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Polymorphisms of the NOS3 gene and risk of myocardial infarction in the Tunisian population
Cytokine 64 (2013) 646–651
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Polymorphisms of the NOS3 gene and risk of myocardial infarction in the Tunisian population q Amani Kallel a, Mohamed Hédi Sbaï a, Yousra Sediri a, Salem Abdessalem b, Mohamed Sami Mourali b, Moncef Feki a, Rachid Mechmeche b, Riadh Jemaa a,⇑, Naziha Kaabachi a a b
Université de Tunis El Manar, Hôpital la Rabta, Service de Biochimie, LR99ES11, 1007 Tunis, Tunisie Université de Tunis El Manar, Hôpital la Rabta, service des Explorations Fonctionnelles et de Reanimations en Cardiologie, Hospital la Rabta, Tunis, Tunisie
a r t i c l e
i n f o
Article history: Received 8 February 2013 Received in revised form 29 July 2013 Accepted 4 September 2013 Available online 3 October 2013 Keywords: NOS3 gene polymorphisms Myocardial infarction Haplotype Risk factors
a b s t r a c t Controversial results regarding the association of eNOS gene (NOS3) polymorphisms with myocardial infarction (MI) have been reported. This study investigated the relationship of the 786T>C (rs2070744), 894G>T (rs1799983) and 4a4b polymorphisms of the NOS3 gene with the presence of MI in the Tunisian population. In addition, we also examined the association of NOS3 gene haplotypes with MI in Tunisian subjects. A total of 303 patients with MI and 225 controls were included in the study. The 894G>T and 786T>C single nucleotide polymorphisms were analyzed by PCR-RFLP, and 4a4b polymorphism just for PCR. There was significant linkage disequilibrium between the three NOS3 polymorphisms (p < 0.0001). The genotype distribution and allele frequency of NOS3 4a4b, but not 786T>C and 894G>T, polymorphism was significantly different between MI patients and controls. The univariate logistic regression analysis showed a significant association of the 4a4b polymorphism and MI according to co-dominant, dominant and recessive models (co-dominant model OR: 4.38, 95%CI: 1.24–15.41; p = 0.021, dominant model OR: 1.66, 95%CI: 1.14–2.42); p = 0.007, and recessive model OR: 3.85, 95%CI: 1.10–13.47; p = 0.035). The multivariate analysis, adjusted for traditional cardiovascular risk factors, revealed that the NOS3 4a4a genotype was an independent predisposing factor to MI, according to the models considered. In addition, a haplotype 7 (C-T-4a), (OR = 12.05, p = 0.010) was a risk factor of MI after controlling for classical risk factors. These finding suggest that the 4a4b polymorphism of the NOS3 gene was associated with MI in Tunisian patients. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Coronary artery disease (CAD) is one of the leading causes of morbidity and mortality in much of the world today. CAD is a complex condition, resulting from genetic and environmental contributions [1]. There is strong experimental and clinical evidence that abnormalities in nitric oxide (NO) availability play an important role in the pathophysiology of CAD. This is consistent with the notion that NO has a major role in the maintenance of endothelial function, causes vasodilatation, prevents platelet and leucocyte
q This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ⇑ Corresponding author. Address: Laboratoire de Biochimie, Hôpital la Rabta, 1007 Jabbari, Tunis, Tunisia. Tel.: +216 71 561 912/98 819 168; fax: +216 71 561 912. E-mail address: [email protected] (R. Jemaa).
1043-4666/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cyto.2013.09.005
adhesion, and inhibits vascular smooth muscle cell proliferation and migration [2], and oxidation of atherogenic low-density lipoprotein [3]. The advances in our understanding of NO biology and clinical relevance have lead to a number of studies investigating whether polymorphisms in the gene encoding the enzyme that synthesizes NO in endothelial cells (endothelial nitric oxide synthase, eNOS) affect NO availability and are associated with CAD [4]. The eNOS gene (NOS3) was cloned and sequenced in 1993 and mapped to chromosome 7q35-36. Spanning 21 kb of genomic DNA, the gene comprises 26 exons that encode a 135-kDa protein containing 1203 aminoacids [5]. Several polymorphisms have been identified in the NOS3 gene. Among them, a common variant located in exon 7 (894G>T) of the NOS3 gene that modifies its coding sequence (Glu298-Asp) has been linked by several groups to the risk for CAD, stroke, and myocardial infarction [6–11], however other studies failed to find any relationship between this variant and atherosclerosis [12–15]. Another genetic variant, a single nucleotide polymorphism (SNP), resulting from a thymine being replaced by a cytosine at nucleotide 786, has been
A. Kallel et al. / Cytokine 64 (2013) 646–651
identified in the promoter region of the NOS3 gene (786T>C) and speculated to influence mRNA transcription and reduce eNOS gene expression. The 786T>C variant has been associated with hypertension [16], coronary spasm [17], and myocardial infarction [18,19], however similar to the 894G>T variant inconsistent associations of the 786T>C polymorphism with CAD, were also shown [15,20]. Finally, a 27-base pair (bp) repeat polymorphism in intron 4 of the NOS3 gene has been linked by several groups to the risk for CAD, and MI in some [21–23] but not all studies [15,24]. Ethnic background is known to influence polymorphism frequencies and their effects on the disease. The great majority of NOS3 studies were done on European or North American Caucasians and few studies have addressed the NOS3 effects in Arabs or their descendents. The aim of the present study was to investigate the possible association between the 894G>T, 786T>C, and intron 4a4b polymorphisms of the NO3 gene and haplotypes and the risk of MI in a sample of the Tunisian male population.
2. Materials and methods 2.1. Study population A total of 528 unrelated Tunisian subjects were included into this case-control study, of which 303 were MI cases and 225 healthy controls. MI patients were enrolled from the Department of Cardiology, Rabta University Hospital of Tunis. The mean age of this group was 54 years (SD 8). Diagnosis of MI was confirmed according to the European Society of Cardiology criteria; a typical rise and fall of serum creatine kinase-MB isoenzyme (CK-MB), with at least one of the following criteria: ischemic symptoms, development of pathologic Q waves on the ECG, ECG changes indicative of ischemia (ST segment elevation or depression). The control group included 225 apparently healthy male subject volunteers, with no history of angina pectoris or MI, and with a normal electrocardiogram. Their mean age was 52 years (SD 8). Controls with familial history CAD and taking medications determined by interviewing, were excluded from the study. Patients and controls were homogeneous Tunisian Arab descendents who resided in Tunisia and all were from North Tunisia. Informed written consent was obtained from all participants and the design of the study was approved by the local ethics committee. Weight and height were measured on the subjects barefooted and lightly clothed. Body mass index (BMI; kg/m2) was calculated and obesity was defined as BMI >30 kg/m2 [25]. Diabetes mellitus was defined as hyperglycemia, requiring antidiabetic drugs or testing blood glucose over 7.0 mmol/L. Hypertension was defined as systolic blood pressure P140 mmHg and/or diastolic blood pressure P90 mmHg, or the use of antihypertensive drug treatment, or a combination of these. Dyslipidemia was defined as a total cholesterol (TC) level >6.47 mmol/L and/or triglyceride (TG) level >2.26 mmol/L. A smoker was defined as a current smoker or an ex-smoker.
2.2. Laboratory analysis Blood samples were obtained after an overnight fast. Plasma levels of TC, TG and HDL-cholesterol (HDL-C) were measured by standardized enzymatic procedures and apo AI and apo B were measured using turbidimetric assay using commercial kits (Roche Diagnostics, Mannheim, Germany) on a Hitachi 912 analyzer. LDL cholesterol (LDL-C) was calculated using the Friedewald equation when the triglyceride concentrations did not exceed 4.8 mmol/L [26].
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2.3. Genetic analysis Genomic DNA was extracted from ethylenediaminetetraacetate anticoagulated whole blood using conventional salting-out procedure [27]. NOS3 894G>T and 786T>C genotype analysis was performed by PCR-RFLP analysis, using MboI and MspI digestion, respectively. For the 894G>T polymorphism (rs1799983), DNA was amplified using the following primers 50 -CATGAGGCT CAGCCCCAGAAC-30 (forward) and 50 -AGTCAATCCCTTTGGTGCTC AAC-30 (reverse). DNA was amplified for 30 cycles, each cycle comprising denaturation at 95 °C for 1 min, annealing at 60 °C for 1 min, extension at 70 °C for 1 min with final extension time of 5 min at 70 °C. The initial denaturation stage was carried out at 95 °C for 5 min. PCR products were digested with the restriction enzyme MboI at 37 °C for 16 h and separated on 2.5% agarose gel an d identified by ethidium-bromide staining. The G allele visualized as a 206-bp band, while the T allele was visualized as 119and 87-bp fragments. Genotypes for the 786T>C polymorphism (rs2070744) in the 5-flanking region of NOS3 gene were determined by PCR amplification using the primers 50 -TGG AGA GTG CTG GTG TAC CCC A-30 (sense) and 50 -GCC TCC ACC CCC ACC CTG TC-30 (antisense). The amplified products were digested overnight at 37 °C with 5U of MspI restriction enzyme, producing fragments of 140 and 40 bp for the wild-type allele (allele T), or 90, 50, and 40 bp in the case of a polymorphic variant (allele C). Fragments were separated by electrophoresis on 2.5% agarose gels with ethidium bromide staining. The genotyping of the NOS3 4a4b polymorphism was determined using the method of Wang et al. [21] with minor modifications. Primer pairs for PCR were: sense, 50 AGG CCC TAT GGT AGT GCC TTT-30 ; and antisense, 50 -TCT CTT AGT GCT GTG GTC AC-30 . The amplified fragments were separated on 2% agarose gels with ethidium bromide staining. A 420-bp band indicated five repeats of the 27-bp (NOS34b allele) and a 393-bp band four repeats (NOS34a allele). An NOS34b allele was the common allele. 2.4. Statistical analysis Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS 10.0 for Windows; SPSS Inc., Chicago, IL, USA). Distributions of continuous variables in groups were expressed as mean ± SD, and compared with unpaired Student’s t-tests. Agreement of genotype frequencies with Hardy–Weinberg equilibrium expectations was tested using v2 goodness-of-fit test with one degree of freedom. The NOS3 gene polymorphisms was first assessed in three genotype categories (wild-type, heterozygote, and homozygote variants) and then grouped into two categories with heterozygotes and homozygote variants combined because of the dominant and the recessive models of inheritance observed for these polymorphisms. Odds ratio (OR) and 95% confidence intervals (CI) were calculated using unconditional logistic regression. Crude and adjusted models for diabetes, dyslipidemia, hypertension, smoking and genotypes were fitted. The coefficient of determination (R2) was also calculated. The degree of linkage disequilibrium (LD) between polymorphism was assessed using the Thesias software (http://genecanva.ecgene.net). Logistic regression analysis models were used to evaluate the haplotypes as possible risk factors. The differences were considered significant if P value was less than 0.05. 3. Results Table 1 summarizes the clinical and biochemical data of MI patients and the control subjects. There are no differences in the mean age, and BMI between the two groups, while the prevalence of hypertension, diabetes mellitus, dyslipidemia and smoking were
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Table 1 Demographic and clinical characteristics of the study population. Variables
Controls (n = 225)
Patients (n = 303)
P
Age (years) BMI (kg/m2) Diabetes (%) Hypertension (%) Obesity (%) Dyslipidemia (%) Smoking (%) TC (mmol/L) TG (mmol/L) LDL-C (mmol/L) HDL-C (mmol/L)
52.8 + ± 8.6 25.9 ± 4.5 13.1 17.1 21.2 15.6 68.4 4.86 ± 0.980 1.57 ± 1.00 2.95 ± 0.82 1.16 ± 0.31
54.1 ± 8.7 25.3 ± 3.5 38.7 31.7 10.1 29.4 94.3 5.05 ± 1.13 1.89 ± 0.81 3.34 ± 1.01 0.85 ± 0.23
0.079 0.065 <0.001 <0 .001 <0.001 <0.001 <0.001 0.055 <0.001 <0.001 <0.001
Data are expressed as means ± SD for some and as percent in others variables.
significantly higher in patients group than in the control group (p < 0.001). The MI group showed higher concentrations of triglycerides, LDL-cholesterol and Apo B, and lower concentrations of HDL – cholesterol and ApoAI compared with the control group. Three types of polymorphism in the NOS3 gene, 894G>T, 786T>C, and 4a4b, were analyzed and their genotype distributions and allele frequencies in MI patients and controls are shown in Tables 2–4. The distribution of the NOS3 genotypes and allele frequencies in MI and control groups were compatible with Hardy–Weinberg equilibrium (all p > 0.05). For the 4a4b polymorphism a significant difference in both genotype distribution and allele frequency between MI patients and controls subjects was observed. The univariate logistic regression analysis showed a significant association of the 4a4b polymorphism and MI according to co-dominant, dominant and recessive models (co-dominant model OR: 4.38, 95%CI: 1.24–15.41; p = 0.021, dominant model OR: 1.66, 95%CI: 1.14–2.42); p = 0.007, and recessive model OR: 3.85, 95%CI: 1.10–13.47; p = 0.035) (Table 2). The multivariate analysis, adjusted for traditional cardiovascular risk factors (diabetes, hypertension, dyslipidemia, and smoking), revealed that the NOS3 4a4a genotype was an independent predisposing factor to MI, according to the models considered (codominant model: R2 = 0.217; p < 0.001; dominant model: R2 = 0.211; p < 0.001 and recessive model: R2 = 0.17; p < 0.001) (Table 1). The MI patient group showed a significant higher frequency of the 4a allele compared to the controls (21% vs. 13%; OR: 1.76, 95%CI: 1.25–2.49, p = 0.0007). No significant difference in NOS3-786T>C and 894G>T polymorphisms genotype distribution and allele frequency was observed between patients and controls. Neither in the co-dominant, nor
in the dominant and recessive models was statistically significant differences found in the distribution of genotypes frequencies between MI patients and controls. MI risk for each of these models was also estimated. Neither in the crude estimations, nor in those adjusted for diabetes, dyslipidemia, hypertension and smoking was a higher MI risk with variant allele found for these polymorphisms (Tables 3 and 4). Linkage disequilibrium analysis (Table 5), as defined by the delta (D0 ) was determined for the three NOS3 SNPs. Linkage disequilibrium was seen for 786T>C and 894G>T (D0 = 0.479, p < 0.0001), 786T>C and 4a4b (D0 = 0.374, p = 0.0002), and 894G>T and 4a4b (D0 = 0.709, p < 0.0001) among patients, and between 786T>C and 894G>T (D0 = 0.475, p < 0.0001), 786T>C and 4a4b (D0 = 0.589, p = 0.0002), and 894G>T and 4a4b (D0 = 0.939, p < 0.0001) among controls. Table 6 shows the results of the haplotype analyses. We did not observe any haplotype which might significantly increase or decrease the risk of MI. Multiple logistic regression analyses were performed to determine independent of MI risk. The analyses revealed that the haplotype 7 (C-T-4a), (OR = 12.05, p = 0.010) was a risk factor of MI after controlling for classical risk factors.
4. Discussion CAD is a multifactorial disease in which genetic and environmental factors play a great role. Associations of DNA markers at the candidate genes for CAD such NOS3 have been found previously in different populations. This phenomenon seems to be population specific as a result of variations in different subsets of genetic and environmental factors predisposing individuals to CAD from different populations and inter-population differences in genetic background. Additionally, there is strong experimental and clinical evidence that abnormalities in nitric oxide (NO) availability play an important role in the pathophysiology of hypertension [28] and diabetic vascular diseases [29], but studies on the association of NOS3 gene polymorphisms with hypertension and diabetes in different populations have yielding conflicting results, with some studies providing evidence for an association [16,30,31] while others have found none [21,32]. In this work, we have undertaken a case-control study to investigate the role of the 894G>T, 786T>C and 4a4b polymorphisms at the NOS3 gene in susceptibility to MI in Tunisians. Both patients with MI and healthy controls belonged to the same ethnic background and all shared a common geographic origin in North Tunisia. Our data showed that the NOS3 gene intron 4a4b
Table 2 Genotype distribution and relative allele frequencies of 4a4b polymorphism at the NOS3 gene among patients and controls and crude and adjusted estimations for MI. Genotypes
MI patients (n = 303)
Controls (n = 225)
Unadjusteda OR [95% CI]
P-valuea
Adjustedb OR [95% CI]
P-valueb
Codominant model 4b4b n (%) 4a4b n (%) 4a4a n (%)
187 (61.7) 101 (33.3) 15 (5.0)
164 (72.9) 58 (25.8) 3 (1.3)
1 1.52 (1.03–2.24) 4.38 (1.24–15.41)
0.031 0.021
1 1.59 (1.02–2.49) 4.66 (1.19–18.28)
0.039 0.027
Dominant model 4b4b n (%) 4a4b + 4a4a n (%)
187 (61.7) 116 (38.3 %)
164 (72.9) 61 (27.1 %)
1.66 (1.14–2.42)
0.007
1 1.75 (1.14–2.71)
0.011
Recessive model 4a4a n (%) 4a4b + 4b4b n (%)
15 (5.0) 288 (95.0)
3 (1.30) 222 (98.7)
3.85 (1.10–13.47)
0.035
1 4.03 (1.03–15.68)
0.044
Allele frequency 4b (%) 4a (%)
79 21
87 13
1.76 (1.25–2.49)
0.0007
MI, myocardial infarction; OR, odds ratio; CI, Confidence interval. a Crude logistic regression model. b Adjusted for diabetes, hypertension, dyslipidemia and smoking.
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A. Kallel et al. / Cytokine 64 (2013) 646–651 Table 3 Genotype distribution and relative allele frequencies of 894G>T polymorphism at the NOS3 gene among patients and controls and crude and adjusted estimations for MI. Genotypes
MI patients (n = 303)
Controls (n = 225)
Unadjusteda OR [95% CI]
P-valuea
Adjustedb OR [95% CI]
P-valueb
Codominant model GG n (%) GT n (%) TT n (%)
163 (53.8) 122 (40.3) 18 (5.9)
128 (56.9) 78 (34.7) 19 (8.4)
1.22 (0.85–1.77) 0.74 (0.37–1.47)
0.272 0.397
1 1.28 (0.83–1.97) 1.29 (0.57–2.94)
0.251 0.530
Dominant model GG n (%) GT + TT n (%)
163 (55.8) 140 (46.2)
128 (56.9) 97 (43.1)
1.13 (0.80–1.60)
0.480
1 1.28 (0.85–1.93)
0.225
Recessive model TT GT + GG
18 (5.9) 285 (94.1)
19 (8.4) 206 (91.6)
0.68 (0.35–1.33)
0.267
1 1.17 (0.52–2.60)
0.695
Allele frequency G (%) T (%)
84 26
85 25
1.02 (0.76–1.35)
0.913
MI: myocardial infarction; OR, odds ratio; CI, Confidence interval. a Crude logistic regression model. b Adjusted for age, diabetes, hypertension, dyslipidemia and smoking.
Table 4 Genotype distribution and relative allele frequencies of 786T>C polymorphism at the NOS3 gene among patients and controls and crude and adjusted estimations for MI. Genotypes
MI patients (n = 303)
Controls (n = 225)
Unadjusteda OR [95% CI]
P-valuea
Adjustedb OR [95% CI]
P-valueb
Codominant model TT n (%) CT n (%) CC n (%)
120 (39.6) 144 (47.5) 39 (12.9)
96 (42.7) 102 (45.3) 27 (12.0)
1.12 (0.78–1.63) 1.15 (0.66–2.02)
0.518 0.612
1 1.16 (0.76–1.78) 1.21 (0.63–2.32)
0.476 0.564
Dominant model TT n (%) CT + CC n (%)
120 (39.6) 183 (60.4)
96 (42.7) 129 (57.3)
1.13 (0.79–1.61)
0.479
1.17 (0.78–1.76)
0.429
Recessive model CC n (%) TT + CT n (%)
39 (12.9) 264 (87.1)
27 (12.0) 198 (88.0)
1.08 (0.64–1.83)
0.765
1.11 (0.60–2.06)
0.720
Allele frequency T(%) C (%)
63 37
65 35
1.09 (0.84–1.42)
0.509
MI: myocardial infarction; OR, odds ratio; CI, Confidence interval. a Crude logistic regression model. b Adjusted for age, diabetes, hypertension, dyslipidemia and smoking.
Table 5 The estimated linkage disequilibrium D’ values for each pairwise combination of the 786T>C, 4a4b and 894G>T polymorphisms among patients and controls. Patients
786T>C and 894G>T 786T>C and intron 4a4b 894G>T and intron 4a4b
Controls
D’
r
P
D’
r
P
0.479 0.374 0.709
0.153 0.224 0.050
<0.0001 0.0002 <0.0001
0.475 0.589 0.939
0.151 0.299 0.050
<0.0001 0.0002 <0.0001
Table 6 Haplotype frequencies in patients and controls. Variable
Haplotypes
Patients
Controls
Unadjusted OR [95% CI]
p
Adjusted OR [95% CI]
P*
H1a
111 112 121 211 212 221 222
0.4254 0.0979 0.1060 0.1426 0.0797 0.1079 0.0359
0.4613 0.0743 0.1094 0.1420 0.0558 0.1493 0.0060
– 1.45 1.06 1.11 1.56 0.81 7.11
– 0.207 0.786 0.629 0.171 0.326 0.057
– 1.56 1.34 1.14 1.40 0.82 12.05
– 0.208 0.305 0.581 0.352 0.450 0.010
H2 H3 H4 H5 H6 H7
OR, odds ratio; CI confidence interval. Covariates are adjusted for HTA, diabetes, dyslipidemia, and smoking. a Haplotype T-G-4b (H1) is chosen to be the reference haplotype.
*
[0.81–2.58] [0.65–1.73] [0.71–1.72] [0.82–2.99] [0.53–1.22] [0.93–53.88]
[0.77–3.16] [0.76–2.37] [0.70–1.87] [0.68–2.84] [0.49–1.36] [1.80–80.54]
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polymorphism was associated with MI in Tunisian patients. This association persisted after adjusting for several potential confounding factors. The 4a allele frequency of the NOS3 gene 4a4b polymorphism was significantly higher in patients with MI than in controls. Several researchers have investigated the association between the NOS3 gene 4a4b polymorphism and CAD, with conflicting results; some studies showing an association, while others not. The relationship between 4a4a genotype and CAD was first described by Wang et al. in Australia [21]. The association was later confirmed in Japanese, in African Americans, in Italian and in Turkish population [33–36]. However, other investigators did not detect a link to CAD [12,15,37–40]. The mechanism by which the NOS34a4b polymorphism confers susceptibility to MI is not yet understood. The 4a4b polymorphism is associated with altered plasma NO levels, influencing both NO and enzyme production [41]. Several studies reported that lower NO plasma levels for 4a allele curriers in Asians and African–Americans but not in others [42,43] Furthermore, data from the literature reported that the variance of NO circulating levels accounted for 25% [44]. It has been demonstrated in a Korean population that the effect of the 4a4b polymorphism on the variance of plasma NO depends on smoking status [45]. Impaired NO function may contribute to the development of atherosclerosis by inhibiting vascular smooth muscle cells proliferation [42], platelet adhesion/aggregation and leukocyte and monocyte adhesion [46,47]. Moreover, NO induces tissue factor expression and the ability of lymphocyte to form coaggregates with platelets. The investigated variant is unlikely to be functional. However, it could be in linkage disequilibrium, with another variant lying elsewhere in the gene. It has been demonstrated by Wang et al. [48] that the 4a allele at intron 4 and the 786C variant at the promoter region which are in linkage disequilibrium, affect the transcription efficiency of the NOS3 gene in a haplotype-dependent fashion. Therefore, these polymorphisms (4a4b and 786T>C) may be functionally involved in increasing MI predisposition or could be a marker in linkage disequilibrium with relevant functional changes. For 894G>T polymorphism, no significant differences in allele frequency or genotype distribution were observed between MI patients and controls in this study. The relation between the 894G>T mutation and MI remains controversial. Our results are in agreement with previous studies in Europeans, Canadian, Australians and Japanese subjects [49,50–52], but at variance with others [6,53–55]. Concerning the 786T>C polymorphism, our data showed that this polymorphism was not associated with MI in Tunisian subjects. The genotype and the allele frequencies of the 786T>C variant were similar between MI and control subjects. This result is similar to previously described by others investigators from different populations [15,20,56–59], but is at variance with some previous studies, which demonstrated association between the 786T>C variant and CAD [18,11,60–63]. The discrepancy between studies could be not only of differences in ethnicity or different sample size, but also, most importantly, of different selection criteria adopted for patients and controls, in particular clinical presentation, extent of disease, age, geographical area, concomitant environmental risk factors and gene–gene and gene–environment interactions [62,63]. Some limitations of our work should be taken into consideration. First, this work was based on a limited number of patients and controls. Second, all patients enrolled in this study were men and coronary angiography was not performed in control subjects, who were without symptoms and without history of any form of vascular events. Finally, we did not measure eNOS activity or NO concentrations to prove the biological effect.
5. Conclusion In the present study, we present a simultaneous analysis of different NOS3 polymorphisms with regards to MI in the Tunisian population. Our finding suggest that 4a4b but not 786T>C, and 894G>T polymorphisms is associated with the risk of MI in Tunisian male population. 5. Competing interests The authors declare that they have no competing interests. No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subjects of this article. Acknowledgment This work was supported by a Grant from the ‘‘Ministry of Higher Education and Scientific Research’’ of Tunisia. References [1] Lusis AJ, Fogelman AM, Fonarow CC. Genetic basis of atherosclerosis: part I. New genes and pathways. Circulation 2004;110:1868–73. [2] Kugiyama K, Yasue H, Okumura K, et al. Nitric oxide activity is deficient in spasm arteries of patients with coronary spastic angina. Circulation 1996;94:266–72. [3] Cooke JP, Dzau VJ. Nitric oxide synthase: role in the genesis of vascular disease. Annu Rev Med 1997;48:489–509. [4] Hingorani AD. Polymorphisms in endothelial nitric oxide synthase and atherogenesis: John French Lecture 2000. Atherosclerosis 2001;154:521–7. [5] Marsden P, Heng HH, Scherer SW, et al. Structure and chromosomal localization of the human constitutive endothelial nitric oxide synthase gene. J Biol Chem 1993;268:17478–88. [6] Hingorani AD, Liang CF, Fatibene J, et al. A common variant of the endothelial nitric oxide synthase (Glu298-Asp) is a major risk factor for coronary artery disease in the UK. Circulation 1999;100:1515–20. [7] Colombo MG, Andreassi MG, Paradossi U, et al. Evidence for association of a common variant of the endothelial nitric oxide synthase gene (Glu298Asp polymorphism) to the presence, extent, and severity of coronary artery disease. Heart 2002;87:525–8. [8] McNamara DM, Holubkov R, Prostava L, et al. Effect of the ASP298 variant of endothelial nitric oxide synthase on survival for patients with congestive heart failure. Circulation 2003;107:1598–602. [9] Rios DL, D’Onofrio LO, Souza JK, et al. Smoking-dependent and haplotypespecific effects of endothelial nitric oxide synthase gene polymorphisms on angiographically assessed coronary artery disease in Caucasian- and AfricanBrazilians. Atherosclerosis 2007;193:135–41. [10] Morray B, Goldenberg I, Moss AJ, et al. Polymorphisms in the paraoxonase and endothelial nitric oxide synthase genes and the risk of early-onset myocardial infarction. Am J Cardiol 2007;99:1100–5. [11] Berdili A, Sekuri C, Cam S, et al. Association between the eNOS (Glu298Asp) and the RAS genes polymorphisms and premature coronary artery disease in a Turkish population. Clin Chim Acta 2005;351:87–94. [12] Via M, Lopez-Alomar A, Valveny N, et al. Lack of association between Enos gene polymorphism and ischemic heart disease in the Spanish population. Am J Med Genet 2003;116A:243–8. [13] Schmoelzer I, Renner W, Paulweber B, et al. Lack of association of the Glu298Asp polymorphism of endothelial nitric oxide synthase with manifest coronary artery disease, carotid atherosclerosis and reactivity in two Austrian populations. Eur J Clin Invest 2003;33:191–8. [14] Jaramillo PC, Munoz MA, Lanas MC, Lana ZF, Salazar LA. Endothelial nitric oxide synthase G894T gene polymorphism in Chilean subjects with coronary artery disease and controls. Clin Chim Acta 2006;371:102–6. [15] Jaramillo PC, Lanas C, Lanas F, Salazar LA. Polymorphisms of the NOS3 gene in Southern Chilean subjects with coronary artery disease and controls. Clin Chim Acta 2010;411:258–62. [16] Jemaa R, Kallel A, Sediri Y, et al. Association between 786TC polymorphism in the endothelial nitric oxide synthase gene and hypertension in the Tunisian population. Exp Mol Pathol 2011;90:210–4. [17] Yoshimura M, Yasue H, Nakayama M, et al. Genetic risk factors for coronary artery spasm: significance of endothelial nitric oxide synthase gene T-786-C and missense Glu298Asp variants. J Invest Med 2000;48:367–74. [18] Nakayama M, Yasue H, Yochimura M, et al. T(-786)C mutation in the 5’flanking region of the endothelial nitric oxide synthase gene is associated with myocardial infarction, especially without coronary organic stenosis. Am J Cardiol 2000;86:628–34.
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Polymorphisms of the NOS3 gene and risk of myocardial infarction in the Tunisian population q Amani Kallel a, Mohamed Hédi Sbaï a, Yousra Sediri a, Salem Abdessalem b, Mohamed Sami Mourali b, Moncef Feki a, Rachid Mechmeche b, Riadh Jemaa a,⇑, Naziha Kaabachi a a b
Université de Tunis El Manar, Hôpital la Rabta, Service de Biochimie, LR99ES11, 1007 Tunis, Tunisie Université de Tunis El Manar, Hôpital la Rabta, service des Explorations Fonctionnelles et de Reanimations en Cardiologie, Hospital la Rabta, Tunis, Tunisie
a r t i c l e
i n f o
Article history: Received 8 February 2013 Received in revised form 29 July 2013 Accepted 4 September 2013 Available online 3 October 2013 Keywords: NOS3 gene polymorphisms Myocardial infarction Haplotype Risk factors
a b s t r a c t Controversial results regarding the association of eNOS gene (NOS3) polymorphisms with myocardial infarction (MI) have been reported. This study investigated the relationship of the 786T>C (rs2070744), 894G>T (rs1799983) and 4a4b polymorphisms of the NOS3 gene with the presence of MI in the Tunisian population. In addition, we also examined the association of NOS3 gene haplotypes with MI in Tunisian subjects. A total of 303 patients with MI and 225 controls were included in the study. The 894G>T and 786T>C single nucleotide polymorphisms were analyzed by PCR-RFLP, and 4a4b polymorphism just for PCR. There was significant linkage disequilibrium between the three NOS3 polymorphisms (p < 0.0001). The genotype distribution and allele frequency of NOS3 4a4b, but not 786T>C and 894G>T, polymorphism was significantly different between MI patients and controls. The univariate logistic regression analysis showed a significant association of the 4a4b polymorphism and MI according to co-dominant, dominant and recessive models (co-dominant model OR: 4.38, 95%CI: 1.24–15.41; p = 0.021, dominant model OR: 1.66, 95%CI: 1.14–2.42); p = 0.007, and recessive model OR: 3.85, 95%CI: 1.10–13.47; p = 0.035). The multivariate analysis, adjusted for traditional cardiovascular risk factors, revealed that the NOS3 4a4a genotype was an independent predisposing factor to MI, according to the models considered. In addition, a haplotype 7 (C-T-4a), (OR = 12.05, p = 0.010) was a risk factor of MI after controlling for classical risk factors. These finding suggest that the 4a4b polymorphism of the NOS3 gene was associated with MI in Tunisian patients. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Coronary artery disease (CAD) is one of the leading causes of morbidity and mortality in much of the world today. CAD is a complex condition, resulting from genetic and environmental contributions [1]. There is strong experimental and clinical evidence that abnormalities in nitric oxide (NO) availability play an important role in the pathophysiology of CAD. This is consistent with the notion that NO has a major role in the maintenance of endothelial function, causes vasodilatation, prevents platelet and leucocyte
q This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ⇑ Corresponding author. Address: Laboratoire de Biochimie, Hôpital la Rabta, 1007 Jabbari, Tunis, Tunisia. Tel.: +216 71 561 912/98 819 168; fax: +216 71 561 912. E-mail address: [email protected] (R. Jemaa).
1043-4666/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cyto.2013.09.005
adhesion, and inhibits vascular smooth muscle cell proliferation and migration [2], and oxidation of atherogenic low-density lipoprotein [3]. The advances in our understanding of NO biology and clinical relevance have lead to a number of studies investigating whether polymorphisms in the gene encoding the enzyme that synthesizes NO in endothelial cells (endothelial nitric oxide synthase, eNOS) affect NO availability and are associated with CAD [4]. The eNOS gene (NOS3) was cloned and sequenced in 1993 and mapped to chromosome 7q35-36. Spanning 21 kb of genomic DNA, the gene comprises 26 exons that encode a 135-kDa protein containing 1203 aminoacids [5]. Several polymorphisms have been identified in the NOS3 gene. Among them, a common variant located in exon 7 (894G>T) of the NOS3 gene that modifies its coding sequence (Glu298-Asp) has been linked by several groups to the risk for CAD, stroke, and myocardial infarction [6–11], however other studies failed to find any relationship between this variant and atherosclerosis [12–15]. Another genetic variant, a single nucleotide polymorphism (SNP), resulting from a thymine being replaced by a cytosine at nucleotide 786, has been
A. Kallel et al. / Cytokine 64 (2013) 646–651
identified in the promoter region of the NOS3 gene (786T>C) and speculated to influence mRNA transcription and reduce eNOS gene expression. The 786T>C variant has been associated with hypertension [16], coronary spasm [17], and myocardial infarction [18,19], however similar to the 894G>T variant inconsistent associations of the 786T>C polymorphism with CAD, were also shown [15,20]. Finally, a 27-base pair (bp) repeat polymorphism in intron 4 of the NOS3 gene has been linked by several groups to the risk for CAD, and MI in some [21–23] but not all studies [15,24]. Ethnic background is known to influence polymorphism frequencies and their effects on the disease. The great majority of NOS3 studies were done on European or North American Caucasians and few studies have addressed the NOS3 effects in Arabs or their descendents. The aim of the present study was to investigate the possible association between the 894G>T, 786T>C, and intron 4a4b polymorphisms of the NO3 gene and haplotypes and the risk of MI in a sample of the Tunisian male population.
2. Materials and methods 2.1. Study population A total of 528 unrelated Tunisian subjects were included into this case-control study, of which 303 were MI cases and 225 healthy controls. MI patients were enrolled from the Department of Cardiology, Rabta University Hospital of Tunis. The mean age of this group was 54 years (SD 8). Diagnosis of MI was confirmed according to the European Society of Cardiology criteria; a typical rise and fall of serum creatine kinase-MB isoenzyme (CK-MB), with at least one of the following criteria: ischemic symptoms, development of pathologic Q waves on the ECG, ECG changes indicative of ischemia (ST segment elevation or depression). The control group included 225 apparently healthy male subject volunteers, with no history of angina pectoris or MI, and with a normal electrocardiogram. Their mean age was 52 years (SD 8). Controls with familial history CAD and taking medications determined by interviewing, were excluded from the study. Patients and controls were homogeneous Tunisian Arab descendents who resided in Tunisia and all were from North Tunisia. Informed written consent was obtained from all participants and the design of the study was approved by the local ethics committee. Weight and height were measured on the subjects barefooted and lightly clothed. Body mass index (BMI; kg/m2) was calculated and obesity was defined as BMI >30 kg/m2 [25]. Diabetes mellitus was defined as hyperglycemia, requiring antidiabetic drugs or testing blood glucose over 7.0 mmol/L. Hypertension was defined as systolic blood pressure P140 mmHg and/or diastolic blood pressure P90 mmHg, or the use of antihypertensive drug treatment, or a combination of these. Dyslipidemia was defined as a total cholesterol (TC) level >6.47 mmol/L and/or triglyceride (TG) level >2.26 mmol/L. A smoker was defined as a current smoker or an ex-smoker.
2.2. Laboratory analysis Blood samples were obtained after an overnight fast. Plasma levels of TC, TG and HDL-cholesterol (HDL-C) were measured by standardized enzymatic procedures and apo AI and apo B were measured using turbidimetric assay using commercial kits (Roche Diagnostics, Mannheim, Germany) on a Hitachi 912 analyzer. LDL cholesterol (LDL-C) was calculated using the Friedewald equation when the triglyceride concentrations did not exceed 4.8 mmol/L [26].
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2.3. Genetic analysis Genomic DNA was extracted from ethylenediaminetetraacetate anticoagulated whole blood using conventional salting-out procedure [27]. NOS3 894G>T and 786T>C genotype analysis was performed by PCR-RFLP analysis, using MboI and MspI digestion, respectively. For the 894G>T polymorphism (rs1799983), DNA was amplified using the following primers 50 -CATGAGGCT CAGCCCCAGAAC-30 (forward) and 50 -AGTCAATCCCTTTGGTGCTC AAC-30 (reverse). DNA was amplified for 30 cycles, each cycle comprising denaturation at 95 °C for 1 min, annealing at 60 °C for 1 min, extension at 70 °C for 1 min with final extension time of 5 min at 70 °C. The initial denaturation stage was carried out at 95 °C for 5 min. PCR products were digested with the restriction enzyme MboI at 37 °C for 16 h and separated on 2.5% agarose gel an d identified by ethidium-bromide staining. The G allele visualized as a 206-bp band, while the T allele was visualized as 119and 87-bp fragments. Genotypes for the 786T>C polymorphism (rs2070744) in the 5-flanking region of NOS3 gene were determined by PCR amplification using the primers 50 -TGG AGA GTG CTG GTG TAC CCC A-30 (sense) and 50 -GCC TCC ACC CCC ACC CTG TC-30 (antisense). The amplified products were digested overnight at 37 °C with 5U of MspI restriction enzyme, producing fragments of 140 and 40 bp for the wild-type allele (allele T), or 90, 50, and 40 bp in the case of a polymorphic variant (allele C). Fragments were separated by electrophoresis on 2.5% agarose gels with ethidium bromide staining. The genotyping of the NOS3 4a4b polymorphism was determined using the method of Wang et al. [21] with minor modifications. Primer pairs for PCR were: sense, 50 AGG CCC TAT GGT AGT GCC TTT-30 ; and antisense, 50 -TCT CTT AGT GCT GTG GTC AC-30 . The amplified fragments were separated on 2% agarose gels with ethidium bromide staining. A 420-bp band indicated five repeats of the 27-bp (NOS34b allele) and a 393-bp band four repeats (NOS34a allele). An NOS34b allele was the common allele. 2.4. Statistical analysis Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS 10.0 for Windows; SPSS Inc., Chicago, IL, USA). Distributions of continuous variables in groups were expressed as mean ± SD, and compared with unpaired Student’s t-tests. Agreement of genotype frequencies with Hardy–Weinberg equilibrium expectations was tested using v2 goodness-of-fit test with one degree of freedom. The NOS3 gene polymorphisms was first assessed in three genotype categories (wild-type, heterozygote, and homozygote variants) and then grouped into two categories with heterozygotes and homozygote variants combined because of the dominant and the recessive models of inheritance observed for these polymorphisms. Odds ratio (OR) and 95% confidence intervals (CI) were calculated using unconditional logistic regression. Crude and adjusted models for diabetes, dyslipidemia, hypertension, smoking and genotypes were fitted. The coefficient of determination (R2) was also calculated. The degree of linkage disequilibrium (LD) between polymorphism was assessed using the Thesias software (http://genecanva.ecgene.net). Logistic regression analysis models were used to evaluate the haplotypes as possible risk factors. The differences were considered significant if P value was less than 0.05. 3. Results Table 1 summarizes the clinical and biochemical data of MI patients and the control subjects. There are no differences in the mean age, and BMI between the two groups, while the prevalence of hypertension, diabetes mellitus, dyslipidemia and smoking were
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Table 1 Demographic and clinical characteristics of the study population. Variables
Controls (n = 225)
Patients (n = 303)
P
Age (years) BMI (kg/m2) Diabetes (%) Hypertension (%) Obesity (%) Dyslipidemia (%) Smoking (%) TC (mmol/L) TG (mmol/L) LDL-C (mmol/L) HDL-C (mmol/L)
52.8 + ± 8.6 25.9 ± 4.5 13.1 17.1 21.2 15.6 68.4 4.86 ± 0.980 1.57 ± 1.00 2.95 ± 0.82 1.16 ± 0.31
54.1 ± 8.7 25.3 ± 3.5 38.7 31.7 10.1 29.4 94.3 5.05 ± 1.13 1.89 ± 0.81 3.34 ± 1.01 0.85 ± 0.23
0.079 0.065 <0.001 <0 .001 <0.001 <0.001 <0.001 0.055 <0.001 <0.001 <0.001
Data are expressed as means ± SD for some and as percent in others variables.
significantly higher in patients group than in the control group (p < 0.001). The MI group showed higher concentrations of triglycerides, LDL-cholesterol and Apo B, and lower concentrations of HDL – cholesterol and ApoAI compared with the control group. Three types of polymorphism in the NOS3 gene, 894G>T, 786T>C, and 4a4b, were analyzed and their genotype distributions and allele frequencies in MI patients and controls are shown in Tables 2–4. The distribution of the NOS3 genotypes and allele frequencies in MI and control groups were compatible with Hardy–Weinberg equilibrium (all p > 0.05). For the 4a4b polymorphism a significant difference in both genotype distribution and allele frequency between MI patients and controls subjects was observed. The univariate logistic regression analysis showed a significant association of the 4a4b polymorphism and MI according to co-dominant, dominant and recessive models (co-dominant model OR: 4.38, 95%CI: 1.24–15.41; p = 0.021, dominant model OR: 1.66, 95%CI: 1.14–2.42); p = 0.007, and recessive model OR: 3.85, 95%CI: 1.10–13.47; p = 0.035) (Table 2). The multivariate analysis, adjusted for traditional cardiovascular risk factors (diabetes, hypertension, dyslipidemia, and smoking), revealed that the NOS3 4a4a genotype was an independent predisposing factor to MI, according to the models considered (codominant model: R2 = 0.217; p < 0.001; dominant model: R2 = 0.211; p < 0.001 and recessive model: R2 = 0.17; p < 0.001) (Table 1). The MI patient group showed a significant higher frequency of the 4a allele compared to the controls (21% vs. 13%; OR: 1.76, 95%CI: 1.25–2.49, p = 0.0007). No significant difference in NOS3-786T>C and 894G>T polymorphisms genotype distribution and allele frequency was observed between patients and controls. Neither in the co-dominant, nor
in the dominant and recessive models was statistically significant differences found in the distribution of genotypes frequencies between MI patients and controls. MI risk for each of these models was also estimated. Neither in the crude estimations, nor in those adjusted for diabetes, dyslipidemia, hypertension and smoking was a higher MI risk with variant allele found for these polymorphisms (Tables 3 and 4). Linkage disequilibrium analysis (Table 5), as defined by the delta (D0 ) was determined for the three NOS3 SNPs. Linkage disequilibrium was seen for 786T>C and 894G>T (D0 = 0.479, p < 0.0001), 786T>C and 4a4b (D0 = 0.374, p = 0.0002), and 894G>T and 4a4b (D0 = 0.709, p < 0.0001) among patients, and between 786T>C and 894G>T (D0 = 0.475, p < 0.0001), 786T>C and 4a4b (D0 = 0.589, p = 0.0002), and 894G>T and 4a4b (D0 = 0.939, p < 0.0001) among controls. Table 6 shows the results of the haplotype analyses. We did not observe any haplotype which might significantly increase or decrease the risk of MI. Multiple logistic regression analyses were performed to determine independent of MI risk. The analyses revealed that the haplotype 7 (C-T-4a), (OR = 12.05, p = 0.010) was a risk factor of MI after controlling for classical risk factors.
4. Discussion CAD is a multifactorial disease in which genetic and environmental factors play a great role. Associations of DNA markers at the candidate genes for CAD such NOS3 have been found previously in different populations. This phenomenon seems to be population specific as a result of variations in different subsets of genetic and environmental factors predisposing individuals to CAD from different populations and inter-population differences in genetic background. Additionally, there is strong experimental and clinical evidence that abnormalities in nitric oxide (NO) availability play an important role in the pathophysiology of hypertension [28] and diabetic vascular diseases [29], but studies on the association of NOS3 gene polymorphisms with hypertension and diabetes in different populations have yielding conflicting results, with some studies providing evidence for an association [16,30,31] while others have found none [21,32]. In this work, we have undertaken a case-control study to investigate the role of the 894G>T, 786T>C and 4a4b polymorphisms at the NOS3 gene in susceptibility to MI in Tunisians. Both patients with MI and healthy controls belonged to the same ethnic background and all shared a common geographic origin in North Tunisia. Our data showed that the NOS3 gene intron 4a4b
Table 2 Genotype distribution and relative allele frequencies of 4a4b polymorphism at the NOS3 gene among patients and controls and crude and adjusted estimations for MI. Genotypes
MI patients (n = 303)
Controls (n = 225)
Unadjusteda OR [95% CI]
P-valuea
Adjustedb OR [95% CI]
P-valueb
Codominant model 4b4b n (%) 4a4b n (%) 4a4a n (%)
187 (61.7) 101 (33.3) 15 (5.0)
164 (72.9) 58 (25.8) 3 (1.3)
1 1.52 (1.03–2.24) 4.38 (1.24–15.41)
0.031 0.021
1 1.59 (1.02–2.49) 4.66 (1.19–18.28)
0.039 0.027
Dominant model 4b4b n (%) 4a4b + 4a4a n (%)
187 (61.7) 116 (38.3 %)
164 (72.9) 61 (27.1 %)
1.66 (1.14–2.42)
0.007
1 1.75 (1.14–2.71)
0.011
Recessive model 4a4a n (%) 4a4b + 4b4b n (%)
15 (5.0) 288 (95.0)
3 (1.30) 222 (98.7)
3.85 (1.10–13.47)
0.035
1 4.03 (1.03–15.68)
0.044
Allele frequency 4b (%) 4a (%)
79 21
87 13
1.76 (1.25–2.49)
0.0007
MI, myocardial infarction; OR, odds ratio; CI, Confidence interval. a Crude logistic regression model. b Adjusted for diabetes, hypertension, dyslipidemia and smoking.
649
A. Kallel et al. / Cytokine 64 (2013) 646–651 Table 3 Genotype distribution and relative allele frequencies of 894G>T polymorphism at the NOS3 gene among patients and controls and crude and adjusted estimations for MI. Genotypes
MI patients (n = 303)
Controls (n = 225)
Unadjusteda OR [95% CI]
P-valuea
Adjustedb OR [95% CI]
P-valueb
Codominant model GG n (%) GT n (%) TT n (%)
163 (53.8) 122 (40.3) 18 (5.9)
128 (56.9) 78 (34.7) 19 (8.4)
1.22 (0.85–1.77) 0.74 (0.37–1.47)
0.272 0.397
1 1.28 (0.83–1.97) 1.29 (0.57–2.94)
0.251 0.530
Dominant model GG n (%) GT + TT n (%)
163 (55.8) 140 (46.2)
128 (56.9) 97 (43.1)
1.13 (0.80–1.60)
0.480
1 1.28 (0.85–1.93)
0.225
Recessive model TT GT + GG
18 (5.9) 285 (94.1)
19 (8.4) 206 (91.6)
0.68 (0.35–1.33)
0.267
1 1.17 (0.52–2.60)
0.695
Allele frequency G (%) T (%)
84 26
85 25
1.02 (0.76–1.35)
0.913
MI: myocardial infarction; OR, odds ratio; CI, Confidence interval. a Crude logistic regression model. b Adjusted for age, diabetes, hypertension, dyslipidemia and smoking.
Table 4 Genotype distribution and relative allele frequencies of 786T>C polymorphism at the NOS3 gene among patients and controls and crude and adjusted estimations for MI. Genotypes
MI patients (n = 303)
Controls (n = 225)
Unadjusteda OR [95% CI]
P-valuea
Adjustedb OR [95% CI]
P-valueb
Codominant model TT n (%) CT n (%) CC n (%)
120 (39.6) 144 (47.5) 39 (12.9)
96 (42.7) 102 (45.3) 27 (12.0)
1.12 (0.78–1.63) 1.15 (0.66–2.02)
0.518 0.612
1 1.16 (0.76–1.78) 1.21 (0.63–2.32)
0.476 0.564
Dominant model TT n (%) CT + CC n (%)
120 (39.6) 183 (60.4)
96 (42.7) 129 (57.3)
1.13 (0.79–1.61)
0.479
1.17 (0.78–1.76)
0.429
Recessive model CC n (%) TT + CT n (%)
39 (12.9) 264 (87.1)
27 (12.0) 198 (88.0)
1.08 (0.64–1.83)
0.765
1.11 (0.60–2.06)
0.720
Allele frequency T(%) C (%)
63 37
65 35
1.09 (0.84–1.42)
0.509
MI: myocardial infarction; OR, odds ratio; CI, Confidence interval. a Crude logistic regression model. b Adjusted for age, diabetes, hypertension, dyslipidemia and smoking.
Table 5 The estimated linkage disequilibrium D’ values for each pairwise combination of the 786T>C, 4a4b and 894G>T polymorphisms among patients and controls. Patients
786T>C and 894G>T 786T>C and intron 4a4b 894G>T and intron 4a4b
Controls
D’
r
P
D’
r
P
0.479 0.374 0.709
0.153 0.224 0.050
<0.0001 0.0002 <0.0001
0.475 0.589 0.939
0.151 0.299 0.050
<0.0001 0.0002 <0.0001
Table 6 Haplotype frequencies in patients and controls. Variable
Haplotypes
Patients
Controls
Unadjusted OR [95% CI]
p
Adjusted OR [95% CI]
P*
H1a
111 112 121 211 212 221 222
0.4254 0.0979 0.1060 0.1426 0.0797 0.1079 0.0359
0.4613 0.0743 0.1094 0.1420 0.0558 0.1493 0.0060
– 1.45 1.06 1.11 1.56 0.81 7.11
– 0.207 0.786 0.629 0.171 0.326 0.057
– 1.56 1.34 1.14 1.40 0.82 12.05
– 0.208 0.305 0.581 0.352 0.450 0.010
H2 H3 H4 H5 H6 H7
OR, odds ratio; CI confidence interval. Covariates are adjusted for HTA, diabetes, dyslipidemia, and smoking. a Haplotype T-G-4b (H1) is chosen to be the reference haplotype.
*
[0.81–2.58] [0.65–1.73] [0.71–1.72] [0.82–2.99] [0.53–1.22] [0.93–53.88]
[0.77–3.16] [0.76–2.37] [0.70–1.87] [0.68–2.84] [0.49–1.36] [1.80–80.54]
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A. Kallel et al. / Cytokine 64 (2013) 646–651
polymorphism was associated with MI in Tunisian patients. This association persisted after adjusting for several potential confounding factors. The 4a allele frequency of the NOS3 gene 4a4b polymorphism was significantly higher in patients with MI than in controls. Several researchers have investigated the association between the NOS3 gene 4a4b polymorphism and CAD, with conflicting results; some studies showing an association, while others not. The relationship between 4a4a genotype and CAD was first described by Wang et al. in Australia [21]. The association was later confirmed in Japanese, in African Americans, in Italian and in Turkish population [33–36]. However, other investigators did not detect a link to CAD [12,15,37–40]. The mechanism by which the NOS34a4b polymorphism confers susceptibility to MI is not yet understood. The 4a4b polymorphism is associated with altered plasma NO levels, influencing both NO and enzyme production [41]. Several studies reported that lower NO plasma levels for 4a allele curriers in Asians and African–Americans but not in others [42,43] Furthermore, data from the literature reported that the variance of NO circulating levels accounted for 25% [44]. It has been demonstrated in a Korean population that the effect of the 4a4b polymorphism on the variance of plasma NO depends on smoking status [45]. Impaired NO function may contribute to the development of atherosclerosis by inhibiting vascular smooth muscle cells proliferation [42], platelet adhesion/aggregation and leukocyte and monocyte adhesion [46,47]. Moreover, NO induces tissue factor expression and the ability of lymphocyte to form coaggregates with platelets. The investigated variant is unlikely to be functional. However, it could be in linkage disequilibrium, with another variant lying elsewhere in the gene. It has been demonstrated by Wang et al. [48] that the 4a allele at intron 4 and the 786C variant at the promoter region which are in linkage disequilibrium, affect the transcription efficiency of the NOS3 gene in a haplotype-dependent fashion. Therefore, these polymorphisms (4a4b and 786T>C) may be functionally involved in increasing MI predisposition or could be a marker in linkage disequilibrium with relevant functional changes. For 894G>T polymorphism, no significant differences in allele frequency or genotype distribution were observed between MI patients and controls in this study. The relation between the 894G>T mutation and MI remains controversial. Our results are in agreement with previous studies in Europeans, Canadian, Australians and Japanese subjects [49,50–52], but at variance with others [6,53–55]. Concerning the 786T>C polymorphism, our data showed that this polymorphism was not associated with MI in Tunisian subjects. The genotype and the allele frequencies of the 786T>C variant were similar between MI and control subjects. This result is similar to previously described by others investigators from different populations [15,20,56–59], but is at variance with some previous studies, which demonstrated association between the 786T>C variant and CAD [18,11,60–63]. The discrepancy between studies could be not only of differences in ethnicity or different sample size, but also, most importantly, of different selection criteria adopted for patients and controls, in particular clinical presentation, extent of disease, age, geographical area, concomitant environmental risk factors and gene–gene and gene–environment interactions [62,63]. Some limitations of our work should be taken into consideration. First, this work was based on a limited number of patients and controls. Second, all patients enrolled in this study were men and coronary angiography was not performed in control subjects, who were without symptoms and without history of any form of vascular events. Finally, we did not measure eNOS activity or NO concentrations to prove the biological effect.
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