For a number of cardiovascular diseases, it has been proposed that both susceptibility to disease and interindividual variability in response to treatment relates in part to genetic variants. β-Adrenergic receptors, possibly the most intensively studied receptors of G-protein-coupled receptors, have a major role in the field of cardiovascular diseases 1. The human heart expresses β1-adrenergic and β2-adrenergic receptors that increase cardiac contractility. In addition, β3-adrenergic receptors have been described to mediate negative inotropic effects, but their role remains uncertain. All three subtypes of ADR appear to occur in cardiomyocytes, and they seem to possess distinct intracellular signaling and functional properties 2.
The human adrenergic receptors are highly polymorphic. The sequence variants ADRB3 (C190T or Trp64Arg, rs4994), ADRB1 (C1165G or Arg389Gly, rs1801253), and ADRA2A (C-1291G, rs1800544) have been found to be associated with a range of cardiovascular diseases and cardiovascular risk factors. The functional relevance of these variants has been extensively evaluated in smooth muscle cells and in transgenic mice 3 and is mostly related to cardiac function 4,5.
The β3-adrenergic receptor (ADRB3) gene has been mapped to chromosome 8p12-p11.2 6. A missense mutation of the human ADRB3 gene replacing tryptophan with arginine at codon 64 (Trp64Arg) has been reported to be associated with cardiovascular risk factors such as obesity 7, insulin resistance 8, earlier onset of noninsulin-dependent diabetes mellitus 9, and elevated blood pressure 10. The glycine (Gly389) substitution of the arginine in position 389 of ADRB1 gene causes the receptor to have loss of contractile performance, further followed by increased myocardial fibrosis, and heart failure 11. Another polymorphism, ADRA2A C-1291G (rs1800544) maps to chromosome 10q24-q26 and is located in the promoter region of ADRA2A gene. The homozygous variant of C-1291G polymorphism alters gene expression and increases receptor density. The altered receptor further influences abdominal obesity and estimates of insulin, glucose, and lipid metabolism and risk of cardiac heart failure.
It is important to note that these receptors are highly expressed throughout the cardiovascular system, in which they regulate coronary vasodilatation 12. Therefore, taking into consideration the pathophysiological consequences of ADRB3, ADRA2A, and ADRB1 receptors in cardiovascular function, the present study was designed to investigate the association between adrenergic receptor polymorphisms and coronary artery disease (CAD) in a North Indian population.
Materials and methods
The present study was performed in a total of 600 CAD patients. All patients were recruited from July 2008 to September 2012 from the Department of Cardiology of Sanjay Gandhi Postgraduate Institute of Medical Sciences (SGPGIMS), Lucknow, Uttar Pradesh, India. All patients had significant CAD, as confirmed by coronary angiography. Further, all the patients underwent coronary angioplasty. The echo assessments of respective patients were noted just before the coronary angioplasty procedure (irrespective of the myocardial infarction event). The detailed clinical history of CAD patients was based on hospital investigations including coronary angiography. Angiographically identified stenoses greater than 70% in the major coronary vessels at the time of the study were used to classify patients as having single-vessel, double-vessel, or triple-vessel disease. The CAD patients having any other cardiac disorders like cardiomyopathies and valvular disease were excluded from the study.
The control (non-CAD) population consisted of 200 individuals (173 men and 27 women) (mean age 54.10±8.30 years) with no clinical evidence of CAD and also without positive family history of CAD or myocardial infarction. Furthermore, the inclusion criteria for controls were absence of prior history of abnormal lipid profile, hypertension, and obesity. Both patients and controls were frequency-matched to age, sex, and ethnicity. To test the possibility for population stratification, the genomic control method was used as described by Devlin and Roeder 13. After obtaining informed consent, all individuals were personally interviewed for information on food habits, occupation, and tobacco usage. The study was approved by local ethical review committees of the institute and the authors followed the norms of the World’s Association Declaration of Helsinki 14.
The clinical data were obtained by reviewing the patient’s medical records. Left ventricle ejection fraction (LVEF) was calculated quantitatively by echocardiography, just before the angiography procedure, using the Simpson’s method 15. Echocardiography was repeated in 10% of patients and results were totally concordant. Hypertension was defined as systolic blood pressure greater than 140 mmHg or a diastolic blood pressure greater than 90 mmHg or patients using antihypertensive drugs. Smoking was classified as smokers (exsmoker and current smokers) and nonsmokers. Similarly, diabetes mellitus was defined as patients with a fasting plasma glucose level greater than 6.9 mmol/l or patients using antidiabetic medication. The height and weight for each patient were recorded to calculate the BMI status, followed by the risk of obesity. All laboratory parameters, as stated in the medical record, were determined in overnight-fasting patients. Total cholesterol, HDL–cholesterol, and triglyceride levels were measured by standard enzymatic methods. LDL-cholesterol concentrations were calculated using the Friedewald’s formula 16.
Genomic DNA was isolated from peripheral blood leukocytes according to a standard salting out method 17. The ADRB3 T190C, ADRA2A C-1291G, and ADRB1 C1165G polymorphisms were determined by PCR-restriction fragment length polymorphism method 18. As a negative control, PCR mix without DNA sample was used to ensure contamination-free PCR product. The digested PCR fragments were separated on 15% polyacrylamide gels, stained with ethidium bromide and observed using an ultraviolet imaging system (BioRad Gel-Doc EZ imager; BioRad, Philadelphia, Pennsylvania, USA). Genotyping was performed without knowledge of the case or control status. To validate the genotyping, 10% of samples were regenotyped by other laboratory personnel and genotyping results were reproducible with no discrepancy.
The sample size was calculated using the QUANTO 1.1 program (http://hydra.usc.edu/gxe) using minor allele frequency data from HapMap (http://www.hapmap.org/). Desired power of study was set at 80%. Descriptive statistics were presented as mean and SD for continuous measures, whereas absolute value and percentages were used for categorical measures. The χ2 goodness of fit test was used for any deviation from Hardy–Weinberg equilibrium in controls. Differences in genotype and allele frequencies between study groups were estimated by χ2-test. Association was expressed as odds ratios (OR) or risk estimates with 95% confidence intervals. In addition, the association between adrenergic β-receptor gene polymorphisms and significant risk factors of CAD was analyzed using binary logistic regression. P-values less than 0.05 were considered significant. All analyses were performed using the SPSS statistical analysis software, version 16.0 (SPSS Inc., Chicago, Illinois, USA).
The putative functional effects were determined in coding region of ADRD3 gene by online web servers FASTSNP (http://fastsnp.ibms.sinica.edu.tw) and F-SNP http://compbio.cs.queensu.ca/F-SNP/19 and for coding region the effect on the protein structure was considered.
Demographic and clinical characteristics of CAD patients are shown in Table 1. There was no significant difference in the mean age of CAD patients and controls. The male/female ratio was comparable in both CAD cases as well as in controls. Evaluation of the defined risk factors showed that 44.8% patients were hypertensive and 29.2% patients were diabetic. Moreover, 24.2% patients were associated with smoking and the calculated BMI corresponded to 24.49±3.10. Patients admitted with stable angina were 28.3% and unstable angina/non-ST-segment elevation myocardial infarction formed 21.9% of the clinical syndrome. ST-segment elevation myocardial infarction patients with anterior wall myocardial infarction and inferior wall myocardial infarction were 28.1 and 21.3%, respectively. Only two patients were found to be affected with lateral wall myocardial infarction (0.4%). The angiographic profile categorized patients with single-vessel disease, double-vessel disease, and triple-vessel disease as 75.8, 19.3, and 4.8%, respectively. The mean ejection fraction was 49.32±10.16. The kurtosis and skewness for LVEF were 0.09 and −0.82. Thus, the data appeared to be normally distributed. Of the total 600 CAD patients, 68.3% showed preserved (>45%) ejection fraction, whereas 31.7% had reduced ejection fractions (≤45%).
Analysis of adrenergic receptor gene polymorphisms between CAD patients and controls
The observed frequencies of the three studied polymorphisms (ADRB3 T190C, ADRA2AC-1291G, and ADRB1 C1165G) in controls were in accordance with Hardy–Weinberg equilibrium (P>0.05). Table 2 shows the risk of CAD in relation to the single nucleotide polymorphisms (SNPs) of adrenergic receptors. On comparing the genotype frequency distribution in CAD patients with that of controls, no significant difference was observed in the distribution of ADRA2A C-1291G and ADRB1 C1165G polymorphisms both at the genotypic and allelic level. However, on comparing the frequency distribution of ADRB3 T190C in CAD patients and healthy controls, significant association was observed with CC genotype of ADRB3 T190C polymorphism (P=0.040, OR=1.5; Table 2). Also, at allelic level C allele of ADRB3 T190C conferred risk for CAD (P=0.005, OR=1.7; Table 2).
Influence of adrenergic receptor genetic polymorphisms on CAD risk due to established risk factors
A case only study was performed to estimate the correlation between the genotypes of ADRB3 T190C, ADRA2AC-1291G, and ADRB1 C1165G and the established risk factors (diabetes mellitus, hypertension, and smoking status and lipid levels) for CAD. The individuals were compared for the distribution of ADRB3 T190C, ADRA2AC-1291G, and ADRB1 C1165G genotypes with risk factors. However, all the three selected genetic variants in adrenergic receptors were not found to modulate the CAD risk by diabetes, hypertension, smoking, and lipid levels (Table 3).
Analysis of adrenergic receptor gene polymorphisms between patients with reduced (LVEF≤45) and preserved (LVEF>45) left ventricular ejection fraction
CAD is an established risk factor for left ventricular dysfunction (LVD) so we segregated the CAD patients on the basis of reduced (≤45%) and preserved (>45%) LVEF and compared their status with ADRB3 T190C, ADRA2AC-1291G, and ADRB1 C1165G polymorphisms. There were no significant differences in the distributions of ADRB3 T190C, ADRA2AC-1291G, and ADRB1 C1165G polymorphisms in both reduced and preserved LVEF (P=0.093, 0.856, 0.595, respectively). Thus, the adrenergic receptor sequence variants conferred no risk of the LVD in CAD patients (Table 4).
In-silico analysis of ADRB3 genetic variants on gene activity
As the SNP of ADRB3 is located in coding sequence, it was plausible that the SNPs may have influence on transcription of the gene. In-silico analysis using FAST-SNP and F-SNP showed change in transcriptional regulation and alternate splicing for the selected SNP (Table 5).
In the present study we used the candidate gene approach to determine whether the genetic variants of ADR genes – ADRB3 T190C, ADRA2A C-1291G, and ADRB1 C1165G are involved in the development of CAD. The main finding of the study indicates significant association of ADRB3 T190C polymorphism with higher risk of CAD. However no association was observed for ADRB1 C1165G and ADRA2A C-1291G polymorphisms with CAD.
Earlier studies have reported that the ADRB3 T190C polymorphism is associated with some cardiovascular risk factors such as obesity 7, insulin resistance 8, and earlier onset of noninsulin-dependent diabetes mellitus 9. However, the data on effect of ADRB3 polymorphisms on lipid metabolism are inconsistent and heterogeneous. Some studies did not find any correlation between 190C allele and serum lipids 8,20. In contrast, a study conducted on a young Danish population showed that the risk genotype had associations with hypertriglyceridemia and increased LDL-cholesterol levels 21. In our study, we observed that ADRB3 190C carriers had low triglyceride levels, but none of lipid parameter attained statistical significance. Such discordant result may be partially explained by ethnicity, age, or population differences in various studies.
The gene product of ADRB3 is localized in human ventricle, coronary arteries, vasculature 22, human skeletal muscle, brown and white adipose tissues 23, and gastrointestinal tract 24 where it is thought to mediate relaxation and increase mucosal blood flow. Beyond metabolic functions, β3-adrenergic receptors regulate cardiac inotropy, angiogenesis, and endothelium-dependent vasorelaxation in the coronary microvasculature 25,26. Experimental data clearly shows that, besides its effect on lipolysis and biological energy production, ADRB3 may modulate peripheral vascular tone and increase the blood pressure 8. Some clinical studies also point out possible relationship between ADRB3 T190C polymorphism 8,27–29 and arterial hypertension as well as higher mortality among hypertensive patients 30. Thus, the ADRB3 T190C polymorphism results in lowered responsiveness to potent agonists including endogenous catecholamines 31. The underlying mechanism for the development of CAD could be that the substitution of T190 to 190C in ADRB3 might result in decreased lipolysis, which might raise the possibility of change in lipid profile in human. Higher lipid levels may cause atherosclerosis and due to decrease of lipolytic activity, plaques formed in arteries are unable to get dissolved and thus setting the stage for atherosclerotic plaque formation. Our in-silico studies also suggest the influence of genetic variants of adrenergic receptor on gene transcription and splicing mechanisms.
Genetic variants of the cardiac ADRB1 and ADRA2A have been linked with heart failure 32. A study by Small et al.33 concluded that the risk for heart failure in blacks was increased for the ADRB1 Arg389 variant when combined with the ADRA2A-receptor deletion polymorphism. These polymorphisms have also been associated with metabolic syndrome, atrial fibrillation, and myocardial ischemia. However in the present study we did not find any significant association of ADRA2A and ADRB1 genetic variants with either CAD or LVEF, which is closely related to heart failure risk. The reason behind this discrepancy could again be the population variation. The allelic frequencies of ADRB1 and ADRA2A polymorphisms were not comparable between the studies. Also, the total number of cases enrolled was relatively small in earlier studies, which may raise the chances of type II β error in those studies. It is also possible that the SNPs of ADRB1 and ADRA2A may not be conferring direct effects on CAD susceptibility and their effects may be mediated through their linkage to some key functional polymorphisms.
In the present study, the sample size is sufficient to yield 80% power but it is limited in subgroup and clinical variable analysis. Therefore, study may require confirmation in larger cohorts. As, this is an association study, we cannot rule out the presence of possible linkage disequilibrium with other neighboring genes that might explain the significant association with CAD phenotypes or adverse prognosis.
Our study showed that ADRB3 T190C confers increased risk for the development of CAD, whereas no association was observed for ADRA2A C-1291G and ADRB1 C1165G polymorphisms either CAD or LVD.
The funding for the study was provided by the Department of Biotechnology, Government of India. Research fellowships to authors A.M., A.S. and T.M. by ICMR, New Delhi are gratefully acknowledged.
Conflicts of interest
There are no conflicts of interest.
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