Abstract
Microvascular abnormalities caused by endothelial dysfunction seem to be responsible for the myocardial ischemia in patients with cardiac syndrome X (CSX). Nitric oxide is a key mediator of endothelial function and is synthesized by endothelial nitric oxide synthase (eNOS). We investigated if the 3 potential polymorphisms of the eNOS gene (VNTR in intron 4, T786C polymorphism in the promoter region, and G894T polymorphism in exon 7) are independent risk factors for CSX. Sixty-nine patients with CSX and 73 healthy controls were studied. Genotypes were determined through polymerase chain reaction with or without restriction endonuclease digestions. Genotype distribution was significantly different between patients with CSX and controls for intron 4aa (allele for 4 repeats of 27 bp), intron 4aa genotype frequency being 3.2% and 6.8%, respectively. The presence of intron 4a is 3.2 (odds ratio) times protective (95% confidence interval, 1.5-6.8) for the risk of CSX disease. The protective effect of intron 4a polymorphism also holds after adjustment for age and sex and when the study group is limited to those without hypertension and hyperlipidemia. No significant difference was observed in genotype distribution of G894T and T786C polymorphism between patients with CSX and controls. In conclusion, intron 4aa genotype of eNOS gene is protective for CSX. No association was found between promoter and exon 7 polymorphisms of eNOS gene and CSX.
Cardiac syndrome X (CSX) is a clinical entity characterized by anginal-like chest pain without flow-limiting stenoses on coronary angiography and noncardiac causes of chest pain.1,2Although multiple mechanisms such as endothelial dysfunction, inflammation, estrogen deficiency, and increased pain sensitivity have been suggested to contribute to pathogenesis of CSX, the exact pathophysiological mechanisms involved in the picture of syndrome have not been fully elucidated.2-6However, recent evidences suggest microvascular ischemia secondary to endothelial dysfunction as a prominent pathophysiological explanation for CSX.2Impaired nitric oxide (NO) release and/or activity and eventual endothelial dysfunction may cause impaired vasodilatation and inappropriate vasoconstriction in the coronary microvasculature leading to ischemia, the presence of which has been demonstrated in many studies.7-12Nitric oxide is the most potent vasodilator factor produced by the endothelium. Besides its role in vasomotor tonus, it also displays important atheroprotective actions via its anti-inflammatory, antiproliferative, anticoagulant, and antioxidant properties.
Nitric oxide is synthesized enzymatically from l-arginine by a family of 3 NO synthases (NOS).13The endothelial NOS (eNOS or NOS3: chromosome 7q35-36) is found primarily in the endothelium and at low levels in platelets. The eNOS gene comprises 26 exons that span 21 kb. Several polymorphisms have been identified in the eNOS gene. Among them, the 3 polymorphisms stand out in reliefs, which are toward the 5' flanking region, a promoter (T786C) and a variable number of tandem repeats (VNTR) within intron 4 and a G894T in exon 7. Although the functional significances of these polymorphisms are not fully elucidated, numerous genetic association studies have been performed, exploring the association of these polymorphisms with clinical coronary artery disease, hypertension, stroke, and preeclampsia.14,15The polymorphisms of the eNOS gene could influence individual susceptibility to CSX by altering the amount of NO generated by the endothelium.16,17
The aim of this study was to present the genotype distributions of the 3 eNOS gene polymorphisms (VNTR in intron 4, T786C in the promoter region, and G894T in exon 7) and the interactions between them in an attempt to answer if those polymorphisms could play a role in microvascular ischemia in patients with CSX and be a marker for the syndrome. Interactions between polymorphisms on the same chromosome are important in determining disease risk, so we tested the multilocus haplotypes to overcome the problems caused by using single polymorphisms in genetic association studies.14
SUBJECTS AND METHODS
Study Population
We enrolled 69 patients with CSX who were admitted to Hacettepe University Cardiology Department with typical anginal-like chest pain. All patients had a history of effort angina, ST-segment depression associated with angina during at least 1 previous exercise stress test or ischemia on myocardial perfusion scintigraphy and angiographically normal epicardial coronary arteries. Mild hypertension was present in 33 patients, but left ventricular hypertrophy was excluded by echocardiography in all of them.
A group of 73 volunteers were served as control subjects. Control subjects did not have any symptomatic or clinically relevant disease, and they all had normal standard 12-lead electrocardiography (ECG), exercise stress test, and 2-dimensional Doppler echocardiography.
All patients were evaluated with a detailed medical story, physical examination, chest radiograph, 12 derivation ECG, complete blood count, and serum biochemistry. The presence of risk factors such as smoking, hypertension, diabetes, and hyperlipidemia was evaluated.
All study patients had undergone noninvasive assessment with treadmill exercise test or myocardial perfusion scintigraphy before coronary angiography. Ischemic changes such as exercise-induced 1-mm ST horizontal or down-sloping ST depressions or higher measured 80 milliseconds from the J point in at least 2 ECG leads on treadmill exercise test or reversible myocardial perfusion defects on myocardial perfusion scintigraphy had been observed in all patients who were recruited as patient group. Only patients whose coronary arteries were angiographically normal were enrolled in the study. Patients with coronary artery stenosis on subsequent coronary angiography were excluded from study.
Patients with diabetes, moderate to severe hypertension or uncontrolled hypertension, previous history of coronary artery disease, valvular heart disease, any kind of cardiomyopathies, and the ones with evidence of left ventricle hypertrophy on echocardiogram and suspected pericarditis were excluded from the study. Other exclusion criteria were chronic obstructive or interstitial pulmonary disease with pulmonary hypertension, chronic liver or renal disease, malignancy, collagen tissue disease, and hyperthyroidism or hypothyroidism.
The study was approved by the local research ethics committee, and written informed consent was obtained from all patients.
Molecular Analysis
Genomic DNA was isolated from the whole blood by using Nucleospin Blood Quick Pure Kit (Machenery-Nagel, Germany). Polymerase chain reactions (PCRs) were carried out for the 3 polymorphism of eNOS gene by using specific primers and conditions.18
Genotyping
VNTR in intron 4
Sense and antisense primers 5'- AGG CCC TAT GGT AGT GCC TTT-3' and 5'- TCT CTT AGT GCT GTG GTC AC-3' were used for PCR, respectively. Polymerase chain reaction was performed in a 40-μL reaction volume containing 400 ng of template DNA, 10 pmol/μL of each primer, 0.2 mmol/L of each dNTP, 4 μL of 10 × PCR buffer, 2.5 mmol/L of MgCl2, and 3 U of Taq DNA Polymerase (Fermentas Life Sciences, Leon-Rot, Germany). The PCR mixtures were heated to 94°C for 30 seconds for denaturation and underwent 35 cycles at 94°C for 30 seconds of denaturation, at 63°C for 30 seconds of annealing, and at 72°C for 1 minute of extension by using ICycler (BioRad, München, Germany). Finally, extension was conducted at 72°C for 5 minutes. The VNTR was determined by separating 4bb (5 repeats of 27 bp; 420 bp) and 4aa (4 repeats of 27 bp; 393 bp) DNA fragments on a 3% NuSieve agarose gel. DNA marker was ØX174 DNA/BsuRI9(HaeIII) (Fermentas Life Sciences).
T786C polymorphism in the promoter region
The sense 5'- TGG AGA GTG CTG GTG TAC CCC A -3' and antisense 5'- GCC TCC ACC CCC ACC CTG TC-3' primers were used in a total volume of 50 μL, containing 400 ng of template DNA, 6.25 pmol/μL of each primer, 0.25 mmol/L of each dNTP, 5 μL of 10 × PCR buffer, 1.5 mmol/L of MgCl2, and 3 U of Taq DNA Polymerase (Fermentas Life Sciences). The reaction conditions used were as follows: 1 step of denaturation at 94°C for 5 minutes followed by 40 cycles comprising 1 minute at 94°C for denaturation, 1 minute at 61°C for annealing, 1 minute at 72°C for extension, and 1 step of extension at 72°C for 5 minutes using ICycler (BioRad). Polymerase chain reaction products were checked on a 2% agarose gel. Amplified products were digested with MspI (Fermentas Life Sciences) for 3 hours at 37°C. Resulting fragments of 140 and 40 bp for the wild type allele (TT) or 90, 50, and 40 bp in the case of polymorphic allele (CC) were determined by separating them on a 2% agarose gel. DNA marker was a 50-bp DNA ladder (New England Biolabs, Frankfurt, Germany).
G894T polymorphism in exon 7
The sequences of the sense and antisense primers were 5'- AAG GCA GGA GAC AGT GGA TGG A-3' and 5'- CCC AGT CAA TCC CTT TGG TGC TCA-3', respectively. Polymerase chain reactions were performed in a total volume of 50 μL, containing 400 ng of template DNA, 6.25 pmol/μL of each primer, 0.25 mmol/L of each dNTP, 5 μL of 10 × PCR buffer, 1.5 mmol/L of MgCl2, and 3 U of Taq DNA Polymerase (Fermentas Life Sciences) with the same cycles as described previously for the promoter region. The resulting 268-bp fragment was digested with MboI (Fermentas Life Sciences) for 3 hours at 37°C, producing 178- and 90-bp fragments (polymorphic allele) (TT) or no digestion (wild type) (GG). Restriction sites were confirmed on 2% agarose gel. DNA marker was ØX174 DNA/BsuRI9(HaeIII) (Fermentas Life Sciences).
Statistical Analysis
The descriptive characteristics were presented as mean and SD for numeric variables and as counts and percentages for the categorical ones. The differences between the 2 study groups were analyzed using the Student t test for age and for χ2 tests with Monte Carlo approximation for exact probability calculations, where appropriate, for the presence of polymorphisms and presence of comorbidities. Risks were calculated in odds ratios (ORs) with 95% confidence intervals. Adjustments for age and sex for the protective effect of the presence of intron 4a in the intron region and the analyses of possible interactions with the presence of other polymorphisims were performed using the logistic regression test. Hardy-Weinberg equilibrium was tested in the control group to investigate a possible significant deviation from the expected genotype frequencies of a population with random mating.
RESULTS
The demographic characteristics and incidences of cardiovascular risk factors of study patients are shown on Table 1. The mean ± SD age distributions are 56.9 ± 8.4 years for patients with CSX and 47.1 ± 8.4 years for the control group. Hypertension and hyperlipidemia prevalence comprises 47.8% and 39.1%, respectively, in the CSX group and 5.5% and 1.4% in the controls.
The electrophoretic results for the 3 eNOS gene polymorphisms are shown in Figure 1 as examples. Regarding the eNOS polymorphisms studied, only the incidence of VNTR in intron 4 was shown to differ significantly between patients and controls (Table 2). The genotype distribution in the control group maintained Hardy-Weinberg equilibrium for all alleles. The presence of intron 4a (4 repeats of 27 bp) of the eNOS gene in any allele is significantly different between patients (22.2%) and controls (47.9%). The presence of intron 4a is 3.2 (OR) times protective (95% CI, 1.5-6.8) for the risk of CSX disease (Table 3). There were no significant differences regarding the frequencies of other eNOS polymorphisms between patients and controls. Also, there were no significant interactions among the 3 loci in CSX risk. Significant difference regarding the prevalence of intron 4a of eNOS gene still persisted after exclusion of patients with hypertension and hyperlipidemia (20.8% in patients vs 46.4% in controls, P = 0.028). The protective effect of intron 4a polymorphism also holds after adjusting for age and sex (OR, 3.6; 95% CI, 1.3-9.7).
DISCUSSION
In our study, we found that intron 4a genotype of eNOS gene is protective for CSX in Turkish patients. The protective effect of intron 4a polymorphism for CSX was observed after adjusting for age and sex. Our results are consistent with the theory suggesting the role of altered release and activity of NO as a cause for angina in these patients.12
It is established that a large proportion of patients with chest pain and normal coronary arteries have both abnormal vasodilator coronary blood flow responses and increased sensitivity of the coronary microcirculation to vasoconstrictor stimuli.8The presence of microvascular dysfunction has been confirmed by several studies, demonstrating an impairment of both endothelium-dependent and endothelium-independent coronary vasodilatation.19The endothelium produces diverse vasoactive substances such as vasodilator NO and vasoconstrictor endothelin and the balanced opposition of these substances determine vascular tone. It has been hypothesized that failure of balanced opposition between these mediators in the presence of endothelial dysfunction may be responsible for microvascular angina in patients with CSX.4,20Genetically, alterations in release and/or activity of these substances may create susceptibility to the development of microvascular angina. Given the importance of endothelial NO in cardiovascular homeostasis, genetic polymorphisms play role in the risk and/or outcome of microvascular angina.
There exist quite limited data in the interactions of eNOS gene polymorphisms and CSX at the time being. Kolasińska-Kloch et al.16also found an association of VNTR in intron of eNOS gene with patients with CSX, similar to our results. Although measuring NO levels has complexity, they revealed lower NO levels in patients with CSX with intron 4a polymorphism and −894T polymorphism in exon 7. The concentration of NO can be estimated by determining both nitrite and nitrate levels in biological samples. However, the limitation is the environmental factors, especially dietary nitrite/nitrate content, which could be a confounding factor for the plasma measurements. Therefore, only a limited number of studies exist describing the functional effects of eNOS polymorphisms. Tsukada et al.17found statistically significant reductions (10%-20%) in mean plasma NO concentrations in homozygotes of intron 4a. Allelic variations may either directly affect the properties of the eNOS enzyme or indirectly be linked to additional variations in eNOS gene that produce direct effects. Mutations within introns could affect rates of eNOS transcription or processing of the primary transcript, thus such mutations could ultimately affect eNOS enzyme levels. It was also demonstrated a cis-acting role of VNTR in intron 4, in eNOS transcription.21With these observations, our findings suggest that intron 4a polymorphism may play a pivotal role in eNOS gene expression and eventual endothelial function in CSX and seems to have a protective role in the development of microvascular angina.
As discussed by the authors, one polymorphism may be in linkage disequilibrium with a regulatory polymorphism on the same haplotype.22Therefore, beside the main effects, we also tested the multiple loci interactions to overcome the problems caused by using single polymorphisms in genetic association studies, which were insignificant.
No association with T786C polymorphism in the promoter region in Turkish patients with CSX was found, whereas Nakayama et al.23found greater incidence of T786C polymorphism in Japanese patients with coronary vasospasm. The pattern of eNOS polymorphisms may vary noticeably among different ethnic groups. Moreover, environmental exposure may modify the influence of eNOS variants on disease risk.24No significant effect of G894T polymorphism in exon 7 on the risk and extent of Turkish patients with CSX was also found, like most studies on cardiovascular disease risks for this polymorphism. Other genetic variants such as VNTR in intron 4 may be relevant.25-29However, reports describing associations of this polymorphism with various other diseases exist. Although G894T in exon 7 polymorphism exists in the coding region and is more susceptible to lower eNOS levels,30-32we could not find any significant role of G894T polymorphism in exon 7 on microvascular angina.
Our study has limitations with regard to the sample size when reporting insignificant associations and the difference in demographic characteristics between the patients and the controls. Statistical adjustments by age and sex were introduced to overcome these pitfalls.
In conclusion, our study demonstrates a protective effect of intron 4a allele of eNOS gene in CSX in Turkish patients. Current evidence suggests that the eNOS G894T polymorphism in exon 7 and T786C polymorphic promoter play no role as a risk factor for CSX in Turkish patients. Further studies using larger samples of eNOS gene polymorphisms in combination with other relevant genes could provide more insight into microvascular angina process.
ACKNOWLEDGMENT
The authors thank the Turkish Society of Cardiology for their support to the study.