GENETIC TESTING AND MOLECULAR BIOMARKERS Volume 19, Number 4, 2015 ª Mary Ann Liebert, Inc. Pp. 1–6 DOI: 10.1089/gtmb.2014.0261
ORIGINAL ARTICLE
Contribution of eNOS Variants to the Genetic Susceptibility of Coronary Artery Disease in a Tunisian Population Marwa Ben Ali, Safia Messaoudi, Houda Ezzine, and Touhami Mahjoub
Nitric oxide (NO), produced by the enzyme endothelial nitric oxide synthase (eNOS), has critical roles in the regulation of vascular homeostasis and prevention of atherogenesis by inhibiting leukocytes, platelet activation, and smooth muscle cell proliferation. There is strong experimental and clinical evidence that abnormalities in eNOS availability play an important role in the pathophysiology of coronary artery disease (CAD). Controversial results regarding the association of eNOS gene polymorphisms with CAD have been reported. The aim of this study is to investigate the relationship of the 894G > T (rs1799983) and 4a/4b (rs61722009) polymorphisms of the eNOS gene with the presence of CAD in the Tunisian population. A total of 332 patients with CAD and 368 controls were included in this study. The 894G > T (rs1799983) single-nucleotide polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism, and 4a/4b (rs61722009) polymorphism just by polymerase chain reaction (PCR). eNOS rs1799983 was significantly associated with CAD under the additive, dominant, but not recessive, models (additive model OR: 2.81; 95% CI [2.05–3.85]; p < 0.001, dominant model OR: 2.84; 95% CI [2.09–3.86]; p < 0.001, and recessive models p = 0.09). This remained significant after adjustment for age, gender, diabetes, smoking, and hypertension. In contrast to eNOS rs1799983, eNOS rs61722009 was not associated with CAD under any of the genetic models tested. These findings suggest that the G894T (rs1799983) polymorphism of the eNOS gene was associated with CAD in Tunisian patients.
taining blood flow and vascular integrity (Draijer et al., 1995; White et al., 1998), vasodilatation (White et al., 2000), and inhibiting smooth muscle proliferation (Garg and Hassid, 1989). Moreover, it has been shown that abnormalities in the endothelial NO pathway are present in humans with atherosclerosis and that eNOS inhibition accelerates atherosclerosis in animal models (Cayatte et al., 1994). Thus, deficiency in NO production has an important role in the pathogenesis of several cardiovascular diseases. Many polymorphisms have been reported as susceptibility genes in various cardiovascular diseases (Marsden et al., 1993). The most studied polymorphisms are eNOS gene intron 4a/4b VNTR (rs61722009) and eNOS 894G > T (rs1799983) polymorphisms (Ichihara et al., 1998; Hingorani et al., 1999). Since eNOS expression is genetically determined, previous studies on the association of the eNOS rs1799983 and rs61722009 with CAD were explored, but with inconclusive findings (Cam et al., 2005; Kerkeni et al., 2006; Yeqing et al., 2012; Abdel Aziz et al., 2013; Ekmekci et al., 2013). Based on the previous data, it has been hypothesized that impaired NO production due to eNOS 894G > T (rs1799983)
Introduction
C
oronary artery disease (CAD) is one of the major causes of mortality and is responsible for *30% of deaths worldwide (Cam et al., 2005; Jaramillo et al., 2006; Kim et al., 2007). The underlying pathogenesis of CAD involves the interaction of either genetic or environmental factors (Salimi et al., 2006). Endothelial dysfunction is an early event in the development of atherosclerosis and a subclinical condition found in most patients with CAD (Brunner et al., 2005; Versari et al., 2009). The main biological feature of endothelial dysfunction is an impaired nitric oxide (NO) bioavailability as a consequence of reduced production by endothelial nitric oxide synthase (eNOS). Nitric oxide, an important endotheliumderived relaxing factor, is synthesized from l-arginine by at least three isoforms of the NOS (inducible NOS, constitutive neuronal NOS, constitutive eNOS) (Palmer, 1993; Gardemann et al., 2002). Several lines of evidence support a key role for NO in the development of CAD and its complications, including main-
Laboratory of Human Genome and Multifactorial Diseases (LR12ES07), Faculty of Pharmacy of Monastir, University of Monastir, Monastir, Tunisia.
1
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BEN ALI ET AL.
polymorphism and eNOS 4a/4b (rs61722009) polymorphism may contribute greatly to the pathogenesis of CAD. The aim of the present case–control study was to investigate the association between 894G > T and 4a/4b eNOS polymorphisms and CAD risk in a population sample of 332 Tunisian patients with CAD and 368 gender- and age-matched healthy subjects. Subjects and Methods Subjects
This retrospective case–control study included 332 CAD patients (231 males and 101 females, mean age 58.17 – 11.75 years). CAD was defined as 50% stenosis in the left main coronary artery (Austen et al., 1975). Diagnosis of myocardial infarction (MI) was confirmed as per the World Health Organization (WHO) criteria (WHO, 1979), which were based on diagnosis of chest pain lasting 20 min or longer and clinical symptoms, elevation in cardiac enzymes (including CK-MB levels more than twice baseline levels) or ECG changes (ST segment elevation or pathologic Q waves). None of the study patients had evidence of significant atherosclerotic vascular diseases, autoimmune, cancer, renal, or hepatic illness. The control group included 368 healthy subjects (229 males and 139 females, mean age 56.54 – 14.60 years), whose coronary arteries were normal (by angiography), and/or when they presented with no history and no symptoms suggestive of CAD, and no electrocardiographic signs of MI. Patients and controls were homogeneous Tunisian Arab descendents who resided in Tunisia and all were from Central Tunisia. A complete clinical history, including cardiovascular risk factors such as hypertension, diabetes mellitus, cigarette smoking, was taken from all participants (Table 1). Diabetes was diagnosed according to fasting blood glucose (WHO criteria [7 mmol/dL]) and/or the use of glucose-lowering drugs (oral hypoglycemic and insulin). Obesity was defined as body–mass index (BMI was calculated as weight/height2 [kg/m2]) of 30 or higher, and hypertension was defined as blood pressure (140/90 mmHg on two separate occasions
and/or the use of antihypertensive therapy). Blood pressure (right arm) was measured twice using a mercury sphygmomanometer with participants in the sitting position following a 5-min rest; the mean of two readings measured 1 min apart was used. LDL-cholesterol, HDL-cholesterol, and triglycerides were measured by routine methods using enzymatic colorimetric assays on different fully automated equipments. All participants were asked to sign a consent form indicating their willingness to participate in the study, and all institutional ethic requirements were met. Finally, the Ethics Committee of Sousse approved the present study. eNOS genotyping
Genomic DNA was extracted from peripheral blood leukocytes using the proteinase K/salting-out method. Genotype for the eNOS 894G > T (rs1799983) polymorphism was determined by PCR-reaction-restriction fragment length polymorphism analysis using specific oligonucleotide primers as follows: forward, 5¢-CATGAGGCTCAGCCCCAGAAC-3¢ and reverse, 5¢-AGTCAATCCCTTTGGTGCTCAC-3¢ (Ezzidi et al., 2008). DNA was amplified for 30 cycles, each cycle comprising denaturation at 95C for 1 min, annealing at 60C for 1 min, extension at 70C for 1 min with final extension time of 5 min at 70C. The initial denaturation stage was carried out at 95C for 5 min. Polymerase chain reaction (PCR) products were digested by MobI and were separated by a 2.5% agarose gel. The resulting 206-bp PCR product was cleaved into two smaller fragments of 119 and 87 bp in the presence of a T nucleotide at 894 (corresponding to Asp 298), but not in its absence. The fragments were visualized after ethidium bromide staining under UV light. The 4a/4b (rs61722009) polymorphism was detected by PCR using the following primers: forward 5¢-CTATGGTA GTGCCTTGGCTGGAGG-3¢ and reverse 5¢-ACCGCCC AGGGAACTCCGCT-3 (Ezzidi et al., 2008). The amplified fragments were separated on 2% agarose gels with ethidium bromide staining. PCR products comprised a 210-bp (4b) and 183-bp (4a) bands.
Table 1. Profile of Patients and Control Subjects Characteristics Gender M/F, n (%) Mean age (years; mean – SD) Mean BMI (kg/m2) Hypertension, n (%) Systolic BP (mmHg) Diastolic BP (mmHg) Diabetes, n (%) Smokers, n (%) Urea (mmol/L) Creatinine (mmol/L) Total cholesterol (mmol/L) Triglycerides (mmol/L) HDL-cholesterol (mmol/L) LDL-cholesterol (mmol/L)
Controls (n = 368)
Patients CAD (n = 332)
pa
229/139 (62.2/37.8) 56.54 – 14.60 27.81 – 3.15 41 (11.4)b 11.63 – 2.96 7.51 – 1.60 17 (4.6) 43 (11.7) 4.96 – 1.73 73.93 – 20.78 4.72 – 0.96 1.70 – 0.98 1.43 – 0.72 1.72 – 0.92
231/101 (69.6/30.4) 58.17 – 11.75 26.98 – 4.34 178 (53.6) 13.26 – 2.89 7.67 – 1.69 174 (52.4) 175 (52.47) 8.01 – 5.97 111.27 – 69.18 4.65 – 1.95 1.88 – 1.70 1.11 – 0.62 2.92 – 1.12
0.53 0.13
ORIGINAL ARTICLE
Contribution of eNOS Variants to the Genetic Susceptibility of Coronary Artery Disease in a Tunisian Population Marwa Ben Ali, Safia Messaoudi, Houda Ezzine, and Touhami Mahjoub
Nitric oxide (NO), produced by the enzyme endothelial nitric oxide synthase (eNOS), has critical roles in the regulation of vascular homeostasis and prevention of atherogenesis by inhibiting leukocytes, platelet activation, and smooth muscle cell proliferation. There is strong experimental and clinical evidence that abnormalities in eNOS availability play an important role in the pathophysiology of coronary artery disease (CAD). Controversial results regarding the association of eNOS gene polymorphisms with CAD have been reported. The aim of this study is to investigate the relationship of the 894G > T (rs1799983) and 4a/4b (rs61722009) polymorphisms of the eNOS gene with the presence of CAD in the Tunisian population. A total of 332 patients with CAD and 368 controls were included in this study. The 894G > T (rs1799983) single-nucleotide polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism, and 4a/4b (rs61722009) polymorphism just by polymerase chain reaction (PCR). eNOS rs1799983 was significantly associated with CAD under the additive, dominant, but not recessive, models (additive model OR: 2.81; 95% CI [2.05–3.85]; p < 0.001, dominant model OR: 2.84; 95% CI [2.09–3.86]; p < 0.001, and recessive models p = 0.09). This remained significant after adjustment for age, gender, diabetes, smoking, and hypertension. In contrast to eNOS rs1799983, eNOS rs61722009 was not associated with CAD under any of the genetic models tested. These findings suggest that the G894T (rs1799983) polymorphism of the eNOS gene was associated with CAD in Tunisian patients.
taining blood flow and vascular integrity (Draijer et al., 1995; White et al., 1998), vasodilatation (White et al., 2000), and inhibiting smooth muscle proliferation (Garg and Hassid, 1989). Moreover, it has been shown that abnormalities in the endothelial NO pathway are present in humans with atherosclerosis and that eNOS inhibition accelerates atherosclerosis in animal models (Cayatte et al., 1994). Thus, deficiency in NO production has an important role in the pathogenesis of several cardiovascular diseases. Many polymorphisms have been reported as susceptibility genes in various cardiovascular diseases (Marsden et al., 1993). The most studied polymorphisms are eNOS gene intron 4a/4b VNTR (rs61722009) and eNOS 894G > T (rs1799983) polymorphisms (Ichihara et al., 1998; Hingorani et al., 1999). Since eNOS expression is genetically determined, previous studies on the association of the eNOS rs1799983 and rs61722009 with CAD were explored, but with inconclusive findings (Cam et al., 2005; Kerkeni et al., 2006; Yeqing et al., 2012; Abdel Aziz et al., 2013; Ekmekci et al., 2013). Based on the previous data, it has been hypothesized that impaired NO production due to eNOS 894G > T (rs1799983)
Introduction
C
oronary artery disease (CAD) is one of the major causes of mortality and is responsible for *30% of deaths worldwide (Cam et al., 2005; Jaramillo et al., 2006; Kim et al., 2007). The underlying pathogenesis of CAD involves the interaction of either genetic or environmental factors (Salimi et al., 2006). Endothelial dysfunction is an early event in the development of atherosclerosis and a subclinical condition found in most patients with CAD (Brunner et al., 2005; Versari et al., 2009). The main biological feature of endothelial dysfunction is an impaired nitric oxide (NO) bioavailability as a consequence of reduced production by endothelial nitric oxide synthase (eNOS). Nitric oxide, an important endotheliumderived relaxing factor, is synthesized from l-arginine by at least three isoforms of the NOS (inducible NOS, constitutive neuronal NOS, constitutive eNOS) (Palmer, 1993; Gardemann et al., 2002). Several lines of evidence support a key role for NO in the development of CAD and its complications, including main-
Laboratory of Human Genome and Multifactorial Diseases (LR12ES07), Faculty of Pharmacy of Monastir, University of Monastir, Monastir, Tunisia.
1
2
BEN ALI ET AL.
polymorphism and eNOS 4a/4b (rs61722009) polymorphism may contribute greatly to the pathogenesis of CAD. The aim of the present case–control study was to investigate the association between 894G > T and 4a/4b eNOS polymorphisms and CAD risk in a population sample of 332 Tunisian patients with CAD and 368 gender- and age-matched healthy subjects. Subjects and Methods Subjects
This retrospective case–control study included 332 CAD patients (231 males and 101 females, mean age 58.17 – 11.75 years). CAD was defined as 50% stenosis in the left main coronary artery (Austen et al., 1975). Diagnosis of myocardial infarction (MI) was confirmed as per the World Health Organization (WHO) criteria (WHO, 1979), which were based on diagnosis of chest pain lasting 20 min or longer and clinical symptoms, elevation in cardiac enzymes (including CK-MB levels more than twice baseline levels) or ECG changes (ST segment elevation or pathologic Q waves). None of the study patients had evidence of significant atherosclerotic vascular diseases, autoimmune, cancer, renal, or hepatic illness. The control group included 368 healthy subjects (229 males and 139 females, mean age 56.54 – 14.60 years), whose coronary arteries were normal (by angiography), and/or when they presented with no history and no symptoms suggestive of CAD, and no electrocardiographic signs of MI. Patients and controls were homogeneous Tunisian Arab descendents who resided in Tunisia and all were from Central Tunisia. A complete clinical history, including cardiovascular risk factors such as hypertension, diabetes mellitus, cigarette smoking, was taken from all participants (Table 1). Diabetes was diagnosed according to fasting blood glucose (WHO criteria [7 mmol/dL]) and/or the use of glucose-lowering drugs (oral hypoglycemic and insulin). Obesity was defined as body–mass index (BMI was calculated as weight/height2 [kg/m2]) of 30 or higher, and hypertension was defined as blood pressure (140/90 mmHg on two separate occasions
and/or the use of antihypertensive therapy). Blood pressure (right arm) was measured twice using a mercury sphygmomanometer with participants in the sitting position following a 5-min rest; the mean of two readings measured 1 min apart was used. LDL-cholesterol, HDL-cholesterol, and triglycerides were measured by routine methods using enzymatic colorimetric assays on different fully automated equipments. All participants were asked to sign a consent form indicating their willingness to participate in the study, and all institutional ethic requirements were met. Finally, the Ethics Committee of Sousse approved the present study. eNOS genotyping
Genomic DNA was extracted from peripheral blood leukocytes using the proteinase K/salting-out method. Genotype for the eNOS 894G > T (rs1799983) polymorphism was determined by PCR-reaction-restriction fragment length polymorphism analysis using specific oligonucleotide primers as follows: forward, 5¢-CATGAGGCTCAGCCCCAGAAC-3¢ and reverse, 5¢-AGTCAATCCCTTTGGTGCTCAC-3¢ (Ezzidi et al., 2008). DNA was amplified for 30 cycles, each cycle comprising denaturation at 95C for 1 min, annealing at 60C for 1 min, extension at 70C for 1 min with final extension time of 5 min at 70C. The initial denaturation stage was carried out at 95C for 5 min. Polymerase chain reaction (PCR) products were digested by MobI and were separated by a 2.5% agarose gel. The resulting 206-bp PCR product was cleaved into two smaller fragments of 119 and 87 bp in the presence of a T nucleotide at 894 (corresponding to Asp 298), but not in its absence. The fragments were visualized after ethidium bromide staining under UV light. The 4a/4b (rs61722009) polymorphism was detected by PCR using the following primers: forward 5¢-CTATGGTA GTGCCTTGGCTGGAGG-3¢ and reverse 5¢-ACCGCCC AGGGAACTCCGCT-3 (Ezzidi et al., 2008). The amplified fragments were separated on 2% agarose gels with ethidium bromide staining. PCR products comprised a 210-bp (4b) and 183-bp (4a) bands.
Table 1. Profile of Patients and Control Subjects Characteristics Gender M/F, n (%) Mean age (years; mean – SD) Mean BMI (kg/m2) Hypertension, n (%) Systolic BP (mmHg) Diastolic BP (mmHg) Diabetes, n (%) Smokers, n (%) Urea (mmol/L) Creatinine (mmol/L) Total cholesterol (mmol/L) Triglycerides (mmol/L) HDL-cholesterol (mmol/L) LDL-cholesterol (mmol/L)
Controls (n = 368)
Patients CAD (n = 332)
pa
229/139 (62.2/37.8) 56.54 – 14.60 27.81 – 3.15 41 (11.4)b 11.63 – 2.96 7.51 – 1.60 17 (4.6) 43 (11.7) 4.96 – 1.73 73.93 – 20.78 4.72 – 0.96 1.70 – 0.98 1.43 – 0.72 1.72 – 0.92
231/101 (69.6/30.4) 58.17 – 11.75 26.98 – 4.34 178 (53.6) 13.26 – 2.89 7.67 – 1.69 174 (52.4) 175 (52.47) 8.01 – 5.97 111.27 – 69.18 4.65 – 1.95 1.88 – 1.70 1.11 – 0.62 2.92 – 1.12
0.53 0.13
