjects. For the subjects with age b64 y, the variant genotypes showed significantly higher risk of CAD than wild-type 198Pro/Pro. However, it was without significance among subjects with age ≥64 y. Our findings are in agreement with the conclusions of the Swedish twin study that when people die of CAD at younger ages, genetic mechanisms play a greater role than in deaths at an older age [30]. In addition, a hypothesis could also be suggested that, as subjects having CAD before 64 y showed higher prevalence of 198Pro/leu or 198Leu/leu genotypes, this polymorphism could be associated with the premature atherosclerosis in humans. Although this is just a hypothesis to interpret the results of our study, these results could be a clue to solve the mystery of the GPX1 gene in human aging and atherosclerosis. And further studies are needed to confirm this hypothesis. Our data also showed that the association between the increased CAD risk and the variant genotypes was more evident in male subjects than in female subjects. Several investigators have reported the sexspecific effects of genotypes [23,25]. However, the results were inconsistent [26]. Zhao et al. [25] reported that a significant decreased risk of recurrence in patients with superficial bladder cancer was found in female subjects with the variant genotypes (adjusted hazard ratio = 0.28, 95%CI = 0.09–0.90) while not in male subjects (adjusted hazard ratio = 0.77, 95%CI = 0.49–1.23) [25]. In addition, RaaschouNielsen et al. [23] showed that the homozygote 198Leu/leu was significantly associated with decreased risk of lung cancer only in female subjects (adjusted incidence rate ratio = 0.35, 95%CI = 0.15– 0.85) but not in male subjects (adjusted incidence rate ratio = 0.96, 95% CI = 0.47–1.94) [23]. However, previous study performed in 630 male subjects by Ratnasinghe et al. [26] showed that the variant genotypes were significantly associated with the increased risk of lung cancer (198Leu/leu vs. 198Pro/Pro: adjusted OR = 2.3, 95%CI = 1.3–3.8; 198Pro/ leu vs. 198Pro/Pro: adjusted OR = 1.8, 95%CI = 1.2–2.8) [26]. The stratified analysis by smoking status revealed that, among non-smokers, those with the variant genotypes had significantly higher risk of CAD, while among smokers, no statistical significance was noted. Smoking has been strongly implicated as a risk factor for atherosclerosis [31]. Multiple evidences support the major pathological mechanism of oxidative stress linking smoking to atherosclerosis [32–35]. Smoking is known to increase lipid peroxidation, which contributes to the atherogenic potential tobacco use [32–34]. Studies have also reported that smokers have an increased susceptibility of LDL to oxidation and that, in smokers there are higher levels of oxidized LDL which participates in early stages of the atherosclerosis [32,34,35]. Importantly, recent study by Ravn-Haren et al. [19] has shown that GPX1 activity was tended to be lower in smokers compared with non-smokers [19]. Thus, we speculated that, the decreased GPX1 activity caused by smoking might mask the effect of the GPX1 198Pro/leu variant among smokers and, with the variant genotypes, the decreased GPX1 activity and enhanced production of reactive oxygen radicals might be more evident in non-smokers. Several limitations in the present study should be acknowledged. Firstly, the relatively small sample size of our study, especially after stratifying by age, sex and smoking status, may limit the statistical power. Nevertheless, the present study provides valuable insights and interesting information and may serve to guide future studies in this area. Secondly, although we selected control subjects who had no history of angina pectoris and no symptoms or signs of other atherosclerotic vascular diseases, we could not exclude the possibility that some of them were affected by CAD without having performed coronary angiography. Finally, the GPX1 198Pro/leu polymorphism associated with CAD in this study may be in linkage disequilibrium with polymorphisms of other nearby genes that actually contributed to the development of CAD.
In conclusion, we conducted a pilot study to reveal the association of GPX1 198Pro/leu polymorphism with susceptibility to CAD. Our data provide evidence that the GPX1 198Pro/leu variant genotypes are associated with the increased risk of CAD in the Chinese population, supporting that the GPX1 198Pro/leu variant is involved in the cardiovascular disease. The potential interactions between the GPX1 198Pro/leu genotype and age, sex or smoking also have been demonstrated in this study. Acknowledgements The project was supported by grants from the National Natural Science Foundation of China (No 30672486), the Natural Science Foundation of Jiangsu Province (No BK2006525), “333 Project” and “Qinglan Project” Funding for the Young Academic Leader of Jiangsu Province to B Wang. References [1] Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med 1999;340:115–26. [2] Harrison D, Griendling KK, Landmesser U, Hornig B, Drexler H. Role of oxidative stress in atherosclerosis. 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