1. Introduction
Coronary heart disease (CHD) has reached epidemic proportions among Indians. India is undergoing a rapid health transition with an increasing burden caused by coronary heart disease [
1]. For adults over 20 years old, the estimated prevalence of CHD is around 4% in rural areas and 10% in urban areas, representing a two-fold rise in rural areas and a six-fold rise in urban areas between 1960 and 2000 [
2]. Also, an increased susceptibility to CHD among Indian migrants in various parts of the world in comparison to the native population studied has been reported [
3,
4]. Several factors are suspected to cause high CHD morbidity and mortality rates among Indians, including genetic, metabolic, early-life, conventional, and non-conventional risk factors. However, the results from the interheart study conclusively established the role of behavioral and conventional risk factors in the prediction of CHD risk among Indians [
5].
The catechol-
O-methyltransferase is coded as the
COMT gene and is one of the enzymes essential for metabolizing circulating serum catecholamines, catalyzes the
O-methylation of various compounds, like catechol estrogens and dietary polyphenols, using S-adenosylmethionine (SAM) as the methyl donor. COMT also has a role in dopamine inactivation and thus is involved in the regulation of vascular resistance. Two versions of this enzyme are synthesized from the gene [
6]. The longer form, called membrane-bound catechol-
O-methyltransferase (MB-COMT), is chiefly produced by nerve cells in the brain. Other tissues, including the liver, kidneys, and blood, produce a shorter form of the enzyme called soluble catechol-
O-methyltransferase (S-COMT). This form of the enzyme helps control the levels of certain hormones. The general function of COMT is the elimination of biologically active or toxic catechols and some other hydroxylated metabolites. During the first trimester of pregnancy, COMT present in the placenta protects the developing embryo from activated hydroxylated compounds [
7,
8]. COMT also acts as an enzymatic detoxicating barrier between the blood and other tissues, shielding against the detrimental effects of xenobiotics. COMT may serve some unique or indirect functions in the kidney and intestinal tract by modulating the dopaminergic tone. The same may be true in the brain, where only one single gene COMT codes for both S-COMT and MB-COMT using two separate promoters. Both rat and human S-COMTs contain 221 amino acids, and their molecular weights are 24.8 and 24.4 kD, respectively. Rat MB-COMT contains 43 and human MB-COMT contains 50 additional amino acids, of which 17 (rat) and 20 (human) are hydrophobic membrane anchors [
9,
10].
The remainder of the MB-COMT molecule is suspended on the cytoplasmic side of the intracellular membrane. The enzyme coding region contains a well-investigated single-nucleotide-polymorphism (SNP) in codon 158 (rs4680), in which valine (Val) is substituted to methionine (Met) through a substitution of guanine to adenine. This common functional A/G polymorphism in the
COMT gene produces three genotype groups (homozygousVal158Val, heterozygous Val158Met, and homozygous Met158Met), which result in different enzyme activities based on changes in thermostability [
11,
12] as depicted in
Table 1. The three phenotypes of the COMT enzyme activities include COMT A/A with low enzyme activity, COMT A/G with medium enzyme activity and COMT G/G with high enzyme activity. The Met allele is associated with low enzymatic activity resulting in higher levels of prefrontal dopamine. Conversely, the Val allele is associated with high enzymatic activity and lower levels of prefrontal dopamine. The Met allele has been associated with several psychiatric disorders such as panic disorder [
13,
14].
COMT-Val158Met-polymorphism has been intensively studied. Associations have been found with the susceptibility and appearance of cognitive phenotypes, psychiatric disorders, changes in brain activation and structure, and cancer susceptibility [
15,
16]. Furthermore, links between COMT genotypes and the increased risk of coronary events or the outcome of patients with ischemic heart disease were revealed [
17,
18]. COMT Val158Met polymorphism is associated with the increased risk of acute coronary events and it may interact with high serum tHcy levels [
19]. Several small population-based studies found genetic variation in
COMT to be associated with coronary heart disease [
20] and hypertension in men [
21,
22,
23]. Based on study findings, the Val/Val genotype appears to be associated with a higher prevalence of increased systolic blood pressure compared with the Met/Met or Met/Val genotypes at the
COMT gene [
24]. In another study, the low activity COMT genotype (Met158Met) was associated with heavy alcohol use [
25]. The influence of COMT G158A polymorphism was studied in some human cancers, such as breast [
26], endometrial [
27], and prostate carcinomas [
28]. The unifying hypothesis is that hyperhomocysteinemia may exert its pathogenic effects largely through metabolic accumulation of S-adenosyl-
l-homocysteine (SAH)a potent non-competitive inhibitor of COMT-mediated methylation metabolism of various catechol substrates. Therefore, we wanted to test the hypothesis that the functional polymorphism Val108Met in the
COMT gene could modify the risk of a coronary event in our population.
2. Materials and Methods
2.1. Study Population
This study was a population-based cohort study designed to investigate risk factors for CAD related to outcomes in middle-aged men and women from India. A total of 200 blood samples were analyzed among which 100 were from CAD patients (96 men and 4 women) and 100 healthy controls.
2.2. Patient Selection Criteria
2.2.1. Inclusion Criteria
Patients undergoing elective angiography for the evaluation of stable chest pain at the Sri Jayadeva Institute of Cardiovascular Science and Research and Karnataka Institute of Diabetology, Bangalore were recruited. Some non-invasive tests were performed including an electrocardiogram (ECG or EKG), ambulatory electrocardiography, Holter monitoring, chest X-ray, echocardiogram (echo), cardiac computed tomography (CCT), exercise stress test and Myocardial Perfusion Imaging (MPI) or Multigated acquisition scan (MUGA). Patients were not selected on the basis of the chest pain characteristics (i.e., whether or not suspicious of angina or myocardial ischemia), but merely that chest pain was a presenting symptom prompting an elective angiography. The cohort was classified based on their coronary angiographic findings as either significant CAD (stenosis ≥ 50%) or ICAD (no stenosis or stenosis < 50%). The exclusion criteria included patients with a history of non-coronary cardiac disorders cases, previously performed coronary bypass surgery, or percutaneous transluminal coronary angioplasty (PTCA) due to their treated coronary status.
2.2.2. Selection Criteria of Healthy Controls
A healthy control cohort was established from participants visiting for routine checkup to Sri Jayadeva Institute of Cardiovascular Science and Research and Karnataka Institute of Diabetology, Bangalore. These participants completed the informed consent form and questionnaire. The healthy control cohort was selected based on self-reported absence of previous heart attack or angina. Some biochemistry tests were also performed. The exclusion criteria included participants with available cardiac history, history of angina and/or myocardial infarction.
2.3. Sample Collection
From each patient, a 4 mL peripheral blood sample was collected in EDTA vials after obtaining informed consent from all patients. The samples were collected only after the approval letter was obtained from the concerned Institutional Ethics Committee.
2.4. Measurement of Serum Lipids and Apolipoproteins
After overnight fasting and prior to coronary angiography, blood was collected from each subject. Serum total cholesterol, triglyceride, high density lipoprotein (HDL), and low density lipoprotein (LDL)-C concentrations and total cholesterol/HDL-C ratios were determined using the standard method.
2.5. Data Collection
The corresponding data from each patient were collected and analyzed for their cholesterol level, diabetes, RBS, LDL, HDL, TGL, and smoking. The patient follow-up was performed regularly and samples were collected on a regular basis.
2.6. Genotyping Analysis
2.6.1. DNA Extraction
Genomic DNA was extracted using a Blood DNA Isolation Kit (GEB100) from Geneaid (Taipei, Taiwan) from the whole blood according to manufacturer’s instructions. The mean optical density (OD) of the DNA samples was determined as 1.8 nm using Nanodrop.
2.6.2. AS-PCR for COMT (Val158Met) gene Polymorphism
The enzyme coding region of COMT contains a well-investigated single nucleotide polymorphism (SNP) (rs4680), with the presence of guanine or adenine at nucleotide 475, encoding a valine (Val) or methionine (Met) at codon 158, producing three genotype groups: homozygous Val158Val or GG, heterozygous Val158Met or G/A, and homozygous Met158Met or AA. The same primers were used for detecting COMT 475A > G gene polymorphism as shown in
Table 2.
COMT (Val158Met) G > A gene polymorphism was identified by AS-PCR. The amplification was accomplished with a 25-μL reaction mixture containing 100 ng template DNA, 0.25 μL of 25 pmol of each primer, 2.5 μL 10 mM dNTP’s 1.5 μL of 20 mM MgCl
2, and 0.3 μL of 5 U/μL Taq polymerase with 2.5 μL of 10X Taq Buffer (Fermantas, Waltham, MA, USA). The thermocycling program involved initial denaturation at 96 °C for 10 min and 30 cycles: 96 °C for 30 s, 56 °C for 45 s, 72 °C for 45 s, and final extension at 72 °C for 5 min. Presence or absence of each allele (PCR products) were observed by electrophoresis on 2% agarose gel. After electrophoresis, amplified products were visualized by ethidium bromide stain using ultraviolet trans-illumination as depicted in
Figure 1. Genotyping was performed without the knowledge of the case/control status of the study subjects.
2.7. Stastical Analysis
CAD patients and controls were compared using statistical analysis performed using the SPSS 16.0 software package. Chi-square analysis and Fisher exact test were used to compare COMT rs4680 G > A gene polymorphism frequency with several clinical aspects, including sex, age, cholesterol, HDL, LDL, TGL, hypertension, diabetes, alcohol, Pan Masala, and smoking. Hardy-Weinberg equilibrium was tested with a χ2 test to compare the observed genotype frequencies within the case-control groups. The p value was considered to be significant when <0.05.
4. Discussion
Catechol-
O-methyltransferase is one of several enzymes that degrade catecholamines such as dopamine, epinephrine, and norepinephrine. As the regulation of catecholamines is impaired in a number of medical conditions, several pharmaceutical drugs target COMT to alter its activity and therefore the availability of catecholamines. Levodopa, a precursor of catecholamines, is an important substrate of COMT. COMT inhibitors, like entacapone, save levodopa from COMT and prolong the action of levodopa. Entacapone is a widely used adjunct drug with levodopa therapy. When given with an inhibitor of dopa decarboxylase (carbidopa or benserazide), levodopa is optimally saved. This “triple therapy” is becoming a standard in the treatment of Parkinson’s disease [
29].
The enzyme coding region contains a well-investigated single-nucleotide-polymorphism (SNP) in codon 158 (rs4680), in which valine (Val) is substituted by methionine (Met) through a substitution of guanine to adenine, which subsequently leads to different enzyme activities based on changes in thermostability. The Met/Met-genotype has a three- to four-fold reduced enzymatic activity compared with the Val/Val-genotype The Val variant catabolizes dopamine at up to four times the rate of its methionine counterpart [
6].
COMT-Val158Met-polymorphism has been intensively studied. Associations have been found with the susceptibility and appearance of cognitive phenotypes, psychiatric disorders, changes in brain activation and structure, and cancer susceptibility. Furthermore, links between COMT genotypes and the increased risk of coronary events or the outcome of patients with ischemic heart disease have been revealed [
8].
The main finding of our study is that common functional Val158Met polymorphism of the
COMT gene is an independent risk factor for coronary artery disease events in the Indian population, which may also interact with serum haemocystein, alcohol, and triglycerides to further increase the risk of coronary events. Men who were homozygous (Met/Met-genotype) for the low activity allele of COMT had increased serum levels of estradiol, which has shown that the altered estrogen status could be involved in this effect. In that study, 74% of study subjects were men and the interaction between COMT genotype and homocysteine were not studied [
26,
27]. Opposite results were reported, in which the low activity genotype of COMT reduced the risk of myocardial infarction.
The possible mechanism behind the protective effect of the low activity COMT genotype on myocardial infarction is not fully understood. However, altered estrogen status could be involved in the protective effect of this genotype, since the low activity COMT genotype less efficiently removes estrogens, which, as a consequence, could lead to more cardiovascular protection from estrogen. Not estrogen itself but its hydroxylated metabolites are thought to at least partially mediate the cardiovascular protection provided by estrogen [
27]. Thus, increased levels of estrogen and/or the hydroxylated metabolites might be involved in the cardioprotective effect of the low activity COMT genotype.
The cardioprotective effect of low activity COMT genotype was most evident in older patients [
30]. The reason for this could be that the level of estrogen might be sufficient to provide cardiovascular protection independent of COMT activity at younger ages. However, with increasing age, the levels decline and approach a critical level, where the low activity COMT enzyme results in protection by slowing down the degradation of estrogen and its metabolites [
31]. Previous studies showed that type I alcoholism is more common among subjects with the low activity COMT genotype (LL), compared with high activity (HH) or heterozygotic (LH) genotypes. Alcoholism and heavy smoking are highly comorbid and are cotransmitted in the Indian population. Catechol-
O-methyltransferase (COMT) functional polymorphism, Val158Met, has been associated with alcoholism in Caucasians [
32]. Our study investigated the association of alcohol, pan masala consumption, and tobacco use with Val158Met polymorphism and results indicated that the low activity COMT genotype may contribute significantly to alcohol intake in the general male population.
Our results require confirmation in an independent population of patients and controls. The frequency of COMT low activity genotype was higher among the subjects with abnormal RBS and diabetes. Association of the
COMT gene with diabetes and nephropathy has been reported in a study conducted on an Asian Indian population in which a genetic variant showed an association with diabetic nephropathy [
33]. An important strength of the present study is that it is a retrospective study with very well characterized subjects and cardiovascular endpoints. Strikingly, the most prominent risk factors for CAD were hypertension, diabetes mellitus, and metabolic syndrome, which are associated with a reduced antioxidant capacity and increased oxidative stress, resulting in endothelial dysfunction, crucial and early events in atherogenesis. Endothelial dysfunction is a major player in the development and progression of vascular pathology in pulmonary arterial hypertension. The alteration of the shear forces alters the biology of the endothelial monolayer and subsequently the susceptibility of vessels to atherosclerosis. Patients with intracranial atherosclerotic disease (ICAD) ICAD often have coexisting systemic atherosclerosis and multiple potential stroke mechanisms that affect their prognosis, suggesting that extensive evaluations of overlapping diseases may allow better risk stratification [
34]. Ion channels that are directly implicated in the development of atherosclerosis have not been identified. This functional polymorphism results in the met variant having a 3–4 fold lower enzymatic activity than the val variant, and is, therefore, inversely correlated with endogenous levels of dopamine and other COMT substrates, both at rest and with exercise or cardiac surgery-induced stress. In addition, our population-based case control study has reported that this genetic variation in
COMT is associated with increased risk of coronary heart disease.