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Genetic variant in visfatin gene promoter is associated with decreased risk of coronary artery disease in a Chinese population
Clinica Chimica Acta 411 (2010) 26–30
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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 e v i e r. c o m / l o c a t e / c l i n c h i m
Genetic variant in visfatin gene promoter is associated with decreased risk of coronary artery disease in a Chinese population Jian-Jun Yan a, Na-Ping Tang a,b, Jian-Jin Tang a, En-zhi Jia a, Ming-Wei Wang a, Qi-Ming Wang a, Jun Zhu a, Zhi-Jian Yang a, Lian-sheng Wang a,⁎, Jun Huang a,⁎ a
Department of Cardiology, First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, 210029, Jiangsu Province, China National Shanghai Center for New Drug Safety Evaluation and Research, Shanghai Institute of Pharmaceutical industry, 199 Guoshoujing Road, Zhangjiang Hi-Tech Park, Pudong, Shanghai 201203, China
b
a r t i c l e
i n f o
Article history: Received 28 March 2009 Received in revised form 27 September 2009 Accepted 29 September 2009 Available online 3 October 2009 Keywords: Coronary artery disease Visfatin Polymorphism
a b s t r a c t Background: Visfatin is a newly identified pro-inflammatory adipokine expressed predominantly in visceral fat. Previous studies have suggested a role for visfatin in low-grade inflammation and regulation of lipid metabolism. Most recently, a genetic polymorphism − 1535C > T located in the visfatin gene promoter has been identified, and suggested to be associated with the regulation of visfatin expression, lipid levels. However, it is unclear whether this polymorphism has a linkage with CAD. Methods: We conducted a hospital-based case-control study with 257 CAD patients and 292 controls to examine the potential association of the Visfatin − 1535C > T polymorphism with CAD. Results: The frequencies of the CC, CT, and TT genotypes in cases were significantly different from those of controls (χ2 = 6.223, P = 0.045). Subjects with the variant genotypes (CT + TT) had a 40% decreased risk of CAD relative to CC carriers (adjusted OR = 0.60, 95%CI = 0.40–0.89). Furthermore, the adjusted OR of a TT genotype for CAD was 0.52 (95%CI = 0.31–0.87). There was a significant association between Visfatin − 1535C > T polymorphism and triglyceride levels in both CAD patients and controls (P = 0.003, 0.018, respectively). In stratified analyses, the T allele was significantly associated with reduced risk of CAD in males, subjects with age < 59 years, and non-smokers. Moreover, a borderline statistical significance (P = 0.058 for trend) was observed between the variant genotypes and severity of CAD. Conclusion: Our results suggested that Visfatin − 1535C > T polymorphism might be associated with reduced risk of CAD in a Chinese population. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Coronary artery disease (CAD) is one of the major causes of death in most countries, including China [1]. Atherosclerosis, the main cause of CAD, is an inflammatory disease in which immune mechanisms interact with environmental and genetic risk factors to initiate, propagate, and activate lesions in the arterial tree, eliciting various acute and sever events such as acute coronary syndromes [2]. Most recently, genetic studies have revealed a series of candidate gene susceptibility variants that may also contribute to the pathogenesis of CAD [3,4]. Visfatin (also known as pre-B cell colony-enhancing factor or PBEF) is a novel pro-inflammatory adipokine expressed markedly in visceral fat [5]. Visfatin is a 52- to 55-kDa protein, which was originally cloned as a growth factor enhancing the effect of stem cell factor and IL-7 on the colony formation activity of early stage B cells [6]. Visfatin has a potential insulin-mimetic action that may cause insulin sensitivity [5], ⁎ Corresponding authors. Tel./fax: +86 25 83724440. E-mail addresses: [email protected] (L. Wang), [email protected] (J. Huang). 0009-8981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2009.09.033
however, which remains controversial [5,7]. Recent studies have indicated that visfatin is involved in the processes of inflammation, an important component of atherosclerosis. Serum visfatin levels were positively correlated with the serum levels of IL-6 and CRP, indicating that circulating visfatin may reflect inflammation status [8,9]. Visfatin has also been shown to be localized to foam cell macrophages within unstable atherosclerotic lesions, which potentially play a role in plaque destabilization [10]. Moreover, it has been suggested that visfatin plays a key part in nicotinamide adenine dinucleotide (NAD) biosynthesis pathway [7,11,12]. Furthermore, visfatin might be an independent CAD risk factor [13]. The visfatin gene is located on chromosome 7q22.2, which is composed of 11 exons and 10 introns, spanning 34.7 kb of genomic DNA. The proximal and distal regions of visfatin gene promoter contain several transcription initiation sites, and binding sites for Sp1 and ubiquitous transcription factors such as nuclear factor 1 (NF-1), AP-1, AP-2 [14]. To date, several single nucleotide polymorphisms in the visfatin gene have been described. The visfatin promoter polymorphisms have been reported as a linkage with low-grade and acute inflammation [15,16]. One of the most frequently studied
J.-J. Yan et al. / Clinica Chimica Acta 411 (2010) 26–30
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polymorphisms, −1535C > T, was reported to be associated with the regulation of visfatin gene expression and serum lipid level [16–18]. However, there were some conflicting results regarding whether this variant is functional or not [16,17,24]. To the best of our knowledge, no data have been published on the association of the − 1535C > T polymorphism with the risk of CAD. We investigate the potential association between the − 1535C > T polymorphism and the risk of CAD in a hospital-based case-control study in a Chinese population.
Biotech Co., Ltd.). Subsequently, LDR products were analyzed by DNA sequencing (Model 377, Applied Biosystems). All assays were conducted blindly without the knowledge of case or control status. Additionally, about 10% of the samples were randomly selected and retested by direct DNA sequencing on a 3730xl DNA analyzer (Applied Biosystems) and the results were 100% concordant.
2. Materials and methods
Statistical analyses were conducted with Stata ver. 8.0 (STATA Corp., College Station, TX) and SPSS 13.0 (SPSS Inc., Chicago, IL). Normality was tested 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 between 2 groups were analyzed by Mann–Whitney U-test. Pearson χ2-test was used to compare allele distribution and qualitative variables represented as frequencies. Hardy–Weinberg equilibrium was assessed for both subjects and controls by a χ2 goodness-of-fit test. Odds ratio (OR) and 95% confidence interval (CI) were calculated to estimate the correlation between the −1535C> T polymorphism and the risk of CAD. A binary logistic regression analysis was used for the evaluation of the independent effect of visfatin 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 −1535C> T polymorphism with CAD severity was evaluated by χ2-test for trend. Two-tailed P < 0.05 were considered as statistical significance.
2.1. Study population The association study enrolled 257 unrelated patients of CAD, who were all admitted to the First Affiliated Hospital of Nanjing Medical University. The diagnosis of CAD was certified by coronary angiography performed with the Judkins technique using a quantitative coronary angiographic system [19]. CAD was defined as an angiography with ≥50% luminal narrowing in one main coronary artery or more. Patients with CAD were divided into 1-, 2-, and 3-vessel disease subgroups according to the number of significantly stenosed vessels with reference to the Coronary Artery Surgery Study classification. Two cardiologists who were unaware of the patients included in this study assessed the angiograms. This study included 292 unrelated control subjects randomly selected from outpatients (staff of local companies and administration agencies) who underwent regular physical examinations during the same time in the same hospital. After considering it unethical to perform coronary angiography to rule out the presence of asymptomatic CAD, those control subjects with a history of angina, symptoms or signs of other atherosclerotic vascular diseases and an abnormal electrocardiogram were excluded. All subjects enrolled in this study were of Han Chinese origin and residing in or near Jiangsu Province. They had no history of significant concomitant diseases including cardiomyopathy, bleeding disorders, renal failure, previous thoracic irradiation therapy and malignant diseases. Hypertension was defined as resting systolic blood pressure >140 mm Hg and/or diastolic blood pressure >90 mm Hg or in the presence of active treatment with antihypertensive agents. Diabetes was defined as fasting blood glucose >7.0 mmol/l or a diagnosis of diabetes needing diet or antidiabetic drug therapy. Dyslipidemia was defined as total cholesterol concentration of >5.72 mmol/l or on drugs. Individuals who formerly or currently smoked ≥10 cigarettes per day for at least 2 years were defined as smokers. This study was approved by the First Affiliated Hospital Ethics Committee of Nanjing Medical University and informed consent was obtained from each participant.
2.3. Statistical analysis
3. Results 3.1. Demographic information The characteristics of our study subjects are given in Table 1. Patients with CAD were much older, smoked more cigarettes, had significantly higher weight, TG, TC , LDL-C, body mass index (BMI), and were more likely to be diabetic, hypertensive and dyslipidemic than the control subjects. In terms of coronary angiographic findings, 100 (38.9%) CAD cases had single-vessel disease, 87 (33.9%) had double-vessel disease and 70 (27.2%) had triple-vessel disease. Table 1 Baseline characteristics of cases and controls. Characteristics
2.2. DNA extraction and genotyping Peripheral venous blood was drawn from each participant. Genomic DNA was extracted using the AxyPrep DNA Blood kit (Axygen Scientific Inc., Union City, CA, USA). The −1535C > T (rs61330082) polymorphism was genotyped by the PCR-LDR Sequencing method, as reported previously [20,21]. A 216 bp DNA fragment (nucleotide positions −1637 to −1422 of the promoter) containing the polymorphic site was amplified by PCR in the ABI 9600 (Applied Biosystems, Foster City, CA) using the forward primer 5′-ACACAGGGAAGATCAACCAA-3′ and the reverse primer 5′-AGACAACCTCAGTCAACACTA-3′. The PCR was carried out in a total volume of 15 μl containing 1.5 μl 10 × PCR buffer, 0.2 μl 10 pmol each primer, 0.3 μl dNTP, 0.25 μl Taq polymerase (MBI fermentas), 2 μl of genomic DNA and 10.75 μl H2O. The PCR cycling parameters were 35 cycles of 30 s at 94 °C, 55 °C for 30 s and 72 °C for 45 s. Ligase Detection Reaction (LDR) was performed in a total volume of 10 μl containing 2 μl PCR product, 1 μl 10 × Taq DNA ligase buffer, 0.125 μl 40 U/μl Taq DNA ligase (NEB), 1 μl 10 pmol probes (0.33 μl each of probe), and 5.875 μl H2O. LDR probes were composed of 1 common probe and 2 discriminating probes (designed by the Shanghai Generay
Age (years) Sex (male), n (%) 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 (%)
Cases
Controls
(n = 257)
(n = 292)
P
63.9 ± 9.6 210 (81.7) 24.9 ± 3.3 128 (49.8) 56 (21.8) 64 (24.9) 96 (37.4) 4.14 ± 1.16 1.73 ± 0.96 1.12 ± 0.43 2.58 ± 0.89 5.86 ± 2.69
59.3 ± 12.4 211 (72.3) 24.1 ± 2.7 98 (33.6) 23 (7.9) 33 (11.3) 42 (14.4) 3.96 ± 1.17 1.42 ± 1.01 1.33 ± 0.35 2.28 ± 0.76 5.00 ± 0.92
< 0.001 0.009 0.002 < 0.001 < 0.001 < 0.001 < 0.001 0.009 < 0.001 < 0.001 < 0.001 < 0.001
100 (38.9) 87 (33.9) 70 (27.2)
– – –
– – –
BMI, body mass index; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; TC, total cholesterol; TG, triglyceride. Age, TC, TG, HDL-C, LDL-C and glucose (expressed as mean ± SD) were abnormal distributed and analyzed by Mann–Whitney U-test. BMI (expressed as mean ± SD) was normally distributed and analyzed by Student's t-test. Other data were expressed as frequencies and percentages and evaluated by χ2-test.
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J.-J. Yan et al. / Clinica Chimica Acta 411 (2010) 26–30
Table 2 Distribution of the visfatin genotype and the risk estimates for the variant visfatin genotypes. Adjusteda
Cases
Controls
Crude
(n = 257)
(n = 292)
OR (95%CI)
P
OR (95%CI)
P
92 (35.8) 118 (45.9) 47 (18.3) 165 (64.2)
78 (26.7) 143 (49.0) 71 (24.3) 214 (73.3)
1.00 0.70 (0.48–1.03) 0.56 (0.35–0.90) 0.65 (0.45–0.94)
0.071 0.017 0.022
1.00 0.64 (0.42–0.98) 0.52 (0.31–0.87) 0.60 (0.40–0.89)
0.042 0.013 0.011
302 (58.8) 212 (41.2)
299 (51.2) 285 (48.8)
– –
– –
– –
– –
b
Genotype , n (%) CC CT TT CT + TT Allelec, n (%) C allele T allele
Distributions of the visfatin genotype in cases and controls were in Hardy–Weinberg equilibrium (P = 0.399 and P = 0.733, respectively). CI, confidence interval; OR, odds ratio. a Adjusted for age, sex, body mass index, smoking status, hypertension, diabetes, dyslipidemia. b Pearson χ2 = 6.223, P = 0.045 for genotype. c Pearson χ2 = 6.300, P = 0.012 for allele.
3.2. Genotypes and allele frequencies of cases and controls and their associations with CAD The allele frequencies of SNP −1535C > T in CAD group and control group were 58.8%/41.2% and 51.2%/48.8%, respectively. The genotype distribution in our study subjects showed no deviation from Hardy– Weinberg equilibrium (P = NS). The distribution of C > T genotypes was also markedly different between cases and controls (P = 0.045) as shown in Table 2. Table 2 also shows the risk estimates for the variant −1535C > T genotypes among CAD patients compared with controls. Overall, after being adjusted for the risk factors including age, sex, BMI, smoking status, hypertension, diabetes and dyslipidemia, the OR for subjects with the variant genotypes (CT and TT) was 0.60 (95% CI= 0.40–0.89, P = 0.011). We also found that TT genotype carriers had a 48% reduction in risk of CAD compared with the CC carriers
Table 3 BMI, lipid profiles and glucose of cases and controls in the visfatin genotypes. Characteristics
BMI (kg/m2) TC (mmol/l) TG (mmol/l) HDL-C (mmol/l) LDL-C (mmol/l) Glucose (mmol/l)
Cases
P
CC
CT + TT
24.9 ± 3.4 4.26 ± 1.20 1.91 ± 0.93 1.11 ± 0.41 2.67 ± 0.90 5.84 ± 2.59
24.9 ± 3.3 4.06 ± 1.14 1.63 ± 0.97 1.12 ± 0.44 2.54 ± 0.88 5.87 ± 2.74
NS NS 0.003 NS NS NS
Controls CT + TT
24.2 ± 2.39 4.04 ± 1.25 1.67 ± 1.35 1.33 ± 0.34 2.45 ± 0.73 5.00 ± 0.81
24.1 ± 2.86 3.94 ± 1.14 1.33 ± 0.83 1.34 ± 0.35 2.21 ± 0.77 5.00 ± 0.95
3.3. BMI, lipid profiles and glucose of cases and controls in the visfatin genotypes Table 3 shows BMI, lipid profiles and glucose levels according to the −1535C> T genotypes in the CAD patients and controls. We found a significant association between the genotypes and serum triglyceride levels in these 2 groups (P = 0.003 and 0.018, respectively). In addition, we also noted that there was a borderline significance between the genotypes and serum LDL-C levels in controls (P = 0.062). However, there were no significant differences between the genotypes and BMI or glucose levels in both groups. 3.4. Stratified analyses of the polymorphism and CAD
P
CC
(adjusted OR = 0.52, 95% CI = 0.31–0.87, P = 0.013). We used a dominant model for analysis in the present study. In addition, we have attempted to conduct analysis with other models. However, no other statistical significance was observed (data not shown).
NS NS 0.018 NS NS NS
BMI, body mass index; CAD, coronary artery disease; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; TC, total cholesterol; TG, triglyceride. TC, TG, HDL-C, LDL-C and glucose (expressed as mean ± SD) were abnormal distributed and analyzed by Mann–Whitney U-test. BMI (expressed as mean ± SD) was normal distributed and analyzed by Student's t-test.
We conducted stratification analyses according to median age of controls (59 years), gender, smoking status (Table 4). We noted that the T allele was significantly associated with a reduction in risk of CAD in male (adjusted OR = 0.49, 95% CI = 0.31–0.77, P = 0.002); subjects with age < 59 years (adjusted OR = 0.43, 95% CI = 0.21–0.88, P = 0.021) and non-smokers (adjusted OR = 0.57, 95% CI = 0.37– 0.87, P = 0.009), while not in female (adjusted OR = 1.27, 95% CI = 0.52–3.08, P = NS); subjects with age ≥ 59 years (adjusted OR = 0.68, 95% CI = 0.41–1.12, P = NS) and smokers (adjusted OR = 0.82, 95% CI = 0.36–1.85, P = NS). Patients with CAD were subclassified into 3 subgroups (single-, double- and triple-vessel disease) on the basis of the number of affected coronary arteries. The frequency of patients with variant genotypes decreased gradually
Table 4 Stratified analyses by age and sex for the variant visfatin genotypes in cases and controls. Variable
Age (median) <59 years ≥59 years Sex Males Females Smoking Yes No
TT + CT/CC
Adjusteda
Crude
Case
Controls
OR (95%CI)
P
OR (95%CI)
P
44/25 121/67
108/38 106/40
0.62 (0.34–1.15) 0.68 (0.43–1.09)
NS NS
0.43 (0.21–0.88) 0.68 (0.41–1.12)
0.021 NS
131/79 34/13
158/53 56/25
0.56 (0.37–0.85) 1.17 (0.53–2.58)
0.006 NS
0.49 (0.31–0.77) 1.27 (0.52–3.08)
0.002 NS
67/29 98/63
31/11 183/67
0.72 (0.31–1.66) 0.58 (0.38–0.89)
NS 0.012
0.82 (0.36–1.85) 0.57 (0.37–0.87)
NS 0.009
CI, confidence interval; OR, odds ratio. a Adjusted for age, sex, body mass index, smoking status, hypertension, diabetes, dyslipidemia.
J.-J. Yan et al. / Clinica Chimica Acta 411 (2010) 26–30 Table 5 Visfatin genotypes and CAD severity. CAD severity
SVD (n = 100) DVD (n = 87) TVD (n = 70)
P for trenda
Genotype, n (%) CC
CT + TT
31 (33.7) 29 (31.5) 32 (34.8)
69 (41.8) 58 (35.2) 38 (23.0)
0.058
CAD, coronary artery disease; DVD, double-vessel disease; SVD, single-vessel disease; TVD, triple-vessel disease. a χ2 = 3.601 for trend.
from single- to triple-vessel disease with a borderline significance (P = 0.058 for trend) (Table 5). 4. Discussion The present case-control study analyzed the association between CAD risk and genetic polymorphism at position −1535 in the promoter of visfatin gene. According to our data, the −1535 C to T variant was associated with risk reduction of CAD development in a Chinese population. Recent studies have also indicated that visfatin, whose plasma concentrations are altered in obesity and obesity related disorders, might be involved in the processes of low-grade inflammation [22], an important component of atherosclerosis. Visfatin might induce endothelial VEGF and MMP production and activity via multiple signalling pathways including MAPK, PI3K/Akt, and VEGFR2, suggesting a vital role of visfatin in the pathogenesis of vascular inflammation [23]. Visfatin has also been shown to be localized to foam cell macrophages within unstable atherosclerotic lesions, which presumptively play a role in plaque destabilization [10]. In addition, plasma visfatin levels are significantly correlated with CAD, particularly ACS, independent of well-known CAD risk factors [13]. The −1535C > T polymorphism is located on the distal promoter region, which contains several CAAT boxes and TATA-like sequences and binding sites for CCAAT/NF-1, NF- B, NF-IL-6, GR, and AP-1 [14]. More importantly, −1535C > T, was reported to be associated with the regulation of visfatin gene expression [16,24] and serum lipid level [17,18]. Then, we hypothesized that the −1535C > T polymorphism might be correlated well with the susceptibility of CAD. In the present study, we conducted a hospital-based case-control study with 257 CAD patients and 292 controls to examine the potential association of Visfatin −1535C> T polymorphism with CAD. A 40% decreased risk of CAD was observed in subjects with −1535CT/TT compared with the CC carriers (adjusted OR= 0.60, 95% CI = 0.40–0.89, P = 0.011), indicating the T allele might confer a protective factor. The study of Ye et al. showed that the T variant in the T-1535C SNP (described as C-1543T in their study) resulted in nearly a 2-fold decrease in the reporter gene expression and transcription of visfatin was reduced in patients with the T variant, suggesting that the −1535C > T polymorphism is associated with a decreased risk of acute respiratory distress syndrome (ARDS) [16]. Thereby, the T allele was expected to have lower levels of visfatin gene transcription and enzymatic activity than C allele. There is a strong likelihood that the C to T substitution at this position may reduce activity of transcription factors (such as NF- B and AP-1), thereby downregulating visfatin gene transcriptional activity. Recently, they also confirmed that, compared to its common −1535C allele, the −1535T variant significantly attenuated its binding to an IL-1β induced unknown transcription factor in pulmonary vascular endothelial cells, which may underlie reduced expression of PBEF and lower susceptibility to acute lung injury [24]. Whereas, another investigation among Japanese suggested that this variation might be not functional [17]. Differences in experimental conditions, ethnicity and geographic variation may account for this discrepancy. Overall, we could not
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exclude the possibility that the association between −1535C > T polymorphism and CAD was due to the effect of this variant on the expression of visfatin gene, however, more studies are needed to confirm this hypothesis. We also estimated the impact of − 1535C > T polymorphism on the BMI, lipid profiles and glucose levels in CAD patients and controls. Our data showed that this promoter variant was associated with a lower serum triglyceride concentration in CAD patients and in controls. Likewise, Tokunaga et al. also found that the − 1535 T/T genotype was associated with lower serum triglyceride levels and higher HDL-C levels in the nondiabetic subjects [17]. Additionally, Jian et al. also reported that the haplotype containing − 1535C > T polymorphism was correlated significantly with lipid metabolism [18]. Bailey et al. observed a remarkable association between the rs7899066 and rs11977021 variants and dyslipidemia associated with insulin resistance [25]. Though the precise mechanism by which this variant influences lipid metabolism remains to be determined, there are still several plausible explanations. First, it has been shown that lipid phenotypes (HDL-C, TG) have a linkage with chromosome 7 [26,27], where the visfatin gene is located. It is likely that the T variant might have an impact on gene transcriptional activity, thus inducing different lipid phenotypes. Second, the −1535C > T may be in linkage disequilibrium with other SNPs such as the rs7899066 and rs11977021 in visfatin promoter, exerting influence on lipid metabolism. Third, visfatin has been reported as the rate limiting component in the mammalian nicotinamide adenine dinucleotide (NAD) biosynthesis pathway [11,12]. As a result, NAD regulated by visfatin has an effect on lipid metabolism. However, our study indicated that there were no significant differences between the genotypes and BMI or glucose levels in CAD patients and controls. This finding is in concordance with the observations made by Tokunaga et al. [17] and Zhang YY et al.[15], revealing that visfatin gene might not be related to glucose metabolism directly. But Jian et al. revealed that haplotype containing − 1535C > T polymorphism was significantly associated with 2-h glucose concentrations [18]. These discrepancies may be explained by several factors: first, differences in confounding factors (such as age, drug therapy and lifestyle) due to different criteria for recruitment; and second, the possibility of this variant's linkage disequilibrium with other SNPs. Nonetheless, additional studies are required to further define the results in detail. According to stratified analyses, we also observed an interaction between the −1535C > T polymorphism and sex. In male subjects, those who carried the variant genotypes had a significant reduction in risk of CAD than CC carriers. Epidemiological data show that the risk of CAD differs between female and male patients. There are several possible mechanisms for sex differences in gene expression, such as imprinting [28] and hormonal effects [29]. Kowalska et al. reported that visfatin remains an independent predictor of serum testosterone and free androgen index in the lean polycystic ovary syndrome (PCOS) subjects [30]. Moreover, testosterone has been found to reduce the expression of proinflammatory cytokines, such as TNF-a, IL-1, and IL-6 in human vascular endothelium and monocytes, and imbalance of sex steroids contributes to adverse cardiac effects in men [31]. Thereby, that testosterone might synergize the influence of the genetic variants, would account for this observation, inducing CAD risk reduction in male subjects. We also noticed that decreased risk of CAD with the variant visfatin genotypes was pronounced in younger subjects (age <59 years), but not in older subjects. Overwhelming accumulated exposure to environmental risks in older individuals may weaken the influence of this polymorphism in our study. Besides, de Luis et al. [32] and Jin et al. [33] also found that age has an inverse correlation with serum visfatin concentrations. It is possible that lower serum visfatin concentrations might also temper the impact of this polymorphism in the older group. We noted that among non-smokers, possession of the variant genotypes had a 43% reduced risk of CAD than CC genotype carriers.
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J.-J. Yan et al. / Clinica Chimica Acta 411 (2010) 26–30
Exposure to tobacco smoke has been clearly accepted as a major risk factor for CAD. Excessive exposure to smoking may veil the protective function of the variant genotypes in our study. Our result suggests that the − 1535C > T polymorphism may not be a protective factor against smoking-related CAD in the Chinese population. Nevertheless, this result might be a coincidence due to the relatively small numbers in the subgroups. Our study has several limitations. Firstly, selection bias in the present study might have affected our results, although, the genotype distribution of patients and controls in our study was compatible with the Hardy–Weinberg expectations. Secondly, the relatively small sample size may underpower the results of our study. Thirdly, our data were obtained at the time of diagnosis, thus prospectively followed-up clinical outcome including severe cardiac events may be required to analyze the association between the − 1535C > T polymorphism and the CAD prognosis. Lastly, our study was performed in a Chinese population. Data should be extrapolated to other regions and ethnic groups cautiously. However, this internally consistent pilot study should provide valuable information to future studies in this area. This study demonstrates that Visfatin –1535C > T polymorphism is associated with reduced risk of CAD. Especially in younger, male subjects and non-smokers, the –1535C > T polymorphism is correlated well with risk reduction of CAD. Acknowledgments Project was supported by grants from the Natural Science Foundation of Jiangsu Province (No. BK2007254), Ministry of Personnel of China for returned student (No. DG216D5021) and the National Natural Science Foundation of China (No. 30871078). References [1] He J, Gu D, Wu X, et al. Major causes of death among men and women in China. N Engl J Med 2005;353:1124–34. [2] Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med 2005;352:1685–95. [3] Wang Q, Rao S, Shen GQ, et al. Premature myocardial infarction novel susceptibility locus on chromosome 1P34–36 identified by genome wide linkage analysis. Am J Hum Genet 2004;74:262–71. [4] Samani NJ, Braund PS, Erdmann J, et al. The novel genetic variant predisposing to coronary artery disease in the region of the PSRC1 and CELSR2 genes on chromosome 1 associates with serum cholesterol. J Mol Med 2008;86(11):1233–41. [5] Fukuhara A, Matsuda M, Nishizawa M, et al. Visfatin: a protein secreted by visceral fat that mimics the effects of insulin. Science 2005;307:426–30. [6] Samal B, Sun Y, Stearns G, et al. Cloning and characterization of the cDNA encoding a novel human pre-B-cell colony-enhancing factor. Mol Cell Biol 1994;14:1431–7. [7] Revollo JR, Körner A, Mills KF, et al. Nampt/PBEF/Visfatin regulates insulin secretion in beta cells as a systemic NAD biosynthetic enzyme. Cell Metab 2007;6(5): 363–75. [8] Oki K, Yamane K, Kamei N, et al. Circulating visfatin level is correlated with inflammation, but not with insulin resistance. Clin Endocrinol 2007;67:796–800. [9] Moschen AR, Kaser A, Enrich B, et al. Visfatin, an adipocytokine with proinflammatory and immunomodulating properties. J Immunol 2007;178: 1748–58. [10] Dahl TB, Yndestad A, Skjelland M, et al. Increased expression of visfatin in macrophages of human unstable carotid and coronary atherosclerosis: possible role in inflammation and plaque destabilization. Circulation 2007;115:972–80.
[11] Rongvaux A, Shea RJ, Mulks MH, et al. Pre-B-cell colony-enhancing factor, whose expression is up-regulated in activated lymphocytes, is a nicotinamide phosphoribosyl transferase, a cytosolic enzyme involved in NAD biosynthesis. Eur J Immunol 2002;32:3225–34. [12] Revollo JR, Grimm AA, Imai S. The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyl transferase regulates Sir2 activity in mammalian cells. J Biol Chem 2004;279:50754–63. [13] Liu SW, Qiao SB, Yuan JS, Liu DQ. Association of plasma visfatin levels with inflammation, atherosclerosis, and acute coronary syndromes in humans. Clin Endocrinol (Oxf) 2009;71(2):202–7. [14] Ognjanovic S, Bao S, Yamamoto SY, et al. Genomic organization of the gene coding for human pre-B-cell colony enhancing factor and experssion in human fetal membranes. J Mol Endocrinol 2002;26:107–17. [15] Zhang YY, Gottardo L, Thompson R, Powers C, et al. A visfatin promoter polymorphism is associated with low-grade inflammation and type 2 diabetes. Obesity 2006;14:2119–26. [16] Ye SQ, Simon BA, Maloney JP, et al. Pre-B-cell colony enhancing factor as a potential novel biomarker in acute lung injury. Am J Respir Crit Care Med 2005;171:361–70. [17] Tokunaga A, Miura A, Okauchi Y, et al. The − 1535 promoter variant of the visfatin gene is associated with serum triglyceride and HDL-cholesterol Levels in Japanese subjects. Endocr J 2008;55:205–12. [18] Jian WX, Luo TH, Gu YY, et al. The visfatin gene is associated with glucose and lipid metabolism in a Chinese population. Diabet Med 2006;23:967–73. [19] Santamore WP, Kahl FR, Kutcher MA, et al. A microcomputer based automated, quantitative coronary angiographic analysis system. Ann Biomed Eng 1988;16: 367–77. [20] Favis R, Day JP, Gerry NP, et al. Universal DNA array detection of small insertions and deletions in BRCA1 and BRCA2. Nat Biotechnol 2000;18:561–4. [21] Xiao Z, Xiao J, Jiang Y, et al. A novel method based on ligase detection reaction for low abundant YIDD mutants detection in hepatitis B virus. Hepatol Res 2006;34: 150–5. [22] Filippatos TD, Derdemezis CS, Gazi IF, et al. Increased plasma visfatin levels in subjects with the metabolic syndrome. Eur J Clin Invest 2008;38:71–2. [23] Adya R, Tan BK, Punn A, et al. Visfatin induces human endothelial VEDF and MMP2/9 production via mark and P13/Akt signaling pathways: novel insights into visfatin-induced angiogenesis. Cardiovasc Res 2008;78:356–65. [24] Liu P, Li H, Cepeda J, et al. Critical role of PBEF expression in pulmonary cell inflammation and permeability. Cell Biol Int 2009;33(1):19–30. [25] Bailey SD, Loredo-Osti JC, Lepage P, et al. Common polymorphisms in the promoter of the visfatin gene (PBEF1) influence plasma insulin levels in a French-Canadian population. Diabetes 2006;55(10):2896–902. [26] Arya R, Blangero J, Williams K, et al. Factors of insulin resistance syndrome-related phenotypes are linked to genetic locations on chromosomes 6 and 7 in nondiabetic mexican-americans. Diabetes 2002;51:841–7. [27] Duggirala R, Balangero J, Almasy L, et al. A major susceptibility locus influencing plasma triglyceride concentrations is located on chromosome 15q in Mexican Americans. Am J Hum Genet 2000;66:1237–45. [28] Reik W, Walter J. Evolution of imprinting mechanisms: the battle of the sexes begins in the zygote. Nat Genet 2001;27:255–6. [29] Sieck GC. Genome and hormones: an integrated approach to gender differences in physiology. J Appl Physiol 2001;91:1485–6. [30] Kowalska I, Straczkowski M, Nikolajuk A, et al. Serum visfatin in relation to insulin resistance and markers of hyperandrogenism in lean and obese women with polycystic ovary syndrome. Hum Reprod 2007;22:1824–9. [31] Choi BG, McLaughlin MA. Why men's hearts break: cardiovascular effects of sex steroids. Endocrinol Metab Clin N Am 2007;36:365–77. [32] de Luis DA, Gonzalez Sagrado M, Conde R, Aller R, Izaola O, Romero E. Effect of a hypocaloric diet on serum visfatin in obese non-diabetic patients. Nutrition 2008;24: 517–21. [33] Jin H, Jiang B, Tang J, et al. Serum visfatin concentrations in obese adolescents and its correlation with age and high-density lipoprotein cholesterol. Diabetes Res Clin Pract 2008;79:412–8.
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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 e v i e r. c o m / l o c a t e / c l i n c h i m
Genetic variant in visfatin gene promoter is associated with decreased risk of coronary artery disease in a Chinese population Jian-Jun Yan a, Na-Ping Tang a,b, Jian-Jin Tang a, En-zhi Jia a, Ming-Wei Wang a, Qi-Ming Wang a, Jun Zhu a, Zhi-Jian Yang a, Lian-sheng Wang a,⁎, Jun Huang a,⁎ a
Department of Cardiology, First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, 210029, Jiangsu Province, China National Shanghai Center for New Drug Safety Evaluation and Research, Shanghai Institute of Pharmaceutical industry, 199 Guoshoujing Road, Zhangjiang Hi-Tech Park, Pudong, Shanghai 201203, China
b
a r t i c l e
i n f o
Article history: Received 28 March 2009 Received in revised form 27 September 2009 Accepted 29 September 2009 Available online 3 October 2009 Keywords: Coronary artery disease Visfatin Polymorphism
a b s t r a c t Background: Visfatin is a newly identified pro-inflammatory adipokine expressed predominantly in visceral fat. Previous studies have suggested a role for visfatin in low-grade inflammation and regulation of lipid metabolism. Most recently, a genetic polymorphism − 1535C > T located in the visfatin gene promoter has been identified, and suggested to be associated with the regulation of visfatin expression, lipid levels. However, it is unclear whether this polymorphism has a linkage with CAD. Methods: We conducted a hospital-based case-control study with 257 CAD patients and 292 controls to examine the potential association of the Visfatin − 1535C > T polymorphism with CAD. Results: The frequencies of the CC, CT, and TT genotypes in cases were significantly different from those of controls (χ2 = 6.223, P = 0.045). Subjects with the variant genotypes (CT + TT) had a 40% decreased risk of CAD relative to CC carriers (adjusted OR = 0.60, 95%CI = 0.40–0.89). Furthermore, the adjusted OR of a TT genotype for CAD was 0.52 (95%CI = 0.31–0.87). There was a significant association between Visfatin − 1535C > T polymorphism and triglyceride levels in both CAD patients and controls (P = 0.003, 0.018, respectively). In stratified analyses, the T allele was significantly associated with reduced risk of CAD in males, subjects with age < 59 years, and non-smokers. Moreover, a borderline statistical significance (P = 0.058 for trend) was observed between the variant genotypes and severity of CAD. Conclusion: Our results suggested that Visfatin − 1535C > T polymorphism might be associated with reduced risk of CAD in a Chinese population. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Coronary artery disease (CAD) is one of the major causes of death in most countries, including China [1]. Atherosclerosis, the main cause of CAD, is an inflammatory disease in which immune mechanisms interact with environmental and genetic risk factors to initiate, propagate, and activate lesions in the arterial tree, eliciting various acute and sever events such as acute coronary syndromes [2]. Most recently, genetic studies have revealed a series of candidate gene susceptibility variants that may also contribute to the pathogenesis of CAD [3,4]. Visfatin (also known as pre-B cell colony-enhancing factor or PBEF) is a novel pro-inflammatory adipokine expressed markedly in visceral fat [5]. Visfatin is a 52- to 55-kDa protein, which was originally cloned as a growth factor enhancing the effect of stem cell factor and IL-7 on the colony formation activity of early stage B cells [6]. Visfatin has a potential insulin-mimetic action that may cause insulin sensitivity [5], ⁎ Corresponding authors. Tel./fax: +86 25 83724440. E-mail addresses: [email protected] (L. Wang), [email protected] (J. Huang). 0009-8981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2009.09.033
however, which remains controversial [5,7]. Recent studies have indicated that visfatin is involved in the processes of inflammation, an important component of atherosclerosis. Serum visfatin levels were positively correlated with the serum levels of IL-6 and CRP, indicating that circulating visfatin may reflect inflammation status [8,9]. Visfatin has also been shown to be localized to foam cell macrophages within unstable atherosclerotic lesions, which potentially play a role in plaque destabilization [10]. Moreover, it has been suggested that visfatin plays a key part in nicotinamide adenine dinucleotide (NAD) biosynthesis pathway [7,11,12]. Furthermore, visfatin might be an independent CAD risk factor [13]. The visfatin gene is located on chromosome 7q22.2, which is composed of 11 exons and 10 introns, spanning 34.7 kb of genomic DNA. The proximal and distal regions of visfatin gene promoter contain several transcription initiation sites, and binding sites for Sp1 and ubiquitous transcription factors such as nuclear factor 1 (NF-1), AP-1, AP-2 [14]. To date, several single nucleotide polymorphisms in the visfatin gene have been described. The visfatin promoter polymorphisms have been reported as a linkage with low-grade and acute inflammation [15,16]. One of the most frequently studied
J.-J. Yan et al. / Clinica Chimica Acta 411 (2010) 26–30
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polymorphisms, −1535C > T, was reported to be associated with the regulation of visfatin gene expression and serum lipid level [16–18]. However, there were some conflicting results regarding whether this variant is functional or not [16,17,24]. To the best of our knowledge, no data have been published on the association of the − 1535C > T polymorphism with the risk of CAD. We investigate the potential association between the − 1535C > T polymorphism and the risk of CAD in a hospital-based case-control study in a Chinese population.
Biotech Co., Ltd.). Subsequently, LDR products were analyzed by DNA sequencing (Model 377, Applied Biosystems). All assays were conducted blindly without the knowledge of case or control status. Additionally, about 10% of the samples were randomly selected and retested by direct DNA sequencing on a 3730xl DNA analyzer (Applied Biosystems) and the results were 100% concordant.
2. Materials and methods
Statistical analyses were conducted with Stata ver. 8.0 (STATA Corp., College Station, TX) and SPSS 13.0 (SPSS Inc., Chicago, IL). Normality was tested 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 between 2 groups were analyzed by Mann–Whitney U-test. Pearson χ2-test was used to compare allele distribution and qualitative variables represented as frequencies. Hardy–Weinberg equilibrium was assessed for both subjects and controls by a χ2 goodness-of-fit test. Odds ratio (OR) and 95% confidence interval (CI) were calculated to estimate the correlation between the −1535C> T polymorphism and the risk of CAD. A binary logistic regression analysis was used for the evaluation of the independent effect of visfatin 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 −1535C> T polymorphism with CAD severity was evaluated by χ2-test for trend. Two-tailed P < 0.05 were considered as statistical significance.
2.1. Study population The association study enrolled 257 unrelated patients of CAD, who were all admitted to the First Affiliated Hospital of Nanjing Medical University. The diagnosis of CAD was certified by coronary angiography performed with the Judkins technique using a quantitative coronary angiographic system [19]. CAD was defined as an angiography with ≥50% luminal narrowing in one main coronary artery or more. Patients with CAD were divided into 1-, 2-, and 3-vessel disease subgroups according to the number of significantly stenosed vessels with reference to the Coronary Artery Surgery Study classification. Two cardiologists who were unaware of the patients included in this study assessed the angiograms. This study included 292 unrelated control subjects randomly selected from outpatients (staff of local companies and administration agencies) who underwent regular physical examinations during the same time in the same hospital. After considering it unethical to perform coronary angiography to rule out the presence of asymptomatic CAD, those control subjects with a history of angina, symptoms or signs of other atherosclerotic vascular diseases and an abnormal electrocardiogram were excluded. All subjects enrolled in this study were of Han Chinese origin and residing in or near Jiangsu Province. They had no history of significant concomitant diseases including cardiomyopathy, bleeding disorders, renal failure, previous thoracic irradiation therapy and malignant diseases. Hypertension was defined as resting systolic blood pressure >140 mm Hg and/or diastolic blood pressure >90 mm Hg or in the presence of active treatment with antihypertensive agents. Diabetes was defined as fasting blood glucose >7.0 mmol/l or a diagnosis of diabetes needing diet or antidiabetic drug therapy. Dyslipidemia was defined as total cholesterol concentration of >5.72 mmol/l or on drugs. Individuals who formerly or currently smoked ≥10 cigarettes per day for at least 2 years were defined as smokers. This study was approved by the First Affiliated Hospital Ethics Committee of Nanjing Medical University and informed consent was obtained from each participant.
2.3. Statistical analysis
3. Results 3.1. Demographic information The characteristics of our study subjects are given in Table 1. Patients with CAD were much older, smoked more cigarettes, had significantly higher weight, TG, TC , LDL-C, body mass index (BMI), and were more likely to be diabetic, hypertensive and dyslipidemic than the control subjects. In terms of coronary angiographic findings, 100 (38.9%) CAD cases had single-vessel disease, 87 (33.9%) had double-vessel disease and 70 (27.2%) had triple-vessel disease. Table 1 Baseline characteristics of cases and controls. Characteristics
2.2. DNA extraction and genotyping Peripheral venous blood was drawn from each participant. Genomic DNA was extracted using the AxyPrep DNA Blood kit (Axygen Scientific Inc., Union City, CA, USA). The −1535C > T (rs61330082) polymorphism was genotyped by the PCR-LDR Sequencing method, as reported previously [20,21]. A 216 bp DNA fragment (nucleotide positions −1637 to −1422 of the promoter) containing the polymorphic site was amplified by PCR in the ABI 9600 (Applied Biosystems, Foster City, CA) using the forward primer 5′-ACACAGGGAAGATCAACCAA-3′ and the reverse primer 5′-AGACAACCTCAGTCAACACTA-3′. The PCR was carried out in a total volume of 15 μl containing 1.5 μl 10 × PCR buffer, 0.2 μl 10 pmol each primer, 0.3 μl dNTP, 0.25 μl Taq polymerase (MBI fermentas), 2 μl of genomic DNA and 10.75 μl H2O. The PCR cycling parameters were 35 cycles of 30 s at 94 °C, 55 °C for 30 s and 72 °C for 45 s. Ligase Detection Reaction (LDR) was performed in a total volume of 10 μl containing 2 μl PCR product, 1 μl 10 × Taq DNA ligase buffer, 0.125 μl 40 U/μl Taq DNA ligase (NEB), 1 μl 10 pmol probes (0.33 μl each of probe), and 5.875 μl H2O. LDR probes were composed of 1 common probe and 2 discriminating probes (designed by the Shanghai Generay
Age (years) Sex (male), n (%) 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 (%)
Cases
Controls
(n = 257)
(n = 292)
P
63.9 ± 9.6 210 (81.7) 24.9 ± 3.3 128 (49.8) 56 (21.8) 64 (24.9) 96 (37.4) 4.14 ± 1.16 1.73 ± 0.96 1.12 ± 0.43 2.58 ± 0.89 5.86 ± 2.69
59.3 ± 12.4 211 (72.3) 24.1 ± 2.7 98 (33.6) 23 (7.9) 33 (11.3) 42 (14.4) 3.96 ± 1.17 1.42 ± 1.01 1.33 ± 0.35 2.28 ± 0.76 5.00 ± 0.92
< 0.001 0.009 0.002 < 0.001 < 0.001 < 0.001 < 0.001 0.009 < 0.001 < 0.001 < 0.001 < 0.001
100 (38.9) 87 (33.9) 70 (27.2)
– – –
– – –
BMI, body mass index; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; TC, total cholesterol; TG, triglyceride. Age, TC, TG, HDL-C, LDL-C and glucose (expressed as mean ± SD) were abnormal distributed and analyzed by Mann–Whitney U-test. BMI (expressed as mean ± SD) was normally distributed and analyzed by Student's t-test. Other data were expressed as frequencies and percentages and evaluated by χ2-test.
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J.-J. Yan et al. / Clinica Chimica Acta 411 (2010) 26–30
Table 2 Distribution of the visfatin genotype and the risk estimates for the variant visfatin genotypes. Adjusteda
Cases
Controls
Crude
(n = 257)
(n = 292)
OR (95%CI)
P
OR (95%CI)
P
92 (35.8) 118 (45.9) 47 (18.3) 165 (64.2)
78 (26.7) 143 (49.0) 71 (24.3) 214 (73.3)
1.00 0.70 (0.48–1.03) 0.56 (0.35–0.90) 0.65 (0.45–0.94)
0.071 0.017 0.022
1.00 0.64 (0.42–0.98) 0.52 (0.31–0.87) 0.60 (0.40–0.89)
0.042 0.013 0.011
302 (58.8) 212 (41.2)
299 (51.2) 285 (48.8)
– –
– –
– –
– –
b
Genotype , n (%) CC CT TT CT + TT Allelec, n (%) C allele T allele
Distributions of the visfatin genotype in cases and controls were in Hardy–Weinberg equilibrium (P = 0.399 and P = 0.733, respectively). CI, confidence interval; OR, odds ratio. a Adjusted for age, sex, body mass index, smoking status, hypertension, diabetes, dyslipidemia. b Pearson χ2 = 6.223, P = 0.045 for genotype. c Pearson χ2 = 6.300, P = 0.012 for allele.
3.2. Genotypes and allele frequencies of cases and controls and their associations with CAD The allele frequencies of SNP −1535C > T in CAD group and control group were 58.8%/41.2% and 51.2%/48.8%, respectively. The genotype distribution in our study subjects showed no deviation from Hardy– Weinberg equilibrium (P = NS). The distribution of C > T genotypes was also markedly different between cases and controls (P = 0.045) as shown in Table 2. Table 2 also shows the risk estimates for the variant −1535C > T genotypes among CAD patients compared with controls. Overall, after being adjusted for the risk factors including age, sex, BMI, smoking status, hypertension, diabetes and dyslipidemia, the OR for subjects with the variant genotypes (CT and TT) was 0.60 (95% CI= 0.40–0.89, P = 0.011). We also found that TT genotype carriers had a 48% reduction in risk of CAD compared with the CC carriers
Table 3 BMI, lipid profiles and glucose of cases and controls in the visfatin genotypes. Characteristics
BMI (kg/m2) TC (mmol/l) TG (mmol/l) HDL-C (mmol/l) LDL-C (mmol/l) Glucose (mmol/l)
Cases
P
CC
CT + TT
24.9 ± 3.4 4.26 ± 1.20 1.91 ± 0.93 1.11 ± 0.41 2.67 ± 0.90 5.84 ± 2.59
24.9 ± 3.3 4.06 ± 1.14 1.63 ± 0.97 1.12 ± 0.44 2.54 ± 0.88 5.87 ± 2.74
NS NS 0.003 NS NS NS
Controls CT + TT
24.2 ± 2.39 4.04 ± 1.25 1.67 ± 1.35 1.33 ± 0.34 2.45 ± 0.73 5.00 ± 0.81
24.1 ± 2.86 3.94 ± 1.14 1.33 ± 0.83 1.34 ± 0.35 2.21 ± 0.77 5.00 ± 0.95
3.3. BMI, lipid profiles and glucose of cases and controls in the visfatin genotypes Table 3 shows BMI, lipid profiles and glucose levels according to the −1535C> T genotypes in the CAD patients and controls. We found a significant association between the genotypes and serum triglyceride levels in these 2 groups (P = 0.003 and 0.018, respectively). In addition, we also noted that there was a borderline significance between the genotypes and serum LDL-C levels in controls (P = 0.062). However, there were no significant differences between the genotypes and BMI or glucose levels in both groups. 3.4. Stratified analyses of the polymorphism and CAD
P
CC
(adjusted OR = 0.52, 95% CI = 0.31–0.87, P = 0.013). We used a dominant model for analysis in the present study. In addition, we have attempted to conduct analysis with other models. However, no other statistical significance was observed (data not shown).
NS NS 0.018 NS NS NS
BMI, body mass index; CAD, coronary artery disease; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; TC, total cholesterol; TG, triglyceride. TC, TG, HDL-C, LDL-C and glucose (expressed as mean ± SD) were abnormal distributed and analyzed by Mann–Whitney U-test. BMI (expressed as mean ± SD) was normal distributed and analyzed by Student's t-test.
We conducted stratification analyses according to median age of controls (59 years), gender, smoking status (Table 4). We noted that the T allele was significantly associated with a reduction in risk of CAD in male (adjusted OR = 0.49, 95% CI = 0.31–0.77, P = 0.002); subjects with age < 59 years (adjusted OR = 0.43, 95% CI = 0.21–0.88, P = 0.021) and non-smokers (adjusted OR = 0.57, 95% CI = 0.37– 0.87, P = 0.009), while not in female (adjusted OR = 1.27, 95% CI = 0.52–3.08, P = NS); subjects with age ≥ 59 years (adjusted OR = 0.68, 95% CI = 0.41–1.12, P = NS) and smokers (adjusted OR = 0.82, 95% CI = 0.36–1.85, P = NS). Patients with CAD were subclassified into 3 subgroups (single-, double- and triple-vessel disease) on the basis of the number of affected coronary arteries. The frequency of patients with variant genotypes decreased gradually
Table 4 Stratified analyses by age and sex for the variant visfatin genotypes in cases and controls. Variable
Age (median) <59 years ≥59 years Sex Males Females Smoking Yes No
TT + CT/CC
Adjusteda
Crude
Case
Controls
OR (95%CI)
P
OR (95%CI)
P
44/25 121/67
108/38 106/40
0.62 (0.34–1.15) 0.68 (0.43–1.09)
NS NS
0.43 (0.21–0.88) 0.68 (0.41–1.12)
0.021 NS
131/79 34/13
158/53 56/25
0.56 (0.37–0.85) 1.17 (0.53–2.58)
0.006 NS
0.49 (0.31–0.77) 1.27 (0.52–3.08)
0.002 NS
67/29 98/63
31/11 183/67
0.72 (0.31–1.66) 0.58 (0.38–0.89)
NS 0.012
0.82 (0.36–1.85) 0.57 (0.37–0.87)
NS 0.009
CI, confidence interval; OR, odds ratio. a Adjusted for age, sex, body mass index, smoking status, hypertension, diabetes, dyslipidemia.
J.-J. Yan et al. / Clinica Chimica Acta 411 (2010) 26–30 Table 5 Visfatin genotypes and CAD severity. CAD severity
SVD (n = 100) DVD (n = 87) TVD (n = 70)
P for trenda
Genotype, n (%) CC
CT + TT
31 (33.7) 29 (31.5) 32 (34.8)
69 (41.8) 58 (35.2) 38 (23.0)
0.058
CAD, coronary artery disease; DVD, double-vessel disease; SVD, single-vessel disease; TVD, triple-vessel disease. a χ2 = 3.601 for trend.
from single- to triple-vessel disease with a borderline significance (P = 0.058 for trend) (Table 5). 4. Discussion The present case-control study analyzed the association between CAD risk and genetic polymorphism at position −1535 in the promoter of visfatin gene. According to our data, the −1535 C to T variant was associated with risk reduction of CAD development in a Chinese population. Recent studies have also indicated that visfatin, whose plasma concentrations are altered in obesity and obesity related disorders, might be involved in the processes of low-grade inflammation [22], an important component of atherosclerosis. Visfatin might induce endothelial VEGF and MMP production and activity via multiple signalling pathways including MAPK, PI3K/Akt, and VEGFR2, suggesting a vital role of visfatin in the pathogenesis of vascular inflammation [23]. Visfatin has also been shown to be localized to foam cell macrophages within unstable atherosclerotic lesions, which presumptively play a role in plaque destabilization [10]. In addition, plasma visfatin levels are significantly correlated with CAD, particularly ACS, independent of well-known CAD risk factors [13]. The −1535C > T polymorphism is located on the distal promoter region, which contains several CAAT boxes and TATA-like sequences and binding sites for CCAAT/NF-1, NF- B, NF-IL-6, GR, and AP-1 [14]. More importantly, −1535C > T, was reported to be associated with the regulation of visfatin gene expression [16,24] and serum lipid level [17,18]. Then, we hypothesized that the −1535C > T polymorphism might be correlated well with the susceptibility of CAD. In the present study, we conducted a hospital-based case-control study with 257 CAD patients and 292 controls to examine the potential association of Visfatin −1535C> T polymorphism with CAD. A 40% decreased risk of CAD was observed in subjects with −1535CT/TT compared with the CC carriers (adjusted OR= 0.60, 95% CI = 0.40–0.89, P = 0.011), indicating the T allele might confer a protective factor. The study of Ye et al. showed that the T variant in the T-1535C SNP (described as C-1543T in their study) resulted in nearly a 2-fold decrease in the reporter gene expression and transcription of visfatin was reduced in patients with the T variant, suggesting that the −1535C > T polymorphism is associated with a decreased risk of acute respiratory distress syndrome (ARDS) [16]. Thereby, the T allele was expected to have lower levels of visfatin gene transcription and enzymatic activity than C allele. There is a strong likelihood that the C to T substitution at this position may reduce activity of transcription factors (such as NF- B and AP-1), thereby downregulating visfatin gene transcriptional activity. Recently, they also confirmed that, compared to its common −1535C allele, the −1535T variant significantly attenuated its binding to an IL-1β induced unknown transcription factor in pulmonary vascular endothelial cells, which may underlie reduced expression of PBEF and lower susceptibility to acute lung injury [24]. Whereas, another investigation among Japanese suggested that this variation might be not functional [17]. Differences in experimental conditions, ethnicity and geographic variation may account for this discrepancy. Overall, we could not
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exclude the possibility that the association between −1535C > T polymorphism and CAD was due to the effect of this variant on the expression of visfatin gene, however, more studies are needed to confirm this hypothesis. We also estimated the impact of − 1535C > T polymorphism on the BMI, lipid profiles and glucose levels in CAD patients and controls. Our data showed that this promoter variant was associated with a lower serum triglyceride concentration in CAD patients and in controls. Likewise, Tokunaga et al. also found that the − 1535 T/T genotype was associated with lower serum triglyceride levels and higher HDL-C levels in the nondiabetic subjects [17]. Additionally, Jian et al. also reported that the haplotype containing − 1535C > T polymorphism was correlated significantly with lipid metabolism [18]. Bailey et al. observed a remarkable association between the rs7899066 and rs11977021 variants and dyslipidemia associated with insulin resistance [25]. Though the precise mechanism by which this variant influences lipid metabolism remains to be determined, there are still several plausible explanations. First, it has been shown that lipid phenotypes (HDL-C, TG) have a linkage with chromosome 7 [26,27], where the visfatin gene is located. It is likely that the T variant might have an impact on gene transcriptional activity, thus inducing different lipid phenotypes. Second, the −1535C > T may be in linkage disequilibrium with other SNPs such as the rs7899066 and rs11977021 in visfatin promoter, exerting influence on lipid metabolism. Third, visfatin has been reported as the rate limiting component in the mammalian nicotinamide adenine dinucleotide (NAD) biosynthesis pathway [11,12]. As a result, NAD regulated by visfatin has an effect on lipid metabolism. However, our study indicated that there were no significant differences between the genotypes and BMI or glucose levels in CAD patients and controls. This finding is in concordance with the observations made by Tokunaga et al. [17] and Zhang YY et al.[15], revealing that visfatin gene might not be related to glucose metabolism directly. But Jian et al. revealed that haplotype containing − 1535C > T polymorphism was significantly associated with 2-h glucose concentrations [18]. These discrepancies may be explained by several factors: first, differences in confounding factors (such as age, drug therapy and lifestyle) due to different criteria for recruitment; and second, the possibility of this variant's linkage disequilibrium with other SNPs. Nonetheless, additional studies are required to further define the results in detail. According to stratified analyses, we also observed an interaction between the −1535C > T polymorphism and sex. In male subjects, those who carried the variant genotypes had a significant reduction in risk of CAD than CC carriers. Epidemiological data show that the risk of CAD differs between female and male patients. There are several possible mechanisms for sex differences in gene expression, such as imprinting [28] and hormonal effects [29]. Kowalska et al. reported that visfatin remains an independent predictor of serum testosterone and free androgen index in the lean polycystic ovary syndrome (PCOS) subjects [30]. Moreover, testosterone has been found to reduce the expression of proinflammatory cytokines, such as TNF-a, IL-1, and IL-6 in human vascular endothelium and monocytes, and imbalance of sex steroids contributes to adverse cardiac effects in men [31]. Thereby, that testosterone might synergize the influence of the genetic variants, would account for this observation, inducing CAD risk reduction in male subjects. We also noticed that decreased risk of CAD with the variant visfatin genotypes was pronounced in younger subjects (age <59 years), but not in older subjects. Overwhelming accumulated exposure to environmental risks in older individuals may weaken the influence of this polymorphism in our study. Besides, de Luis et al. [32] and Jin et al. [33] also found that age has an inverse correlation with serum visfatin concentrations. It is possible that lower serum visfatin concentrations might also temper the impact of this polymorphism in the older group. We noted that among non-smokers, possession of the variant genotypes had a 43% reduced risk of CAD than CC genotype carriers.
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J.-J. Yan et al. / Clinica Chimica Acta 411 (2010) 26–30
Exposure to tobacco smoke has been clearly accepted as a major risk factor for CAD. Excessive exposure to smoking may veil the protective function of the variant genotypes in our study. Our result suggests that the − 1535C > T polymorphism may not be a protective factor against smoking-related CAD in the Chinese population. Nevertheless, this result might be a coincidence due to the relatively small numbers in the subgroups. Our study has several limitations. Firstly, selection bias in the present study might have affected our results, although, the genotype distribution of patients and controls in our study was compatible with the Hardy–Weinberg expectations. Secondly, the relatively small sample size may underpower the results of our study. Thirdly, our data were obtained at the time of diagnosis, thus prospectively followed-up clinical outcome including severe cardiac events may be required to analyze the association between the − 1535C > T polymorphism and the CAD prognosis. Lastly, our study was performed in a Chinese population. Data should be extrapolated to other regions and ethnic groups cautiously. However, this internally consistent pilot study should provide valuable information to future studies in this area. This study demonstrates that Visfatin –1535C > T polymorphism is associated with reduced risk of CAD. Especially in younger, male subjects and non-smokers, the –1535C > T polymorphism is correlated well with risk reduction of CAD. Acknowledgments Project was supported by grants from the Natural Science Foundation of Jiangsu Province (No. BK2007254), Ministry of Personnel of China for returned student (No. DG216D5021) and the National Natural Science Foundation of China (No. 30871078). References [1] He J, Gu D, Wu X, et al. Major causes of death among men and women in China. N Engl J Med 2005;353:1124–34. [2] Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med 2005;352:1685–95. [3] Wang Q, Rao S, Shen GQ, et al. Premature myocardial infarction novel susceptibility locus on chromosome 1P34–36 identified by genome wide linkage analysis. Am J Hum Genet 2004;74:262–71. [4] Samani NJ, Braund PS, Erdmann J, et al. The novel genetic variant predisposing to coronary artery disease in the region of the PSRC1 and CELSR2 genes on chromosome 1 associates with serum cholesterol. J Mol Med 2008;86(11):1233–41. [5] Fukuhara A, Matsuda M, Nishizawa M, et al. Visfatin: a protein secreted by visceral fat that mimics the effects of insulin. Science 2005;307:426–30. [6] Samal B, Sun Y, Stearns G, et al. Cloning and characterization of the cDNA encoding a novel human pre-B-cell colony-enhancing factor. Mol Cell Biol 1994;14:1431–7. [7] Revollo JR, Körner A, Mills KF, et al. Nampt/PBEF/Visfatin regulates insulin secretion in beta cells as a systemic NAD biosynthetic enzyme. Cell Metab 2007;6(5): 363–75. [8] Oki K, Yamane K, Kamei N, et al. Circulating visfatin level is correlated with inflammation, but not with insulin resistance. Clin Endocrinol 2007;67:796–800. [9] Moschen AR, Kaser A, Enrich B, et al. Visfatin, an adipocytokine with proinflammatory and immunomodulating properties. J Immunol 2007;178: 1748–58. [10] Dahl TB, Yndestad A, Skjelland M, et al. Increased expression of visfatin in macrophages of human unstable carotid and coronary atherosclerosis: possible role in inflammation and plaque destabilization. Circulation 2007;115:972–80.
[11] Rongvaux A, Shea RJ, Mulks MH, et al. Pre-B-cell colony-enhancing factor, whose expression is up-regulated in activated lymphocytes, is a nicotinamide phosphoribosyl transferase, a cytosolic enzyme involved in NAD biosynthesis. Eur J Immunol 2002;32:3225–34. [12] Revollo JR, Grimm AA, Imai S. The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyl transferase regulates Sir2 activity in mammalian cells. J Biol Chem 2004;279:50754–63. [13] Liu SW, Qiao SB, Yuan JS, Liu DQ. Association of plasma visfatin levels with inflammation, atherosclerosis, and acute coronary syndromes in humans. Clin Endocrinol (Oxf) 2009;71(2):202–7. [14] Ognjanovic S, Bao S, Yamamoto SY, et al. Genomic organization of the gene coding for human pre-B-cell colony enhancing factor and experssion in human fetal membranes. J Mol Endocrinol 2002;26:107–17. [15] Zhang YY, Gottardo L, Thompson R, Powers C, et al. A visfatin promoter polymorphism is associated with low-grade inflammation and type 2 diabetes. Obesity 2006;14:2119–26. [16] Ye SQ, Simon BA, Maloney JP, et al. Pre-B-cell colony enhancing factor as a potential novel biomarker in acute lung injury. Am J Respir Crit Care Med 2005;171:361–70. [17] Tokunaga A, Miura A, Okauchi Y, et al. The − 1535 promoter variant of the visfatin gene is associated with serum triglyceride and HDL-cholesterol Levels in Japanese subjects. Endocr J 2008;55:205–12. [18] Jian WX, Luo TH, Gu YY, et al. The visfatin gene is associated with glucose and lipid metabolism in a Chinese population. Diabet Med 2006;23:967–73. [19] Santamore WP, Kahl FR, Kutcher MA, et al. A microcomputer based automated, quantitative coronary angiographic analysis system. Ann Biomed Eng 1988;16: 367–77. [20] Favis R, Day JP, Gerry NP, et al. Universal DNA array detection of small insertions and deletions in BRCA1 and BRCA2. Nat Biotechnol 2000;18:561–4. [21] Xiao Z, Xiao J, Jiang Y, et al. A novel method based on ligase detection reaction for low abundant YIDD mutants detection in hepatitis B virus. Hepatol Res 2006;34: 150–5. [22] Filippatos TD, Derdemezis CS, Gazi IF, et al. Increased plasma visfatin levels in subjects with the metabolic syndrome. Eur J Clin Invest 2008;38:71–2. [23] Adya R, Tan BK, Punn A, et al. Visfatin induces human endothelial VEDF and MMP2/9 production via mark and P13/Akt signaling pathways: novel insights into visfatin-induced angiogenesis. Cardiovasc Res 2008;78:356–65. [24] Liu P, Li H, Cepeda J, et al. Critical role of PBEF expression in pulmonary cell inflammation and permeability. Cell Biol Int 2009;33(1):19–30. [25] Bailey SD, Loredo-Osti JC, Lepage P, et al. Common polymorphisms in the promoter of the visfatin gene (PBEF1) influence plasma insulin levels in a French-Canadian population. Diabetes 2006;55(10):2896–902. [26] Arya R, Blangero J, Williams K, et al. Factors of insulin resistance syndrome-related phenotypes are linked to genetic locations on chromosomes 6 and 7 in nondiabetic mexican-americans. Diabetes 2002;51:841–7. [27] Duggirala R, Balangero J, Almasy L, et al. A major susceptibility locus influencing plasma triglyceride concentrations is located on chromosome 15q in Mexican Americans. Am J Hum Genet 2000;66:1237–45. [28] Reik W, Walter J. Evolution of imprinting mechanisms: the battle of the sexes begins in the zygote. Nat Genet 2001;27:255–6. [29] Sieck GC. Genome and hormones: an integrated approach to gender differences in physiology. J Appl Physiol 2001;91:1485–6. [30] Kowalska I, Straczkowski M, Nikolajuk A, et al. Serum visfatin in relation to insulin resistance and markers of hyperandrogenism in lean and obese women with polycystic ovary syndrome. Hum Reprod 2007;22:1824–9. [31] Choi BG, McLaughlin MA. Why men's hearts break: cardiovascular effects of sex steroids. Endocrinol Metab Clin N Am 2007;36:365–77. [32] de Luis DA, Gonzalez Sagrado M, Conde R, Aller R, Izaola O, Romero E. Effect of a hypocaloric diet on serum visfatin in obese non-diabetic patients. Nutrition 2008;24: 517–21. [33] Jin H, Jiang B, Tang J, et al. Serum visfatin concentrations in obese adolescents and its correlation with age and high-density lipoprotein cholesterol. Diabetes Res Clin Pract 2008;79:412–8.